Ethylene biosynthesis in Regnellidium diphyllum and Marsilea quadrifolia

The pathway of ethylene biosynthesis was examined in two lower plants, the semi-aquatic ferns Regnellidium diphyllum Lindm. and Marsilea quadrifolia L. As a positive control for the ethylene-biosynthetic pathway of higher plants, leaves of Arabidopsis thaliana (L.) Heynh. were included in each experiment. Ethylene production by Regnellidium and Marsilea was not increased by treatment of leaflets with 1-aminocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene in higher plants. Similarly, ethylene production was not inhibited by application of aminoethoxyvinylglycine and -aminoisobutyric acid, inhibitors of the ethylene biosynthetic enzymes ACC synthase and ACC oxidase, respectively. However, ACC was present in both ferns, as was ACC synthase. Compared to leaves of Arabidopsis, leaflets of Regnellidium and Marsilea incorporated little [¹⁴C]ACC and [¹⁴C]methionine into [¹⁴C]ethylene. From these data, it appears that the formation of ethylene in both ferns occurs mainly, if not only, via an ACC-independent route, even though the capacity to synthesize ACC is present in these lower plants.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AdoMet S-adenosyl-l-methionine - AIB -aminoisobutyric acid - AVG aminoethoxyvinylglycine This research was supported by the U.S. Department of Energy through grant No. DE-FG02-91ER20021 and, in part, by a fellowship of the National Engineering and Research Council of Canada to Jacqueline Chernys.     

Translocation profiles of 11C-assimilates in the petiole of Marsilea quadrifolia L.

An array of three multipliers with BGO-crystals arranged along the petiole has been used to study the movement of ¹¹C-assimilate in Marsilea quadrifolia L. The leaf blades were exposed to a 3–6 min pulse of ¹¹CO₂ under continuous light. Data were recorded with 10-s counting intervals and corrected for background, sensitivity of detectors, and ¹¹C-decay. The speed of translocation could be obtained from the time difference between the straight lines of ¹¹C-increase monitored at different positions along the petiole. The speed was within the range of 60 to 150 cm h^-1. Cutting as well as cold treatment at definite locations on the petiole led to an immediate stop of ¹¹C-movement. Both inhibition and—in the case of chilling—restoration of translocation were detectable within minutes. When the assimilation chamber was flushed with nitrogen gas, the normal increase of ¹¹C at the monitored sites slowed down which, after prolonged N₂-treatment, even resulted in a decrease. With a time-delay down the petiole, replacement of N₂ by an air-stream led to complete restoration of the ¹¹C-slope. These and similar shortterm effects on translocation could not be detected by former methods. This technique is also useful in with species other than Marsilea qu., which was chosen as a test plant because of the anatomical simplicity of its petiole.     

Determination of endogenous abscisic acid levels in immature cereal embryos during in vitro culture

Levels of endogenous abscisic acid (ABA) in immature wheat (Triticum aestivum cv. Timmo) and barley (Hordeum vulgare cv. Golden Promise) embryos have been determined by enzyme-linked immunosorbent assay. Embryos of both cereal species showed an increase in ABA content during development on the parent plant. Immature embryos were excised and cultured in vitro on nutrient media that led to precocious germination or on media containing 9% (w/v) mannitol that maintained their developmental arrest. Barley and wheat embryos responded to these culture conditions in an identical manner with respect to changes in morphology, fresh weight, protein and lectin content. However, in complete contrast, the ABA content of barley embryos increased by an order of magnitude during culture on mannitol, whereas that of wheat embryos showed no significant change. The results are discussed within the context of the role of ABA in the regulation of embryo development.Abbreviations ABA abscisic acid - BGA barley-germ agglutinin - dpa days post anthesis - ELISA enzyme-linked immunosorbent assay - GC-MS gas chromatography-mass spectrometry - WGA wheat-germ agglutinin     

Effects of NaCl and KNO3 concentrations on the abscisic acid content of Dunaliella sp. (Chlorophyta)

Abscisic acid (ABA) is a hormone which has a number of roles during the life cycle of a plant. We demonstrated the occurrence of ABA in a halotolerant green alga, Dunaliella sp. isolated from a salt pond near Adelaide, South Australia, using thin layer chromatography (TLC), high performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The variation of cellular ABA and protein content during the growth of an axenic clonal culture of Dunaliella sp. was investigated under different concentrations of NaCl and KNO₃.Experimental results can be summarized as follow: (1) ABA content was changed with the growth stage of culture: A rapid increase in ABA content was observed in the logarithmic phase. After this, the content rapidly decreased to very low values. (2) ABA content was also affected by the NaCl concentration. The content had a minimum value at the NaCl concentration (15%) where growth rate was maximal, and higher values at higher or lower concentrations of NaCl. (3) The ABA content also increased with decreasing nitrogen concentration of the medium.     

