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.     

Abscisic Acid Alters the Metabolism of Indole-3-Acetic Acid in Senescing Flowers of Cucumis melo L

Experiments were conducted to investigate indole-3-acetic acid (IAA) and abscisic acid (ABA) metabolism associated with postanthesis senescence of ovaries from nonpollinated muskmelon (Cucumis melo L.) flowers. Flowers attached to the vine were allowed to senesce for 4 days after anthesis or were harvested at full anthesis and aged for the same time interval with or without access to water. The IAA ester, amide-linked forms of IAA, free ABA, and ABA esters increased in senescent ovaries from flowers left attached to the vine. Detaching flowers from the vine resulted in an accumulation of free and amide-linked IAA in the senescing ovary but suppressed accumulation of ester IAA. Free ABA failed to increase in ovaries detached from the vine. Subjecting detached flowers to water stress had no effect on the endogenous level of free ABA but resulted in the accumulation of ABA ester and suppression of any increase in free IAA. However, detached flowers treated with 0.1 millimolar ABA accumulated 75% less free IAA and initiated the synthesis of ester IAA. Detached flowers treated with ABA also accumulated high levels of ester ABA. These results suggest that the metabolism of free IAA in muskmelon ovary tissue is regulated in situ and not the consequence of external synthesis and importation. ABA appears to be transported into the senescing ovary from an external source and alters the IAA metabolism in such a manner as to suppress the level of free IAA while stimulating accumulation of the ester IAA.     

Endogenous Auxin and Ethylene in the Lichen Ramalina duriaei

Indole-3-acetic acid (IAA) levels and ethylene evolution rates were measured in a fruticose lichen Ramalina duriaei collected from carob trees growing in northeast Israel. IAA levels were estimated by gas liquid chromatography with electron capture detection of the pentafluorobenzyl ester and also by enzyme-linked immunosorbent assay following methylation. The identity of the isolated IAA was confirmed by gas chromatography-mass spectrometry of both the methyl and the pentafluorobenzyl ester. IAA levels in lichens 1 year after transplanting to an air-polluted urban site were found to be lower than in the control thalli left at a nonpolluted, rural site. The material from the latter contained about 2.5 micrograms per gram fresh weight free IAA and 0.5 microgram per gram fresh weight conjugated IAA, while the urban material contained 0.3 microgram per gram each of free and conjugated IAA. Ethylene production rate was 1.0 nanoliter per gram fresh weight per hour in the material from the rural site and 1.5 nanoliters per gram fresh weight per hour in material from the urban site.     

Measurement of Indole-3-Acetic Acid in Peach Fruits (Prunus persica L. Batsch cv Redhaven) during Development

The amount of indole-3-acetic acid (IAA) was measured in peach fruits by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution assay with [¹³C₆]IAA as an internal standard throughout the growing season. Ethylene evolution of the fruit was also measured. IAA levels were 25 nanograms per gram fresh weight, 18 days after anthesis. Both IAA levels and rates of ethylene evolution declined to their lowest levels (7 nanograms IAA per gram fresh weight and 0.01 nanoliter ethylene per gram per hour) in the second stage of fruit growth. Endogenous levels of free-IAA and ethylene evolution increased in the last stage of peach fruit development to 32 nanograms per gram fresh weight and 0.27 nanoliter per gram per hour, respectively. IAA amounts peaked in the ovules 67 days after anthesis.     

