CURWATURES IN TIMOTHY ROOTS INDUCED BY ULTRAVIOLET RADIATION 1 Robert T. Brumfield ULTRAVIOLET RADIATION penetrates most living materials for such short distances that studies of its effects have been limited largely to unicellular or- ganisms and tissues of multicellular ones grown in culture. Cell division and elongation can be ob- served directly in the surface cells of living roots of the small-seeded grasses. These roots are of suffi- ciently small diameter (130–2000) to be suitable material for studies of the effects of radiations of limited penetration. With these facts in mind it was considered worth while to investigate the effect of ultraviolet on cell division and elongation in tim- othy (Phleum pratense) roots. During the course of the experiments it was found that ultraviolet radiation induced curvatures in the root, which is the subject of this report. MATERIALS AND METHODS.—The type of moist chamber used for observation is shown in fig. 1. It consists of a rectangular piece of lucite 2 × 3 in. and /8 in. thick, having a rectangular opening in the center that can be covered with a 1 × 2 in. cover slip. Cover slips bearing strips of filter paper affixed to both sides form a thin moist-chamber, and a groove 4/3 in. wide and 1/16 in. deep extends from one edge to the opening. Water is added through the groove, which is left open to allow for gaseous interchange. The primary root adheres to the inner surface of the cover slip and is abundantly supplied with water from the droplets condensed on it. When the chamber is placed on the stage of a microscope in a horizontal position (stage vertical), the root grows downward along the surface of the cover slip and, therefore, can be observed directly. Under these conditions the root may grow 9–10p/min. at 27–29°C. and reach the bottom of the chamber in 2–3 days. Photomicrographs taken at successive in- tervals give a permanent record of the behavior of the root. Since visible light apparently has no ap- preciable effect on the growth of these roots (Good- win and Stepka, 1945), the image of the root can be projected directly into sensitized paper, thus obvi- ating the use of a negative. A camera is not needed when the apparatus is set up in a dark room and the light source screened to avoid stray light. When handled in this manner, the microscope acts as a photoenlarger. A Bausch and Lomb ribbon fila- ment lamp with a copper sulfate cell provides suffi- cient illumination. A X10 objective and a X5 ocular were used in the experiments reported here, and the distance from the microscope to the paper holder was adjusted to give a magnification of X100. 1 Received for publication July 16, 1953. * This work was performed under Contract No. W-7405- eng-26 for the Atomic Energy Commission. UNIVERSITY OF MICHIGAN ºf 3 9015 O8647 5665 The source of radiation was a 15-w. General Elec- tric germicidal lamp which delivers more than 80 per cent of its energy in the 2537-A region. All exposures were made at a distance of 50 cm., the intensity being 8 ergs/mm.*/sec. as measured by a General Electric germicidal ultraviolet intensity meter. The roots were exposed by directing the groove in the chamber toward the lamp; thus the whole seedling, with the possible exception of the upper part of the coleoptile, was unilaterally irradi- ated. In all experiments seedlings having roots 5–7 mm. in length were exposed to the same dosage simultaneously, four chambers being used with one seedling in each. The experiments were carried out at room temperatures, 27–29°C. RESULTS.—Exposure to ultraviolet radiation for 1 min. had no evident effect. Two minutes of irra- diation induced curvature in the roots as shown in fig. 2a–e, which is a series of photographs of the same root taken at the intervals following irradia- tion indicated in the description of the figure. The unilateral exposure was given to the right-hand side of the root. The 2-min. dosage induced a “nega- tive” curvature (away from the source) which reached a maximum some 60–80 min. after irradia- tion, the center of the bend being about 0.6–0.7 mm. behind the tip. Four-midate exposures induced tWO CLIrVatureS as shown in fig. 3 Øe. First a “posi- tive” curvature (toward t eºsource) developed about 20 min. afterºirradia fon, its' center being about 1 mm. behind *\root tip. Káter a “nega- § rº & ‘J C s . S NS / N I --|--|-- / K | *- : s *.* -: * - - r _- - *º- * - *- - - *-ºs tº- º 24- - - - * - - - - t- º tº- - - - Fig. 1. Diagram of moist chamber used for observation and exposure of living roots to ultraviolet. L = lucite rectangle; C = cover slip; F = filter paper; S = seedling root; W = water. - --- ?  *:::: *ś K.: - $ §tièáššāºš *::::- 3.<> -º,-3. 