Potential Role of Abscisic Acid in Cotton Fiber and Ovule Development

Fibers and ovules of a cotton cultivar (Gossypium hirsutum L. Trambak-108) were analyzed for growth and free abscisic acid (ABA) content by indirect enzyme immunoassay. An inverse correlation between fiber elongation and ABA content was observed. In the seed, accumulation of ABA was observed during secondary thickening and the maturation phase. The potential role of ABA in fiber and seed development is discussed. Received June 25, 1997; accepted October 15, 1997     

Levels of Cytokinins in the Ovules of Cotton Mutants with Altered Fiber Development

Endogenous levels of cytokinin and abscisic acid (ABA) were determined in ovules of normal cotton (TM-1) and four fiber differentiation mutants (n2, Ligon lintless, H10, and Xu142) before and after flowering by enzyme-linked immunosorbent assays. The fluctuation patterns of ABA levels in ovules of normal cotton and mutants were similar. At the fiber elongation stage, ABA content was low, and from 1 day after flowering, the ABA content decreased steadily. On the other hand, the peaks of isopentenyladenine and isopentenyladenosine in ovules of TM-1 were observed 1 day before flowering. The level of cytokinins decreased after flowering in TM-1, whereas in the mutants it increased steadily. These results indicate that endogenous ABA is probably not the main inhibitor for fiber elongation and that endogenous cytokinins likely play a dual role in fiber development. Before flowering, cytokinins function as one of the stimuli for the initiation of fibers, but after flowering, cytokinins inhibit fiber growth. Received February 18, 1997; accepted June 11, 1997     

Fluctuation in levels of endogenous plant hormones in ovules of normal and mutant cotton during flowering and their relation to fiber development

The contents of indole-3-acetic acid (IAA), gibberellins (GAs), abscisic acid (ABA), and cytokinins were determined in ovules of normal cotton (Tm-1) and a kind of fiber differentiation mutant (Xin) before and after flowering by enzyme-linked immunosorbent assays. It was found that 24 h before flowering, a peak of IAA content was observed in ovules of Tm-1, whereas in ovules of Xin, a low level of IAA was determined. From –1 day (1 day before flowering) to +3 days (3 days after flowering), GA₁₊₃ levels in ovules of Xin were 40–70% lower than those of Tm-1; GA₄₊₇ levels were very low, and there was no visible difference in GA₄₊₇ content between normal and mutant cotton. The ABA content in ovule of Tm-1 decreased by 70% 3 days after flowering, whereas that of Xin only decreased by 20%. The levels of cytokinins in ovules of Tm-1 decreased after flowering, and those of Xin kept up a steady increase.Abbreviations IAA indole-3-acetic acid - GA gibberellin - ABA abscisic acid - ELISA enzyme-linked immunosorbent assay - FW fresh weight - PBS phosphate-buffered saline - iPA isopentenyladenosine - ZR zeatin riboside - DHZR dihydrozeatin riboside - CTK cytokinin     

Effect of phytohormones on fiber initiation of cotton ovule

In order to study the effect of phytohormones on cotton fiber initiation, contents of four endogenous phytohormones and activities of four related enzymes in ovules (in vivo) of a fuzzless–lintless mutant (fl) and its wild-type (FL) line were measured from 4 days before anthesis (day −4) to 4 days after anthesis (day 4). The results showed that contents of indole-3-acetic acid, gibberellic acid (GA), and zeatin riboside in fl ovules were lower than those in FL ovules. Therefore, indole-3-acetic acid, GA, and zeatin riboside were thought to be the promoters of fiber initiation. Although abscisic acid (ABA) content in fl ovule was slightly higher than that in FL ovule on day 0, which might imply that ABA inhibited fiber initiation. Fiber initiation could also be influenced by enzyme through regulating synthesis and degradation of related phytohormones since fl ovules were significantly higher in activities of indole-3-acetic acid oxidase, cytokinin oxidase and peroxidase, but lower in activity of tryptophan synthetase than those in FL ovules. To test the above hypothesis, exogenous plant growth regulators were also applied for the culture of ovules from fl and FL in vitro. When no regulators were added, no fiber was induced on fl ovule, but a few fibers were induced in FL ovule. Higher total fiber units (TFU) were observed when indole-3-acetic acid and gibberellic acid (GA₃) were applied either separately or in combination to media. TFU did not increased with zeatin riboside alone, but the highest TFU was achieved when zeatin riboside was applied together with indole-3 acetic acid and GA₃, which implied that fiber initiation could be promoted by them as additive.     

