Changes in endogenous polyamines during flower development in two diverse species of rose

Changes in the concentrations of endogenous free, conjugated and bound polyamine were determined in petals of two different species of rose, viz. Rosa damascena and Rosa bourboniana, from small bud (stage 1) till full bloom (stage 8). High free putrescine and spermidine concentrations were associated with early stages of flower development and then decreased in R. damascena. At full bloom, the concentration of free putrescine was higher than rest of the polyamines measured. A steady increase in conjugated putrescine, spermidine and spermine was observed during entire period of flower development with predominance of conjugated putrescine at full bloom. In R. damascena the bound spermine was higher than rest of the polyamines during full bloom. In R. bourboniana, during the early stages of flower development, similar situation was observed, however, at full bloom, free spermidine concentration was higher than rest of the polyamines. In this species, the concentration of conjugated and bound spermine was higher than rest of the polyamines during full bloom. Polyamine concentrations were generally lower in the petals of R. bourboniana than R. damascena which may be due to genotypic differences. The possible roles of the observed polyamines are discussed in relation to flower development.IHBT Communication no, 0345.     

Post-harvest alterations in polyamines and ethylene in two diverse rose species

Changes in the concentrations of endogenous polyamines and ethylene were determined in two diverse species of rose viz. Rosa damascena and Rosa bourboniana during post-harvest periods. At full bloom, the concentrations of free putrescine was significantly higher than rest of the polyamines, i.e. spermine and spermidine in both the species. The concentrations of all the polyamines decreased during subsequent periods upto 48 h after full bloom. Similar situation was also observed in conjugated fraction but in bound fraction, during full bloom, the concentration of spermine was higher than rest of the polyamines. In both the species, ethylene showed higher levels during full bloom with maximum in R. damascena, which increased, during post-harvest periods. The possible significance of polyamines, ethylene and their interactions is discussed during post-harvest periods in flowers.     

The effect of polyamines on leaf senescence in two diverse rose species

Polyamines (PAs) retarded the senescence of leaf discs of two diverse speciesof rose viz., Rosa bourboniana andRosa damascena, while polyamine biosynthetic inhibitorsdifluoromethylornithine (DFMO), difluoromethylarginine (DFMA), methylglyoxal-bis(guanylhydrazone) (MGBG) and abscisic acid (ABA) promoted senescence. Sperminewas significantly the most effective polyamine in retarding senescence inR. bourboniana while MGBG and DFMA were more prominent inaccelerating senescence in R. damascena and R.bourboniana respectively. Protein and RNA content were significantlyhigher in polyamine treated leaf discs compared to those treated with polyaminebiosynthetic inhibitors and ABA. Total and reducing sugars decreased under alltreatments while the starch content increased significantly only in polyaminetreated leaf discs. Peroxidase and cellulase activities were retarded bypolyamine treatments and accelerated by polyamine biosynthetic inhibitors andABA. The role of PAs is discussed in relation to senescence.     

Xylem and Phloem Derived Polyamines during Flowering in Two Diverse Rose Species

Polyamine contents in xylem (root) and phloem (leaf) exudates in two diverse species of rose, viz. Rosa damascena Mill and Rosa bourboniana Desport, were analyzed before, during, and after flowering in the main flowering season, that is, April–May. Only free putrescine (Put) was detected in the xylem and phloem exudates at these time points, and it was high during the peak flowering period. In phloem, Put content was significantly higher in R. bourboniana than in R. damascena at all three stages; whereas in the xylem exudate it was relatively higher in R. damascena at the peak flowering period. A spray of α-difluoromethylornithine (DFMO), an irreversible inhibitor of the putrescine biosynthetic inhibitor ornithine decarboxylase (ODC), markedly decreased the flowering. This effect was reversed by application of Put alone or in combination with DFMO. The significance of this finding is discussed in light of polyamine translocation during flowering. *IHBT Communication: 0354     

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     

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     

Plant regeneration from Bulgarian rose callus

Plant regeneration capacity of Bulgarian rose callus tissue was examined. Adventitious bud formation could be successfully attained, depending on the kinds of mineral salts used in the medium, auxin and cytokinin used. When callus tissues were cultured on the medium without ammonium nitrate and contained indoleacetic acid and benzyladenine, buds were formed in the callus. The number of buds were significantly increased by the simultaneous addition of calcium ionophore. When the cultures were transferred to the medium without cytokinin, roots were formed in the basal part of the buds.Abbreviations BA benzyladenine - IAA indoleacetic acid - K kinetin - NAA naphthaleneacetic acid     

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.     