3 & #3 #2, # Nº sº * £º. ºf &: 8 at ºs. § tº 4% $ tº º & # 3 -3, 4:3 43%.5% cºlºz [Vol. 40 .." BOTANY AMERICAN JOURNAL 616 ºr , October, 1953] tive” curvature appeared and reached a maximum about 100 min. after irradiation. The center of the negative curvature was much nearer the apex than the positive one, being about 0.5 mm. behind it. Eight-minute exposures to the same intensity also induced positive and negative curvatures (fig. 4a– e), but both curvatures were much more extreme than in the 4-min. exposure. The positive curvature developed about 20 min. after irradiation and was about 1 mm. behind the tip, while the negative cur- vature reached a maximum ca. 100 min. following exposure and was ca. 0.4 mm. behind it. It can be seen from fig. 2e, 3e, and 4e that the curvatures tended to straighten out ca. 120–140 min. after ir- radiation. This was probably a geotropic response since the roots were kept in a vertical position after irradiation. There is some variation in the magnitude of the curvature induced in different roots by all exposures and there is also variation in the time after irradia- tion at which the maximum curvature is reached. A total of 8 roots were exposed to each dosage and the figures shown were chosen as representative of the behavior of the group. DISCUSSION.—Curvatures in plants result from the unequal expansion of cells on opposite sides of the growing organ. It is improbable that the elon- gation of cells on the nonirradiated side of the root is affected by the ultraviolet, since its penetration is limited. Therefore negative curvatures (away from the source of radiation) are caused by a stim- ulation of cell elongation on the irradiated side; positive curvatures result when the elongation of cells on the irradiated side is depressed. The rate of cell elongation in the root is very closely controlled by some mechanism, this control being somehow associated with the distance from the cell to the tip of the root. The effect of the higher doses of ultraviolet is stimulatory to cell elongation in one part of the root and inhibitory in BRUMFIELD––CUşVATURES IN TIMOTHY ROOTS 617 another. This differential effect is possibly related to different processes taking place in different parts of the root, or to an effect on some substance or substances present in graded quantities along the axis of the root. It is well known that auxins play an important part in the control of growth in plants, and it may be that the effect of ultraviolet is medi- ated through the inactivation of auxin. Up to the present time, however, attempts to modify the re- sponse of the root to ultraviolet by treatment with indole-acetic acid before and after irradiation have given inconclusive results. Tang and Bonner (1947) have described an en- zyme in pea epicotyls which inactivates auxin b oxidation, and Galston and Baker (1951) found a naturally occurring inhibitor of the enzyme in pea epicotyls. If this system is present in timothy roots it may be that the effect of ultraviolet is mediated through the inactivation of part of this system. Pos- sibly negative curvatures are induced by an effect on one part of this mechanism and positive curva- tures by an effect on another. An understanding of the agent or agents through which ultraviolet in- fluences cell elongation, thereby inducing curvatures in these roots, will be an important addition to our knowledge of the factors controlling growth and development. SUMMARY Curvatures are induced in roots of Phleum pra- tense when exposed unilaterally to ultraviolet radia- tion. Low doses induce a curvature away from the source of radiation. Higher doses induce two cur- vatures, one toward the source and occurring soon after exposure in the basal part of the growing point, and one away from the source and occurring later. The latter is closer to the root apex. LITERATURE CITED GALSTON, A. W., AND ROSAMOND S. BAKER. 1951. Studies on the physiology of light action. III. Light activation of a flavoprotein enzyme by reversal of a naturally oc- curring inhibition. Amer. Jour. Bot. 38: 190-195. GOODWIN, R. H., AND W. STEPKA. 1945. Growth and dif- ferentiation in the root tip of Phleum pratense. Amer. Fig. 2–4 show effect of ultraviolet radiation in inducing curvatures in Phleum roots.-Fig. 2a shows the shape of the root immediately after exposure for 2 min.—Fig. 2b, 2c, 2d, and 2e show the curvature of the same root at 40, 60, 80, and 120 min. after irradiation, respectively.—Fig. 3a shows the shape of another root immediately after exposure for 4 min.— Fig. 3b-e show its curvature at 20, 60, 100, and 140 min. after irradiation, respectively.—Fig. 4a–e show the curvatures induced by 8-min. exposure, 4a being taken immediately after exposure, and 4b, 4c, and 4e showing its curvature 20, 60, BIOLOGY DIVISION, OAK RIDGE NATIONAL LABORATORY, OAK RIDGE, TENNESSEE Jour. Bot. 32: 36-46. TANG, Y. W., AND J. BonneR. 1947. The enzymatic inactiva- tion of indoleacetic acid. I. Some characteristics of the enzyme contained in pea seedlings. Arch. Biochem. 13: 11-25. 100, and 140 min. after exposure. X41. l