/n Milan Introduction In salt-affected environments sodium usually is the principal problem ion. It is excluded from the tops of most crop plants (Brown et al. 1953, Bernstein et al. 1956).

Jacoby (1964) and Pearson (1967) studied sodium exclusion in beans and reported that the roots retained large amounts of sodium. Sodium which was translocated found its way into the stems. A so-called "antagonism" between sodium and calcium has been noted as far back as 1902 when Kearney and Cameron (1902) reported that the addition of calcium would "neutralize" the harmful effects of sodium on various plants. Subsequent reports by Kearney and Har- ter (1907) and Harris et al. (1923) served to substantiate and extend the findings of Kearney and Cameron. Ratner (1935) suggested that the toleration by soil- grown plants of high levels of sodium was related to the availability of calcium. At high concentrations of sodium, crops failed to grow because of a breakdown in the cal- cium regime of the soil, resulting in an insufficiency of 15 Homework due On Flarch 4th calcium available as a plant nutrient. Thorne (1945) and Bower and Turk (1946) performed experiments similar to those of Ratner and reached similar conclu- sions. Using excised barley roots, Epstein (1961) demonstra- ted the essentiality of calcium for selective cation absorption. Jacobson et al. (1961), Handley et al. (1965), Rains and Epstein (1967), and Elzam and Epstein (1969 b), all using excised roots, demonstrated that calcium in the absorption medium depresses the absorption of sodium. Hyder and Greenway (1965) studied calcium-sodium relationships in solution culture and noted an increase in dry weight of barley and subterranean clover as the ratio of calcium to sodium was increased. LaHaye and Epstein (1969) reported that beans, normally sensitive to high concentrations of sodium chloride, grew well in nutrient solutions containing 50 mM sodium chloride if sufficient calcium was present. This paper describes additional experiments on the role of calcium in the salt relations of bean plants. Materials and Methods Greenhouse experiments Seeds of brittle wax bush bean (Phaseolus vulgaris L.) were germinated for 5 days in the dark at 24°C in wet paper towels. The seedlings were then transferred onto stainless steel screens over 0.1 concentration Johnson nutrient solution (Johnson et al. 1957) and placed under a "Gro-Lux" lamp (Sylvania Electronics, Danvers, Mas- sachusetts). After two days the plants were transferred to the greenhouse and transplanted into 42-liter tanks containing the identical nutrient solution; there were twelve plants per tank. The plants grew in these tanks for one week, and were then transplanted into the ex- 214 DRY WEIGHT, g 0.5 F 04 0.3-0 0.28 هکار زد 0.1 oh 0.1 WEET in LEAVES ROOTS STEMS 10 Co", mM Figure 1. The effect of external calcium concentration on the dry weight of 1-week old bean plants cultured in nutrient solutions with 50 mM NaCl. Fresh weights of stems, leaves, and roots of all plants were determined on harvesting. In these experiments leaf petioles were treated as stem tissue. The leaves, stems, and roots were dried at 70°C for 96 hours and their dry weights were recorded. They were then ground in a Wiley mill. Fifty mg samples were ashed at 480°C in a muffle furnace, the ash put into solution in 50 ml of 0.1 N HCl, and sodium contents determined with a flame spectrophotometer. Calcium determinations were done by atomic absorption spectrophotometry with La as an interference supressor, at a concentration of 4000 mg/l in 0.125 N HCI. Each treatment was replicated four times. Na, mol/mg dry weight Experiments with excised roots The technique used was essentially the "tea bag" technique of Epstein et al. (1963). Beans were allowed to germinate in the dark in the presence of 0.5 mM CaSO4, at 24°C for 6 days. The roots were cut off 2 cm below the point where they joined the stem. They were then washed three times in distilled-deionized water. The roots were thoroughly mixed in a beaker of water. They were then blotted on clean cheesecloth and one gram samples were quickly weighed on a torsion balance. After weighing, the roots were transferred to open-weave cheesecloth "tea bags" and suspended in a 0.5 mM 1.6 1.20 0.80 0.40 0 Pogo P 3 1 DAY perimental solutions. Average high daytime greenhouse temperature was 33°C. The noontime light intensity in the greenhouse was 54,000 lux. Nutrient solutions used in experiments of one week's duration or less consisted of 0.1 Johnson solution lacking Ca(NO3)2 and containing NaCl at 50 mM. Calcium sul- fate was added to give concentrations ranging from 0 to 10 mM. Control plants were grown in 0.1 Johnson solu- Caso, solution. The solution was aerated and main- tained at 30°C. tion containing the Ca gradients but no NaCl. The pH of the solutions varied from 5.2 to 5.5. Long-term experi- ments (more than 1 week) were done with 0.5 Johnson solution lacking Ca(NO3)₂ to which NaCl and Caso, had been added. The solutions were renewed every fourth day. ROOTS 5 Ca²*, mM Figure 2. Sodium content of bean roots and stems as a func- tion of the external calcium concentration. The plants were cultured in nutrient solutions with 50 mM NaCl for 1 day. STEMS Experimental solutions consisted of 50 mM NaCl labeled with 22Na. The concentration of calcium (as CaSO) in the experimental solutions was varied from 0 to 10 mM. The volume of all experimental solutions was 200 ml and the pH was 5.3. Before they were transferred into the experimental solutions, the "tea bags" were given an additional rinse in approximately 150 ml of 0.5 mM CaSO4 solution. They were twirled to remove excess solution