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Relatively little is known about changes in the cytosolic free calcium ion concentration ([Ca(2+)](c)) in monocotyledonous plants. Therefore, we produced transgenic winter wheat lines stably expressing the calcium-sensitive photoprotein aequorin constitutively in the cytosol. [Ca(2+)](c) was detected in vivo by luminometry, and [Ca(2+)](c) elevations were imaged at video rate. Experiments with the transgenic seedlings focused on potential changes in [Ca(2+)](c) during cold exposure. Temperature-induced changes in [Ca(2+)](c) were found to be more dependent on the change in temperature (dT dt(-1)) than on the absolute value of temperature. [Ca(2+)](c) increased only at cooling rates higher than 8 degrees Cmin(-1), indicating that an overall cellular [Ca(2+)](c) increase is of minor relevance as a signal for cold acclimation in wheat under ecological conditions. The results are discussed with regard to the so-called 'calcium signature hypothesis'.

Cold acclimation of plants affects many aspects of metabolism. Changes in plasma membrane lipids have always been considered to be important for development of frost resistance and survival at subzero temperatures. We studied different cultivars of winter wheat (Triticum aestivum L.) that differed in frost resistance induced either by cold acclimation or treatment with the plant hormone abscisic acid (ABA). Plasma membranes were isolated from non-acclimated and cold- as well as from ABA-acclimated plants, and were subjected to detailed lipid analysis. Cold acclimation affected virtually all plasma membrane lipid components and their constituents, resulting in both increases and decreases, which varied between the three groups of plants investigated. Including the cold-induced variations observed in the few plant species studied in detail previously, cerebrosides were the only components reduced by cold acclimation in all plants. In wheat, more uniform and consistent patterns were obtained when considering colligative parameters such as total free sterols, phospholipids or glycolipids, either as the proportion of total lipids or based on plasma membrane protein. The parameter which changed most significantly in parallel to the increase of inducible frost resistance in the three groups of plants was the ratio of free sterols/glycolipids, which increased. ABA treatment resulted in qualitatively similar effects in only one cultivar, but in general these changes were less pronounced. Compared to changes in transcription rates of several cold-induced genes and in the concentration of various compatible solutes reported for other plants, the observed changes in plasma membrane lipids are minor ones. This may indicate that acclimation-induced changes can be accomplished by posttranscriptional regulation of enzymatic activities, which is in agreement with the failure to detect significant changes in transcription of the corresponding genes during cold induction.

Four-week-old potted sunflower plants (Helianthus annuus L.) were exposed to drought for up to two days by withholding irrigation. During the stress treatment and after rewatering, xylem sap was collected from decapitated hypocotyls by pressurising the root system. The water potential (Ψ w ) of the hypocotyl, the diffusive resistance of the second leaf pair, total transpiration and the concentration and flux rates of ABA and trans-zeatin riboside (ZR), identified by combined gas chromatography-mass spectrometry (GC-MS) as the dominant cytokinin in xylem sap, were determined. ABA contents were also analysed in root and leaf tissue. When Ψ w of the hypocotyl decreased, the concentration and flux rate of ZR decreased drastically after a transient rise. A significant rise in ABA concentration and flux rate in xylem sap as well as a parallel rise in leaf diffusive resistance occurred as soon as Ψ w reached values of -0.4 MPa and lower. Root ABA concentration began to rise at the same water potential parallel to the rise in xylem sap, whereas the ABA concentration in leaves began to rise only at Ψ w values lower than -0.6MPa. Treatment of the root system with norflurazon prior to drought stress suppressed the increase in the ABA concentration in xylem sap and caused higher transpiration rates. Watering the drought-stressed plants led to a rapid decrease in ABA content of the xylem sap within three hours, whereas the decrease in leaf diffusive resistance was somewhat slower. The ZR concentration in the xylem sap rose continuously after rewatering, reaching a 60-fold increase after five hours, and declined again afterwards. Studies in which ZR and ABA were applied to cut shoots in concentrations similar to those in xylem sap of well-watered plants (ZR) and drought-stressed plants (ABA) showed that ZR, even in very low concentrations, antagonised the effect of ABA on transpiration. The results are discussed with regard to a possible antagonistic interaction of ZR and ABA as non-hydraulic root-to-shoot signals, and with regard to their interplay with hydraulic signalling.