Digital image analysis of expansion growth of cultured cotton ovules with fibers and their responses to ABA

Cotton ovule cultures have obvious advantages over whole plants when experimental protocols call for inhibitors, radio-labeled precursors or controlled environmental conditions to be tested. The responses of ovule expansion growth and attached fiber elongation to external factors require accurate measurement techniques. This paper presents a new method for digital image analysis of the growth area of cotton ovules with fibers at high resolution. The method was characterized under constant conditions and during dynamic responses to different levels of ABA (abscisic acid) treatment. The growth area was treated as area occupied within the outline of the Petri dish image of the growing ovule with fibers. Growth area increase showed the same trends as fiber length increase and was significantly correlated with the fiber length increase under different levels of ABA treatment (r ² = 0.97). This new analysis method provides a simple, noninvasive, and more accurate approach for growth analysis in the cotton ovule culture system. Using this method, the effects of ABA on expansion growth of ovule with fibers were characterized.     

EFFECTS OF PLANT GROWTH SUBSTANCES ON IN VITRO FIBER DEVELOPMENT FROM UNFERTILIZED COTTON OVULES

Fertilization of cotton ovules was prevented by removal of styles and stamens on the morning of anthesis. Forty-eight hr later ovaries were harvested and ovules were aseptically transferred to liquid culture medium supplemented with various plant growth substances. In the absence of phytohormones, ovules browned and failed to increase in size or produce fibers. Indoleacetic acid and gibberellic acid provided for ovule growth and fiber development. Kinetin provided for ovule growth only. The ovule's capacity for indoleacetic acid- or gibberellic acid-stimulation of fiber development was reduced by high concentrations of kinetin or abscisic acid. Low concentrations of kinetin partially reversed the inhibitory effect of abscisic acid.     

棉花胚珠纤维发育的研究

将未受精的棉花胚珠漂浮培养在加有不同植物激素的BT培养基上,培养48小时或96小时后,用扫描电镜观察纤维发育情况,以及测定胚珠内IAA氧化酶活性变化及内源ABA的含量变化,并和同一时期的大田生长的胚珠进行比较。结果表明:IAA+GA_3是最佳激素组合。在这种激素组合的培养基中培养的未受精胚珠,在纤维发育、酶活性变化等方面,均与大田生长的胚珠相似。这一激素组合还能抑制离体胚珠内源ABA的增长,但同一时期的大田生长的胚珠,其内源ABA含量却相对要高。     

Osmoregulation in Cotton Fiber: Accumulation of Potassium and Malate during Growth

Kinetics and osmoregulation of cotton (Gossypium hirsutum L.) fiber growth (primarily extension) have been studied. Growth is dependent on turgor pressure in the fiber. It is inhibited when a decrease in the water potential of the culture medium due to an addition of Carbowax 6000, equals the turgor pressure of the fiber. Potassium and malate accumulate in the fiber and reach peak levels when the growth rate is highest. Maximum concentrations of potassium and malate reached in the fiber can account for over 50% of the osmotic potential of the fiber. As growth slows down, levels of potassium and malate decrease and turgor pressure declines. Cotton ovules are capable of fixing H¹⁴CO₃⁻ in the dark, predominantly into malate. Fiber growth is inhibited by the absence of potassium and/or atmospheric CO₂. We suggest that potassium and malate act as osmoregulatory solutes and that malate, at least in part, arises from dark CO₂ fixation reactions.     

The effect of Cl- upon the sensitivity of starch-containing and starch-deficient stomata and guard cell protoplasts towards potassium ions,fusicoccin and abscisic acid

Chloride ions are necessary to compensate for the positively charged potassium ions imported into guard cells of Allium cepa L. during stomatal opening. Therefore an external Cl^- supply of intact Allium plants is important. But high levels of chloride have been found to reduce the sensitivity of the starch-lacking stomata and isolated guard cell protoplasts (GCPs) from Allium to potassium ions, fusicoccin and abscisic acid. Furthermore, with high levels of chloride, malate anions disappear from the guard cells of Allium, a finding which contrasts with situation in Vicia where the stomatal sensitivity to K⁺ ions, fusicoccin and ABA is not influenced by Cl^- ions and malate levels are unaffected. It is suggested that the absence of malate as a proton yielding primer inhibits the mechanism of H⁺/K⁺ exchange in Allium.Abbreviations ABA abscisic acid - FC fusicoccin - GCPs guard cell protoplasts     