Changes in Free and Conjugated Indole 3-Acetic Acid and Abscisic Acid in Young Cotton Fruits and Their Abscission Zones in Relation to Fruit Retention during and after Moisture Stress

Experiments were conducted with field-grown cotton (Gossypium hirsutum L.) in 1985 and 1986 to determine effects of water deficit on levels of conjugated indole 3-acetic acid (IAA) and abscisic acid (ABA) in young fruits (bolls) and their abscission zones in relation to boll retention. Tissues were harvested three times during an irrigation cycle in 1985. They were harvested twice during an irrigation cycle and once after irrigation in 1986 to determine extent of recoveries of measured parameters. As reported earlier, the free IAA content of abscission zones decreased with moisture stress. Irrigation caused a partial recovery in free IAA content of abscission zones and caused a partial recovery in rate of boll retention. In contrast to free IAA, conjugated IAA increased with water deficit, both in 3-day-old bolls and in their abscission zones. Bolls contained much more ester IAA than their abscission zones. Some, but not all, of the increase in ester IAA in bolls during moisture stress could have come from a conversion of amide-linked IAA. Amide IAA decreased slightly during stress and increased after irrigation, but the concentration was low relative to ester IAA. Free and conjugated ABA both increased during stress and decreased after irrigation. However, the concentration of conjugated ABA remained relatively high in abscission zones. Ester IAA, being more resistant than free IAA to enzymic destruction during stress, may hasten recovery of fruit retention after relief of stress by providing a source of free IAA in abscission zones to inhibit continued abscission.     

Free and conjugated indole-3-acetic Acid in developing bean seeds

The changes in conjugated indole-3-acetic acid (IAA) levels compared to the levels of free IAA have been analyzed during the development of bean (Phaseolus vulgaris L.) seed using quantitative mass spectrometry. Free and ester-linked IAA levels are both relatively high in the early stages of seed development but drop during seed maturation. Concomitantly, the amide-linked IAA becomes the major form of IAA present as the seed matures. In fully mature seed, amide IAA accounts for 80% of the total IAA. The total IAA pool in the seed is maintained at approximately the same level (150-170 nanograms/seed) once the level of free IAA has attained its maximum. Thus, the amount of amide IAA conjugates that accumulate in mature seed is closely related to the amounts of free and ester-linked IAA that disappeared from the rapidly growing seed. Analysis of developing bean pods, from which the seeds were taken for analysis, showed very low levels of both ester and amide-linked IAA conjugates. The pattern of changes seen in the levels of free and conjugated IAA in developing bean seed supports our prior hypothesis suggesting a role of IAA conjugates in the storage of the phytohormone in the seed.     

Endogenous IAA levels and development of coffee flower buds from dormancy to anthesis

Dormant coffee (Coffea arabica L.) flower buds require water stress to stimulate regrowth. A xylem specific water-soluble dye, azosulfamide, was used to quantify water uptake of buds after their release from dormancy by water stress. In non-stressed flower buds, the rate of water uptake was generally slower and variable compared to stressed flower buds, where the rate of uptake tripled from 1 to 3 days after rewatering and preceded the doubling of fresh and dry weight of buds. Free, ester and amide IAA levels of developing flower buds were measured by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution technique with [¹³C₆]IAA as an internal standard. Throughout development, the majority of IAA was present as amide IAA. The proportions of amide and free IAA increased one day after plants were released from water stress, and preceded the doubling of fresh and dry weight. Free and conjugated IAA content per bud remained stable during the period of rapid flower growth until one day before anthesis.Abbreviations FW fresh weight - IAA indole 3-acetic acid - HPLC high performance liquid chromatography - GC-MS-SIM gas chromatography-mass spectrometry selected ion monitoring - NAA naphthalene acetic acid - IBA indole butyric acid     

Amide-Linked Indoleacetic Acid Conjugates May Control Levels of Indoleacetic Acid in Germinating Seedlings of Phaseolus vulgaris

We have shown that amide-linked IAA (indole-3-acetic acid) conjugates accumulated to high levels during maturation of bean seeds (K. Bialek and J.D. Cohen [1989] Plant Physiol 91: 775-779). In the present study, we were interested in the fate of these and other IAA conjugates during seed germination. The content of amide-linked conjugates of IAA in cotyledons declined dramatically during the first hours of imbibition. The rate of decline slowed markedly during the period of the resumption of axis growth. The level of amide-linked IAA conjugates in cotyledons remained relatively high after almost 1 week of germination. The decline of IAA conjugates in cotyledons was followed by a steady increase in the content of both free and amide-linked IAA in the embryonic axes. Amide-linked IAA conjugates were also present in the axes cultured on agar after the cotyledons were removed, which suggests that de novo production of these IAA conjugates occurs in the axis of germinating bean seedlings. A comparison of relative amounts of free and conjugated IAA in the axes of intact seedlings and axes cultured on agar showed lower levels of free IAA and higher levels of conjugated IAA in much slower growing isolated axes. These results suggest a more general role for IAA conjugates in the control of seedling growth than simply to serve as a seed storage form of auxin.     

