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Bound auxin formation in growing stems.

The term "bound auxin" is herein used to describe auxin conjugates insoluble in organic solvents which dissolve indoleacetic acid (IAA) and its derivatives, but hydrolyzable by NaOH to release IAA. Bound auxin from pea stems was fractionated into water-soluble, water-insoluble/NaOH-hydrolyzable, and insoluble components. Formation of bound auxin commenced with 15 minutes of applying exogenous labeled IAA, and progressively increased in amount, relative to IAA uptake, over 6 hours. Formation was not restricted to any particular zone of the stem and occurred in both light- and dark-grown stems. A greater quantity of bound auxin was formed in light-grown stems, reaching 4.2 and 7.7%, of the IAA taken up, in the water-soluble and water-insoluble/NaOH-hydrolyzable fractions after 6 hours. The presence of sucrose, during either the IAA treatment or an aging pretreatment had no effect, though 6 hours aging did cause a subsequent increase in the water-insoluble fraction of the bound auxin. Bound auxin formation in light-grown stems was dependent on respiratory metabolism, being reduced by KCN. It was also reduced, compared to total uptake, by inhibitors of RNA, and protein synthesis (6-methylpurine and cycloheximide) but only when the inhibitors preceded auxin addition and were present for a 4-hour period. Addition of inhibitors following auxin had no effect, suggesting an early inductive effect of auxin on bound auxin formation. Inhibitors of cell elongation had no effect. Deoxyglucose, an inhibitor of glucan synthesis, had only a small effect on the water-soluble fraction. Bound auxin is an important auxin product in growing plants. Its function is unknown, but some possibilities are discussed.

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A Mung Bean Assay for Malformin-induced Growth Stimulation.

A bioassay employing green or etiolated cuttings of Phaseolus aureus Roxb. was developed for determining malformin-induced growth stimulation in light. Growth enhancement of green cuttings was more rapid and relatively greater than that of etiolated cuttings. Cuttings from green seedlings responded less as seedlings aged; those from etiolated seedlings responded more. Malformin also stimulated the growth of green or etiolated seedlings in light. Most growth enhancement induced by malformin occurred in the upper 1 cm of the stem. Using green cuttings, malformin stimulated stem elongation relatively more when cotyledons, leaves, or especially apical buds were removed. Although malformin failed to stimulate elongation of 2-cm stem sections "floated" on solutions in Petri dishes, it stimulated elongation of sections when they were upright. High concentrations of indoleacetic acid inhibited growth enhancement by malformin. When gibberellin and malformin were combined, growth enhancement was nearly additive.

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Metabolism of Poly(A) in Plant Cells: Discrete Classes Associated with Free and Membrane-bound Polysomes.

In the subapical region of dark-grown pea epicotyls about 40% of the total polysomes are associated with membranes. The presence of poly(A) in polysomal mRNA was detected by hybridization of unlabeled RNA with (3)H-poly(U). Both free mRNA and messenger ribonucleoprotein particles in polysomes hybridize with (3)H-poly(U) quantitatively. The binding of (3)H-poly(U) to polysomes is increased by treatment with the detergent sodium dodecyl sulfate. Since detergent influenced the (3)H-poly(U) binding more in membrane-bound polysomes than in free, there may be more protein(s) associated with the poly(A) portion of the mRNA in membrane-bound polysomes. Analysis of the poly(A) segments isolated from the mRNA of these two classes of polysomes indicates that there are discrete classes of poly(A) and they appear to be differentially associated with free and membrane-bound polysomes. Mean size distribution of poly(A) in free polysomes is larger than in membrane-bound polysomes.Following treatment (2 days) with the plant growth hormone indoleacetic acid, there is a gradual decrease in the mean length of total poly(A), which appears to correspond to a decrease in the size of the polysomes and their associated mRNA.

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Anomalous Temperature Dependence of Seedling Development in Some Soybean (Glycine max [L.] Merr.) Cultivars: Role of Ethylene.

