Significance of the zygotic seed coat on quiescence and desiccation tolerance of Medicago sativa L. somatic embryos

Summary The influence of the zygotic seed coat on precocious germination and desiccation tolerance of somatic embryos has been studied using alfalfa (Medicago sativa L.). When cultured in contact with somatic embryos, seed coats at certain developmental stages inhibited precocious germination and induced desiccation tolerance in the somatic embryos. Germination of somatic embryos was inhibited by seed coats at the age of 16–26 days after pollination (DAP) and desiccation tolerance was induced after 20–26 DAP. Both phenomena were related to the synthesis of abscisic acid in the seed coat. The absence of a quiescent phase and desiccation tolerance in alfalfa somatic embryos may be related to the lack of developmental control by the seed coat.Abbreviations ABA Abscisic acid - DAP Days after pollination     

Abscisic acid levels and metabolism in the leaf epidermal tissue of Tulipa gesneriana L. and Commelina communis L.

We have shown the presence of abscisic acid (ABA) in abaxial epidermal strips taken from Tulipa gesneriana and Commelina communis and that the ABA level rises in the epidermis when leaves are water stressed. ABA levels had risen 50% in the abaxial epidermis of C. communis 30 min after the leaves lost 10% of their fresh weight. Epidermis from both T. gesneriana and C. communis metabolize [¹⁴C]ABA to several products probably including phaseic acid (PA) and dihydrophaseic acid (DPA).Abbreviations ABA abscisic acid - RIA radioimmunoassay - PA phaseic acid - DPA dihydrophaseic acid - TLC thin-layer chromatography - GC gas chromatography     

Evidence of a role for tyrosine dephosphorylation in the control of postgermination arrest of development by abscisic acid in Arabidopsis thaliana L

In the present paper evidence is presented indicating that tyrosine dephosphorylation is a key regulatory mechanism in postgermination arrest of Arabidopsis thaliana L. seed development mediated by abscisic acid (ABA). By using phenylarsine oxide (PAO), an inhibitor of tyrosine phosphatases, the sensitivity to the inhibitory effect of ABA on seed germination is enhanced. Consistent with this finding, we demonstrate that the ABA-responsive gene, RAB18, is hyperinduced in seeds imbibed in ABA plus PAO, compared with seeds imbibed only with ABA.     

Abscisic Acid Inhibition of Kinetin Nucleotide Formation in Germinating Lettuce Seeds

Imbibing ‘Grand Rapids’ lettuce (Lactuca saliva L.) seeds take up ¹⁴C-kinetin, and metabolize this cytokinin to the 5′-nucleotide. The identity of the labeled nucleotide in seed extracts was verified by Sephadex LH-20 column chromatography, paper and thin layer chromatography, and high voltage paper electrophoresis. Incubations with kinetin in the presence of abscisic acid lead to an apparent specific inhibition of kinetin nucleotide formation. ABA has no effect on kinetin uptake, and does not inhibit kinetin nucleotide synthesis in vitro by a cell-free preparation from lettuce seeds. Additionally, ABA does not inhibit adenylate synthesis from exogenously supplied adenine. These results represent a specific cytokinin-ABA interaction, which might play a significant role in the hormonal regulation of lettuce seed germination.     

Changes in phenolic acids and abscisic acid in sugar pine seed coats during stratification

The phenolic acids and abscisic acid (ABA) of sugar pine ( Pinus lambertiana Dougl.) seeds coats, separated by high-pressure liquid chromatography, were analyzed during 90 days stratification of the seeds. Although levels of seed coat phenolic acids and ABA declined significantly during, stratification, this decrease did not appear to be responsible for the loss of dormancy due to stratification. Lack of improved germination following washing, cracking, or removal of the seed coats, plus additional evidence, did not support a significant role for the seed coat in the dormancy of sugar pine seeds.     

