A Simple Purification of Indole-3-Acetic Acid and Abscisic Acid for GC-SIM-MS Analysis by Microfiltration of Aqueous Samples through Nylon

A simple procedure was developed for the partial purification of plant tissue samples to be analyzed simultaneously for indole-3-acetic acid (IAA) and abscisic acid (ABA). The procedure relies on removal of contaminants by filtration through nylon and partitioning into dichloromethane. This procedure successfully purified both IAA and ABA from muskmelon, cotton, and broccoli tissue. Twenty individual samples can be purified and methylated in 8 h for analysis of free IAA and ABA with gas chromatography-selected ion monitoring-mass spectrometry. The use of microfiltration of aqueous samples through nylon offers new opportunities for improving the efficiency of existing sample purification procedures.     

Changes in Abscisic Acid and Indoleacetic Acid before and after Anthesis Relative to Changes in Abscission Rates of Cotton Fruiting Forms

Cotton (Gossypium hirsutum L.) fruiting forms exhibit pronounced changes, with age, in their probability of abscission. Large floral buds rarely abscise, but after anthesis the young fruits (bolls) have a high probability of abscising. Abscission rate reaches a peak about 5 to 6 days after anthesis and then gradually decreases. An experiment was conducted to try to determine the reason for the rapid and pronounced increase in probability of abscission just after anthesis. Cotton was grown in the field and fruiting forms of various ages from 9 days before to 9 days after anthesis were all harvested the same day and subsequently analyzed for ABA and IAA. The concentration of ABA decreased slightly at anthesis and increased gradually thereafter. In contrast, the concentration of IAA was high before anthesis and then decreased at anthesis to about one-fifth the previous concentration. IAA remained low for at least 4 days after anthesis and then increased rapidly between 7 and 9 days after anthesis. The high concentration of IAA in floral buds before anthesis is probably a major factor in their resistance to abscission. Likewise, the low concentration of IAA at anthesis and for about 4 days thereafter may promote fruit abscission during the young boll stage.     

Purification and measurement of abscisic Acid and indoleacetic Acid by high performance liquid chromatography

A procedure was selected for the simultaneous extraction and purification of abscisic acid (ABA) and indoleacetic acid (IAA). Unnecessary steps were eliminated and an accumulation of aqueous phase was avoided. The superior performance of diethyl ether (compared to ethyl acetate) for bulk purification and the superior resolution provided by 250 millimeter columns packed with 5-micrometer spherical particles of strong anion exchanger and octadecylsilane (C18) greatly facilitated the purification of samples. A fixed-wavelength (254 nanometer) ultraviolet detector and a fluorescence detector connected in series on a high performance liquid chromatograph permitted nondestructive monitoring and measurement of ABA and IAA. Derivatization was not necessary for chromatography or for detection. Isocratic elution with simple mobile phases gave sharp peaks. A few simple precautions minimized losses. Recoveries through the entire procedure averaged about 75% for ABA and about 50% for IAA. Purified ABA and IAA fractions were usually free of interfering contaminants. Identities were confirmed by gas chromatography-mass spectrometry.     

Abscisic Acid and cutout in cotton

A decline in growth, flowering, and boll (fruit) retention is referred to as cutout in cotton (Gossypium hirsutum L.). Fruit load affects cutout, possibly through hormonal effects. Experiments were conducted to test the hypothesis that fruits are a source of abscisic acid (ABA) that moves into fruiting branches and growing points where it inhibits growth, flowering, and boll retention. Removal of the flower or young boll at the first node of fruiting branches did not decrease the ABA content of fruiting branches or the abscission zone at the second node. Effects on ABA content of the boll at the second node varied. In one field test, ABA content of bolls at the second node decreased with successive harvests as bolls were removed from first node positions of several fruiting branches. Thus, the effect was cumulative and was not limited to individual branches. Removal of the flower or boll at the first node increased boll retention at the second node. Removal of all flowers during the first 3 weeks of flowering delayed the decreases in growth, flowering, and boll retention that occurred as fruit load increased. But, the ABA content of fruiting branches and mainstem apices was not decreased by early defruiting and did not increase with increasing fruit load. The results do not support the hypothesis that fruits are a source of ABA that moves into fruiting branches and growing points where it then inhibits growth, flowering, and boll retention.     