Cytokinin Activity in Lupinus albus L: IV. Distribution in Seeds

Endogenous levels of cytokinin activity were examined in Lupinus albus L. seed at intervals of 2 weeks after anthesis using the soybean callus bioassay. High levels of cytokinin activity per gram seed material were present in the seeds at 2, 4, and 6 weeks after anthesis. The cytokinin activity per gram seed material was low at 8 and 10 weeks after anthesis. Cytokinin activity associated with each seed was greatest at 6 weeks after anthesis. The majority of the activity in the seeds at 4, 6, and 8 weeks after anthesis was in the endosperm. Cytokinin activity was also detected in the testas and embryos at 4, 6, 8, and 10 weeks, and the suspensors at 4 weeks. Column chromatography of extracts of the different seed fractions on Sephadex LH-20 indicated that the cytokinins present coeluted with zeatin, zeatin riboside, and the glucoside cytokinins. It is suggested that cytokinins are accumulated in the seeds and are stored in the endosperm mainly in the form of ribosides and glucosides of zeatin. The reduction in cytokinin activity in the seed coincides with the reduction in endosperm volume and embryo growth and suggests that these compounds are utilized during the course of seed maturation.     

Changes in the level of free and ester indol-3yl-acetic Acid in growing maize roots

The levels of free and ester-linked indole-3-acetic acid (IAA) in different parts of the maize root were measured using gas chromatography-mass spectrometry (selected ion monitoring). In roots of 2-day-old plants, the distribution of free and ester IAA differed both along the root and between stele and cortex. The levels of IAA and IAA esters were then measured in whole roots and in the elongation zone using roots of different ages. The level of ester IAA decreased steadily with time. In contrast, the level of free IAA in the elongation zone was found to increase after a few days of culture at which time the rate of root growth was decreasing.     

Levels of Indole-3-Acetic Acid in Lemna gibba G-3 and in a Large Lemna Mutant Regenerated from Tissue Culture

Large changes in indole-3-acetic acid (IAA) levels occur during growth of Lemna gibba G-3 in sterile culture. The levels of IAA were measured in plants during a 45 day growth cycle using HPLC and isotope dilution analysis followed by selected ion current monitoring GC-MS analysis with ¹³C₆-IAA as the internal standard. Even though the rate of plant growth remained constant over the entire growth period, IAA levels ranged from a high of 222 to a low of 6 nanograms per gram fresh weight. A Lemna mutant (jsR₁) which has a giant phenotype was obtained by regeneration from primary callus cultures. Microspectrofluorometry of diamidino-2-phenylindole stained cells showed that jsR₁ has the same amount of DNA per nucleus as the parent line (PL). All jsR₁ cell types measured are about 1.5 times larger than in PL. The endogenous levels of IAA per gram fresh weight were higher in jsR₁ at several stages of the plant culture cycle as compared to PL. This difference ranged from 1.2 to over 100 times as much. While PL showed only one high peak at day 9, jsR₁ had IAA levels of 480 and 680 nanograms per gram fresh weight at days 9 and 45, respectively. Throughout the midculture stage of the growth cycle (20-28 days) both jsR₁ and PL had IAA levels in the range of 9 to 14 nanograms per gram fresh weight. In contrast to PL, at day 45, jsR₁ had no detectable ester or amide conjugates of IAA. These changes in IAA levels were determined in sterile plant cultures and thus cannot be attributed to bacterial or fungal activity.     

