Endogenous ABA Levels Research Articles

Endogenous Levels of Abscisic Acid in Embryogenic Cells, Nonembryogenic Cells and Somatic Embryos of Carrot (Daucus carota L.)

Summary The levels of endogenous ABA were measured by ELISA in embryogenic cells, nonembryogenic cells and somatic embryos of carrot. Embryogenic cells were estimated to contain a 67.2 times higher level of ABA than non-embryogenic cells, and a 2.5 times higher level than somatic embryos. The correlation between the endogenous level of ABA and embryogenic competence of carrot cultured cells is discussed.

Seed Development in the Recalcitrant SpeciesQuercus roburL: Water Status and Endogenous Abscisic Acid Levels

Water status and endogenous ABA levels were measured during the development of the embryonic axis and cotyledons in fruits of Quercus robur L. As dry matter accumulated in the embryo during development, both moisture content and osmotic potential (ψ,) declined. Measured by combined gas chromatography-mass spectrometry, ABA concentration in the axis remained high until near shedding, when it declined. In the cotyledons there was a well-defined, transient peak in ABA concentration coinciding with the start of their rapid increase in starch content. At shedding, the cotyledons had a lower moisture content and lower ψ, than the axis. After shedding, axis dry weight increased and moisture content declined in storage, suggesting a late continuation of development. Prematurely harvested fruits, stored in the absence of water, also showed continued axis development, and subsequently germinated without prior desiccation. There was no evidence of post-maturation drying in the embryo, although there was a small net loss of water from the cotyledons before shedding. Embryo development in Q. robur, therefore, differed significantly from that in orthodox seeds of other dicotyledonous species.

Interaction between Maturation and High Relative Humidity Treatments and their Effects on Germination of Sitka Spruce Somatic Embryos

Summary A high relative humidity treatment (HRH) stimulated frequency of root emergence and improved post-germinative growth and development from somatic embryos of sitka spruce (Picea sitchensis (Bong) Carr.). Comparison between temporal changes in embryo water content during the HRH treatment and its subsequent effects on germination revealed that, although the majority of water was lost during the first week of HRH treatment, gradual and significant improvements in germination were evident throughout a 5-week treatment period. Somatic embryos transferred directly from maturation conditions onto growth regulator-free medium showed rapid depletion of major storage proteins whereas those transferred to the HRH treatment (growth regulator-free and partial water loss) showed no loss in these proteins. Apparently, the partial drying prevented at least some metabolic events associated with early stages of germination. Longer periods of HRH treatment were required by embryos matured on 80 μM ABA to obtain germination equivalent to those embryos produced on 40 or 60 μM ABA, suggesting that there may be a relationship between endogenous ABA levels and the effects of the HRH treatment on germination. Extending the HRH treatment for longer periods increased the percentage of embryos that developed into high quality emblings (those exhibiting root emergence and an elongated, non-vitrified hypocotyl). ≪High quality emblings≫ acclimatized well to ex vitro conditions and, subsequently, developed into vigorous plants similar to typical sitka spruce seedlings.

Changes in water relations and endogenous abscisic acid content of wheat and barley grains and embryos during development

Abstract. Immature cereal embryo development can be controlled by in vitro culture on media containing ABA, or by media of low osmotic potential. To assess the possible in vivo roles of these factors, endogenous ABA levels and water relations of embryos and grains of wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) were determined during development. ABA concentrations remained consistent with those required to inhibit precocious germination in vitro of early stage embryos but not of more mature embryos. With increasing maturity, a difference in water potential developed between grain and embryo, suggestive of an in vivo role for water status in controlling the development of the embryo.

