The Inhibition by Cytokinin of Auxin‐Promoted Elongation in Excised Soybean Hypocotyl

Vanderhoef, Larry N.; Stahl, Catherine A.; Siegel, Ned R.; Zeigler, R. S.

doi:10.1111/j.1399-3054.1973.tb04804.x

Cited by 37 publications

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“…Soybean hypocotyl elongation cannot continue by this first wall-loosening auxin action alone. This very important point was first established with experiments which employed cytokinin, an elongation inhibitor (20,22). Auxin must have another activity in its regulation of cell elongation, very likely the regulation of the production of compounds necessary for sustained cell elongation.…”

Section: Discussionmentioning

confidence: 99%

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Auxin-regulated Wall Loosening and Sustained Growth in Elongation

Vanderhoef

Dute²

1981

Plant Physiol.

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It is proposed that auxin regulates and coordinates both wail loosening and the supply of wall materials in elongation. The Experiments reported herein support the proposal that auxin coordinates wall loosening and the supply of wall materials in its regulation of cell elongation. MATERIALS AND METHODSSoybean seedlings (Glycine max L. Merr. var. Wayne) were germinated in the dark for 3 days, and the elongating segment of the hypocotyl was excised as described (20,22). Pea seedlings, Pisum sativum var. Alaska, were germinated in the dark in moist vermiculite at 25 C for 4 days. Elongating segments were excised from the first internode of the epicotyl immediately below the hook. Hypocotyl and epicotyl extension were measured continuously with a linear position-sensitive transducer in an apparatus modified after that reported (19), except that the clamped segments were immersed in 25 C medium. The pH 6 medium, 5 mM Kphosphate, and the pH 4 medium, 5 iim K-citrate, contained 30 mm sucrose. IAA was added to a final concentration of 45 ,uM in the soybean experiments.The mode of action of auxin-regulated elongation is not known. Recently, two hypotheses have guided research in this area. In the 1960s, the gene expression hypothesis proposed that auxin regulated wall loosening and steady-state elongation by action at gene transcription or translation (9,14). However, recognition of the short lag between auxin application and detectable elongation rate increase (10,12,25) by Evans and Ray (6) was thought to rule out primarily mediation at gene expression in auxin-regulated wall loosening (11). In 1971, the wall-acidification hypothesis (4, 13) was independently proposed by Hager et al. (7) and Cleland (3). This hypothesis, which proposes that auxin regulates wall loosening by causing a pH drop in the area of the cell wall, has survived a series of rigorous challenges (4). The molecular mechanism of auxin-caused wall acidification remains to be described.In 1975, we commented on the unusual early kinetics which are seen when auxin stimulates elongation in auxin-depleted (hence, slowly growing), excised elongating segments (20). We suggested, and others have confirmed (8), that auxin-induced elongation could be separated into two phases, the early burst of growth (simulated by lowering the pH from 6 to 4) and a later phase associated with long-term, steady-state growth. Subsequent experiments showed that elongation during the first phase was biochemically distinct from elongation during the second phase (15,16,21,23 Figure 1. The figure is based on the data that prove the existence of separable responses, on the data which support the wall-acidification and gene expression hypotheses and on the consideration of what very likely happens to an excised rapidly elongating segment which is preincubated and then treated with acid or auxin. The figure depicts two auxin activities in the control of growth, wail loosening and the regulation of the supply of wall materials. During steady-state growth in the intact plant, t...

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Section: Discussionmentioning

confidence: 99%

“…var. Wayne) were germinated in the dark for 3 days, and the elongating segment of the hypocotyl was excised as described (20,22). Pea seedlings, Pisum sativum var.…”

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confidence: 99%

Auxin-regulated Wall Loosening and Sustained Growth in Elongation

Vanderhoef

Dute²

1981

Plant Physiol.

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“…plant tissues, but effects are often opposite in different species or growth situations and they do not bear any consistent relationship to total RNA levels. It has been suggested (19,27) that the ability of auxins and cytokinins to stimulate or to inhibit RNA metabolism depends in part on relative hormone concentrations. Another source of confusion may be the fact that, in most studies to date where RNase activity has been studied during plant development, only total levels are measured or, in cases where tissue is fractionated, only one fraction is assayed.…”

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Generation and Suppression of Microsomal Ribonuclease Activity after Treatments with Auxin and Cytokinin

Birmingham

Maclachlan

1972

Plant Physiol.

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plant tissues, but effects are often opposite in different species or growth situations and they do not bear any consistent relationship to total RNA levels. It has been suggested (19,27) that the ability of auxins and cytokinins to stimulate or to inhibit RNA metabolism depends in part on relative hormone concentrations. Another source of confusion may be the fact that, in most studies to date where RNase activity has been studied during plant development, only total levels are measured or, in cases where tissue is fractionated, only one fraction is assayed. This fails to take into account the likelihood that more than one RNase exists in growing tissues and that the various activities are associated with either soluble or membrane-bound fractions (18,21,28,30). A thorough investigation of the effects of different growth regulators on total and subcellular levels of RNA and RNase activity and their relation to growth in a well characterized tissue has not been reported. This paper describes effects on these parameters mainly of the auxin indoleacetic acid (IAA) and the cytokinin benzyladenine, applied alone or together to growing regions of decapitated pea epicotyls. In this semi-intact system, auxin treatment has been shown (5,8,16) to result in lateral cell expansion and massive increases in RNA and protein in various cell subfractions, including microsomes. Growing regions of peas are well known to yield RNase activity in soluble and microsomal fractions (11, 15, 18).Many plant and animal growth hormones evoke synthesis of messenger and ribosomal RNA, and the evidence is convincing that new polysomal species are needed for synthesis of enzymes that help to bring about particular morphogenetic events (6,9,13,22,25 growth regulator, and seedlings were allowed to continue growth in darkness at 22 C. Three days later, at least 50 segments per treatment were detached, lanolin was wiped from the surface, and length and fresh weight were recorded (8).Isolation of "Light" and "Heavy" Microsomes. Segments (approximately 5 g fresh weight per treatment) were ground in a mortar at 4 C in 2 volumes of extraction medium consisting of 0.4 M sucrose (RNase free), 5 mm Mg acetate, 100 mM tris-HCl (pH 7.5 at 22 C), 20 mm KCl, and 5 mM 8t-mercaptoethanol. The brei was squeezed through Miracloth (Calbiochem) to remove cell walls and debris (wall fraction) and the exudate was centrifuged at 16,000g for 10 min in order to sediment mitochondria and nuclei (particulate fraction). The supernatant was divided into two 6-ml aliquots and layered over 6 ml 50% (w/v)