Effect of Endosperm Removal on 7 Normal NaOH-Labile Indole-3-acetic Acid Conjugates in Shoots and Roots of Zea mays Seedlings

The pool of amide-linked indole-3-acetic acid (amide IAA) in the shoot of growing etiolated seedlings of Zea mays increases between the 3rd and 5th day of germination to equal the amount of free IAA and two-thirds the amount of ester IAA. Deseeding the germinant changes the pool size of free and amide IAA in a manner suggestive of conversion of endogenous free IAA to amide IAA. Deseeding also caused an almost total disappearance of amide IAA from the root, demonstrating that the pool of amide IAA is not inert and can be actively metabolized in young Z. mays seedlings.     

Euphorbia escula L. Root and Root Bud Indole-3-Acetic Acid Levels at Three Phenologic Stages

Endogenous indoleacetic acid (IAA) levels of Euphorbia esula L. primary root and root buds were examined at three phenologic stages. High performance liquid chromatography coupled with fluorescence detection and gas chromatography-mass spectrometry, using ¹³C₆[benzene ring]-indole-3-acetic acid as internal standard, were used to measure root bud free and bound IAA levels in vegetative, full flower, and post-flower plants. Highest levels of free IAA (103 nanograms per gram fresh weight) were found in root buds during full flower. Esterified and amide IAA increased significantly in root buds of full flower and post-flower plants, but were not detectable in root buds of vegetative plants. Primary rootfree IAA was highest in vegetative and full flower plants (34.5 nanograms per gram fresh weight) and decreased by 50% in post-flower plants.     

Relationship of endogenous abscisic Acid to sucrose level and seed growth rate of soybeans

It has been proposed that abscisic acid (ABA) may stimulate sucrose transport into filling seeds of legumes, potentially regulating seed growth rate. The objective of this study was to determine whether the rate of dry matter accumulation in seeds of soybeans (Glycine max L.) is correlated with the endogenous levels of ABA and sucrose in those sinks. The levels of ABA and sucrose in seed tissues were compared in nine diverse Plant Introduction lines having seed growth rates ranging from 2.5 to 10.0 milligrams dry weight per seed per day. At 14 days after anthesis (DAA), seeds of all genotypes contained less than 2 micrograms of ABA per gram fresh weight. Levels of ABA increased rapidly, however, reaching maxima at 20 to 30 DAA, depending upon tissue type and genotype. ABA accumulated first in seed coats and then in embryos, and ABA maxima were higher in seed coats (8 to 20 micrograms per gram fresh weight) than in embryos (4 to 9 micrograms per gram fresh weight. From 30 to 50 DAA, ABA levels in both tissues decreased to less than 2 micrograms per gram fresh weight. Levels of sucrose were also low early in development, less than 10 milligrams per gram fresh weight at 14 DAA. However, by 30 DAA, sucrose levels in seed coats had increased to 20 milligrams per gram fresh weight and remained fairly constant for the remainder of the filling period. In contrast, sucrose accumulated in embryos throughout the filling period, reaching levels greater than 40 milligrams per gram fresh weight by 50 DAA. Correlation analyses indicated that the level of ABA in seed coats and embryos was not directly correlated to the level of sucrose measured in those tissues or to the rate of seed dry matter accumulation during the linear filling period. Rather, the ubiquitous pattern of ABA accumulation early in development appeared to coincide with water uptake and the rapid expansion of cotyledons occurring at that time. Whole tissue sucrose levels in embryos and seed coats, as well as sucrose levels in the embryo apoplast, were generally not correlated with the rate of dry matter accumulation. Thus, it appears that, in this set of diverse soybean genotypes, seed growth rate was not limited by endogenous concentrations of ABA or sucrose in reproductive tissues.     

