Comparative Effects of Hydrogen Ions, Carbon Dioxide, and Auxin on Pea Stem Segment Elongation

The cellular adjustment of the pH of the external environment of soybean (Glycine mar) hypocotyl elongating cells, frequently assumed to be hydrogen ion secretion when the pH is lowered, is unaffected by auxin. These elongating cells actively adjust the external hydrogen ion concentration (from any pH in the range of 4-8) to pH 5.4 + 0.2. This pH adjustment occurs in a medium which does not contain potassium. Growth-optimum auxin concentrations have no effect on cellular pH adjustment of the external medium, whether added at the beginning of the experiment or after the equilibrium pH is attained. The pH adjustment by the cells occurs rapidly and in spite of the presence of a cutide.The acid growth hypothesis (3, 9, 19) is clearly supported by evidence that coleoptile (2, 7, 8, 18), pea (1, 6, 14, 15), and soybean segments (20) are induced to increase their elongation rate when the pH is decreased, e.g. from pH 6 to pH 4. Data comparing the kinetics of auxin-induced medium acidification to the kinetics of auxin-induced elongation, also described in support of the hypothesis, come from experiments with corn (12), 19), and pea (12).These experiments characterize medium pH adjustment by segments from the elongating region of soybean hypocotyl, and the effect of auxin on this process. It was determined that pH adjustment is not affected by the hormone. These data and the determination of the accompanying report (20) do not support the acid growth hypothesis of auxin action. MATERIALS AND METHODSSoybean seedlings (Glycine max L. Merr. var. Wayne) were germinated in the dark and the elongating segment excised as previously described (23).Medium pH was measured with a Beckman pHasar I pH meter. Hypocotyl segments were incubated (one segment/2 ml) for 1 hr at various pH values in distilled H2O continually adjusted to the desired pH with HCI or KOH. Segments were then transferred to KOH-or HCI-adjusted distilled H2O of identical pH (10 1-cm segments/ml) and the pH recorded every 2 min (see Fig. 1). In other experiments (Figs. 2-4), for easier comparison to previous work, segments were preincubated (one segment/ 2ml) in 1 mm K-phosphate (pH 6) for 1 hr (preincubation periods of 0.5-3 hr gave identical results) then transferred to 1 mm K-phosphate (pH 6) (10 segments/ml). The Electron microscopy was used to determine the presence of a cuticle on these elongating segments (see Fig. 5). One-mm segments were excised from the elongating region of light-grown (900 ft-c) and dark-grown soybean seedlings. The segments were fixed (2 hr at 4 C) in 1 % glutaraldehyde (Polysciences, Warrington, Pa.) buffered with 85 mm Na-phosphate (pH 6.8), washed with 100 mm Na-phosphate (pH 6.8) and postfixed (1.5 hr at 25 C) with 2% OSO4 in 50 mm Na-phosphate (pH 6.8). The segments were dehydrated in a graded acetone series and embedded in a mixture of low viscosity resins. Sections were cut with a diamond knife on a LKB-Huxley Mark 2 ultramicrotome, poststained with 2% aqueous uranyl acetate followed by lead citrate, and observed with a...

mentioning

confidence: 71%

“…The acid growth theory of auxin action has been previously challenged (1,11,16,17). In one case (1), methodology was a problem (6).…”

Section: Discussionmentioning

confidence: 99%

Auxin Has No Effect on Modification of External pH by Soybean Hypocotyl Cells

Vanderhoef

Findley²,

Burke³

et al. 1977

“…The involvement of malate accumulation in auxin-induced elongation may account for the long term growth stimulations in response to CO2. Rapid short term effects of CO2 may arise from its acidifying effect (see also 2,20).…”

Section: Resultsmentioning

confidence: 99%

“…In this respect it may appear to be pertinent that IAA-1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft. 2 The result of this paper has been communicated orally at the 1974 conference of the Deutsche Botanische Gesellschaft, Wurzburg, September 26, 1974. promoted growth is accompanied by consumption of CO2 (25) and that IAA enhances the fixation of H'4C03-by Avena coleoptile cells (4).The present paper demonstrates that IAA-induced elongation growth is paralleled by a stoichiometric potassium and malate accumulation. …”

mentioning

confidence: 99%

Stoichiometric Correlation of Malate Accumulation with Auxin-dependent K⁺-H⁺ Exchange and Growth in Avena Coleoptile Segments

Haschke

Lüttge²

1975

The action of auxin in the promotion of growth has been suggested in the literature to depend on cell wall acidification. In a former investigation by the present authors the electrochemical balance in auxin-induced proton extrusion was shown to be maintained by potassium net uptake. The present paper reports data demonstrating that the elongation of Avena coleoptile segments is accompanied by an accumulation of malate, which is stoichiometrically correlated with potassium uptake. We concluded that this malate accumulation is required in a mechanism regulating intracellular pH.Auxin (IAA)-induced elongation of shoot cells is accompanied by a rapid release of protons from the cells (5, 21). External media of low pH (3-5) can mimic the action of auxin (3, 9, 25). These results have led to the suggestion that hydrogen ions play a role as second messengers in auxin action (9): acidification of the cell wall enhances its extensibility either by activating cell wall-loosening enzymes or by breaking some acid-labile links.Indoleacetic acid is also known to stimulate the uptake of K+ and Rb+ ions into different tissues (14-16). In a previous paper (10) we demonstrated that IAA-promoted proton efflux is electrochemically balanced by a stoichiometric influx of K+ ions. The presence of alkali, and, in particular, of K+ ions synergistically stimulates IAA-induced growth (11).A proton efflux of the observed magnitude must be associated with a mechanism regulating cytoplasmic pH. In many tissues a proton-cation exchange (or antiport), e.g., during excess cation uptake, is associated with synthesis and subsequent accumulation of malic acid by dark C02-fixation via P-enole-pyruvate carboxylases (12). A similar mechanism is reported to be involved in K+-H+-exchange regulating stomatal guard cell movement (1, 18). On the basis of these and other results, Davies (6) and Raven and Smith (19) proposed a common mechanism for stabilization of intracellular pH, which is based on synthesis and breakdown, respectively, of organic acids, especially of malate. In this respect it may appear to be pertinent that IAA-1 This work was supported by a grant from the Deutsche Forschungsgemeinschaft. 2 The result of this paper has been communicated orally at the 1974 conference of the Deutsche Botanische Gesellschaft, Wurzburg, September 26, 1974. promoted growth is accompanied by consumption of CO2 (25) and that IAA enhances the fixation of H'4C03-by Avena coleoptile cells (4).The present paper demonstrates that IAA-induced elongation growth is paralleled by a stoichiometric potassium and malate accumulation. MATERIAL AND METHODSPlant Material. Seeds of Avena sativa (cv. "Flemings Krone") were dehusked and soaked for 3 hr in three changes of aerated 0.5 mM CaSO4. Subsequently, the seeds were placed on a steel screen floated on aerated 0.5 mm CaSO4. Germination, preparation of tissues, and experiments were carried out under dim green light at 28 + 1 C, except for a 3-hr exposure to red light at the beginning of the germination period.Exp...

“…This would occur where some of the wall materials (either already made or destined to be made "in the pipe" at the time of excision) do not get into the wall during the postexcision preincubation. Examination of the kinetics of several species (2,23) suggested that pea epicotyl should show this phenomenon. This could be predicted because of the type of elongation kinetics which occur when exogenous auxin is added to auxin-depleted elongating pea epicotyl segments.…”

Section: Methodsmentioning

confidence: 99%

Auxin-regulated Wall Loosening and Sustained Growth in Elongation

Vanderhoef

Dute²

1981

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...