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“…Vanderhoef and Key (16) and Vanderhoef et al (17) showed that cytokinin inhibited auxin-promoted elongation more severely than control elongation. On the other hand, Helgeson et al (6) have shown that T-2 toxin can decrease cytokinin-controlled growth rates in the tobacco callus After a 1-hr preincubation of 1-cm hypocotyl sections in toxin, tissue was transferred to the growth chamber.…”

Section: Resultsmentioning

confidence: 99%

“…Similar results of toxicity were reported for other toxins of this type by Brian et al (3). To determine whether the alterations in growth might be related to hormone-mediated control of cell growth, we have analyzed these effects of T-2 toxin on growth in the excised soybean hypocotyl system (10,16,17) using the fast growth apparatus, modified from the original design of Evans and Ray (4).…”

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Fusarium tricinctum T-2 Toxin Inhibits Auxin-promoted Elongation in Soybean Hypocotyl

et al. 1973

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T-2 toxin, a mycotoxin produced by Fusarium tricinctum, inhibited elongation of excised hypocotyl sections of Glycine max var. Hawkeye 63. Auxin-promoted elongation was inhibited more severely than was control elongation, and a 1 hour preincubation of 5 AM toxin prevented the induction of a faster rate of elongation by auxin. While the inhibition of elongation by cytokinin was similar to that of the toxin, the mode of action of the two compounds appeared to be different, i.e. their effects on elongation were additive, and only kinetin promoted radial enlargement. Toxin treatment did not diminish cytokinin-induced radial enlargement. The properties of the plasma membrane, as measured by electrolyte leakage, were not affected by the toxin.Fusarium tricinctum (Cda.) Synd. and Hans. produces a potent phytotoxin, 4, 15-diacetoxy-8-(3-methylbutyryloxy)-12, 13-epoxy-A9-trichothene-3-ol (T-2 toxin, see Bamburg and Strong, ref. 2). This toxin is one of over twenty 12,13-epoxytrichothene toxins that affect both plants and animals (2, 3,11 (3). To determine whether the alterations in growth might be related to hormone-mediated control of cell growth, we have analyzed these effects of T-2 toxin on growth in the excised soybean hypocotyl system (10, 16, 17) using the fast growth apparatus, modified from the original design of Evans and Ray (4). 1 cm) of water-saturated vermiculite. For the first 48 hr, the dishes were covered with Saran wrap with 12 razor slits for air circulation. The Saran wrap was then removed, the dish was shaken gently, and the seedlings were watered and allowed to grow 24 hr more. Growth conditions: darkness, 30 C, 80% relative humidity. MATERIALS AND METHODS SeedsThe toxin from Fusarium tricinctum, strain T-2, was the generous gift of E. B. Smalley (Department of Plant Pathology, University of Wisconsin, Madison). It was prepared according to the methods of Marasas et al. (11) and references therein. Analysis and molecular weight determination have shown the crystalline (sheaves of white needles) product to be pure. Gas and paper chromatography of the toxin showed a single peak.For 8-hr incubations, 1 cm hypocotyl sections containing elongating cells (0.5-1.5 cm from cotyledon) were removed with razor blades and stored on ice for not longer than 2 hr in 1% (w/v) sucrose, at which time they were washed with distilled water. Twenty sections were incubated in 25 ml Erlenmeyer flasks with vented metal caps in 4 ml of basal medium containing 5 mM KHYPO4, pH 6.0, 30 mm sucrose, and a bacteriostat (80 ,uM chloramphenicol) at 30 C in a reciprocating shaking water bath. When present, 45 !M auxin (2,4-D) was used. Toxin concentrations are reported for each experiment. After 8 hr the tissue sections were washed with distilled water, blotted dry, and the increases in weight and length were determined. There were two replicates of each treatment in all experiments, and all experiments were repeated twice. Data are presented as the mean + (t for 0.01 X SD).For short term experiments, an apparatus similar ...

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The Inhibition by Cytokinin of Auxin‐Promoted Elongation in Excised Soybean Hypocotyl

Cited by 37 publications

References 19 publications

Auxin-regulated Wall Loosening and Sustained Growth in Elongation

Auxin-regulated Wall Loosening and Sustained Growth in Elongation

Generation and Suppression of Microsomal Ribonuclease Activity after Treatments with Auxin and Cytokinin

Fusarium tricinctum T-2 Toxin Inhibits Auxin-promoted Elongation in Soybean Hypocotyl

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