In Clark and Shelby soybean (Glycine max [L.] Merr.) seedlings, hypocotyl elongation was inhibited and hypocotyl swelling and root dry weight were increased by a temperature of 25 C. At 20 and 30 C, development was normal, as was development of Hawkeye and Mandarin soybean seedlings at all three temperatures. Dry matter distribution at 9 days indicates that inhibition of hypocotyl elongation is not due to a lack of translocation from cotyledons, but to a diversion of dry matter from hypocotyl to root. Ethylene evolution by Clark seedlings at 25 C exceeds that at 20 and 30 C. At all three temperatures, Mandarin seedlings' ethylene evolution is at the same low rates as those of Clark at 20 and 30 C. Clark's enhanced rate at 25 C precedes the deceleration of hypocotyl elongation occurring at 5 days. The abnormal effects of a temperature of 25 C on Clark seedlings' development is partially reversed by CO(2) and GA(3) and can be duplicated in Mandarin by applying ethylene. In Clark, effects of the temperature can be further accentuated by indoleacetic acid, which stimulates ethylene evolution, and by applying ethylene to the seedlings. It is concluded that the temperature-induced symptoms, similar in most respects to the well known "triple response" of legume seedlings, are caused by abnormally high levels of ethylene in tissues of the anomalous cultivars.

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Rapid Hormone-induced Hyperpolarization of the Oat Coleoptile Transmembrane Potential.

The effects of the plant growth substances indoleacetic acid (IAA) and fusicoccin on the transmembrane potential of Avena coleoptile cells (at 27-29 C) were studied. Fusicoccin caused hyperpolarization of the membrane potential which started after a lag of less than 20 seconds, and which on average reached -49 mv at an external K(+) concentration of 1 mm and -75 mv at 0.1 mm K(+). IAA caused a hyperpolarization of -25 mv starting after a lag of 7 to 8 minutes. These results suggest that fusicoccin and IAA both activate electrogenic H(+) extrusion.

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Patterns of Ethylene and Carbon Dioxide Evolution during Cotton Explant Abscission.

The relationship between abscission and the evolution of ethylene and CO(2) was examined in explants and explant segments of cotton seedlings (Gossypium hirsutum L. cv. Acala SJ-1) under both static and flow system conditions, and in the presence and absence of mercuric perchlorate. Explant excision was immediately followed by increased ethylene evolution (wound ethylene); senescence was also accompanied by increased ethylene evolution (senescence ethylene). One or two ethylene peaks were found to interrupt the low background rate of ethylene evolution during the period between excision and senescence. The first intermediate ethylene peak coincided with a rise in CO(2) evolution; however, precedence could not be established. No statistical correlations were discovered between either intermediate ethylene peak and abscission. The best statistical correlation was found between wound ethylene and abscission at 12 hr after excision. No positive correlations were found between senescence ethylene and abscission. Implications of these results for the understanding of the role of ethylene in explant abscission are discussed.Relationships between a number of different explant treatments and ethylene evolution were also examined. Ethylene production in response to indoleacetic acid applications, abscisic acid applications, and different types of wounding is summarized. It was concluded that the results of the standard abscission bioassay (conducted in Petri dishes) have not been influenced by unnatural ethylene accumulations.

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Auxin Transport as Related to Leaf Abscission during Water Stress in Cotton.

Plant water deficits reduced the basipetal transport of auxin in cotyledonary petiole sections taken from cotton (Gossypium hirsutum L.) seedings. A pulse-labeling technique was employed to eliminate complications of uptake or exit of (14)C-indoleacetic acid from the tissue. The transport capacity or the relative amount of radioactivity in a 30-minute pulse which was basipetally translocated was approximately 30% per hour in petioles excised from well watered seedlings (plant water potentials of approximately -4 to -8 bars). No cotyledonary leaf abscission took place in well watered seedlings. Plant water potentials from -8 to -12 bars reduced the transport capacity from 30 to 15% per hour, and although the leaves were wilted, cotyledonary abscission did not increase appreciably at these levels of stress. The threshold water potential sufficient to induce leaf abscission was approximately -13 bars and abscission increased with increasing stress while the auxin transport capacity of the petioles remained relatively constant (15% per hour). The basipetal transport capacity of well watered petioles tested under anaerobic conditions and acropetal transport tested under all conditions were typically less than basipetal transport under the most severe stress conditions. Cotyledonary abscission took place during and 24 hours after relief of stress with little or no abscission taking place 48 hours after relief of stress. Although the water potential returned to -4 bars within hours after rewatering the stressed plants, partial recovery of the basipetal transport capacity of the petioles was not apparent until 48 hours after rewatering, and at least 72 hours was required to return the transport capacity to near normal values. These data support the view that decreased levels of auxin reaching the abscission zone from the leaf blade influence the abscission process and further suggest that the length of time that the auxin supply is maximally reduced is more critical than the degree of reduction.

