Effects of water stress on cellular ultrastructure and on concentrations of endogenous abscisic acid and indole-3-acetic acid in Fatsia japonica leaves

Ultrastructural alterations in mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in water-stressed field-grown plants of Fatsia japonica. Under water deficit cellular membranes were modified and an increase in vesicles was observed. The main damage to the chloroplasts included thylakoid swelling and disruption of the chloroplast envelope. Concomitant variations in abscisic acid and indole-3-acetic acid were observed. Despite the expected increased in endogenous ABA concentration in relation to water stress, after the highest concentration of ABA, observed at predawn in severely stressed plants (29-1), there was a sharp decline from 2768 pmol g fw^–1 to 145 pmol g fw^–1; thus in severely stressed plants ABA levels were not related to changes in bulk leaf ABA contents. Water stress did not influence the concentrations of indole-3-acetic acid, although the increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.Abbreviations ABA abscisic acid - IAA indole-3-acetic acid - leaf water potential     

Model systems for the immunolocalisation of cis,trans abscisic acid in plant tissues

To explore the feasibility of immunolocalisation of endogenous abscisic acid (ABA), model systems were developed for testing quantitatively the sensitivity of the second antibody peroxidase/antiperoxidse (PAP) method for immunolocalisation of ABA on plant tissues. Exogenous (±)ABA was fixed to carrot sections on glass slides or to homogenised pea cotyledon material on microtitre plates, either directly by carbodiimide fixation or by glutaraldehyde fixation of ABA-protein conjugates linked through the C1 carboxyl by 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride (EDC). Backgrounds were decreased by including 0.1% normal goat serum in the incubations, by including 0.1% Triton X-100 as a wetter, by including glycine in the rinses after EDC fixation and by using low-pH rinses after incubation with the primary antibody. Serum antibodies recognising the peptide bond between the protein and abscisic acid were removed by preincubating the serum with acetic acid conjugated to protein. Positives were only accepted when they could be eliminated by adding an excess of ABA-protein conjugate in the primary antiserum. By using a soluble peroxidase reaction product to facilitate quantitation, the limit of reliable exogenous ABA detection was found to be only of the order of 1 pmol. For the histochemical immunolocalisation of endogenous ABA, better antisera and lower backgrounds will be required.The efficiency of fixation of exogenous ABA was determined using [³H] or [¹⁴C]ABA. When aqueous EDC or di-isopropyl carbodiimide (IPC) were used the fixation efficiency was low (up to 5%), but much higher efficiencies (up to 80%) were obtained using IPC vapour with freeze-dried material. Similarly efficient fixation of endogenous ABA in pea cotyledon material, as determined by gas chromatography-mass spectrometry analysis, was obtained using the same technique. The PAP method failed to detect fixed endogenous ABA in pea cotyledons, even though the total tissue amounts present exceeded 1 pmol, evidence that not enough of the ABA was accessible to the antibody.Abbreviations ABA abscisic acid - ACE-ALP acetic acid-alkaline phosphatase - EDC 1-ethyl-3(3-dimethyl-amino-propyl) carbodiimide hydrochloride - GC-MS gas chromatographymass spectrometry - IgG Immunoglobulin G - HSA humanserum albumin - IPC dinsopropyl carbodiimide - LINK goat anti-rabbit IgG - OD optical density - PAP peroxidase/rabbit antiperoxidase complex     

Immunoelectron-microscopy localization of abscisic acid with colloidal gold on Lowicryl-embedded tissues of Chnopodium polyspermum L.

