Inhibition of Abscission by Calcium

Abstract

An inhibition of abscission in bean petiole explants is reported for additions of calcium salts, especially at concentrations between 10-' and 10-1 M. Magnesium is less effective, and other commmon macronutrients are ineffective in inhibiting abscission. Evidence from timing experiments indicates that the calcium inhibition may act on the stage I or the "juvenile" stage of the explant, and that the inhibition of abscission may result from a retardation of senescence development in the pulvinar tissues of treated explants. The abscission of leaves, flowers, and fruits is presumed to be brought about through the weakening of the cell walls in the abscission zone; and this weakening may have two components , a solubiizing of the cell wall cementing substances and a hydrolysis of the structural components of the wall. A major part of the cementing properties of walls is presumed to be through the binding of pectic substances by double salt formation with Ca ions. The experiments reported here are an effort to study the role of Ca in leaf abscission. Sampson (10) observed a loss of Ca from the cells distal to the abscission zone, and he was able to enhance the rate of abscission by adding oxalate to trap Ca ions. Rasmussen and Bukovac (8) have made precise measurements of this Ca loss. Tagawa and Bonner (12) have reported that the application of Ca or Mg solutions to coleoptile tissues resulted in a hardening of the tissues which was interpreted as a wall hardening. We will report here an inhibition of abscission by the application of Ca, and an analysis of the manners in which the inhibition may be achieved. METHODS AND MATERIALS The experimental methods were slightly modified from those of dela Fuente and Leopold (3). Bean seeds (Phaseolus vulgaris L. var. Red Kidney) were germinated in plastic flats containing vermiculite. The plants were grown in a growth chamber (2000 ft-c, 16 hr daily) at a temperature close to 22 C. The plants were used at 14 to 16 days after planting, when the primary leaf blade had reached its maximal expansion. About 75 uniform plants were obtained from each flat. The leaf blade was cut off with a razor at the juncture of the pulvinus and the blade, and the petiole was cut 6 mm from the abscission zone, to provide approximately a 10-mm explant. These explants were placed basal end down in a Petri dish of 'Journal Paper 4913, Agricultural Experiment Station, Purdue University, Lafayette, Ind. 47907. 1 % agar, about 30 explants per dish. The open dishes were arranged in transparent plastic boxes in 100 ft-c of light with water-saturated air continually passing through, maintaining a uniform gas medium for all treatments. Chemical treatments were carried out by immersing the ex-plants in solution for 60 to 90 min. These explants were then transferred to agar which was prepared with the same concentration of chemical. a-Naphthaleneacetic acid treatment was applied as a 5-Il droplet directly on the pulvinus. Explants were treated with ethylene by placing the dishes of explants in a desiccator after they were aged for 10 to 12 hr, and ethylene was injected with a plastic syringe to provide 10 ,u/liter concentration. After 10 to 12 hr of ethylene treatment the dishes were transferred to transparent plastic boxes with water-saturated air continually passing through. Measurement of the abscission breaking force was done with a Ametek force gauge, model L-500, after the ethylene treatment. Each explant is held at the basal end with a pair of blunt forceps. The force is applied across the pulvinus 30 to 40 hr after cutting. The force at which break of the abscission layer occurred was recorded as the abscission breaking force, and values for 25 to 30 explants were used for each concentration. Data are plotted with standard errors. Chlorophyll was extracted with ethanol from 30 explants after severing the pul-vinus from petiole tissue. Absorbance was measured at 665 nm. Ethylene formation was measured by placing the explants in agar as described above, in a 25-ml Erlenmeyer flask. The flask was sealed with a serum cap for 12 hr. One-milliliter gas samples were injected into the gas chromatograph, and the analysis was repeated several times. RESULTS The net effect of solutions of CaClh on the time course of abscission of bean petiole explants is illustrated in Figure 1. In this case, 10 ,ul/liter ethylene was added for a 12-hr period beginning 8 hr after cutting. It can be seen from the figure that 50% abscission was obtained after about 45 hr from cutting , and that the addition of 0.01 or 0.03 M CaCl, delayed 50% abscission by about 35 or 45 hr; 0.1 M completely prevented abscission. Similar experiments carried out in the absence of added ethylene give slower abscission, but as shown in Figure 2 there was about a 20-hr delay of 50% abscission time with 0.03 M and a complete prevention of abscission by 0.1 M CaCl2. There was no evidence of tissue damage by the concentrations of CaCl, reported here, and the explants appeared turgid and healthy after 120 hr. Experiments in which the break-strength of the abscission zone is measured provide a more quantitative manner of evaluating abscission development (2, 6), and a concentration curve for CaCl, effects on abscission using the break-strength type of measurement is shown in Figure 3. In this experiment, 3 X 1 0' M CaCl2 gave nearly a 10-fold increase in the force needed to break the abscission zone. Comparison of the Ca 848