THE EFFECTS OF PLANT GROWTH SUBSTANCES ON IN VITRO FIBER DEVELOPMENT FROM FERTILIZED COTTON OVULES

Cotton ovules were aseptically removed from ovaries 48 hr after anthesis and floated on the surface of liquid medium. Plant growth substances were filter sterilized and added to the medium in which glucose was the principal source of carbohydrate and KNO₃ the sole source of nitrogen. The amount of total fiber produced from the ovule surface was determined colorimetrically from the intensity of a destaining solution to which the ovules had been transferred following their 15-second emersion in a solution of toluidine blue O. Gibberellic acid induced a marked stimulation and kinetin and abscisic acid a marked inhibition of fiber production from fertilized cotton ovules. Indoleacetic acid afforded only a trend of stimulation of fiber production. Gibberellic acid overcame the inhibition of total fiber production induced by both kinetin and abscisic acid. Indoleacetic acid did not overcome the inhibition of total fiber production induced by kinetin but did, to a great extent, overcome the inhibition due to abscisic acid.     

GA,ABA, phenol interaction in the control of growth: Phenolic compounds as effective modulators of GA-ABA interaction in radish seedlings

Abscisic acid, a potent growth inhibitor inhibits hypocotyl growth ofRaphanus sativus seedlings. Phenolic compounds,viz., trans-cinnamic acid, chlorogenic acid, ferulic acid, salicylic acid, tannic acid and quercetin when applied with ABA, antagonize ABA action and restore normal seedling growth. Gibberellic acid promotes hypocotyl growth and on combined application with ABA, the ratio of their concentrations determines the course of the resultant growth. This interaction can be modulated by phenolic compounds. Phenolic compounds in low concentrations when present together with GA and ABA, favour GA-induced growth by antagonizing the inhibitory influence of ABA. The inhibitory action of abscisic acid on a wide range of growth processes is so far known to be reversed only by growth promoting hormones,viz., IAA, GA and cytokinins. Antagonistic action of phenolic compounds towards ABA, and increasing the action of GA when present together with GA and ABA, establishes a dual role to this class of compounds; balancing the effect of both growth promoting and growth inhibiting hormones. Part I.     

A novel cottong ovule culture: Induction, growth, and characterization of submerged cotton fibers (Gossypium hirsutum L.)

Summary The growth of submerged cotton (Gossypium hirsutum L.) fibers from cultured ovules has been investigated. The results indicate that exogenous plant hormone levels regulate the induction of submerged fiber growth. The age of ovules at induction is also important. Cell diameter, wall thickness, and cell length of submerged fibers were measured and compared with air-grown fibers and fibers grown in vivo (produced by cotton plants grown in the greenhouse). Various cellwall thickening patterns were observed among submerged fibers, while only one predominant cell-wall deposition pattern was produced in air-grown fibers and in fibers produced in vivo. The diameter of submerged fibers was about the same as that of air-grown fibers but about 22% less than that of fibers grown, in vivo. It appears that the secondary cell wall thickenings are initiated earlier in submerged fibers. The cell-wall thickness of submerged fibers, at 41 d post anthesis (DPA), was 51% greater than that of fibers grown in vivo, whereas the cell-wall thickness of air-grown fibers was 42% less than that of fibers produced in vivo. The cell length of submerged fibers was approximately half that of fibers grown in vivo. and the air-grown fiber length was about two-thirds of fibers grown in vivo. The age of ovules at induction affects the outcome of the air-grown fiber-cell length, but does not appear to affect the length of submerged fiber cells. To produce submerged fiber growth, we found that the optimal age of ovules at induction was 0 DPA, and the optimal medium (with a GA₃ of 0.5 μM and an IAA range of 5-20 μM) depends on the time of ovule induction (−2 to+2DPA). We conclude that conditions leading to submerged cotton fiber growth have great potential for (a) direct monitoring of growth and making precise, detailed measurements during fiber growth and development; (b) producing cellulose and fibers in vitro more efficiently than earlier ovule-culture methods; and (c) using these unique cultures to obtain a better understanding of signal transduction and gene expression leading to growth, development, and programmed cell death in the life history of the cotton fiber.     