Further study on the localization of abscisic acid (ABA) has been undertaken at the ultrastructural level in Chenopodium polyspermum L. Axillary-bud-bearing nodes on the main axis were fixed with soluble 1-(3-dimethylaminopropyl)-3 ethyl carbodiimide, then postfixed with paraformaldehyde and embedded in Lowicryl K4M at-20° C. Ultrathin sections mounted on grids were successively incubated with rabbit anti-ABA antibodies and with gold-labelled goat anti-rabbit anti-bodies (40 nm particle size). Control sections treated with preimmune rabbit serum and ABA-preabsorbed antibodies were devoid of label. The background staining was very low with this technique. Quantitative analysis of the immunolabelling showed that two main sites of ABA accumulation could be defined: first, plastids in cortical cells and vascular parenchyma cells associated with sieve elements and xylem vessels; second, the cell cytoplasm and nucleus in the axillary bud tip and in procambial strands. In vascular bundles, the cambial cells showed no immunoreactivity. These observations support the hypothesis for the cytoplasmic synthesis of ABA which is subsequently trapped in plastids as cells mature.Abbreviations ABA abscisic acid - EDC 1-(3-dimethyl-aminopropyl)-3-ethyl carbodiimide - GAR 40 goat anti-rabbit antibodies labelled with colloidal gold of particle size 40 nm - IgG immunoglobulin G     

Does abscisic acid influence proline accumulation in stressed leaves?

Root treatments of barley (Hordeum distichum L.) plants with 10^-7 to 10^-4 M abscisic acid (ABA) caused an increase in proline content, especially at higher concentrations, within 2–3 h. Even 3 h after the removal of ABA from the medium the plants continued to accumulate proline. The higher the concentration of the ABA, the higher was the proline level at 6 h. When the highest ABA concentration, 10^-4 M, was tested with polyethylene glycol (PEG) (-5.0 bars) in the medium, the ABA treatment resulted in a higher proline content than in control plants. The treatments PEG alone and PEG + ABA resulted in heavy accumulation of proline, especially, 3 h after releasing the plants from the stress. The proline content in PEG+ABA-treated plants was always higher than plants treated with PGE or ABA alone. In peas (Pisum sativum L. cv Alaska) the same trend occurred although to a lesser degree. These findings indicate an influence of ABA on proline accumulation in water-stressed plants.Abbreviations ABA abscisic acid - PEG polyethylene glycol - RWC relative water content     

The monoclonal antibody JIM19 modulates abscisic acid action in barley aleurone protoplasts

A panel of hybridoma products generated against pea (Pisum sativum L.) guard-cell protoplasts has been assayed for anti-abscisic acid (ABA) biological activity in barley (Hordeum vulgare L.) aleurone protoplasts. The effects of the antibodies on ABA-induced accumulation of mRNA transcribed from RAB-16, a gene responsive to ABA, were determined. Most of the antibodies, and culture medium, had no effect, but five monoclonal antibodies (MAbs) were found to inhibit ABA-induced RAB-16 gene expression and one MAb enhanced it. The effects of one inhibitory MAb, JIM19, were studied in some detail. These effects were specific to ABA-induced events, as incubation with JIM19 had no effect on the expression of a constitutively-expressed gene, GAPDH, encoding glyceraldehyde-3-phosphate dehydrogenase, and only a slight effect on the production of -amylase induced by gibberellic acid. Increasing concentrations of ABA in the incubation medium partly overcame the inhibitory effect of JIM19. Immunolabelling and biological activity remained together during immuno-purification of JIM19 from hybridoma culture supernatant. Immunoblotting of JIM19 to membrane preparations from barley aleurone protoplasts revealed that JIM19 recognised a number of proteins.Abbreviations ABA abscisic acid - GA gibberellin - GA₃ gibberellic acid - GAPDH gene encoding glyceraldehyde-3-phosphate dehydrogenase - GCP guard-cell protoplast - MAb monoclonal antibody - RAB (gene) responsive to ABA We thank the Agricultural and Food Research Council and The Nuffield Foundation for financial support, Professor Keith Roberts (John Innes Institute, Norwich, UK) for advice and generous use of his laboratory and Jan Peart (John Innes Institute) for animal cell culture. S.J.N. is grateful to Professor Colin Hawkes (University of the West of England, Bristol) for his continued support of this project.     

