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

Abstract

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...

Comparison of Auxin-induced and Acid-induced Elongation in Soybean Hypocotyl

“…Addition of auxin after the equilibrium pH had been attained did not alter the pH, but it did increase the rate of elongation, eliciting a normal auxin response. It was concluded that hydrogen ions do not mediate in long term auxin-induced elongation in soybean hypocotyl.The hypothesis that H+ ions act as a second messenger for auxin-promoted elongation (4, 13, 25) is supported by evidence that coleoptile (3,8,10,20,25,26), pea (1,7, 18,19), and soybean (29) segments elongate more rapidly at low pH, Avena and Helianthus wall extensibility is increased at low pH (3, 26), auxin-promoted elongation is preceded by auxin-promoted H+ ion extrusion in Avena (5,6,13,25) (29); (c) after acid-stimulated elongation has subsided, auxin is still capable of increasing the rate of elongation in Avena coleoptile (10, 25, 16) and soybean hypocotyl segments (29); (d) wall-bound glycosidases which have low pH optima, described in support of the hypothesis (16), can be inhibited without inhibiting auxin-promoted elongation in lupin (22) and Avena (9); and (e) auxin does not affect proton secretion in lupin (21) and soybean (28).These data have led us to examine the relationship of HI ionpromoted and auxin-promoted elongation. The accompanying manuscript demonstrates the lack of an auxin effect on medium pH adjustment (28).…”

Auxin-regulated Wall Loosening and Sustained Growth in Elongation

“…That is, exogenous auxin should induce just the second response. Since it has previously been shown (17) that acid-induced growth mimics the auxin-induced first response (wall loosening), the experiment was feasible. Segments were excised from the rapidly elongating region of the soybean hypocotyl and mounted directly into the growth-measuring apparatus.…”

Low Proton Conductance of Plant Cuticles and Its Relevance to the Acid-Growth Theory

“…Acidic solutions promote the growth of plant stem (16,21) and coleoptile sections (5,14), but it is not known whether the protons entered the tissues through the cuticle or simply through the cut ends. The inability to detect significant auxin-induced acidification of the external medium with sections of some dicot stems (12,19) where the direction of proton movement is outside to inside, but we have conducted sufficient experiments in which the direction of proton movement was reversed so as to demonstrate that the direction of proton movement has little effect on its conductance (data not shown).…”

“…Vanderhoef et al (19) have pointed out, however, that a low proton conductance of the cuticle has never been demonstrated, and concluded from their studies on intact soybean segments that 'Supported by United States Department of Energy Contract DE-AT06-76ER76019.…”

“…The excreted protons might be expected to acidify the external solution, too, but it has been difficult to demonstrate any auxin-induced acidification (11,12,19) unless the cuticle is abraded (3,9,16) or removed (1,13). This led us to suggest (1,13,15) that proton conductance of the cuticle is so low that protons excreted into the wall solution are trapped within the tissue unless they escape through cut surfaces.…”