THE USE OF RADIOACTIVE PHOSPHORUS IN TRANSLOCATION STUDIES

ONE OF the most difficult problems in the studv of rapid solute transport in plants is to limit the solute being studied to a single conducting tissue so that one can accurately observe the factors determining its rate and direction of movement. The work of Mason and Maskell (1928) and of Stout and Hoagland (1939) indicates that a ready transfer of solutes may take place between xylem and phloem by cell-to-cell diffusion accelerated by protoplasmic streaming. By the same means solutes may move longitudinally at rates as high as 2 cm. per hour (Crafts, 1939) through channels entirely distinct from the sieve tubes and vessels that are regarded as the normal conduits for rapid longitudinal movement. Failure to distinguish this accelerated cell-tocell diffusion from rapid longitudinal transport has led to many questionable interpretations from experiments designed to elucidate the mechanics of translocation. For this reason Caldwell's translocation studies (1936) using viruses have been criticized; also Schumacher's fluorescein experiments (1933, 1937), the work of Fischer (1936), and Both (1937) with nitrogen compounds, and Gustafson and Darken's translocation studies (1937) using radioactive isotopes. All these experiments have been criticized (Crafts, 1939) because rates greater than those of cell-tocell diffusion accelerated by protoplasmic streaming were not demonstrated, so that conclusions relevant to the mechanism of rapid solute transport were not justified. Several workers have used radioactive isotopes as indicators in experiments on solute transport in plants. Gustafson and Darken (1937) have alreadv been mentioned. Stout and Hoagland (1939) used oiled paper to separate bark from wood for a short distance along stems of willow and geranium. They found that radioactive isotopes of potassium, sodium, or phosphorus introduced in the solution around the roots of these plants subsequently appeared in the wood but not in the bark of the separated region. Wherever the phloem was left attached to the xylem, however, it contained the test element almost as soon as the xylem itself. These observations point to the xylem as the sole channel for rapid upward transport of water and salts in the stem below the principal transpiring region. They also indicate the rapidity with which salts diffuse radially from xylem to phloem. More recently Biddulph (1941) has studied the migration of injected radiophosphorus from bean leaves. He injected the terminal leaflet of the second alternate leaf of each plant as follows. While the blade was held under water he dissected out the 1 Received for publication July 14, 1942. The writer is indebted to A. S. Crafts, D. R. Hoagland, and J. P. Bennett for their advice, and to John Lawrence for generously providing the radioactive phosphorus used in these studies. principal lateral vein of the leaflet to form a flap. This flap was immediately dipped into a small container of radiophosphorus, left for five minutes, and then removed to a special container of water. After a few hours he ashed various fractions of the plant, analyzed them with a Geiger counter, and found radioactivity in the stem both above and below the node of the treated leaf. Since the plant was actively transpiring, he concluded that the radiophosphorus had been transported upward in the xylem and downward in the phloem. The work of Strasburger and Dixon on dye movement in the xylem, however, and more recent work on xylem injection indicate that with Biddulph's injection method no clear differentiation can be made between xylem and phloem movement. His experiments are, therefore, of doubtful significance with respect to the direction, localization, or mechanism of normal solute movement in plants. In using radioactive isotopes in physiological experimentation one should remember that they may affect cell permeability. As early as 1926 Fricke (1926) showed that beta and gamma rays can denature proteins. Barnett (1939) points out that electrically asymmetric lipoidal molecules such as those found in cell membranes may change their polar properties when bombarded with electrons or gamma rays, and that, according to Wilbrandt (1935), any such change will alter the permeability of the membrane. Crane (1939) asserts, "It is unnecessary to consider the local effect of a particular radioactive atom upon a cell which happens to be close to it." He further states that the effect on permeability depends upon the magnitude of the generalized dose of ionization delivered to the material throughout its volume. The beta rays emitted by radiophosphorus must affect permeability much as x-rays do, since the latter produce rapidly moving electrons. Heilbrunn and Mazia (1936) have reviewed experiments dealing with the effect of x-rays on permeability. In general no effect is noted below an activity equivalent to about 106 disintegrations per cc. per second, which is roughly equivalent to a strength of 20 microcuries of radiophosphorus per cc. of solution. In the experiments to be described, solutions having strengths as high as 100 microcuries of radiophosphorus per cc. were placed on the leaf. By its effect on cell permeability this radioactivity may have facilitated the penetration of radiophosphorus into the leaf, but so little of this substance eventually migrated out of the leaf in these experiments on phloem transport that there was probably little effect on permeability in the conducting elements themselves, if Crane's values are correct. Crane has apparently neglected, however, to consider the probability that plant cells will accumulate radioactive salts from the bathing solution and by thus concen-