The effect of plant growth regulator treatments on the levels of ethylene emanating from excised turgid and wilted wheat leaves

Abscisic acid (ABA) inhibits the production of ethylene induced by water stress in excised wheat leaves and counteracts the stimulatory effect of 6-benzyladenine (BA) on this process. The stimulatory effect of BA and the inhibitory effect of ABA were equally pronounced whether external or endogenous ethylene levels were determined. When leaves were sprayed or floated on solutions of BA, indole-3-acetic acid (IAA), gibberellic acid (GA₃), or ABA, the relative activities of these growth regulators on stress-induced ethylene at 10^-4 mol l^-1 were BA>IAA >GA₃>controls>ABA. In non-stressed leaves, however, where the levels of ethylene produced were 2–20 times smaller, the relative activities were IAA >BA>GA₃>controls>ABA. The effects of BA and ABA spray treatment on water stress induced ethylene were closely similar whether the solutions were applied 2 or 18 h prior to the initiation of water stress. The relationships between the levels of endogenous growth regulators in the plant and ethylene release induced by water stress are discussed.Abbreviations BA 6-benzyladenine - IAA indole-3-acetic acid - GA₃ gibberellic acid - ABA abscisic acid - GLC gas-liquid chromatography - _leaf leaf water potential     

Hormonal regulation of cotton ovule and fiber growth: Effects of bromodeoxyuridine,AMO-1618 and p-chlorophenoxyisobutyric acid

The effects of 5-bromo-2-deoxyuridine (BUdR, thymidine analogue), AMO-1618 (2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride), a growth retardant, and p-chlorophenoxyisobutyric acid (PCIB, an antiauxin) on growth (dry weight increase) and fiber development in unfertilized cotton (Gossypium hirsutum L.) ovules grown in vitro have been studied. BUdR (5 M) causes about 70% inhibition of fiber production, with little effect on ovule growth, if applied during the first 6 d of culture in the presence of GA₃ and IAA. AMO-1618, when used with GA₃ alone, causes only a small reduction in both dry weight and fiber production, but when used with IAA alone reduces both fiber production and dry weight, the effect on the latter being predominant. In the presence of both IAA and GA₃, AMO-1618 causes a small decrease in fiber production but a major decrease in dry weight. PCIB completely inhibits fiber growth but has little effect on dry weight, especially when GA₃ is present. These results indicate that GA₃ mainly promotes ovule growth while IAA is largerly responsible for fiber growth.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine carboxylate methyl chloride - BUdR 5-bromo-2-deoxyuridine - GA₃ gibberellic acid - IAA indole-3-acetic acid - PCIB p-chlorophenoxyisobutyric acid - TFU total fiber units     

Effect of water stress on transport of [2-14C]abscisic acid in intact plants ofPhaseolus coccineus L.

Summary Eight- to 10-day-old plants ofPhaseolus coccineus, which grow on vermiculite with a water content of 12–17% of the water-holding capacity, stop growing completely, whereas water potential and relative water content are almost unaffected. [2-¹⁴C]Abscisic acid, which is applied to the midrib of a primary leaf, is transported especially to the roots and the apical bud, but not to the second primary leaf and the cotyledons. Water-stressed plants, however, export only negligible amounts of ABA from the donor leaf to the plant. Thus an accumulation of ABA occurs in the donor leaf. Consequently water stress can increase ABA concentration in leaves not only by stimulating ABA synthesis but also by inhibiting ABA transport. Recovery of growth and ABA transport after reirrigation is very weak. Water stress has no effect on ABA metabolism in bean plants.Abbreviations ABA abscisic acid - GA gibberellic acid - IAA indoleacetic acid - RWC relative water content in plants - TLC thin-layer chromatography     

The isolation of abscisic acid (ABA) deficient mutants by selection of induced revertants in non-germinating gibberellin sensitive lines of Arabidopsis thaliana (L.) heynh.