Why are the effects of abscisic acid on photosynthate unloading so variable? A possible answer from a sourcelimited experimental system

The hypothesis that defoliation might lower seed ABA levels with related changes in the effects of exogenous ABA on photosynthate unloading within seeds was tested. Defoliation of dwarf bean ( Phaseolus vulgaris L. cv. Masterpiece) plants markedly reduced ABA concentrations in seeds although reduction occurred in embryos rather than testas. Results from three experiments examining the effects of ABA on 14 C-photosynthate unloading from excised testas were variable but showed that the promotory effect of ABA was diminished as a result of defoliation pretreatment. The data are consistent with the viewpoint that the variability commonly seen in experiments investigating ABA involvement in photosynthate unloading is caused by fluctuating endogenous ABA levels due, in turn, to the growing conditions of experimental plants.

The effect of 4-chlororesorcinol on the endogenous levels of IAA, ABA and oxidative enzymes in cuttings

Treatment of bean cuttings with 4-chlororesorcinol (4-CR), known to increase the number of roots and extend their distribution, prevented the accumulation of free indol-3-yl-acetic acid (IAA) in the hypocotyls within 24 h after cutting preparation. In mung bean there was no change in the distribution (upper half vs. 1 ower half of the hypocotyl) of IAA within the hypocotyl as a result of the treatment. In bean cuttings the treatment with 4-CR prevented the accumulation of IAA in the bottom of the cutting. Oxidation of IAA as a measure of IAA oxidase activity in bean was enhanced appreciably by 4-chlororesorcinol. The level of abscisic acid in mung bean, on the other hand, remained 3–4 fold higher than in the control, yet still about 50% lower than the zero time level. In untreated mung bean cuttings the activity of peroxidase increased after cutting preparation. In contrast, the activity of peroxidase in 4-Cr-treated cuttings was consistently lower. In order to relate to the effect of exogenously applied auxin the level of peroxidase was measured also in indol-3-yl-butyric acid-treated cuttings. The overall peroxidase activity in IBA-treated cuttings was not affected. However, when assaying for the different isozymes the drop in peroxidase activity was most evident in the inducible basic isoperoxidases both in 4-CR and IBA treatments. It appears that the exposure to 4-CR exerts an effect that is similar to that of exogenously applied auxin, affecting the activity of basic peroxidases and enhancing the oxidation of endogenous IAA, thus allowing the organization of the primordia.

Endogenous levels of abscisic acid and indole-3-acetic acid during in vitro rooting of Wild Cherry explants produced by micropropagation

Levels of endogenous ABA and IAA were quantified during the first week of in vitro rooting of Wild Cherry (Prunus avium L.) using IBA in the culture medium. Hormones were measured in the apical, median and basal parts of the explants using an avidin-biotin based enzyme linked immunosorbent assay (ELISA), after a purification of the methanolic extracts by high-performance liquid chromatography (HPLC). Root primordia started to differentiate from day 5 at the basal part of the explants. ABA and IAA showed considerable changes and high levels were detected during the first week of culture. ABA levels increased transiently mainly in the apical part during root formation. Exogenous IBA was possibly transformed into IAA mainly in the basal part of the explants.

Seed Dormancy in Acer: The Relationship between seed Dormancy, Embryo Dormancy and Abscisic Acid in Acer platanoides L.

Summary Whole seeds and isolated embryos from freshly harvested fruits of Acer platanoides were deeply dormant and even excised embryonic axes from such fruits germinated poorly. Storage of fruits at inductive chilling temperatures (5 °C) for several weeks was necessary for the removal of dormancy from the intact seeds, but dormancy was lost from the embryo and the embryonic axis even during fruit storage at non-inductive temperatures (17 °C). ABA suppressed the increased germination potential that developed in embryos and embryonic axes during the early stages of post-harvest storage, but became less effective after longer storage periods, indicating a loss of sensitivity to ABA in the embryo tissue. The presence of the cotyledons following short periods of fruit storage was also inhibitory to germination of the isolated embryo and the possible role of the cotyledons as a reservoir of ABA is discussed. Endogenous ABA levels in the embryo fell markedly as a result of fruit storage at either inductive or non-inductive temperatures and the speed at which this occurred is compared with the rate at which embryo dormancy was lost. It is concluded that ABA is closely implicated in the regulation of embryo dormancy in this seed, but that the breakage of dormancy in whole seeds is more dependent on other factors concerned with the presence of an intact testa.