Changes in abscisic acid and its glucose ester in Phaseolus vulgaris L. during chilling and water stress

Levels of free and conjugated abscisic acid (ABA) were determined in leaves and roots of intact bean (Phaseolus vulgaris L., cv. Mondragone) seedlings under chilling (3C) and drought as well as during recovery from stress. Abscisic acid-glucose ester (ABAGE) was the only conjugate releasing free ABA after alkaline hydrolysis of the crude aqueous extracts. During the first 20–30 h chilled plants rapidly dehydrated and wilted without any change in ABA and ABAGE levels. Subsequently, leaf and root ABA levels increased and plants regained turgor. ABAGE concentration showed a slight increase in leaves but not in roots. Upon recovery from chilling a transient, but significant, rise in leaf ABA content was observed, while no appreciable change in ABAGE was found. Drought triggered ABA accumulation in leaves and roots, while a rise in ABAGE content was detected only in leaf tissues. Recovery from stress caused a drop in ABA levels without a correspondent increase in ABAGE concentration. We conclude that ABAGE is not a source of free ABA during either chilling or water stress and that only a small proportion of the ABA produced under stress is metabolised to ABAGE during recovery.Abbreviations ABA = abscisic acid - ABAGE = abscisic acid-glucose ester - DW = dry weight - FW = fresh weight - RIA = radioimmunoassay - RWC = relative water content - w = water potential - o = osmotic potential - p = turgor potential     

Radioimmunoassay for the determination of free and conjugated abscisic acid

The characterization and application of a radioimmunoassay specific for free and conjugated abscisic acid (ABA) is reported. The antibodies produced against a bovine serum albumin-(±)-ABA conjugate have a high affinity for ABA (K_a=1.3x10⁹l mol^-1). Trans, trans-ABA and related compounds, such as xanthoxin, phaseic acid, dihydrophaseic acid, vomifoliol or violaxanthin do not interfere with the assay. The detection limit of this method is 0.25x10^-12 mol ABA, the measuring range extends to 20x10^-12 mol, and average recoveries are 103%. Because of the high specificity of this immunoassay, no extract purification steps are required prior to analysis. Several hundred plants can be analyzed per day in a semi-automatic assay performance. ABA has been detected in all higher plant families examined, but was absent in the blue-green alga, Spirulina platensis, the liverwort Marchantia polymorpha, and two species of fungi.Abbreviations ABA abscisic acid - BHT 2.6-di-t-butyl-4-methyl phenol - TLC thin-layer chromatography - HSA human serum albumin Part 7 in the Series: Use of Immunoassay in Plant Science     

Growth,graviresponsiveness and abscisic-acid content of Zea mays seedlings treated with Fluridone

Ten-d-old seedlings of Zea mays L. cv. Tx 5855 treated with 1-methyl-3-phenyl-5-(3-[trifluoromethyl]phenyl)-4-(1H)-pyridinone (Fluridone) were analyzed for abscisic acid (ABA) content using high-performance liquid chromatography with an analysis sensitivity of 2.5 ng ABA g^-1 fresh weight (FW). Seedlings were divided into three portions: leaves, detipped roots, and root tips (terminal 1.5 mm). Control plants (water treatment only; no Fluridone) were characterized by the following amounts of ABA: leaves, 0.114±0.024 (standard deviation) g ABA g^-1 FW; detipped roots, 0.260±0.039±g ABA g^-1 FW; root tips, no ABA detected. We did not detect any ABA in tissues of Fluridone-treated plants. Primary roots of treated and untreated seedlings were strongly graviresponsive, with no significant differences between the curvatures or the growth rates of primary roots of Fluridone-treated and control seedlings. These results indicate that 1) Fluridone completely inhibits ABA synthesis, and 2) ABA is not necessary for positive gravitropism by primary roots of Zea mays.Abbreviations ABA abscisic acid - Fluridone 1-methyl-3-phenyl-5-(3-[trifluoromethyl]phenyl)-4-(1H)-pyridinone - FW fresh weight - SD standard deviation     

Synthesis and metabolism of abscisic acid in detached leaves of Phaseolus vulgaris L. after loss and recovery of turgor

