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

Abstract

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...

Auxin Effect on the Transmembrane Potential Difference of Wild-Type and Mutant Tobacco Protoplasts Exhibiting a Differential Sensitiity to Auxin

“…Only one recent study (1) reports IAA-induced hyperpolarization (-20 mV) using two concentrations (1 and 1 00 M), and it was suggested that the response was concentration independent. In contrast we show here that the NAA-induced Em changes were described by a bell-shaped curve as a function of auxin concentration similar to that described for IAA effects on membrane thickness (22) and calcium binding properties (3) of soybean membranes.…”

“…Ultrastructural alterations were induced by auxin in isolated and in situ soybean plasma membranes (22). Modifications of the physical properties of membranes after auxin treatment have been reported.…”

Association between Elemental Content and Fruit Ripening in rin and Normal Tomatoes

“…These results are suggestive of better preserved membranes which may contribute to the delay in senescence observed in rin mutant. Ca has been reported to maintain the integrity of membranes (6,8,13,14) and delay senescence (14,25). The mutant failed to show any increase in membrane permeability during fruit development and maturation (18) which normally occurs during ripening (2,22) of fruits.…”

“…Divalent cations contribute to membrane stability by altering the structure of the membrane (13,24). For example, Jones and Lunt (8) in their review pointed out that electron microscope studies have shown extensive disintegration of mitochondria, ER, and cytoplasmic membranes in various Ca-deficient plants.…”

Effect of Plant Growth Regulators on Calcium-stimulated Serine Transport into Tobacco Cells

“…One potential explanation of this observation is that most of the binding sites are saturated at I jig/ml. Second, transport was not affected by 2,6-D at or below 10 ,ug/ml and 4-D was less inhibitory than 2,4-D at 10 ,ug/ml and was not inhibitory at 1 jg/ml. All three compounds were inhibitory at 50 ,ug/ml.…”