Four inbred maize lines differing in chilling tolerance were used to study changes in water status and abscisic acid (ABA) levels before, during and after a chilling period. Seedlings were raised in fertilized soil at 24/22°C (day/night), 70% relative humidity. and a 12-h photoperiod with 200 μmol m -2 s -1 from fluorescent tubes. At an age of 2 weeks the plants were conditioned at 14/12°C for 4 days and then chilled for 5 days at 5/3°C. The other conditions (relative humidity, quantum flux, photoperiod) were unchanged. After the chilling period the plants were transferred to the original conditions for recovery. The third leaves were used to study changes in leaf necrosis, ion efflux, transpiration, water status and ABA accumulation. Pronounced differences in chilling tolerance between the 4 lines as estimated by necrotic leaf areas, ion efflux and whole plant survival were observed. Conditioning significantly increased tolerance against chilling at 5/3°C in all genotypes. The genotypes with low chilling tolerance had lower water and osmotic potentials than the more tolerant genotypes during a chilling period at 5/3°C. These differences were related to higher transpiration rates and lower diffusive resistance values of the more susceptible lines. During chilling stress at 5/3°C ABA levels were quadrupled. Only a small rise was measurable during conditioning at 14/12°C. However, conditioning enhanced the rise of ABA during subsequent chilling. ABA accumulation in the two lines with a higher chilling tolerance was triggered at a higher leaf water potential and reached higher levels than in the less tolerant lines. We conclude that chilling tolerance in maize is related to the ability for fast and pronounced formation of ABA as a protective agent against chilling injury.

The effect of abscisic acid (ABA) on assimilate transport in barley was investigated in two parallel experiments. First, the effect upon [(14)C]sucrose transport from the flag leaf to the ear of a single ABA application made at different stages of growth of the fruits was investigated; the effect was measured 24 h after treatment. Second, the effect of a single application of ABA made at the same stages of growth as above on grain weight of the mature plant was investigated. In both types of experiments ABA was applied once to the ear of different plants as an aqueous solution (10(-3)-10(-5) M), one to five weeks after anthesis. [(14)C] sucrose was applied by means of agar blocks. Parallel to these experiments, the endogenous content of ABA was investigated in the developing grains. When ears were treated with ABA two or four weeks after anthesis, an increase of up to 70% in the (14)C-transport from the flag leaf to the ear was observed within a 24-h period after treatment (short duration experiments). At these growth stages the endogenous concentrations of ABA were low. In sharp contrast, ABA, especially in a concentration of 10(-3) M, decreased (14)C-import from the flag leaf when applied three weeks after anthesis. At this stage the endogenous ABA content had reached its maximum. Long duration experiments with a single application of ABA to the car two weeks after anthesis resulted in a marked increase of weight per thousand kernels. ABA applications made earlier or later than two weeks after anthesis either reduced the grain weight or had no effect. It is concluded that ABA is involved in the regulation of assimilate transport from the leaves to the grains, possibly by influencing the unloading of sieve tubes in the ears. Promotion or inhibition of assimilate import by exogenously applied ABA may depend on the developmental stage of the grains and on the endogenous ABA level.

The intial phases of auxin-induced growth in coleoptile segments of Avena sativa L. were investigated using a high resolution growth recording technique, based on an angular position sensing transducer. The first response to the hormone is a slight, transient reduction of the growth rate lasting about 5 min. After this phase growth rate increases to a maximum. The duration of the increase and the maximum clearly depend on the concentration of the hormone. With increasing auxin concentration the duration of the growth rate increase is reduced from about 80 min in 10(-9) M indoleacetic acid (IAA) to about 14 min in 10(-4) M IAA. After the maximum the growth rate declines. Looking at the maximum of the growth rate, we obtained a dose-response curve with a sharp increase between 10(-9) M and 10(-6) M IAA and a slight decline between 10(-6) M and 10(-4) M IAA. This result is confirmed by growth rates measured one and two hours after the application of the hormone.