Metabolism of abscisic acid (ABA) was studied after wilting and upon recovery from water stress in individual, detached leaves of Phaseolus vulgaris L. (red kidney bean). Loss of turgor was correlated with accumulation of ABA and its metabolites, resulting in a 10-fold increase in the level of phaseic acid (PA) and a doubling of the level of conjugated ABA. The level of conjugated ABA in turgid leaves was no higher than that of the free acid. These results indicate that accumulation of ABA in wilted leaves resulted from a stimulation of ABA synthesis, rather than from a release from a conjugated form or from inhibition of the metabolism of ABA. The rate of synthesis of ABA was at its maximum between 2.5 and 5 h after turgor was lost, and slackened there-after. In wilted leaves, the rate of conversion of ABA to PA climbed steadly until it matched the rate of synthesis, after about 7.5 h. Upon rehydration of sections from wilted leaves, the rate of synthesis of ABA dropped close to zero within about 3 h, while the rate of conversion to PA accelerated. Formation of PA was two to four times faster than in sections maintained in the wilted condition; it reached a rate sufficient to convert almost one-half of the ABA present in the tissue to PA within 1 h. In contrast, the alternate route of metabolism of ABA, synthesis of conjugated ABA, was not stimulated by rehydration. The role of turgor in the stimulation of the conversion of ABA to PA was investigated. When leaves that had been wilted for 5 h were rehydrated to different degrees, the amount of ABA which disappeared, or that of PA which accumulated during the next 3 h, did not depend linearly on the water potential of the rehydrated leaf. Rather, re-establishment of the slightest positive turgor was sufficient to result in maximum stimulation of conversion of ABA to PA.Abbreviations ABA abscisic acid - DPA dihydrophaseic acid - PA phaseic acid - _leaf leaf water potential - osmotic pressure     

Endogenous plant hormones of the broad bean,Vicia faba L. (-)-jasmonic acid,a plant growth inhibitor in pericarp

(-)-Jasmonic acid was identified as a plant growth inhibitor of the pericarp of Vicia faba by means of gas-liquid chromatography, high resolution mass spectrometry, ¹H-nuclear magnetic resonance (¹H-NMR), and ¹³C-NMR. Additionally, the pericarp contains very small amounts of abscisic acid (ABA) and 4-dihydrophaseic acid. The highest level of jasmonic acid was reached prior to full pericarp length. This amount (3 g g^-1 fresh weight) is similar to the maximal ABA content in the developing seed. Jasmonic acid is a plant growth inhibitor possessing a relative activity in the wheat seedling bioassay of 1–2.5%, compared to ABA. Contrary to ABA, jasmonic acid does not cause retardation of leaf emergence. The possible physiological role of jasmonic acid in the pericarp is discussed and compared with the assumed function of ABA in developing seeds.Abbreviations ABA abscisic acid - DPA 4-dihydrophaseic acid - DPAMeTMS methyl ester trimethylsilyl ether of DPA - EtOAc ethyl acetate - Et₂O ether - MS mass spectrometry - NMR nuclear magnetic resonance - GLC gas-liquid chromatography - TLC thin-layer chromatography - UV ultraviolet light     

Gas chromatography-mass spectrometric determinations of abscisic acid levels in the cap and the apex of maize roots

Quantitative analyses of abscisic acid (ABA) in different parts of maize root tips (Zea mays L. cv. Kelvedon 33) were performed by mass fragmentography using the hexadeuterated analog of ABA as internal standard. It was found that the cap and the apex contained 36.1 g and 66.5 g ABA kg^–1 fresh weight, respectively. The possibility that the growth regulator formed in the cap and inhibiting the elongation of the extending zone of the root is ABA is discussed.Abbreviations ABA abscisic acid - ABA-D₆ hexadeuterated ABA - ABA-Me and ABA-D₆-Me methyl esters of ABA and ABA-D₆, respectively - GC-MS gas chromatograph(y)-mass spectrometry/spectrometer - IAA indol-3-yl-acetic acid - MF mass fragmentography - TMS trimethylsilyl     