Dormancy in somatic embryos and seeds ofVitis: changes in endogenous abscisic acid during embryogeny and germination

Abscisic acid (ABA) in extracts of somatic embryos and seeds of Gloryvine (Vitis vinifera L.xV. rupestris Scheele) was measured by gas chromatography-mass spectrometry-selected ion monitoring using deuterated ABA, (±)-[C-3Me-²H₃]ABA, ([²H₃]ABA) as internal standard. The ABA content increased rapidly during embryogeny (0.035 ng/embryo at the globular stage to 0.22 ng/embryo at the mature stage). The level of ABA in the tissues of somatic embryos, expressed in ng/mg dry weight, decreased from the globular stage (0.76 ng/mg) to the mature stage (0.25 ng/mg). Chilling (4° C) induced normal germination of seeds and mature somatic embryos and precocious germination of globular, heart-shaped and torpedoshaped somatic embryos. In all cases chilling led to a marked reduction in endogenous ABA. Exogenous (±)-ABA inhibited the germination of chilled somatic embryos.Abbreviations ABA abscisic acid - [²H₃]ABA (±)-[C-3Me-²H₃]-abscisic acid - BHT 2,6-di-t-butyl-4-methylphenol - GC-MS gas chromatography-mass spectrometry - Me-ABA and Me-[²H₃]ABA methyl esters of ABA and [²H₃]ABA, respectively - SIM selected ion monitoring     

Hydrogen sulfide counteracts chlorophyll loss in sweetpotato seedling leaves and alleviates oxidative damage against osmotic stress

Sawgrass (Cladium jamaicense) is the predominant plant and vegetation community in the Florida Everglades. Germination of sawgrass seeds in the laboratory or nursery has been difficult and problematic, yet little is known about the physiological mechanistic regulation of the sawgrass seed germination process. In the present study, we examined the factors and mechanisms that influence sawgrass seed germination. We found that removal of seed husk and bracts, pre-soaking with bleach (hypochlorite), breaking the seed coat, or combinations of these treatments promoted the rate and success of germination, whereas presence of seed-encasing structures or treatment with husk/bract extract inhibited germination. We further detected the presence of abscisic acid (ABA) in the husk and bract. Experiments with ABA and gibberellin biosynthesis inhibitors fluridone and tetcyclacis suggested that ABA already presented in the pre-imbibed seeds, and not derived through post-dormancy de novo synthesis, contributed to the inhibition of seed germination. Examination of bleach and mechanical treatments indicated the physical barrier presented by the seed-encasing structures provided additional mechanism for the long-term delay of seed germination. Based on the results of this study and others, we discussed the implications of sawgrass seed dormancy and germination in relation to its natural habitat and proposed a hypothesis that the protracted seed dormancy in sawgrass offered an adaptive advantage in the pre-anthropogenic Everglades environment, but may become a liability in the current man-managed Everglades water system.     

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     

A monoclonal antibody to (S)-abscisic acid: its characterisation and use in a radioimmunoassay for measuring abscisic acid in crude extracts of cereal and lupin leaves

A monoclonal antibody produced to abscisic acid (ABA) has been characterised and the development of a radioimmunoassay (RIA) for ABA using the antibody is described. The antibody had a high selectivity for the free acid of (S)-cis, trans-ABA. Using the antibody, ABA could be assayed reliably in the RIA over a range from 100 to 4000 pg (0.4 to 15 pmol) ABA per assay vial. As methanol and acetone affected ABA-antibody binding, water was used to extract ABA from leaves. Water was as effective as aqueous methanol and acetone in extracting the ABA present. Crude aqueous extracts of wheat, maize and lupin leaves could be analysed without serious interference from other immunoreactive material. This was shown by measuring the distribution of immunoreactivity in crude extracts separated by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), or by comparing the assay with physicochemical methods of analysis. Analysis of crude extracts by RIA and either, after TLC purification, by gas chromatography using an electron-capture detector or, after HPLC purification, by combined gas chromatography-mass spectrometry (GC-MS) gave very similar ABA concentrations in the initial leaf samples. However, RIA analysis of crude aqueous extracts of pea seeds resulted in considerable overestimation of the amount of ABA present. Determinations of ABA content by GC-MS and RIA were similar after pea seed extracts had been purified by HPLC. Although the RIA could not be used to analyse ABA in crude extracts of pea seeds, it is likely that crude extracts of leaves of several other species may be assayed successfully.Abbreviations ABA abscisic acid - DW dry weight - FW fresh weight - GC-ECD gas chromatography using an electron capture detector - GC-MS combined gas chromatographymass spectrometry - HPLC high-performance liquid chromatography - McAb monoclonal antibody - PVP soluble polyvinylpyrrolidone - RIA radioimmunoassay - TLC thin-layer chromatography     