Changes in the abscisic acid (ABA) content of epidermis and mesophyll of isolated Commelina communis L. leaves were followed and compared to changes in diffusion resistance during a water stress treatment. Diffusion resistance increased within 15 min before a rise in the ABA content of the epidermis could be determined. By application of (14)C-labelled ABA to isolated leaves via the transpiration stream it was found that the minimal amount of ABA to induce stomatal closure was in the range of 0.4 fmol per stomatal complex. Isolated epidermis was treated with tritiated ABA and subjected to microautoradiography. The stomatal complexes showed an accumulation of radioactive material. This accumulation was observable in living as well as in non-living, heat- or cold-treated epidermis. The results are discussed in relation to the hypothesis that ABA regulates stomatal closure in water-stressed leaves., (Copyright © 1985 Gustav Fischer Verlag, Stuttgart. Published by Elsevier GmbH.. All rights reserved.)

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-(14)C]ABA, spec. activity 1.7·10(8)d s(-1)mmol(-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.

1. An already published test method for detecting bactericidal substances in paper chromatograms was further improved. 2. In cultures of Cosmarium impressulum free from bacteria, two bactericidal substances were found in the ether extracts from the algae and two others in the extracts of the culture medium. The are active against some or all bacteria testes (Serratia marcescens, Pseudomonas fluorescens; Aerobacter aerogenes or Bacillus pumilus). 3. If the culture medium of Cosmarium or another desmidiale was inoculated with the test bacteria, a clear bactericidal effect was never observed. 4. Because the activity of the bactericidal substances of Cosmarium is only small and the formation is not constant, it is concluded that the water-soluble bactericidal substances of the alga are not the cause that epiphytic bacteria do not grow normally on Cosmarium.

When pea seedlings lose about 5% of their water content the abscisic acid ((+)-ABA) level of the shoots increases ca. 20 times and the level of bound ABA, in all probability ABA-glucose, ca.7-10 times. After watering both ABA and bound ABA contents decrease within 24-48 h to the level in the control plants.After application of (±)-[2-(14)C] ABA to wilted pea shoots at the time of watering radioactive substances appear in the water-soluble, ether-insoluble fraction of ethanolic extracts and increase with time whereas radioactivity in the acidic ether fraction decreases. The neutral ether fraction remains free of radioactivity. Three radioactive zones, A, B, and C, are seen on chromatograms of the water-soluble fraction. A increases considerably within the entire experimental time, whereas B increases in the first 4-8 h after application and subsequently decreases. The third substance, C, which releases free ABA after hydrolytic treatment, does not change during the experiment. Chromatograms of the acidic ether fraction yield ABA and a substance staying at the origin, possibly phaseic acid and/or dihydrophaseic acid. Only the activity of ABA decreases during the experiment.

The role of abscisic acid (ABA) in the transport of water and ions from the root to the shoot of sunflower plants (Helianthus annuus) was investigated by application of ABA either to the root medium or to the apical bud. The exudation at the hypocotyl stump of decapitated seedlings was measured with and without hydrostatic pressure (0-0.3 MPa) applied to the root. All ABA concentrations tested (10(-10)-10(-4) mol·l(-1)) promoted exudation. Maximal amounts of exudate (200% of control) were obtained with ABA at 10(-6)·mol·l(-1) and an externally applied pressure of 0.1 MPa. The effect was rapid and long-lasting, and involved promotion of ion release to the xylem (during the first hours) as well as an increase in hydraulic conductivity. Abscisic acid applied to the apical bud had effects similar to those of the rootapplied hormone. Increased rates of exudation were also obtained after osmotic stress was applied to the root; this treatment increased the endogenous level of ABA in the root as well as in the shoot. Water potentials of the hypocotyls of intact plants increased when the roots were treated with ABA at 5°C, whereas stomatal resistances were lowered. The results are consistent with the view that ABA controls the water status of the plant not only by regulating stomatal transpiration, but also by regulating the hydraulic conductivity of the root.