Changes in cell ultrastructure and endogenous abscisic acid and indole-3-actic acid concentrations in Fatsia japonica leaves under polyethylene glycol-induced water stress

Ultrastructural alterations in epidermal and mesophyll cells as well as variations in bulk leaf endogenous ABA and IAA concentrations were studied in PEG-treated plants of Fatsia japonica Decne & Plank. Under stress induced by PEG vesicles containing fibrous material and electron-dense bodies associated with plasma membranes were observed. Cytochemical examination indicated that electron-dense bodies corresponded to lipids and the fibrous material of the vesicles were polysaccharides. Chloroplasts, mitochondria, nuclei and Golgi apparatus also showed modifications. A strong relationship was found between increasing PEG-induced water stress, increasing endogenous ABA and ultrastructural changes. In relation with leaf ontogeny and ABA concentration a higher ABA level was observed in younger than in older leaves. The differences in the endogenous concentrations of indole-3-acetic acid are unclear, except after 7 days of PEG-treatment. The increase in the endogenous abscisic acid concentration could be related with the ultrastructural changes.Abbreviations ABA = abscisic acid - IAA = indole-3-acetic acid - PEG = polyethylene glycol - = leaf water potential - TEM = transmission electron microscope     

Changes in the ratio of cis-trans to trans-trans abscisic acid during ripening of apple fruits

Immediately after harvest, abscisic acid (ABA) extracted from fruits of the apple cultivar Golden Delicious comprised solely the cis-trans isomer. During postharvest ripening, however, trans-trans ABA accumulated and finally exceeded the level of cis-trans ABA. The two geometrical isomers were separated and identified by high-performance liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry. After purification by HPLC the putative trans-trans isomer yielded considerable quantities of cis-trans ABA, when irradiated with UV light. This isomerization was more rapid than the reverse reaction. The physiological significance of the accumulation of trans-trans ABA is discussed, as well as the applications of these results in the use of trans-trans ABA as an internal standard during the extraction and quantification of ABA from plant tissues.Abbreviations ABA 2-cis-4-trans abscisic acid - t-ABA 2-trans-4-trans abscisic acid - ECD electron capture detector - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - PVP water insoluble polyvinylpyrroli-done - UV ultraviolet     

Abscisic acid and proline improve desiccation tolerance and increase fatty acid content of celery somatic embryos

Desiccation tolerance of celery (Apium graveolens L.) somatic embryos was increased by supplementation of embryo-production medium with 1 M abscisic acid (ABA) or 1 mM proline, with highest survival obtained with a combination of 1 M ABA and 1 mM proline. Addition of ABA and proline increased fatty acid accumulation by somatic embryos; the effect on fatty acid composition was inconsistent. Somatic embryos capable of germination differed from mature zygotic embryos by greater size, lower fatty acid level, and substantially lower proportion of oleic acid (18:1) as compared to linoleic acid (18:2).     

The effects of abscisic acid on the maturation ofBrassica napus somatic embryos

Summary A comparison of embryos, cultured for increasing periods of time with and without abscisic acid (ABA), was undertaken to investigate, at the ultrastructural level, the influence of this growth regulator on the maturation of rapeseed (Brassica napus) somatic embryos. In the absence of ABA, the embryos germinated precociously while lipid bodies (LB), which were not numerous, soon degraded, as revealed by a depletion process associated with the appearance of morphologically mature glyoxysomes and an increase in the number of mitochondria. Moreover, a lack of protein bodies indicated that storage protein accumulation was not initiated under these conditions. On the contrary, the addition of ABA (10 M) induced marked modification of embryo metabolism. Indeed, ABA completely prevented precocious embryo germination and inhibited lipid reserve catabolism. Moreover, the formation of small vacuoles and proliferation of rough endoplasmic reticulum in their vicinity suggested the onset of storage protein accumulation. After 15 days in the presence of ABA, the embryos contained abundant lipid and protein bodies. Nevertheless, these somatic embryos were not exactly the same as their mature zygotic counterparts since differences were found in chloroplasts, amyloplasts, and nuclear structures. These observations suggest that additional factors might be required to obtain fully mature somatic embryos.Abbreviations ABA abscisic acid - ABM ABA medium - BM basal medium - LB lipid bodies - MS Murashige and Skoog (1962) - PB protein bodies - RER rough endoplasmic reticulum     