Hormonal Regulation of Cotton Fibre Elongation in Gossypium arboreum L. in vitro and in vivo

Changes in endogenous levels of IAA, GA 3 and ABA during in vivo fibre growth of Gossypium arboreum L. cv. LD 230 revealed an overriding influence of ABA in restricting the rate of fibre elongation. No relationship between IAA levels and extent of fibre elongation was discernible. The fibre growth of unfertilized cotton ovules cultured in vitro was promoted by the application of fluridone, an inhibitor of ABA biosynthesis. Fluridone resulted in a marked reduction of ABA concomitant with an increase in IAA and GA3 levels of cultured cotton ovules. The relationship of ABA with IAA and gibberellin is shown to have regulatory influence in determining the extent of cotton fibre elongation.

Reduction of Turgor Induces Rapid Changes in Leaf Translatable RNA

The turgor of pea (Pisum sativum) leaves was reduced by exposing excised pea shoots to a stream of 23 degrees C air for 20 min. Poly(A)(+) RNA was isolated from control and wilted shoots, translated in vitro and radiolabeled translation products separated by electrophoresis on two-dimensional (isoelectric focusing-sodium dodecyl sulfate) polyacrylamide gels. This analysis showed that the levels of several poly(A)(+) RNAs increased in wilted plants. Most of the poly(A)(+) RNAs induced in wilted plants did not accumulate in response to heat shock or exogenously applied ABA even though endogenous ABA levels were found to increase in shoots 30 min after wilting and by 4 h had increased 50-fold (1 versus 0.02 microgram per gram fresh weight). A lambdagt10 cDNA library was constructed using poly(A)(+) RNA from wilted shoots which had been incubated for 4 hours. Differential screening of the library identified four clones corresponding to poly(A)(+) RNAs which are induced in wilted shoots.

Measurement of endogenous ABA levels in chilled somatic embryos of carrot by immunoassay

Somatic embryos of carrot, Daucus carota L. 'Royal Chantenay', were chilled at 4°C for the last 3 days of development in order to harden torpedo stage embryos to increase embryo survival during desiccation. ABA levels in chilled and non-chilled embryos were measured using a polyclonal radioimmunoassay and a monoclonal enzyme-linked immunosorbent assay (ELISA). The monoclonal ELISA is the preferred technique due to superior sensitivity and specificity. ABA levels, measured by either technique, were similar in chilled and non-chilled embryos. The relative water content was lower in chilled embryos than in non-chilled embryos and chilling altered protein secretion of one cell line.

Endogenous Growth Regulators in Leaves and Tissue Cultures of Pennisetum purpureum Schum.

Summary Endogenous levels of major plant growth regulators were measured in leaves and tissue cultures of Napiergrass ( Pennisetum purpureum Schum.) either by high-performance liquid chromatography or radioimmunoassay. The developmentally young, basal region of the tightly furled innermost four young leaves are highly embryogenic (45% of explants produce embryogenic callus) as compared to the more mature middle (28%) and distal (5%) parts of the same leaves. The basal region contained, on a fresh-weight basis, higher levels of both indole-3-acetic acid (IAA; 19 ngg -1 ) and abscisic acid (ABA; 862 ngg -1 ) as compared to middle (11 ngg -1 and 282 ngg -1 , respectively) and distal (13 ngg -1 and 97 ngg -1 , respectively) regions. Segments from fully expanded “mature” leaves did not form any callus and contained lower levels of endogenous IAA (6 ngg -1 fresh weight) and ABA (364 ngg -1 fresh weight) compared to the basal portion of young leaves. Accumulation of high levels of cytokinins (zeatinriboside, dihydrozeatinriboside, 2-isopentenyladenine) was detected in explants after 10d of culture, at the time of visible callus formation. In comparison to the non-embryogenic callus the embryogenic callus consistently contained higher levels of endogenous IAA (5- to 20-fold) and ABA (3- to 4-fold). Levels of cytokinins in non-embryogenic callus were at least two times that in embryogenic callus. These trends were reproducible during regular maintenance for over a year by subculture at two- to three-week intervals. Callus cultures maintained without subculture lost their embryogenic competence after six weeks and contained only low or nondetectable levels of IAA and ABA. The role of endogenous plant growth regulators in determining the morphogenic competence of leaf explants is discussed.