Polyamines,indole-3-acetic acid and abscisic acid in rice phloem sap

Putrescine, spermidine, spermine and cadaverine have been identified and quantified in rice phloem sap and shoot extracts by HPLC. It is suggested that diamines, putrescine and cadaverine, easily migrate into the phloem, while movement of a triamine, spermidine, and a tetramine, spermine, tend to be restricted. Spermine especially seems to be the most immobile among polyamines. Thus it is indicated that movement of polyamines into phloem is decreased with increasing number of amino groups. Indole-3-acetic acid and abscisic acid in rice phloem sap were also analyzed by HPLC and it is suggested that indole-3-acetic acid is transported freely into phloem, while abscisic acid is much more actively exuded into phloem.     

Apoptosis in barley aleurone during germination and its inhibition by abscisic acid

During germination of barley grains, DNA fragmentation was observed in the aleurone. The appearance of DNA fragmentation in the aleurone layer, observed by TUNEL staining in aleurone sections, started near the embryo and extended to the aleurone cells far from the embryo in a time dependent manner. The same spatial temporal activities of hydrolytic enzymes such as -amylase were observed in aleurone. DNA fragmentation could also be seen in vitro under osmotic stress, in isolated aleurone. During aleurone protoplast isolation, a very enhanced and strong DNA fragmentation occurred which was not seen in protoplast preparations of tobacco leaves. ABA was found to inhibit DNA fragmentation occurring in barley aleurone under osmotic stress condition and during protoplast isolation, while the plant growth regulator gibberellic acid counteracted the effect of ABA. Addition of auxin or cytokinin had no significant effect on DNA fragmentation in these cells. To study the role of phosphorylation in ABA signal transduction leading to control of DNA fragmentation (apoptosis), the effects of the phosphatase inhibitor okadaic acid and of phenylarisine oxide on apoptosis were studied. We hypothesize that the regulation of DNA fragmentation in aleurone plays a very important role in spatial and temporal control of aleurone activities during germination. The possible signal transduction pathway of ABA leading to the regulation of DNA fragmentation is discussed.     

Changes in abscisic acid and phenols during flower development in two diverse species of rose

Changes in endogenous abscisic acid (ABA) and phenols were determined in petals of two diverse species of rose, viz., Rosa damascena Mill and Rosa bourboniana Desport during flower development. A progressive increase in free ABA was observed during flower development till full bloom in both the species with higher content of free ABA in Rosa damascena. While bound ABA level increased in Rosa damascena till stage 6, in Rosa bourboniana it continued to increase till full bloom. Acidic phenols increased slowly in both the species till stage 4, but sharply afterwards and no significant differences were noticed during full bloom period. Bound phenols content was higher in Rosa damascena during full bloom period. The significance of these changes in relation to flowering in the two diverse species of rose is discussed.     

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     

Abscission-promoting activities of abscisic acid and five abscisic acid analogs in cotton seedlings and explants

The abscission-promoting activities of abscisic acid (ABA) and 5 ABA analogs were examined in cotton (Gossypium hirsutum L. cv LG102) seedlings and cotyledonary node explants. The analogs tested included a series of acetylenic derivatives that differ in the oxidation state of the C-1 atom, a 2,3 dihydro-derivative of ABA and a 2,3 dihydro-derivative of an acetylenic analog with a C-1 carboxyl moiety. ABA and all five analogs were active in stimulating petiole abscission in explants. Following treatment with 100,µM ABA or analog, 50% abscission of explants was observed after 29 h and complete abscission occurred within 40 h. With one exception, none of the treatments resulted in an increase in explant ethylene production. Pretreatment of the explants with the ethylene antagonist silver thiosulfate completely abolished the abscission-promoting activities of ABA and all of the analogs. Daily application of ABA or any of the analogs had no effect on cotyledon abscission in intact seedlings. The implications of the results with respect to the development of a commercial ABA-like regulator as well as to ABA structure-activity studies are discussed.Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

2′-hydroxymethyl abscisic acid: A major catabolite of (±)-abscisic acid in leaves of Hordeum vulgare L.

Radioactive (±)-abscisic acid (ABA), supplied via the transpiration stream to light-grown leaves of Hordeum vulgare was catabolized to 2′-hydroxymethyl ABA. Identification was made by capillary gas chromatography-mass spectrometry (GC-MS).     