Summary By selecting for germinating seeds in the progeny of mutagen-treated non-germinating gibberellin responsive dwarf mutants of the ga–1 locus in Arabidopsis thaliana, germinating lines (revertants) could be isolated. About half of the revertants were homozygous recessive for a gene (aba), which probably regulates the presence of abscisic acid (ABA). Arguments for the function of this gene were obtained from lines homozygous recessive for this locus only, obtained by selection from the F₂ progeny of revertant X wild-type crosses. These lines are characterized by a reduced seed dormancy, symptoms of withering, increased transpiration and a lowered ABA content in developing and ripe seeds and leaves.Abbreviations ABA Abscisic acid - GA₄₊₇ Mixture of gibberellin A₄ and A₇ - EMS Ethylmethanesulfonate - NG Non-germinating - G Germinating     

Effects of abscisic acid,gibberellic acid and fusicoccin on the transmembrane potential during the early phases of germination in radish (Raphanus sativus L.) seeds

During germination, the transmembrane electric potential (PD) of cortical cells of the embryonal axis of radish seeds (Raphanus sativus L.) rises from-120 mV initially to a maximum of-150 mV after 5 h incubation, then falls again to stable values of around-120 mV. Treatments inhibiting germination block the transitory PD increase. Administration of uncoupling agents or low temperatures, during the process of germination, produces a marked fall of the PD transitory increase. Abscisic Acid has a parallel inhibitory effect on PD and germination, while fusicoccin produces a rise in both; administration of abscisic acid with fusicoccin inhibits germination, while the PD remains at the high levels given by fusicoccin. These results are discussed in relation to ion exchange at membrane level.Abbreviations ABA abscisic acid - FC fusicoccin - GA₃ gibberellic acid - PD electric potential difference (between the vacuole and the external medium) - CH cycloheximide - DNP dinitrophenol - FCCP (p-trifluormethoxy)-carbonylcyanide-phenylhydrazone - DCCD N,N-dicyclohexylcarbodiimide     

Effect of abscisic acid, osmolarity and partial desiccation on the development of recalcitrant mango somatic embryos

Inhibition of mango somatic embryo growth was inducedin vitro by treatments for 4 or more weeks with abscisic acid (0–100 M ABA) with and without high osmolarity provided by mannitol (0–10%). High osmolarity and ABA significantly affected somatic embryo length, precocious germination and the production of good quality secondary somatic embryos. High osmolarity also affected root elongation. Abscisic acid was more effective in suppressing growth and development of 0.5 cm-length somatic embryos than smaller somatic embryos. Development beyond the heart stage was significantly inhibited by both ABA and mannitol treatments. The recovery of good quality somatic embryos was enhanced by high levels of ABA (100 M) with and without mannitol (0–5%). Somatic embryos that had been pulsed with ABA were partially desiccated at different relative humidities. Weight loss was affected only by relative humidity; and ABA did not enhance desiccation tolerance.Abbreviations ABA Abscisic acid - 2,4-D 2,4-Dichlorophenoxyacetic acid - MM1 Mango maturation medium - RH Relative humidity     

The Effect of Photosynthetic Enhancement on Photorespiration in Sinapis alba

High external concentrations of potassium were found to promote light-induced growth of cucumber cotyledons similarly to the effects previously observed for the growth induced by cytokinins. At 40 mM KCl, the response to white light was 3.6 times greater than in the absence of KCl. The promotive effect of calcium on the growth induced by cytokinin was not observed for light. In 40 mM KCl and 10 mM CaCl₂, the responses to light and cytokinin were similar and additive. Both near-red and far-red light induced growth at low intensities. The response to white light at low intensities was sharply increased with higher intensity up to 24 μE m^-2 s^-1 and only slightly increased above that level. Abscisic acid was found to inhibit strongly the responses to light and cytokinin. The inhibition was greater in the presence of KCl than in its absence and thus abscisic acid appeared to inhibit primarily by its interference with potassium uptake. Kinetic analysis found the light response promoted by potassium to be inhibited competitively by abscisic acid, with the light response having a K_m of 34 mM KCl and a V_max of 115 mg/cotyledon × 4 days. The inhibition of the cytokinin response by abscisic acid was noncompetitive in relation to potassium, having a K_m of 1 mM KCl and a V_max of 50 mg/cotyledon × 4 days. It is suggested that cytokinin, light and abscisic acid have primary properties affecting membrane permeability and that their interaction with potassium is an explanation of many similarities between light and cytokinin responses and their inhibition by abscisic acid.