Expansion of Bean Leaves and its Suppression by Salinity

Abstract

Salinity has the curious faculty to limit plant growth without causing overt injury. Leaves of affected plants are characteristically small, but often otherwise normal in appearance. They seem to have about the same metabolic capability, in proportion to their size, as normal, mature leaves (16). It has not been established whether their small size is the result of smaller cells or of fewer cells, that is, whether salinity primarily affects cell division or cell enlargement. The expansion of 'dicotyledenous leaves is frequently attributed to cell enlargement. However, several recent investigations (2, 14, 22) indicate that it is largely the result of an increase in cell number. The purpose of the present investigation was to find out A) which process, cell enlargement or cell division , is primarily responsible for the expansion of beaan leaves, and B) how salinity limits the expansion of these leaves. Materials and Methods Several hundred uniform seeds of dwarf red kidney bean (Phaseolus vulgaris L.) were germinated in flats of washed expanded vermiculite. The flats were kept in a controlled-temperature room on a 12-hour light-and-dark cycle (300 in the light, 210 in the dark). The height of the fluorescent-plus-tungsten lamps was adjusted to maintain about 1500 ft-c at the top of the pllants. On the eighth day after planting the seed, 4 uniform seedlings were transferred to each of eight 23-liter crocks in the controlled-temperature room. Each crock contained 20 liters of aerated nutrient solution of the following composition in mmoles per liter: Ca(NO3)2, 2.5; KNO3, 3.0; MgSO4, 1.5; KH.,PO4, 0.5; plus, 1 mg per liter of iron as ferric citrate and 0.25 mg per liter of boron and manganese as H3BO3 and MnSO4, respectively. Half of the crocks were salinized by adding 24 meq of NaCl per liter of nutrient solution (1 atmosphere osmotic pressure) every 24 hours, starting on the day that the seedlings were transferred to the crocks, to a total of 72 imeq of NaCl per liter (3 atmospheres osmotic pressure). The pH of the solution was kept at about 6.5 by the addition of HNO3 or KOH. Additional iron was added at weekly intervals, and the volume of the solution was maintained by additions of de-mineralized water. The first trifoliolate leaf of both 1 Received April 15, 1964. control and salt-treated plants emerged from the apical bud on the tenth day after planting the seed, the same day that the third and final increment of salt was added. This date was taken as time zero for the leaf-expansion study. Growth measurements were confined to the lateral leaflets of the first trifoliolate leaf. They were begun on the morning of the second (lay after emergence of the leaf from the apical bud. Both lateral leaflets of 2 plants on each culture were detached from the petiolules and measured. The 4 leaflets from each culture were combined into 1 sample, weighed, and frozen. The terminal leaflet of each sampled leaf was weighed before and after drying as a measure of the moisture percentage. Past experiments had shown that the moisture percentage is tthe same for the lateral and terminal leaflets. The sampled plants were removed, leaving 2 plants per culture. By the fifth day, when the second sample was taken, the lateral leaflets were large enough to be analyzed individually , and so each was treated as a separate sample, measured, weighed, and frozen. The terminal leaflets were again used to determine the moisture percentage. The number of plants sampled wvas reduced to 2 for the control and 2 for the salt treatment. This same sampling procedure was repeated again on the eighth, twelfth, and nineteenth days. The dates for sampling were guided 1by the rate of leaflet elongation. The DNA and RNA content of the frozen lateral leaflets was determined (17) by a modified Schmidt-Thannhauser procedure. A comparison of the DNA content of bean leaflets with actual cell counts, to be described in a later paper, showed that the DNA content is normally a very good indicator of cell number. The protein extracted with 0.3 NaOH (17) was determined by the biuret reaction (11). Kjeldahl nitrogen determinations indicated that this extract contained between 86 and 90 % of the total protein nitrogen of the leaves, 10 to 14 c remaining with the cell wall material. The area of the lateral leaflets was calculated front their length by means of the relation 5.97 ± 0.13 cnm2 area per cm length. This relation was established in earlier experiments by measuring the length and area of 43 lateral leaflets of differentt size taken from both control and salt-treated plants. The water content of the lateral leaflets was calculated from their fresh weight using the moisture percentage of the terminal leaflets. 156