Abscisic acid metabolism —vacuolar/extravacuolar distribution of metabolites

The biotransformation of abscisic acid (ABA) was studied in cell suspension cultures of Lycopersicon esculentum. The ABA was converted by the cells to phaseic acid, nigellic acid, dihydrophaseic acid, abscisic acid--D-glucopyranosyl ester (ABA-Glc) and other ABA and phaseic acid conjugates. Investigation of their cellular distribution showed that the conjugated forms were located only in the vacuoles whereas ABA and its acidic metabolites were found mainly in the extravacuolar fractions. Our results, together with a number of studies on the increase of ABA-Glc as a response to stress, allow us to propose that ABA-Glc is irreversibly compartmented in the vacuoles of plant cells.Abbreviations ABA abscisic acid - ABA-Glc -D-glucopyranosyl ester of ABA - DPA 4-dihydrophaseic acid; nigellic acid=3-methyl-5-(1-hydroxy-2-hydroxymethyl-6-dimethyl-4-oxo-cyclohex-2-enyl)-penta-2Z, 4E-dienoic acid - PA phaseic acid     

Identification by combined gas chromatography-mass spectrometry of phaseic acid and dihydrophaseic acid and characterization of further abscisic acid metabolites in pea seedlings

Seven day old seedlings of Pisum sativum L., cv. Kleine Rheinländerin, were wilted for 3 days. After partially removing the roots, they were rewatered and at the same time radioactive abscisic acid([1-¹⁴C]ABA, spec. activity 1.7·10⁸d s^-1mmol^-1) was applied for 1 h via the xylem of the roots. After 24 h, 4 days, and 12 days the seedlings were extracted and the metabolites of ABA were analyzed by means of thin-layer and gas chromatography in combination with mass spectrometry, autoradiography, and scintillation counting. Phaseic acid (PA) and dihydrophaseic acid (DPA) were identified as metabolites of ABA. The presence of another ABA-metabolite was also demonstrated. From its mass spectrum it has been postulated that this metabolite is 4-desoxy-ABA. In addition to these substances, several other metabolites, which are more polar than ABA and its known degradation products, were present in the seedlings. The quantity and number of these unknown metabolites increased with time.Abbreviations ABA abscisic acid - PA phaseic acid - DPA dihydrophaseic acid - TLC thin-layer chromatography - GC gas chromatography - PPO 2,5-diphenyloxazole - POPOP 2,2-p-phenylen bis(5-phenyloxazole)     

Development and storage-protein synthesis in Brassica napus L. embryos in vivo and in vitro

Immature embryos of Brassica napus were cultured in vitro with and without various concentrations of germination inhibitors, and the progress of embryogeny was monitored by comparing accumulation of storage proteins in culture with the normal accumulation in seeds. The two major B. napus storage proteins (12S and 1.7S) were purified from seed extracts and analyzed by rocket immunoelectrophoresis (12S protein) or by sodium lauryl sulfate polyacrylamide gel electrophoresis (1.7S protein). During embryo development within seeds both the 12S and 1.7S proteins were first detected when the cotyledons were well developed (embryo dry weight, 0.4 mg), and each storage protein accumulated at an average rate of 26 g d^-1 during maximum deposition. Accumulation of the 1.7S protein stopped when the water content of the embryo began to decline (embryo DW, 2.7 mg), but accumulation of the 12S protein continued until seed maturity (embryo DW, 3.6 mg). At the end of embryo development the 12S and the 1.7S proteins comprised approx. 60 and 20% of the total salt-soluble protein, respectively. When embryos were removed from seeds at day 27, just as storage protein was starting to accumulate, and placed in culture on a basal medium, they precociously germinated within 3d, and incorporation of amino acids into the 12S storage protein dropped from 3% of total incorporation to less than 1%. If 10^-6 M abscisic acid (ABA) was included in the medium, amino-acid incorporation into the 12S protein increased from 3% of total incorporation when embryos were placed into culture to 18%, 5d later, and the accumulation rate (27.1±2.6 g embryo^-1 d^-1) matched the maximum rate observed in the seed. High osmotica, such as 0.29 M sucrose or mannitol, added to the basal medium, also inhibited precocious germination, but there was a lag period before 12S-protein synthesis rates equaled the rates on ABA media. These results indicate that some factor in the seed environment is necessary for storage-protein synthesis to proceed, and that ABA is a possible candidate.Abbreviations ABA abscisic acid - PAGE polyacrylamide gel electrophoresis - PMSF phenylmethylsulfonylfluoride - SDS sodium lauryl sulfate     

