Timing of the Auxin Response in Etiolated Pea Stem Sections

Barkley, Grant M.; Evans, Michael L.

doi:10.1104/pp.45.2.143

Cited by 53 publications

(31 citation statements)

References 19 publications

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“…it suggests that the ethylene-producing system requires continued synthesis of a protein, as suggested in the case of growth by Barkley and Evans (3,11,12). They proposed that the primary action of auxin in growth does not involve synthesis of a certain specific protein coupled to the growth process, for the lag period of the auxin stimulation is relatively short (10 min), and cycloheximide, and actinomycin D pretreatments do not significantly affect its length.…”

Section: Auxin-induced Etirmentioning

confidence: 94%

Mechanism of Auxin-induced Ethylene Production

1971

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Indoleacetic acid-induced ethylene production and growth in excised segments of etiolated pea shoots (Pisum sativum L. var. Alaska) parallels the free indoleacetic acid level in the tissue which in turn depends upon the rate of indoleacetic acid conjugation and decarboxylation. Both ethylene synthesis and growth require the presence of more than a threshold level of free endogenous indoleacetic acid, but in etiolated tissue the rate of ethylene production saturates at a high concentration and the rate of growth at a lower concentration of indoleacetic acid. Auxin stimulation of ethylene synthesis is not mediated by induction of peroxidase; to the contrary, the products of the auxin action which induce growth and ethylene synthesis are highly labile.It has been suggested that auxin may stimulate ethylene production by inducing the enzymes involved in the formation of the gas (1). This theory is based primarily upon the finding that inhibitors of protein and RNA synthesis prevent auxininduced ethylene production, and also on the observation that stimulation of ethylene production by auxin has a substantial lag period (1,5,7,18,19). Extracts of pea seedlings contain an enzyme that converts methional to ethylene in a cell-free system (20). The enzyme has been identified as a peroxidase (30), a protein which also catalyzes a number of other reactions including oxidation of IAA (13,14,26). The present study presents evidence which shows the peroxidase content not to play a role in the regulation of ethylene biosynthesis in pea seedlings. Growth, ethylene production, the endogenous IAA content, exogenous IAA level, and rate of metabolism of growth hormone were continuously determined and compared in an attempt to determine the mechanism by which auxin induces ethylene production. (29) which is approximately 100% efficient. The radioactivity remaining in the tissue after alcohol extraction was determined after squashing the sections and drying them on a planchet, under which condition self-absorption was negligible. Enzyme Assay for Ethylene Synthesis. Sections of subapical internodes or plumular hooks were homogenized in 50 mm potassium phosphate buffer at pH 7.8 (10 sections/ml), strained through cheesecloth, and centrifuged at lOOOg for 15 min. The preparation could be further purified by dialysis and ammonium sulfate precipitation as described by Ku et al. for the pea enzyme (20), but a variable loss in activity occurred at each step, making the crude extract preferable for quantitative studies. The resulting supernatant (enzyme preparation) was assayed in 5 ml of reaction mixture described by Yang (30), consisting of 10 mM potassium phosphate buffer (pH 7.8), 0.6 mm methional, 3 em manganese sulfate, 80 mm resorcinol, 2 ytM ethylenediaminetetraacetic acid, 80 fM sodium hydrogen sulfite, with 1 ml of enzyme extract. The mixture was incubated at 25 C in a 25-ml Erlenmeyer flask sealed with a vaccine cap, and typically 1-ml samples of air were withdrawn from the flask at 30-min intervals, and ethylene was ...

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Section: Auxin-induced Etirmentioning

confidence: 94%

Mechanism of Auxin-induced Ethylene Production

1971

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“…It has been shown that inhibitors of protein synthesis have a rapid effect on IAA-induced growth (1,5,9,13,16,19). This has led to the suggestion that an unstable protein, which has been called growth-limiting protein (5), is necessary for auxin-induced cell expansion.…”

Section: Introductionmentioning

confidence: 99%

“…It was shown that there is a sizeable growth promotion by auxin after protein synthesis has essentially ceased. It is concluded that the initial phases of auxin action do not require protein synthesis but that its action depends on the existing pool of growth-limiting proteins which is rapidly depleted, and protein synthesis is then required for continued elongation.It has been shown that inhibitors of protein synthesis have a rapid effect on IAA-induced growth (1,5,9,13,16,19). This has led to the suggestion that an unstable protein, which has been called growth-limiting protein (5), is necessary for auxin-induced cell expansion.…”

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confidence: 99%

Growth-limiting Proteins in Relation to Auxin-induced Elongation in Lupin Hypocotyls

Penny

1971

Plant Physiol.

