Altered Cell Microfibrillar Orientation in Ethylene-treated Pisum sativum Stems

The roots and stems of plants typically swell when they are exposed to ethylene (1-3, 5, 8), or supraoptimal auxin (2, 3, 5, 8, 20), and root hairs are induced to form (1, 5, 6, 8). When supraoptimal IAA causes pea subapical stem sections to swell, the optical birefrigence pattern of the cell wall changes, and newly deposited wall microfibrils tend to be oriented in a longitudinal rather than radial direction (20, 21). Ethylene also alters the birefrigence of pea stem subapical cell walls when it causes the tissue to swell (3, 4, 9), producing a characteristic banded pattern. This same pattern occurs in pea stem tissue which has swelled after treatment with supraoptimal IAA (4, 9). The similar effects of ethylene and supraoptimal IAA on pea tissue have been attributed to the fact that supraoptimal IAA induces ethylene formation (2, 3). According to the multinet hypothesis (13, 19), cells elongate predominantly in a longitudinal direction because of the restraining influence of newly deposited, radially oriented microfibrils. If a cell is to expand radially under the influence of ethylene or supraoptimal IAA, newly deposited microfibrils no longer should be oriented in a radial direction. The present communication describes the effect of ethylene on microfibrillar organization in various regions of the cell wall, and shows it to be similar to that resulting from auxin treatment (20, 21). Pea seedlings were grown in vermiculite for 7 days in complete darkness and then treated continuously in a desiccator with 50 ,tl/liter ethylene for 24 to 96 hr, except for one complete aeration each day. Within 24 hr the seedlings displayed a typical triple response to ethylene and thereafter swelling intensified progressively throughout the 96-hr experimental period. Subapical stem pieces were fixed with 3% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2, for 12 hr, rinsed with buffer, postfixed with 1% osmium tetroxide in phosphate buffer for 1 hr, dehydrated with an ethanol series, and embedded in Epon-Araldite epoxy resin. After sectioning, the tissue was stained with uranyl acetate and lead citrate. For light microscope studies, freehand sections were made from swollen subapical tissue without fixation, and the sections were stained with 0.05 % toluidine blue 0 (16). The effects of ethylene, IAA, kinetin, BIA,2 BA, and colchicine on the growth of excised 1-cm subhook sections from 7-day-old control plants were studied singly and in combination by incubating lots of 10 sections in 125-ml Erlenmeyer flasks containing 10 ml of 2% sucrose, 5 mm potassium phosphate buffer, pH 6.8, and 5 mm cobalt chloride solution plus the added hormone or hormones. All manipulations were carried out under dim green light, the flasks were sealed with vaccine caps, ethylene injected with a syringe when required, and tissue incubated in the dark with gentle shaking for 18 hr at 23 C. Ethylene production was then determined by gas chromatographing 5 ml of air from each flask (2), and the tissue was weighed and measured. The concentrations of coichicine, BIA, kinetin, and BA used in these studies were selected to provide maximum swelling with little or no growth inhibition; higher concentrations inhibited growth markedly and did not induce significantly more ethylene production. Control tissue from intact seedlings had the wall structure typical of elongating cells (Fig. 1 a), with radially oriented microfibrils at the inner wall surface, more randomly distributed microfibrils in the center of the wall, and longitudinally oriented microfibrils in the outer region of the wall. The cell depicted in Figure la is an epidermal cell, but similar wall structure was noted in cortical and pith cells. After 24 hr of treatment with ethylene a very different wall structure appeared uniformly in epidermal, pith, and cortical cells of the swollen subapical zone. This structure is illustrated in Figure lb. The microfibrils at the inner surface of the wall are oriented in a predominantly longitudinal direction, whereas those in the center of the wall are radially oriented. The newly deposited longitudinal microfibrils at the inner surface should restrict longitudinal expansion and allow the cell to grow in a radial direction. Those microfibrils originally laid down in a radial direction before ethylene was applied are now located nearer to the center of the wall; they are maintained in a radial configuration by the forces created during radial cell expansion. The microfibrils in the outer portion of the wall had already been pulled into a predominantly longitudinal orientation before ethylene was applied. However, the gas greatly slows cellular elongation, and therefore any further tendency for these fibrils to be pulled into a longitudinal pattern. Instead, because of the forces created during radial expansion, these microfibrils tend now to be pulled into a radial orientation. The wall microfibrillar pattern observed in control tissue was not changed during 48 hr of additional growth of the seedlings; the pattern observed in ethylene-treated tissue was similar at 24 and 48 hr. The experiment was repeated 3 times, and in each case the subapical zones from five control and five ethylene-treated seedlings were fixed and prepared for electron microscopy. Several randomly selected sections were examined from each subapex, and without exception the ethylene-treated and control cells had the distinctly different wall structures depicted in Figures la and lb. 1 Present address: Department of Horticulture, Michigan State University, East Lansing, Michigan 48823. 2 Abbreviations: BIA: benzimidazole; BA: benzyladenine.