and immer- sed in the aerated experimental solutions at 30°C. This procedure was repeated until all 1.00 gram samples were so immersed. Treatments were duplicated. After a 60 minute absorption period, the "tea bags" were removed from the solutions and immediately im- mersed in a cold (5°C) solution containing 5 mM (un- labeled) NaCl and 0.5 mM CaSO4. The samples were washed three times in this solution and suspended in 4000 ml of identical solution for 30 minutes. Results When this desorption period was complete the roots were given an additional rinse in water, air dried, stuffed into planchets, and ashed at 500°C in a muffle furnace. The ash was put into solution with a few drops of water. A drop of detergent was added as a dispersal agent and the samples were evaporated to dryness under an infra- red lamp. The samples were assayed for 22Na using a thin-window gas-flow counter. No, umol/mg dry weight No, pmol/mg dry weight 0.80 1.200 0.40 1.60 concentration or 240 2.00 1.60 1.20 0.80 Figure 3. As for Figure 2, except that the plants were cultured in nutrient solutions with 50 mM NaCl for 2 days. O 1 3 3 5 Ca², mM STEMS STEMS 2 DAYS 7 5 Co², mM 7 DAYS ROOTS 7 ROOTS WEE 10 2 11 10 Figure 4. As for Figure 2, except that the plants were cultured in nutrient solutions with 50 mM NaCl for 7 days. Na, umol/mg dry weight Co², mol/mg dry weight 1.20 1.00 0.80 0.60 1.00 0.407 0.50 0.10 0.05 0.01 0 1 3 Figure 5. Sodium content of bean leaves as a function of the external calcium concentration. The plants were cultured in nutrient solutions with 50 mM NaCl for 7 days. Note the logarithmic scale of the ordinate. 7 DAYS. LEAVES 5. 7 Ca", mM 7 DAYS 2 DAYS 1 DAY 10 7 5 Co": mM 215 LEAVES 10 Figure 6. The effect of the external calcium concentration on the calcium content of the leaves of bean plants. The plants were cultured in nutrient solutions with 50 mM NaCl for the periods indicated. Figure 7. The condition of a typical bean plant after having grown in 0.5 Johnson solution containing 50 mM NaCl and 10 mM CaSO4 for 6 weeks. DRY WEIGHT, g O 10 216.** O TRIFOLIATE LEAVES 2 3 WEEKS 4 STEMS ROOTS 6 Figure 8. Increase in dry weight of bean leaves, stems, and roots during culture in 0.5 Johnson solution containing 50 mM NaCl and 10 mM CaSO4. TOTAL No, mg 160 140 (20 100 80 60 40 20 0 0 r = 0.955 16 20 4 8 12 TOTAL PLANT DRY WEIGHT, g 24 Figure 9. Total sodium of bean plants as a function of the total dry weight of the plants. Plants were harvested after 1, 2, 3, 5, and 6 weeks of growth in 0.5 Johnson solution containing 50 mM NaCl and 10 mM CaSO4. Each point represents a single plant. No ABSORBED, μmol/g.h 10 0.5 2 Ca², mM 6 8 10 Figure 10. The effect of calcium concentration on the rate of absorption of sodium by excised bean roots. The roots were 6 days old and the period of absorption was 1 hour. IS ts g d ld ts ht ar ht Ve $1S 217 tion dropped to about 50 % of the control (no calcium) about the same reduction in sodium uptake observed in the greenhouse experiments with intact plants. Bonds and O'Kelly (1969) demonstrated that in the absence of calcium or strontium very small concentra- tions of NaCl inhibited the elongation of the primary root of corn, Zea mays. Sorokin and Sommer (1940) had earlier shown marked effects of even low concentrations of calcium on the growth of Pisum sativum. For general discussions of the role of calcium see Burström (1968) and Jones and Lunt (1967). Under our experimental conditions (50 mM NaCl), it was observed that those plants grown at CaSO, concentrations of 1 mM and below showed necrotic root tips. Necrosis was progres- s) sively worse at lower calcium levels. Greater root length and greater fresh and dry weights were obtained with ed increasing calcium concentrations. This sodium-calcium m. relationship does not seem to be solely a replacement of li-sodium by calcium or vice versa. There is no close reci- at procity between the sodium and calcium concentrations of the leaves at different external calcium concentra- tions (cf. Figures 5 and 6). It. re he be [m ESS TER Discussion The presence of appropriate concentrations of calcium increases the ability of an otherwise susceptible species, the bean, to withstand the effects of high concentrations of sodium chloride. In the presence of inadequate con- centrations of calcium the plants are unable to exclude sodium. Plants with poor root systems, as a result of inadequate calcium, transfer large quantities of sodium into the tops. Even a low concentration of calcium in the solutions (0.1 mM) resulted in a great improvement in both the appearance of the roots and the ability of the plants to cope with salt. Epstein (1961) and Rains et al. (1964), and Läuchli and Epstein (1970) demonstrated that calcium is essential for selective ion transport by plant cells and Epstein (1965) insisted that a solution containing calcium at a low concentration represents a minimal physiological saline' for plant tissue." Hanson (1960), Marinos (1962), Marschner and Günther (1964), Foote and Hanson (1964), Elzam and Epstein (1969 a) and LaHaye and Epstein (1969) proposed that calcium is an integral part of the plasmalemma, governing its normal impermeabi- lity to and transport of ions. A deficiency of calcium, they proposed, leads to an impairment of the membrane structure, increasing cell permeability. Chemical analysis of the plants indicates that in- creasing concentrations of calcium depress not only the absorption of sodium by the roots, but also its transloca- he tion to the leaves. These results agree in part with those of Jacoby (1964) who showed that sodium is retained in the bean along the route of its ascent through the root