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The role of protein synthesis in auxin-induced cell elongation in lupin hypocotyl segments was studied using cycloheximide. Cycloheximide inhibited protein synthesis by 9 minutes. Experiments adding cycloheximide at various times before and after indolyl-3-acetic acid are reported. Estimates of the relative amounts of growth-limiting protein(s), and a first order rate constant for the apparent turnover of the growthlimiting protein(s) were made. It was shown that there is a sizeable growth promotion by auxin after protein synthesis has essentially ceased. It is concluded that the initial phases of auxin action do not require protein synthesis but that its action depends on the existing pool of growth-limiting proteins which is rapidly depleted, and protein synthesis is then required for continued elongation.It has been shown that inhibitors of protein synthesis have a rapid effect on IAA-induced growth (1,5,9,13,16,19). This has led to the suggestion that an unstable protein, which has been called growth-limiting protein (5), is necessary for auxin-induced cell expansion. It is not possible, however, to determine from these studies with inhibitors whether auxin acts on the synthesis of GLP' or whether the requirement for protein synthesis is a secondary (albeit rapid) effect of auxin action.Other evidence indicates that auxin acts on pre-existing macromolecules and that protein synthesis is not necessary for the initial action of auxin. This is the rapid change of growth rate by: (a) the methyl ester of indolyl-3-acetic acid (21, 22); (b) high temperature at high auxin concentration (14); and (c) the initial decline in growth rate before the auxin-induced stimulation (22). Although we have tried, without success, to obtain these results with lupin hypocotyl segments, they do illustrate the point for coleoptiles at least.In this paper the quantitative study of the effect of cycloheximide leads to a similar conclusion that protein synthesis is not required for the initial action of auxin on cell elongation in lupin hypocotyl. The measurements of elongation were made every minuLte in the apparatus described by Penny (17). Briefly, a segment was held in a chamber which was held on the stage of a microscope. The desired solution flowed through the chamber, and the flow rate of solution was maintained by a Watson Marlow model MHRE flow inducer. Tris-maleate buffer, pH 6.1, 20 mm, was used throughout; the concentration of IAA (when used) was 30 pM and the concentration of CH, when used, was 10 jug/ml. The solutions were aerated and the light energy at the surface of the segments was 3 wm-2 supplied by an incandescent lamp. Temperatures, except where otherwise stated, were maintained at 28 C. There was a tendency for the temperature to increase over the course of a long experiment. but the total difference in temperature during the experiment was never more than 0.5 C. MATERIALS AND METHODS SeedlingsFor measurement of protein synthesis, groups of 120 segments were pretreated in buffer for 2 hr and then blotted a...

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“…There is precedent for interaction of auxin hormones with the plasma membrane (4, 17). The latter findings (4) form a basis for integration of the seemingly disparate fast reactions of auxin action (1,3,12,16) and the delayed transcriptional responses (7,8,11) into a postulated mechanism involving a single master reaction (15). This study provides evidence from high resolution electron microscopy which, in conjunction with fluorescence probe experiments of the accompanying paper (5) were soaked in water overnight, planted in moist vermiculite, and germinated in the dark at 29 C in a constant temperature, high humidity chamber (4).…”

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confidence: 99%

Ultrastructural Alteration of Plant Plasma Membranes Induced by Auxin and Calcium Ions

Morré

Bracker²

1976

Plant Physiol.

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Ultrastructural changes in isolated and in situ plasma membranes of etiolated soybean hypocotyls (Glycine max L. cv. Wayne) were induced by indole-3-acetic acid (IAA), other auxins, and calcium chloride. Fixed and embedded preparations were stained by a phosphotungstate-chromate procedure to identify and accentuate plasma membrane. Measurements were on micrographs obtained with an electron optical system calibrated and corrected for reproducible and accurate size measurements. Plasma membranes treated for 20 minutes with 1 ,UM IAA were 10 to 15% thinner than controls. The response to IAA was rapid, reproducible, auxin-specific, temperature-dependent, and reversible.Comparable responses were obtained with isolated and in situ membranes. Membranes treated with 0.5 M calcium chloride for 20 minutes were 15 to 20% thicker than controls. Multiple cycles of alternating calcium and IAA treatments yielded membranes with dimensions that reflected the last treatment of the series. The findings show a direct response of plasma membranes to growth regulating agents and provide evidence for a cell-free response of isolated plasma membranes to a hormone.The precise mode of action of plant hormones is unknown. This is true in particular for hormones of the auxin type such as IAA, and its synthetic counterpart, 2,4-D. One explanation of hormone action focuses on cell wall loosening as the mechanism of action (2), whereas other findings implicate effects on nucleic acid metabolism (7,8,11). A role for the plasma membrane in regulating cell wall properties is implicit from the physical closeness of the two structures. There is precedent for interaction of auxin hormones with the plasma membrane (4, 17). The latter findings (4) form a basis for integration of the seemingly disparate fast reactions of auxin action (1,3,12,16) and the delayed transcriptional responses (7,8,11) into a postulated mechanism involving a single master reaction (15). This study provides evidence from high resolution electron microscopy which, in conjunction with fluorescence probe experiments of the accompanying paper (5) were soaked in water overnight, planted in moist vermiculite, and germinated in the dark at 29 C in a constant temperature, high humidity chamber (4). Four days after planting, hypocotyls were excised and used for membrane isolations.Membrane Isolations. About 10 g of hypocotyl tissue were homogenized at 0 to 4 C in 10 ml of one of the following media: medium A: 0.1 M K phosphate in freshly prepared and filtered coconut water and containing 20 mm EDTA and 0.5 M sucrose, final pH 7 (4, 17); medium B: deionized water, final pH of homogenate 5.8 ± 0.2; medium C: 0.5 M CaC12, pH 5.9 ± 0.1.Hypocotyls were homogenized for 45 sec with a Polytron 20ST (Kinematica, Lucerne, Switzerland) (9) operating at about 5,000 rpm. The homogenates were filtered through a single layer of Miracloth (Miracloth Sales, Chicopee Mills, N. Y.) to remove whole cells, tissue fragments, and cell walls. The filtered homogenates were divided among 8 to 10 luster...

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Timing of the Auxin Response in Etiolated Pea Stem Sections

Cited by 53 publications

References 19 publications

Mechanism of Auxin-induced Ethylene Production

Mechanism of Auxin-induced Ethylene Production

Growth-limiting Proteins in Relation to Auxin-induced Elongation in Lupin Hypocotyls

Ultrastructural Alteration of Plant Plasma Membranes Induced by Auxin and Calcium Ions

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