Chilling injury and changes in adenosine triphosphate of cotton seedlings

Young Gossypium hirsutum L. seedlings chilled at 5° showed a continual decrease in ATP concentration with time of chilling. Chilled plants returned to optimum conditions were able to restore the initial ATP concentration when chilled only 1 day, but not when chilled 2 days. The decrease in ATP with chilling was prevented by hardening the seedlings at 15° for 2 days (14-hr-day-length) immediately before chilling. The ATP level of hardened plants was higher than of unhardened plants. When hardened plants were chilled at 5°, the ATP level increased in the leaves but decreased in the roots.     

Influence of water deficits on the abscisic Acid and indole-3-acetic Acid contents of cotton flower buds and flowers

A field experiment was conducted during the summer of 1988 to test the hypothesis that water deficit affects the abscisic acid (ABA) and indole acetic acid (IAA) concentrations in cotton (Gossypium hirsutum L.) flower buds in ways that predispose young fruits (bolls) that subsequently develop from them to increased abscission rates. Water deficit had little effect on the ABA content of flower buds but increased the ABA content of flowers as much as 66%. Water deficit decreased the concentrations of free and conjugated IAA in flower buds during the first irrigation cycle but increased them during the second cycle. Flowers contained much less IAA than buds. Water deficit slightly increased the conjugated IAA content of flowers but had no effect on the concentration of free IAA in flowers. Because water deficit slightly increased the ABA content but did not decrease the IAA content of flowers, any carry-over effect of water deficit on young boll shedding might have been caused by changes in ABA but not from changes in IAA.     

A short history of nuclear activation analysis

The major developments in the field of nuclear activation analysis, from 1936 to 1989, are discussed. The developments are grouped into five consecutive time periods. The impact of various scientists on the development of the field in the first 35 years is also discussed.     

Effects of some organic solvents on ethylene evolution from young cotton bolls

The presence of promoter(s) of ethylene biosynthesis in young cotton (Gossypium hirsutum L.) fruits (bolls) was demonstrated by injection of an aqueous extract from bolls into other bolls and measurement of a 3-fold increase in rate of ethylene evolution. Injection of methionine did not affect rate of ethylene production, indicating that the promoter extracted from bolls was not methionine. Injection of the ethoxy analog of rhizobitoxine inhibited ethylene production, indicating that methionine is a precursor of ethylene in cotton bolls. Injection of organic solvents altered membrane permeability, as indicated by decreased resistance to electric current at 1,000 Hz, and stimulated ethylene evolution. The less polar solvents caused large increases in ethylene evolution, major loss of resistance, and visible evidence of membrane damage. The results support the hypothesis that membrane integrity affects rate of ethylene biosynthesis.     

Activity and flexibility of alcohol dehydrogenase in organic solvents

The oxidation of cinnamyl alcohol to cinnamaldehyde by horse liver alcohol dehydrogenase (LADH) was carried out in nearly anhydrous organic solvents and in solvents containing from 0.1 to 10% added water. In nearly anhydrous solvents containing less than 0.02% water, the oxidation rate increased as the water solubility in the solvent decreased, but the reaction did not require active LADH. Moreover, the highest activity in nearly anhydrous heptane was obtained by lyophilizing the enzyme from a solution of pH 2.0, even though LADH exhibits virtually no enzymatic activity in water at this pH. The catalytic activity of LADH was restored and increased dramatically as small amounts of water were added to each solvent. In conjunction with the activity measurements, electron paramagnetic resonance (EPR) spectroscopy and two active-site directed spin labels were used to examine solvent-dependent structural features of LADH. The EPR spectra indicated that LADH became more rigid as the dielectric constant of the solvent decreased. The degree of rigidity also depended on the pH from which the enzyme was lyophilized, indicating that the ionization state of the enzyme can have an important influence on its dynamics in organic solvents. Finally, adding 1% water to organic solvents had no apparent effect on the enzyme's conformation or flexibility near the spin label, even though enzyme activity was an order of magnitude higher when 1% water was present.