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Indoleacetaldehyde in cucumber seedlings.

The presence of indoleacetaldehyde in cucumber (Cucumis sativus L.) cotyledons was demonstrated by thin layer chromatographic R(F) values in three solvent systems, by the formation and hydrolysis of a bisulfite adduct, and by chemical reduction to indoleethanol and oxidation to indoleacetic acid. Bioassays indicated a minimum indoleacetaldehyde content in etiolated cotyledons of 0.7 mug per kg fresh weight. Tissue samples from all parts of both green and etiolated cucumber seedlings reduced exogenously supplied indoleacetaldehyde to indoleethanol.

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Time-dependent Changes in the Auxin Sensitivity of Coleoptile Segments: Apparent Sensory Adaptation.

When segments are excised from corn (Zea mays L.) coleoptiles they exhibit a very low rate of elongation for about 3.5 hours. A strong increase in growth rate (the spontaneous growth response) then occurs and persists for many hours. During the latent period preceding the spontaneous growth response there is an apparent increase with time in the sensitivity of the segments to indoleacetic acid (IAA). This increase in sensitivity is expressed as a 2- to 3-fold increase in the magnitude of the growth response to low levels of IAA and a 3-fold decrease in the latent period of the response during the first 3 hours following excision. A similar increase in sensitivity to low levels of IAA is noted if application of IAA is timed from the point of termination of a previous exposure to the hormone. Since the increase in responsiveness to low levels of IAA is not paralleled by an increase in the rate of uptake of the hormone, the data may be interpreted as evidence for a type of time-dependent sensory adaptation to auxin. The IAA dose-response relationship also changes with time, and there is indirect evidence that an auxin-dependent inhibitor may influence the expression of the apparent sensory adaptation to auxin.

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Induction of resistance to dark abscission by malformin in white light.

When cuttings or seedlings of Phaseolus aureus were treated proximally with malformin for 2 days in continuous white light, resistance to subsequent leaf abscission in the dark resulted. The amount of resistance diminished as the concentration of malformin decreased from 10 to 0.1 micromolar. Resistance to dark abscission persisted for 7 days in continuous light. Little resistance was obtained when cuttings were taken from seedlings grown under low irradiance and short photoperiods, but resistance gradually increased as the photoperiod increased. Resistance to dark abscission induced by malformin in light differs from inhibition of abscission by indoleacetic acid because when malformin is applied in the dark it stimulates abscission after distal or proximal application. Malformin induces resistance only in conjunction with light treatment.

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Pattern of cell division and wound vessel member differentiation in coleus pith explants.

When pith parenchyma explants are taken from Coleus blumei plants and cultured on an agar medium containing sucrose and indoleacetic acid wound vessel members differentiate in 10 days. The time course of wound xylem appearance and an auxin requirement suggest that this uncomplicated system is responding in a manner comparable to wounded Coleus plants and cultured stem segments.Histological examination and cell size comparisons confirm that parenchyma cells divide before differentiating. When colchicine is used to prevent mitosis no tracheary elements differentiate. Following the time course of this cytodifferentiation histologically shows that xylem differentiates from cells that are the products of several cell divisions.

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Red light and auxin effects on rubidium uptake by oat coleoptile and pea epicotyl segments.