Sucrose metabolism and IAA and ethylene production in muskmelon ovaries

Changes induced by the pollination of ovaries may be mediated by phytohormones and involve sudar-mediated by phytohormones and involve sugar-metabolizing enzymes. In order to further explore these relationships, soluble sugars, sucrose-phosphate synthase (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), acid and neutral invertases (EC 3.2.1.26), indole-3-acetic acid (IAA), and ethylene were investigated in muskmelon (Cucumis melo L.) ovaries sampled before, during, and after anthesis. The fresh weight of ovaries increased 100% within 48 h after pollination, but did not change significantly in the absence of pollination. While sugar content per ovary increased after pollination, sugar content per mg protein was unaffected. Sucrose was not detected in nonpollinated ovaries 48 h after anthesis. Free IAA content was highest in ovaries sampled 48h before anthesis. Pollination had no immediate effect on IAA content per mg protein in postanthesis ovaries. Although detected in all ovaries sampled, ethylene production increased significantly only in nonpollinated ovaries. Activity of sucrose-phosphate synthase was the same at all stages. The specific activities of sucrose synthase and the invertases were highest in nonpollinated ovaries. The increase in rate of sugar import into ovaries following pollination was not accompanied by an increase in the specific activity of any enzyme assayed, but was coincident with an increase in the total activity per ovary of surcose synthase and acid invertase. There appears to be no direct relationship between sucrose-metabolizing enzymes, IAA or ethylene in developing pollinated ovaries but the increase in sucrose cleavage activity in nonpollinated ovaries may be related to the increase in ethylene production.     

Transport of indole-3-butyric acid and indole-3-acetic acid in Arabidopsis hypocotyls using stable isotope labeling

The polar transport of the natural auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) has been described in Arabidopsis (Arabidopsis thaliana) hypocotyls using radioactive tracers. Because radioactive assays alone cannot distinguish IBA from its metabolites, the detected transport from applied [3H]IBA may have resulted from the transport of IBA metabolites, including IAA. To test this hypothesis, we used a mass spectrometry-based method to quantify the transport of IBA in Arabidopsis hypocotyls by following the movement of [13C1]IBA and the [13C1]IAA derived from [13C1]IBA. We also assayed [13C6]IAA transport in a parallel control experiment. We found that the amount of transported [13C1]IBA was dramatically lower than [13C6]IAA, and the IBA transport was not reduced by the auxin transport inhibitor N-1-naphthylphthalamic acid. Significant amounts of the applied [13C1]IBA were converted to [13C1]IAA during transport, but [13C1]IBA transport was independent of IBA-to-IAA conversion. We also found that most of the [13C1]IBA was converted to ester-linked [13C1]IBA at the apical end of hypocotyls, and ester-linked [13C1]IBA was also found in the basal end at a level higher than free [13C1]IBA. In contrast, most of the [13C6]IAA was converted to amide-linked [13C6]IAA at the apical end of hypocotyls, but very little conjugated [13C6]IAA was found in the basal end. Our results demonstrate that the polar transport of IBA is much lower than IAA in Arabidopsis hypocotyls, and the transport mechanism is distinct from IAA transport. These experiments also establish a method for quantifying the movement of small molecules in plants using stable isotope labeling.     

Indole-3-acetic acid and indole-3-acetylaspartic acid isolated from seeds of Heracleum laciniatum Horn

Seeds from mature flowers of Heracleum laciniatum were collected locally (Tromsø, Norway). Seed coats were removed and the seeds were analyzed for their content of free, free plus ester-conjugate, and total indole-3-acetic acid (IAA) by quantitative gas chromatography-mass spectrometry. Seeds contained high levels of free and amide-linked IAA relative to other dicotyledonous seeds for which values have been published. The major amide conjugate in this material was identified as indole-3-acetylaspartate by gas chromatography-mass spectrometry of its bis-methyl ester.     