Dormancy termination of western white pine (<Emphasis Type="Italic">Pinus monticola</Emphasis> Dougl. Ex D. Don) seeds is associated with changes in abscisic acid metabolism

Western white pine (Pinus monticola) seeds exhibit deep dormancy at maturity and seed populations require several months of moist chilling to reach their uppermost germination capacities. Abscisic acid (ABA) and its metabolites, phaseic acid (PA), dihydrophaseic acid (DPA), 7-hydroxy ABA (7OH ABA) and ABA-glucose ester (ABA-GE), were quantified in western white pine seeds during dormancy breakage (moist chilling) and germination using an HPLC–tandem mass spectrometry method with multiple reaction monitoring and internal standards incorporating deuterium-labeled analogs. In the seed coat, ABA and metabolite levels were high in dry seeds, but declined precipitously during the pre-moist-chilling water soak to relatively low levels thereafter. In the embryo and megagametophyte, ABA levels decreased significantly during moist chilling, coincident with an increase in the germination capacity of seeds. ABA catabolism occurred via several routes, depending on the stage and the seed tissue. Moist chilling of seeds led to increases in PA and DPA levels in both the embryo and megagametophyte. Within the embryo, 7OH ABA and ABA-GE also accumulated during moist chilling; however, 7OH ABA peaked early in germination. Changes in ABA flux, i.e. shifts in the ratio between biosynthesis and catabolism, occurred at three distinct stages during the transition from dormant seed to seedling. During moist chilling, the relative rate of ABA catabolism exceeded ABA biosynthesis. This trend became even more pronounced during germination, and germination was also accompanied by a decrease in the ABA catabolites DPA and PA, presumably as a result of their further metabolism and/or leaching/transport. The transition from germination to post-germinative growth was accompanied by a shift toward ABA biosynthesis. Dormant imbibed seeds, kept in warm moist conditions for 30 days (after an initial 13 days of soaking), maintained high ABA levels, while the amounts of PA, 7OH ABA, and DPA decreased or remained at steady-state levels. Thus, in the absence of conditions required to break dormancy there were no net changes in ABA biosynthesis and catabolism.Abbreviations ABA abscisic acid - ABA-GE abscisic acid glucose ester - DPA dihydrophaseic acid - 7OH ABA 7-hydroxy abscisic acid - 8OH ABA 8-hydroxy abscisic acid - MRM multiple reaction monitoring - PA phaseic acid     

Genotype and medium affect shoot regeneration of melon

The effects of basal media, growth regulators and gelling agents on the morphogenetic response of eleven Cucumis melo L. var. reticulatus and inodorus genotypes were examined. Regeneration was achieved from cultured cotyledons of all genotypes and the morphogenic response was affected by the genetic background. Among the combination of factors tested, MS basal medium supplemented with 2.8 M 6-benzyladenine (BA) and 1.0 M abscisic acid (ABA) solidified with agar gave the highest frequency of shoot regeneration.Abbreviations ABA abscisic acid - BA 6-benzyladenine - TDZ thidiazuron