Apical segments of etiolated oat (Avena sativa L. cv. Victory) coleoptiles showed enhanced uptake of [(86)Rb(+)] when tested 30 minutes after a 5-minute red irradiation. The response was partly reversible by far red light. Uptake was sensitive to carbonyl cyanide m-chlorophenyl hydrazone, but not to isotonic mannitol. Indoleacetic acid (10(-7) molar) caused a very pronounced and rapid stimulation of uptake. Basal coleoptile segments also exhibited a red light-enhanced uptake, but not an effect of red light on changes in the pH of the medium. The [(86)Rb(+)] uptake of third internode segments from etiolated peas (Pisum sativum L. cv. Alaska) was not affected by either red light or auxin. This tissue also showed no red light effect on acidification of the medium. It is concluded that alteration of [(86)Rb(+)] flux is not a general feature of phytochrome action.

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Independent control of fiber development and nitrate reduction in cultured cotton ovules.

Several lines of evidence implicate ammonium as an important factor in the growth and development of cotton (Gossypium hirsutum L.) ovules cultured in vitro. For example, ovules cultured at 28 C require indoleacetic acid (IAA) and either ammonium or gibberellic acid (GA(3)) in the medium for fiber development, whereas ovules cultured at 34 C require only IAA. Because of this effect of ammonium supply, it seemed possible that hormones or increased temperature were also promoting the availability of reduced nitrogen by induction of increased nitrate reductase activity in the ovules. This possibility was tested.In vivo, where ovules received mostly reduced nitrogen and very little nitrate, they did not display appreciable nitrate reductase activity even when nitrate was forced into the ovary wall by transpiration. After initiation of culture, nitrate became freely available to ovules and their nitrate reductase activity increased rapidly. Treatment with ammonium, GA(3), IAA, or increased temperature had no effect upon this induction. It is concluded that ammonium, hormone, and temperature effects on fiber development are independent of the availability of reduced nitrogen as a general substrate for growth.

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Cell-density-dependent Changes in the Metabolism of Chloronema Cell Cultures: I. Relationship between Cell Density and Enzymic Activities.

In the growing chloronema cell suspension cultures of the moss Funaria hygrometrica Hedw., activities of several enzymes have been found to be cell-density-dependent. Cyclic nucleotide phosphodiesterase (cNPDE), nitrate reductase (NR), and protein kinase showed highest activity at a low cell density (1 to 2 milligrams per milliliter) while indoleacetic acid (IAA) oxidase and peroxidase were highest at a high cell density (>10 milligrams per milliliter). 3'-Nucleotidase and the glycolytic enzymes (aldolase, hexokinase, phosphofructokinase, phosphoglucoisomerase, pyruvate kinase, and triose phosphate isomerase) showed no significant dependence on the cell density. Alternatively, if the NR and peroxidase activities were determined as a function of time in batch cultures, their levels were maximal 60 to 70 and 320 hours after subculture, respectively, the corresponding cell densities being 1 to 2 and 23 milligrams per milliliter. The relationship between cell density and NR and peroxidase activities is the same, whether these enzymes are measured in batch cultures during a growth cycle or in the cells cultured at different initial inoculum densities for a constant time. Conventionally enzymic changes have been correlated with growth phases; however, it is felt that the pattern of enzymic activities can also be interpreted as cell-density-dependent.In moss protonema, the dependence of cNPDE, IAA oxidase, and peroxidase on cell density may play an important role in modulating the endogenous levels of IAA and cAMP, both of which regulate the differentiation of specific cell types (Johri and Desai 1973 Nature New Biol 245: 223-224; and Handa and Johri 1976 Nature 259: 480-482).

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Auxin-induced Ethylene Production and Its Inhibition by Aminoethyoxyvinylglycine and Cobalt Ion.