Variations in sterol and triterpene contents of developing Sorghum bicolor grains

The free, esterified and glycosylated sterols and the pentacyclic triterpene esters of developing Sorghum bicolor grains were analysed by GLC and GC-MS. All the pentacyclic triterpenes were completely esterified but were not detected until 24 days after anthesis. Lupanol, multiflorenol, α-amyrin and isoarborinol were identified in the mature grains as components of the triterpene fraction but no 4,4-dimethylsterols could be found at any stage of development. A sixfold increase in total sterol per grain occurred during development. At 8 days after anthesis, 28-isofucosterol was found to be the second most abundant steryl ester. Campesterol was the major steryl glycoside and obtusifoliol was the major 4-monomethylsterol.     

Photosynthetic and Respiratory Activity of Fruiting Forms within the Cotton Canopy

The supply of photosynthates by leaves for reproductive development in cotton (Gossypium hirsutum L.) has been extensively studied. However, the contribution of assimilates derived from the fruiting forms themselves is inconclusive. Field experiments were conducted to document the photosynthetic and respiratory activity of cotton leaves, bracts, and capsule walls from anthesis to fruit maturity. Bracts achieved peak photosynthetic rates of 2.1 micromoles per square meter per second compared with 16.5 micromoles per square meter per second for the subtending leaf. However, unlike the subtending leaf, the bracts did not show a dramatic decline in photosynthesis with increased age, nor was their photosynthesis as sensitive as leaves to low light and water-deficit stress. The capsule wall was only a minor site of ¹⁴CO₂ fixation from the ambient atmosphere. Dark respiration by the developing fruit averaged −18.7 micromoles per square meter per second for 6 days after anthesis and declined to −2.7 micromoles per square meter per second after 40 days. Respiratory loss of CO₂ was maximal at −158 micromoles CO₂ per fruit per hour at 20 days anthesis. Diurnal patterns of dark respiration for the fruit were age dependent and closely correlated with stomatal conductance of the capsule wall. Stomata on the capsule wall of young fruit were functional, but lost this capacity with increasing age. Labeled ¹⁴CO₂ injected into the fruit interior was rapidly assimilated by the capsule wall in the light but not in the dark, while fiber and seed together fixed significant amounts of ¹⁴CO₂ in both the light and dark. These data suggest that cotton fruiting forms, although sites of significant respiratory CO₂ loss, do serve a vital role in the recycling of internal CO₂ and therein, function as important sources of assimilate for reproductive development.     

Sucrose metabolism and IAA and ethylene production in muskmelon ovaries

Changes induced by the pollination of ovaries may be mediated by phytohormones and involve sudar-mediated by phytohormones and involve sugar-metabolizing enzymes. In order to further explore these relationships, soluble sugars, sucrose-phosphate synthase (EC 2.4.1.14), sucrose synthase (EC 2.4.1.13), acid and neutral invertases (EC 3.2.1.26), indole-3-acetic acid (IAA), and ethylene were investigated in muskmelon (Cucumis melo L.) ovaries sampled before, during, and after anthesis. The fresh weight of ovaries increased 100% within 48 h after pollination, but did not change significantly in the absence of pollination. While sugar content per ovary increased after pollination, sugar content per mg protein was unaffected. Sucrose was not detected in nonpollinated ovaries 48 h after anthesis. Free IAA content was highest in ovaries sampled 48h before anthesis. Pollination had no immediate effect on IAA content per mg protein in postanthesis ovaries. Although detected in all ovaries sampled, ethylene production increased significantly only in nonpollinated ovaries. Activity of sucrose-phosphate synthase was the same at all stages. The specific activities of sucrose synthase and the invertases were highest in nonpollinated ovaries. The increase in rate of sugar import into ovaries following pollination was not accompanied by an increase in the specific activity of any enzyme assayed, but was coincident with an increase in the total activity per ovary of surcose synthase and acid invertase. There appears to be no direct relationship between sucrose-metabolizing enzymes, IAA or ethylene in developing pollinated ovaries but the increase in sucrose cleavage activity in nonpollinated ovaries may be related to the increase in ethylene production.Mention of a specfic product does not imply an endorsement by the United States Depertment of Agriculture or Texas Aricultural Experiment Station over other suitable products.