The Regulation by 2,4-dichlorophenoxyacetic Acid of Endogenous Abscisic Acid Levels in Seedlings of Cucurbita pepo L.

Summary At the concentrations used 2,4-D caused marked increases in the levels of endogenous ABA during the first seven days of growth of Cucurbita pepo seedlings. These increases in ABA were not associated in time with germination itself, which was in any case largely unimpaired by 2,4D treatment, but corresponded with the period when maximum hypocotyl extension was occurring in the water controls. This 2,4-D effect did not appear to be mediated through the endogenous IAA balance. The relationship between endogenous ABA production and the observed inhibition of hypocotyl extension by 2,4-D are discussed.

ABA Levels and Sensitivity in Developing Wheat Embryos of Sprouting Resistant and Susceptible Cultivars

A sprouting-resistant and a sprouting-susceptible wheat cultivar were utilized to examine the role of ABA levels and sensitivity responses in wheat embryonic germination. Endogenous embryonic ABA levels were measured in both cultivars throughout grain maturation utilizing a new and sensitive ABA immunoassay. Embryonic ABA levels of each cultivar were similar with the sprouting-susceptible cultivar having about a 25% lower ABA level than that of the sprouting-resistant cultivar. Larger differences between the cultivars were noted in sensitivity to ABA, as measured by capability of ABA to block embryonic germination. ABA inhibited embryonic germination much more effectively in the sprouting-resistant cultivar.

An analysis of the role of abscisic acid on the differential expansion and chlorophyll synthesis of the two cotyledons of dormant apple embryo in culture

In dormant embryos of Pyrus malus L. cv. Golden delicious laid flat on water agar and cultured under light, inhibition of growth, chlorophyll synthesis and pro‐plastid development in the upper cotyledon was complete, whereas they occurred to a notable degree in the cotyledon in contact with the medium particularly between days 5 and 9 of culture. Radioimmunoassay of endogenous ABA showed a marked difference between the two cotyledons during the first days of culture: on day 2 the level in the upper cotyledon could be 8 times higher than in the lower cotyledon. Experiments using isolated cotyledons showed that this early difference may be responsible for the subsequent divergence in development of the two cotyledons. The effects of exogenous ABA differed depending on the physiological state of the material: when applied at the start of culture ABA inhibited subsequent development, but if applied after 5 days of incubation, when plastid differentiation was already established, ABA no longer prevented growth and only partially inhibited chlorophyll production. The results suggest a direct causal relationship between the levels of endogenous ABA and cotyledon development.

Chemical ionization mass spectrometry of indol-3yl-acetic acid and cis-abscisic acid: Evaluation of negative ion detection and quantification of cis-abscisic acid in growing maize roots

Mass spectra of the derivatives of indol-3yl-acetic acid and cis-abscisic acid were obtained in electron impact and chemical ionization positive ion and negative ion modes. The respective merits of methane, isobutane, and ammonia as reagent gases for structure determination and sensitive detection were compared using the methyl esters. From one to 10 fluorine atoms were attached to IAA to improve the electron-capturing properties of the molecule. The best qualitative information was obtained when using positive ion chemical ionization with methane. However, the most sensitive detection, with at least two ions per molecule, was achieved by electron impact on the IAA-HFB-ME derivative and by negative ion chemical ionization with NH3 on the ABA-methyl ester derivative. p ]Quantitative analyses of ABA in different parts of maize (Zea mays cv. LG 11) root tips were performed by the latter technique. It was found that the cap and apex contained less ABA than the physiologically older parts of the root such as the elongation zone and the more differentiated tissues. This technique was also used to show a relation between maize root growth and the endogenous ABA level of the elongation zone and root tip: there is more ABA in the slowly growing roots than in the rapidly growing ones.