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     

Cryopreservation of immature spring wheat zygotic embryos using an abscisic acid pretreatment

Spring wheat (Triticum aestivum L.) zygotic embryos were successfully cryopreserved, without the addition of exogenous cryoprotectants, using only an abscisic acid (ABA) pretreatment. Optimum survival was obtained when embryos were cultured in vitro for 10 days on semisolid Murashige and Skoog (MS) nutrient medium supplemented with 0.5 mg/L (±) ABA prior to cryopreservation. The embryos resumed growth within three days when returned to MS medium devoid of ABA but containing 2mg/L 2,4-dichlorophenoxyacetic acid. The embryogenic calli produced from these embryos exhibited normal plant regeneration on auxin-free media. Changes in dw/fw ratio, as well as the esterified fatty acid and sucrose concentrations correlated positively with the development of tolerance to cryopreservation.NRCC Publication No. 33519     

Antibodies raised against yeast HSP 104 cross-react with a heat-and abscisic acid-regulated polypeptide in rice

Antibodies raised against yeast heat shock protein (HSP) 104 recognized a heat-inducible polypeptide with a molecular mass of 110 kDa in shoot tissue of young rice seedlings. Root tissue of the same age showed no immuno-reaction with yeast HSP 104 antibodies. The 110 kDa polypeptide of rice was also shown to be abscisic acid-inducible in young seedlings. Though this polypeptide was seen to be constitutively present in the flag leaf of 90-day-old field-grown plant, it was not much affected by either heat shock or abscisic acid in this case.     

Modifications of abscisic acid level in winter oilseed rape leaves during acclimation of plants to freezing temperatures

An almost twofold increase in abscisic acid (ABA) content was observed in the leaves of winter oilseed rape plants (Brassica napus L., var. oleifera L., cv. Jantar) grown in the cold (>0°C). This ABA increase took place during the first three days of cold treatment. After 6 days of plant growth in the cold, the level of ABA started to decline or remained constant, depending on the calculation basis: dry weight or disc area units, respectively. The exposure of cold-acclimated plants to night frost (–5°C for 18 h) induced a further increase (65%) in the ABA level, which begun during the first few hours after thawing. The comparison of time courses of frost resistance increments and ABA content changes showed that modifications of ABA level in the cold-treated leaves preceded those of frost resistance, whereas in the frost-pretreated tissues the ABA increase occurred later than that of frost tolerance. Possible interrelations between ABA content, frost tolerance and tissue water potential modifications in the low temperature-affected tissues are discussed.     

Immunolocalization of abscisic acid by monoclonal antibodies in Lavandula stoechas L. leaves

A monoclonal antibody was used to localize abscisic acid in Lavandula stoechas L. plants treated with 1 M abscisic acid. ABA localization was performed using EDC as the only fixative, which preserves antigenicity and improves the specificity of monoclonal antibodies. A relationship was observed between the effect of abscisic acid on chloroplast ultrastructure and ABA accumulation. Gold particles accumulated on swollen chloroplasts. Our findings provide cytochemical evidence that the chloroplast is a trapping compartment, and yield valuable information on the relation between chloroplast ultrastructure and ABA accumulation.Abbreviations ABA abscisic acid - BSA bovine serum albumin - EDC 1-(3-dimethyl-aminopropyl)-3 ethyl carbodiimide - IgG immunoglobulin G - mAB monoclonal antibodies - pAB polyclonal antibodies     

Further evidence of a role for abscisic acid in conversion of somatic embryos ofDaucus carota

Summary Somatic embryogenesis has been shown to be an imperfect recapitulation of stages involved to form embryos from vegetative tissues. Although abscisic acid has been implicated in normalizing development, studies that specifically investigate conversion (vegetative leaf initiation) in somatic embryos are lacking. This report documents a follow-up of a study that implicated abscisic acid as a vital factor in allowing embryos ofDaucus carota to progress to the plantlet stage. Abscisic acid was determined to enhance conversion at doses ranging from 1 to 50 µM. Younger embryo stages were more responsive to abscisic acid application with regards to plantlet recovery. Pulses of abscisic acid were shown to elicit more rapid response with younger embryo stages, indicating more plastic development. Fluridone, an abscisic acid synthesis inhibitor, was shown to dramatically reduce conversion, even at low doses (<5µM). When abscisic acid was applied concurrently with fluridone, partial restoration of conversion was observed. Histologically, fluridone was seen to cause pronounced vacuolation in the shoot apical notch which resulted in the loss of meristematic cells, negating conversion capacity. Quantitation of total cytoplasmic area showed that abscisic acid reduced vacuolar intrusion into the apical notch, while fluridone caused a significant increase in vacuolation of cells in this region. This report documents further evidence of a role for abscisic acid in plantlet establishment from somatic embryos ofDaucus carota.     

Gibberellin and abscisic acid effects on the activity of hydrolytic enzymes in de-embryonated barley seeds

The production of hydrolytic enzymes by embryo-less barley seeds in response to various gibberellins and abscisic acid was investigated. The data support the hypothesis that plant growth substances may affect the mechanisms of hydrolase production and secretion in cereal seeds in different ways and at different point.