Effects of abscisic acid,gibberellic acid and fusicoccin on the transmembrane potential during the early phases of germination in radish (Raphanus sativus L.) seeds

During germination, the transmembrane electric potential (PD) of cortical cells of the embryonal axis of radish seeds (Raphanus sativus L.) rises from-120 mV initially to a maximum of-150 mV after 5 h incubation, then falls again to stable values of around-120 mV. Treatments inhibiting germination block the transitory PD increase. Administration of uncoupling agents or low temperatures, during the process of germination, produces a marked fall of the PD transitory increase. Abscisic Acid has a parallel inhibitory effect on PD and germination, while fusicoccin produces a rise in both; administration of abscisic acid with fusicoccin inhibits germination, while the PD remains at the high levels given by fusicoccin. These results are discussed in relation to ion exchange at membrane level.Abbreviations ABA abscisic acid - FC fusicoccin - GA₃ gibberellic acid - PD electric potential difference (between the vacuole and the external medium) - CH cycloheximide - DNP dinitrophenol - FCCP (p-trifluormethoxy)-carbonylcyanide-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide     

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)     

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     

Abscisic acid relationships in the chill-related dormancy mechanism in apple seeds

Abscisic acid (ABA) levels in seeds from three cultivars of apple (Malus domestica Borkh.) which have substantially different chilling requirements were investigated by gas chromatography mass-spectrometry selected ion monitoring (GCMS-SIM) during stratification. The ABA content of dormant unchilled seeds was similar in the three cultivars, suggesting no relationship between the chilling requirement of those seeds and their ABA status. That chilling is not related to ABA changes during stratification was confirmed by warm (20°C) and cold (5°C) stratification experiments. ABA content dropped rapidly and nearly identically under both temperature regimes, but only cold stratification promoted germination. The decline in ABA during stratification was due in large part to leaching from the seed coat and nucellar membrane; the ABA content of the embryo remained nearly constant. The radicle in intact seeds stratified at 5°C began growing 20–30 days after the ABA in the seed coat and nucellar membrane had nearly disappeared. Radicle growth did not occur in unchilled seeds, even though ABA had leached from them as well. It is possible that the leaching of ABA from the seed allows certain promotive forces to develop, but if so, these can develop only at chilling temperatures. Studies were also conducted on 2-trans ABA relationships to apple seed dormancy, but no association was evident.Report No. 12, Department of Fruit and Vegetable Science, Cornell University.     

Effect of abscisic acid on galactomannan degradation and endo-β-mannanase activity in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

