Changes in Amide-Linked and Ester Indole-3-Acetic Acid in Cotton Fruiting Forms during Their Development

Abstract

The concentration of free indoleacetic acid (IAA) is high in cotton (Gossypium hirsutum L.) fruiting forms before anthesis, but is low at and for a few days after anthesis. Amide-linked and ester IAA were measured in fruiting forms at 9, 6, and 3 days before anthesis; at anthesis; and at 2, 4, 7, and 9 days after anthesis to determine if free IAA decreased because it was converted to a conjugated form. Cotton (Gossypium hirsutum L.) fruiting forms rarely abscise during the week before anthesis (12, 22), but are most susceptible to abscission during the week after anthesis (16,22). Abscission rate then decreases with increasing fruit (boll) age until it is essentially zero at 18 d after anthesis (15,16).Because exogenous IAA delays or prevents abscission (2, 7), we measured the concentration of free IAA in fruiting forms from 9 d before until 9 d after anthesis (18). The concentration of free IAA decreased about 80% during the 6 d before anthesis, remained low for the next 4 d, and then increased rapidly between 7 and 9 d after anthesis. Abscission rate increased rapidly after anthesis to a maximum at 5 and 6 d postanthesis. it then decreased as free IAA increased. Because these changes in free IAA were inversely correlated with changes in fruiting-form abscission, we suggested that the high concentration of IAA in large flower buds inhibited abscission before anthesis, whereas the low concentration at and for 4 d after anthesis promoted abscission of young bolls (18).A decrease in the concentration of free IAA could result from decreased synthesis, increased destruction, export, conversion to another form, or a combination of these possible events. We measured the concentrations of ester and amidelinked IAA before, at, and after anthesis to see if the decrease in free IAA was associated with a similar increase in either of the conjugated forms. MATERIALS AND METHODS Plant MaterialCotton (Gossypium hirsutum L.), cv Deltapine 61,' was grown in a field at the Western Cotton Research Laboratory in Phoenix as reported earlier (18). Flower buds were harvested 9, 6, and 3 before anthesis; flowers were harvested at anthesis; and bolls were harvested 2, 4, 7, and 9 d after anthesis. All fruiting forms (buds, flowers, and bolls) were harvested the same day to minimize possible differences due to any change in environment. The fruiting forms were rinsed in cold water, quickly frozen at -80°C, and lyophilized. The dry tissue was ground to pass a 40-mesh screen and stored under N2 at -80°C.Analyses of Ester and Amide IAA Samples of 200 or 500 mg each were extracted overnight in 70% acetone that contained antioxidants (19). '4C-IAA was added as an internal standard. The samples plus three 10-mL rinses with 70% acetone were filtered and the volume reduced to about 9 mL by rotary evaporation at 45°C. Ester IAA was hydrolyzed by adding 1 ml of 10 N KOH, mixing, and incubating 1 h at room temperature (3). Amide-linked IAA was hydrolyzed in 7 N NaOH during 3 h at about 100°C (3). All samples were hydrolyzed in a nitrogen a...

Influence of Water Deficits on the Abscisic Acid and Indole-3-Acetic Acid Contents of Cotton Flower Buds and Flowers

“…Large flower buds, however, contain high concentrations of free and amide-linked IAA (15,16) and are resistant to abscission just before they reach anthesis (7,9,21,23). The high concentrations of IAA in large flower buds decreased rapidly to low levels at anthesis and remained low in young bolls for at least 4 d after anthesis (15,16). Low levels of IAA in young bolls are probably a major factor in the high probability of shedding during the young-boll stage of development.…”

“…Analysis of ABA and IAA Free ABA and free and total IAA were analyzed by HPLC by a modification ofprocedures reported earlier (15)(16)(17). Fifty to 200-mg samples of lyophilized tissue were extracted overnight in 80% methanol (free ABA and free IAA) or in 70% acetone (for total IAA) that contained 100 mg Na ascorbate and 200 mg butylated hydroxytoluene per L as antioxidants.…”

“…Hydrolyzed and free IAA and ABA were extracted from the acidified aqueous phase with CH2Cl2, concentrated to an aqueous phase, and partially purified with nylon filters and C 18 cartridges as reported earlier (6,16 (24) were added as internal standards at the start of extraction and the samples then subjected to the same extraction and bulk purification as outlined above. After elution of ABA and IAA from the C1 8 cartridge with methanol, they were methylated with diazomethane (4) and analyzed with a Hewlett-Packard 5970 GC-MS as described earlier (6).…”