Auxin is known to stimulate greatly both C(2)H(4) production and the conversion of methionine to ethylene in vegetative tissues, while amino-ethoxyvinylglycine (AVG) or Co(2+) ion effectively block these processes. To identify the step in the ethylene biosynthetic pathway at which indoleacetic acid (IAA) and AVG exert their effects, [3-(14)C]methionine was administered to IAA or IAA-plus-AVG-treated mung bean hypocotyls, and the conversion of methionine to S-adenosylmethionine (SAM), 1-amino-cyclopropane-1-carboxylic acid (ACC), and C(2)H(4) was studied. The conversion of methionine to SAM was unaffected by treatment with IAA or IAA plus AVG, but active conversion of methionine to ACC was found only in tissues which were treated with IAA and which were actively producing ethylene. AVG treatment abolished both the conversion of methionine to ACC and ethylene production. These results suggest that in the ethylene biosynthetic pathway (methionine --> SAM --> ACC --> C(2)H(4)) IAA stimulates C(2)H(4) production by inducing the synthesis or activation of ACC synthase, which catalyzes the conversion of SAM to ACC. Indeed, ACC synthase activity was detected only in IAA-treated tissues and its activity was completely inhibited by AVG. This conclusion was supported by the observation that endogenous ACC accumulated after IAA treatment, and that this accumulation was completely eliminated by AVG treatment. The characteristics of Co(2+) inhibition of IAA-dependent and ACC-dependent ethylene production were similar. The data indicate that Co(2+) exerts its effect by inhibiting the conversion of ACC to ethylene. This conclusion was further supported by the observation that when Co(2+) was administered to IAA-treated tissues, endogenous ACC accumulated while ethylene production declined.

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Stress-induced Ethylene Production in the Ethylene-requiring Tomato Mutant Diageotropica.

Ethylene synthesis in vegetative tissues is thought to be controlled by indoleacetic acid (IAA). However, ethylene synthesis in the diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) was much less sensitive to IAA than in the normal variety (VFN8). Yet, mechanical wounding stimulated ethylene production by the mutant. The dgt tomato provides an opportunity to study the regulation of stress ethylene independent of IAA effects. Waterlogging (i.e. anaerobic stress) stimulated production of the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid (ACC), in the roots. The ACC was transported to the shoot where it was converted to ethylene. The dgt mutant efficiently utilized ACC for ethylene synthesis under aerobic conditions. The results confirm that the genetic lesion in dgt is located at a step prior to the formation of ACC. Furthermore, induction of ethylene synthesis by anaerobic or mechanical stresses in this mutant is independent of IAA action.

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Evidence that Auxin-induced Growth of Soybean Hypocotyls Involves Proton Excretion.

The role of H(+) excretion in auxin-induced growth of soybean hypocotyl tissues has been investigated, using tissues whose cuticle was rendered permeable to protons or buffers by scarification (scrubbing). Indoleacetic acid induces both elongation and H(+) excretion after a lag of 10 to 12 minutes. Cycloheximide inhibits growth and causes the tissues to remove protons from the medium. Neutral buffers (pH 7.0) inhibit auxin-induced growth of scrubbed but not intact sections; the inhibition increases as the buffer strength is increased. Both live and frozen-thawed sections, in the absence of auxin, extend in response to exogenously supplied protons. Fusicoccin induces both elongation and H(+) excretion at rates greater than does auxin. These results indicate that H(+) excretion is involved in the initiation of auxin-induced elongation in soybean hypocotyl tissue.

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Hormonal Regulation of Lateral Bud (Tiller) Release in Oats (Avena sativa L.).

Stem segments containing a single node and quiescent lateral bud (tiller) were excised from the bases of oat shoots (cv. ;Victory') and used to study the effects of plant hormones on release of lateral buds and development of adventitious root primordia. Kinetin (10(-5) and 10(-6) molar) stimulates development of tillers and inhibits development of root primordia, whereas indoleacetic acid (IAA) (10(-5) and 10(-6) molar) causes the reverse effects. Abscisic acid strongly inhibits kinetin-induced tiller bud release and elon-gation and IAA-induced adventitious root development. IAA, in combination with kinetin, also inhibits kinetin-induced bud prophyll (outermost leaf of the axillary bud) elongation. The IAA oxidase cofactor p-coumaric acid stimulates lateral bud release; the auxin transport inhibitor 2,3,5-triiodo-benzoic acid and the antiauxin alpha (p-chlorophenoxy)-isobutyric acid inhibit IAA-induced adventitious root formation. Gibberellic acid is synergistic with kinetin in the elongation of the bud prophyll. In intact oat plants, tiller release is induced by shoot decapitation, geostimulation, or the emergence of the inflorescence. Results shown support the apical dominance theory, namely, that the cytokinin to auxin ratio plays a decisive role in determining whether tillers are released or adventitious roots develop. They also indicate that abscisic acid and possibly gibberellin may act as modulator hormones in this system.

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