Kinetics of Endogenous Cytokinin, IAA and ABA Levels in Relation to the Growth and Morphology of Tobacco Crown Gall Tissue

The kinetics of endogenous cytokinin, IAA and ABA levels during the growth cycle of a wild-type tobacco crown gall (W38-B6S3) were compared with that of a shoot-inducing (Shi-) mutant. In both tumor types, high IAA and cytokinin (essentially ribosyl-trans-zeatin and its corresponding glucoside) levels were built up by the end of the linear growth phase and maintained during the greater part of the exponential growth period. The stationary phase was preceded by a very drastic decrease in the endogenous level of both hormones. Quantitatively, the wild-type tumour showed a higher IAA level and a reduced cytokinin level compared with the Shi- mutant. No significantly different endogenous ABA pattern was observed. The reduced cytokinin level might correspond to the ratio of transformed/untransformed cells in the wild-type tumour whereas the reduced IAA level in the Shi- mutant may be correlated with the deletion of gene 2 in the T-DNA of the pGV 2206 Ti plasmid. The elevated cytokinin/IAA ratio induced shooting mainly of the untransformed cells in the Shi- mutant tissue whereas in the wild-type, the shoot suppression was compatible with the reduced cytokinin/IAA ratio.

Effets de L'acide abscissique exogène sur la levée de dormance au froid de tubercules de Topinambour cultivés in vitro

In vitro cultured Helianthus tuberoses L. tubers were treated at low temperatures on a medium supplemented with ABA 1.7 × 10−5 M. Endogenous levels of ABA were measured by gas-liquid chromatography and electron capture. The existence of large quantities of this inhibitor does not appreciably modify the release of dormancy and subsequent germination at 24°C. At low temperatures, breaking of dormancy occurs with endogenous ABA levels of about 200–600 μg/kg fresh matter. Germination proceeds at 24°C with about 50–100 μg/kg fresh matter. The breaking of dormancy and the installation of the non dormant state by the low temperatures seem to be independent of the level of ABA in these tubers. The «boulage»: formation of tubers on a mother tuber, which is a perpetuation of the dormant state, is associated with a decreasing level of endogenous ABA. After 6 months, this level decreases to about that measured in non-dormant tubers. In our material, the ABA level does not appear to be involved in the perpetuation of the dormant state.

Effects of Abscisic Acid and Water Stress on the Embryo and Plantlet Formation in Anther Culture of Nicotiana tabacum cv. Samsun

Summary Effects of short treatments of tobacco anthers with ABA and mannitol on pollen embryogenesis were studied. Three-day treatment with ABA (10 −5 M) had a stimulatory effect on the rate of anthers producing plantlets as well as on the number of plantlets formed per anther. Water stress on the anthers, given by adding mannitol (0.5 M) in medium, showed a clear stimulatory effect on plantlet formation when the rate of which in the corresponding control was low. The water stress, imposed on the anthers for 1 day increased the endogenous ABA level of pollen grains from 0.059 ng/grain to 0.120 ng/grain when determined by gas chromatography. Possible roles of ABA and water stress on the pollen embryogenesis were discussed.

FACTORS AFFECTING ABSCISSION IN SPIRODELA OLIGORHIZA (LEMNACEAE)

SUMMARY0.1 M and 0.2 M sucrose do not induce abscission and significantly reduce abscission induced by ultraviolet radiation whereas higher concentrations (0.4 M) induce abscission even without UV radiation. The effect of different concentrations of sucrose on endogenous ABA levels and on ethylene release suggest that sucrose‐induced abscission occurs when ethylene release is accompanied by a rise in the endogenous ABA level. When plants are floated on low concentrations of ABA (0.1 ppm), they release a considerable amount of ethylene but do not abscise. On higher concentrations of ABA (10 ppm), they abscise, even though ethylene release is lower.