The presence of abscisic acid (ABA) in methanol extracts from tomato fruits was determined by thinlayer chromatography, UV absorption, optical rotatory dispersion (ORD) and biological activity in different bioassays. In two growth periods (1968 and 1969), quantitative changes of the ABA content in growing fruits of the variety "Moneymaker" were measured by Milborrow's "racemate dilution technique". The absolute content of ABA (μg/l fruit) was increased during fruit development, reached a maximum, and then decreased in ripening fruits. The ABA concentration (μg/kg) was also highest in unripe fruits and decreased during ripening. Similar results were obtained with the same variety and with the variety "Haubners Vollendung" by means of ORD and UV measurement only, without application of Milborrow's technique.

Twelve-day old pea seedlings were treated with gibberellic acid (GA3). The hormone was applied to the apical bud in quantities of 0.05, 0.5, and 5 μg in 5 μl droplets.After 2 and 4 days the contents of indoleacetic acid (IAA) and abscisic acid (ABA) of the apical parts (apical bud including the 5th internode) and of the basal parts (3rd and 4th internode and leaves) were estimated by means of paper chromatography of ether extracts and bioassay with avena coleoptile segments. In addition, the contents of ABA were checked by spectropolarimetric measurement.Apical parts of the gibberellin treated plants contain more IAA, basal ones less IAA than the untreated controls. On the other hand, the contents of ABA are higher in basal parts and lower in apical parts. Only the seedlings treated with 5 μg GA3 and analysed after 2 days do not follow this rule. Their apical parts contain not only more IAA but also more ABA than the untreated plants, and their basal parts contain more IAA than the controls.It is concluded, therefore, that GA3 causes changes not only in the contents of these hormones but also in the distribution of hormones within the plant.Moreover, GA3 decreases the contents of chlorophyll a and b and of the carotenoids. The changes in carotenoid contents do not seem to be related to the changes in abscisic acid contents.

By paper chromatographic analysis with isopropanol-water-ammonia two fractions which inhibit growth and germination and two fractions which promote straight growth but have no effect on germination were isolated from ether extracts of green pea shoots. The four fractions have the following properties:Rf 0.35; acid; promotes Avena straight growth; has no effect on germination of Lepidium;Rf 0.6; acid; inhibits straight growth and germination;Rf 0.75; acid; behaves like fraction Rf 0.35 but is less active;Rf 0.85; neutral or alkaline; acts similarly to fraction Rf 0.6.The activity of the two inhibiting fractions Rf 0.6 and 0.85 in inhibiting Avena straight growth and Lepidium germination is not changed when a neutral solvent (isopropanol-water) is used instead of the alkaline solvent isopropanol-water-ammonia.There is no evidence for an alkaline germination-inhibiting substance with a Rf-value of 0.55, the so-called correlation inhibitor reported by LIBBERT and LIEBENOW (1964).The behavior of the four fractions was investigated especially in two different bud growth tests. 1. Lateral buds from decapitated pea seedlings are prevented from outgrowth by application of lanolin pastes containing the two straight growth and germination inhibitors. The fraction Rf 0.6 is more active in this test than the fraction Rf 0.85. The inhibition is partly cancelled by the simultaneous addition of gibberellic acid. The fractions Rf 0.35 and 0.75 are inactive in this test. 2. In a second bud growth test isolated nodes with a single bud were used. These test organs are placed horizontally with the buds upward into aqueous solutions containing the chromatographic fractions. Bud growth is inhibited by fraction Rf 0.35 and (to a lesser extent) by fraction Rf 0.75; the fractions Rf 0.6 and 0.85 are inactive. The different action of these growth regulators in the described bud growth tests and their possible role in correlative bud inhibition in vivo is discussed.

Some sugars, especially sucrose and glucose, and some inorganic salts have been found to enhance in low concentrations the germination inhibiting effect of abscisic acid (ABA) in lettuce. There is evidence from gas chromatographic analysis and from bioassays of seed extracts that this stimulation is due to increased uptake of ABA from solution in the presence of glucose and sucrose.