Seeds of Sesbania virgata (Cav.) Pers. (Leguminosae) have an endosperm which accumulates galactomannan as a storage polysaccharide in the cell walls. After germination, it is hydrolysed by three enzymes: α-galactosidase (EC 3.2.1.22), endo-β-mannanase (EC 3.2.1.78) and β-mannosidase (EC 3.2.1.25). This work aimed at studying the effect of abscisic acid (ABA) on galactomannan degradation during and after germination. Seeds were imbibed in water or in 10⁻⁴ M ABA, and used to evaluate the effect of exogenous and endogenous ABA. Tissue printing was used to follow biochemical events by detecting and localising endo-β-mannanase in different tissues of the seed. The presence of exogenous ABA provoked a delay in the cellular disassembly of the endosperm and disappearance of endo-β-mannanase in the tissue. This led to a delay in galactomannan degradation. The testa (seed coat) of S. virgata contains endogenous ABA, which decreases ca. fourfold during storage mobilisation after germination, permitting the galactomannan degradation in the endosperm. Furthermore, endo-β-mannanase was immunolocalised in the testa, which has a living cell layer. The ABA appears to modulate storage mobilisation in the legume seed of S. virgata, and a cause–effect relationship between ABA (probably through testa) and activities of hydrolases is proposed.     

A radioimmunoassay for abscisic acid

We have developed a radioimmunoassay (RIA) for abscisic acid (ABA) in the 0.1 ng to 2.5 ng range. Antibodies were obtained from rabbits immunized with ABA bound via its carboxyl group to bovine serum albumin. Cross-reactivity studies indicate that ABA esters are completely cross-reactive with ABA, while trans, trans abscisic acid (t-ABA) phaseic acid (PA) and dihydrophaseic acid (DPA) have much lower but significant cross-reactivities. Purification methods which reduce the levels of cross-reacting substances are described.Abbreviations RIA radioimmunoassay - DPA 4-dihydrophaseic acid - PA phaseic acid - GC gas chromatography - HPLC high performance liquid chromatography - TLC thin-layer chromatography - BSA bovine serum albumin - ABA abscisic acid - t-ABA trans, trans abscisic acid - IAA indoleacetic acid     

Effect of light on abscisic acid content in photosensitive Scots pine (Pinus sylvestris L.) seed

Dormancy-breaking treatment of the photosensitive Scots pine (Pinus sylvestris L.) seed by white light incubation or a 15-min exposure to red light decreased the abscisic acid content prior to radicle protrusion. Incubation in the dark or exposure to red light followed by a 5-min far-red light irradiation did not cause as great a decrease in abscisic acid content nor was the dormancy relieved. The ability of the far-red light to keep the ABA level high and to prevent germination gradually disappeared as the length of the dark period between the red and far-red treatments was increased to 24 h. ABA was quantified on a gas chromatograph with an electron capture detector.     

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     

Periodicity of response to abscisic acid in lateral buds of willow (Salix viminalis L.)

Aseptically cultured lateral buds of Salix viminalis L. collected from field-grown trees exhibited a clear periodicity in their ability to respond to exogenous abscisic acid (ABA). Buds were kept unopened by ABA only when the plants were dormant or entering dormancy. Short days alone did not induce bud dormancy in potted plants but ABA treatment following exposure to an 8-h photoperiod prevented bud opening although ABA treatment of buds from long-day plants did not. Naturally dormant buds taken from shoots of field-grown trees and cultured in the presence of ABA opened following a chilling treatment. In no cases were the induction and breaking of dormancy and response to ABA correlated with endogenous ABA levels in the buds.Abbreviations ABA abscisic acid - GA₃ gibberellic acid - HPLC high-performance liquid chromatography - LD long day - MeABA methyl ABA - PAR photosynthetically active radiation - SD short day     

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     

Abscisic acid and related compounds in phloem exudate of Yucca flaccida haw. and coconut (Cocos nucifera L.)

Phloem sap collected from Yucca and coconut inflorescence stalks was shown to contain abscisic acid (ABA) and trace amounts of 2-trans ABA. In coconut sap, two compounds probably derived from ABA with mass spectra consistent with their being dihydrophaseic acid and either hydroxyphaseic acid or oxo-dihydrophaseic acid were also found to be present.Abbreviations ABA abscisic acid - TMSi trimethylsilyl - GLC(EC) gas chromatography (electron capture) - GC-MS gas chromatography=mass spectrometry