Relationship between gibberellin and cytokinin activity and flowering in Rosa damascena Mill.

Kinetin at 10 mg l^–1 increased the number of flowers produced on Rosa damascena plants while GA₃ inhibited flowering. In the leaves of non-flowering plants GA-like activity was high while specific cytokinin activity (fraction-II) was significantly higher in flowering plants. A novel compound 10- methyldihydrozeatin riboside and isopentenyl-adenine were identified from TLC fraction-II while TLC fraction-I yielded zeatin and 2-hydroxy-6-methylaminopurine.Abbreviations TLC thin layer chromatography - BA N⁶-benzyladenine - GA₃ gibberellic acid CIMAP communication No. 92-40J     

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.     

Ferricyanide reductase of rose plasma membranes is regulated by nitrogen supply

Summary The ability of suspension-cultured rose (Rosa damascena Mill. cv Gloire de Guilan) cells to reduce ferricyanide is decreased by 50% during an overnight incubation in a low-nutrient (1 mM CaCl₂, 0.1 mM KCl) solution. This loss is not observed when nitrate and/or glutamate is added to the low-nutrient medium, but it occurs in medium containing all the components needed for normal growth except nitrate plus glutamate. Thus, the cells possess both constitutive and inducible enzymes for the reduction of ferricyanide, and nitrate or glutamate is both necessary and sufficient to stimulate the production of the inducible enzyme.     

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     

Changes in abscisic acid levels during dormancy release in freesia corms

A high level of free-abscisic acid (ABA) was detected when corms were still in deep dormancy. The level of free-ABA decreased as the corm dormancy disappeared and increased temporarily after complete release from dormancy. A gradual slight increase of bound-ABA was observed during dormancy release.Treatment of dormant corms with benzyladenine (BA) increased sprouting but the sprouts did not show normal growth. Ethylene treatment induced complete sprouting and subsequent normal growth. Changes in ABA levels and ethylene production are discussed in relation to dormancy release in freesia corms.     

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     

Indoleacetic acid, abscisic acid and phenolic substances during development of hazel leaves

The levels of indoleacetic acid (IAA), abscisic acid (ABA) and phenolic substances were measured monthly in leaves of hazel ( Corylus avellana L. cv. Negreta) in different stages of development from April till abscission in November. IAA and ABA were quantified by flame ionization detector gas-chromatography (GLC), and the total phenolic content by a colorimetric technique. During spring, IAA levels were the highest, coinciding with the greatest expansion of the leaves; after which IAA gradually decreased as the leaves became older, although minor increases were observed in July-August and November. The ABA level in hazel leaves was low throughout the period of leaf existence, with a maximum in July and a second increase in November, coinciding with the last month in which hazel bears leaves. The phenols of the neutral fraction showed a minimum in May and a pronounced increase in June, remaining at this level during summer and decreasing in November. The acid and alkaline fractions showed a maximum in August, which could be related to the decrease in the growth rate of leaves. The residual fraction remained at a relatively low level until September, increasing rapidly in October and November.     

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     

Endogenous cytokinins in rose petals and the effect of exogenously applied cytokinins on flower senescence

In extracts from rose petals cytokinin activity was detected by Amaranthus bioassay in HPLC eluates corresponding to the standards: Z, ZR, 2iP and 2iPA; subsequently, the presence of two groups of endogenous cytokinins was confirmed by ELISA.Measurements of senesence indicators (cell sap osmolarity and conductivity) and observations of flower vase-life indicated that when the above cytokinins were applied as holding solutions they delayed flower senescence by 34–56% and prolonged rose longevity.Abbreviations B.H.T. 2.6-di-t-buytl-4-methyl phenol - ELISA Enzyme linked Immunosorbent Assay - HPLC High Performance Liquid Chromatography - 2iP isopentenyladenine - 2iPA isopentenyladenosine - Z trans-zeatin - ZR trans-zeatin riboside     

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.     

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.