. —-— v *— THE EFFECT or GRAVETY AND CENTEEEFUGAL [mass GK THE mvmsm a? AUXENfi' m CQLEQFTELE sscnohzs 05 gg HAYS E... Y’heats 5cm Hm Dogma of M. 5. MICHIGAN STATE UNIVERSITY. Rajnikorn Ouitrakul 3969 --__4.u .._._ -. - v- __‘. LIBRAR Y _ hiicfliggtrt State Uni‘vcmty -l“._l —-. 4,?A _._ ,._- .oo—. .4 . , ABSTRACT THE EFFECT OF GRAVITY AND CENTRIFUGAL FORCES ON THE MOVEMENT OF AUXINS IN COLEOPTILE SECTIONS OF g;5,ygz§ L. By Rajnikorn Ouitrakul Several effects of gravity on auxin movement have been documented: a lateral downward displacement when the coleoptile is placed horizontally and an inhibition of basipetal transport after inversion of the tissue. The purpose of this work was to investigate in corn coleOptiles the magnitude and time course of effects of gravity (1 x g) on axial and lateral transport of 3- indoleacetic acid (IAA) and l-napthalene acetic acid (NAA) by comparing the transport in and against the direction of the gravitational force. A study of the effects on axial auxin tranSport of centrifugal forces higher than 1 x g also seemed to be useful, since such experiments might help to approach the transduction mechanism of geotrOpism. The influence of gravity on axial transport was shown to be a quick effect. Inversion decreased the rate of IAA-luC basipetal transport by 10-20 percent and the rate of NAA-luC basipetal transport by 30-50 percent. The inhibitory effect was significant within 30 min after Rajnikorn Ouitrakul inversion. The effect was constant at least over the subsequent 3 hours. NAA-luC transport increased after returning the section to an upright position and the higher steady state wasreestablishedquickly (within 20 min). The decrease in basipetal transport rate upon in- version under gravitational stimulus was manifested to- gether with a decrease in velocity of the NAA-luC front. The uptake of IAA-luC or NAA-luc was also inhibited, but to a lesser extent than transport. Centrifugal forces of 5 x g and 10 x g in the direction of transport increased the rate of basipetal IAA-luC exit over the l x g control. Testing transport against the direction of the applied forces, 10 x g decreased the transport rate more than 1 x g. Acropetal movement of IAA-luC in the direction of the applied force (1 x g and 5 x g) seems to be higher than the movement against the force. The rate of acropetal auxin exit, however, was very low and highly variable. The geostimulation of lateral NAA-luc transport was established within 10 min of horizontal stimulation. The lateral NAA-luc transport in the physically downward direction was significantly higher than the upward move- ment. An oscillation of IAA-luC transport was observed, with a period length of more than 60 min. It can be concluded that auxin movement is higher in Rajnikorn Ouitrakul the direction of gravitational and/or centrifugal force than it is in the direction against the force, and that the gravity effects of these forces are expressed quickly. Centrifugal forces of 5 x g or 10 x g provide more effective stimuli than does gravity. Basipetal auxin transport, of NAA at least, is not at a maximum rate under the normal gravitational field. These re— sults suggest that the geosensor may act by a "pressure" mechanism. THE EFFECT OF GRAVITY AND CENTRIFUGAL FORCES ON THE MOVEMENT OF AUXINS IN COLEOPTILE SECTIONS OF ZEA NAYS L. By Rajnikorn Ouitrakul A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1969 ozz5>67 Acrmowisoosmrmr I wish to thank Dr. Rainer Hertel, my major professor, for his guidance and encouragement thro ghout the course of this work. Special thanks are also due to Dr. Barbara Filner for valuable discussion and criticisms. The interest, and encouragement of my academic advisor, Dr. Hans J. Kende, and my guidance committee Drs. Anton Lang, Robert S. Bandurski and Joseph E. Varner are greatly appreciated. I further thank Dr. Aleksander Kivilaan for the trans- lation of some important papers and his interest in this work. The excellent technical assistance of Fan is acknowledged with pleasure. 3 work was supported by U. S. Atomic Energy o—j‘ H o T; Commission Contact No. AT (ll-l)—1339 and by a UNESC . “ 1"- .q ~3- . n. ‘-~- r.- ~“ - : m r 1p U. N. DeveIOpmeno Flogramme-Thailand). f...) C) }_J i“ ‘2 1" L l :5. I.“ Ho TABLE OF CONTENTS INTRODUCTION AND LITERATURE REVIEW . . . . . . . . . . . a. b. C- d. Auxin Transport 0 o o o o o o o o o o o o o o o Gravity Effect on Axial and Lateral Auxin . Movement The Problem of Geosensors and Gravity Transduction o c o o o o o o o o o o o o o o o Aim Of the Work to be Reported o o o o o o o 0 MATERIAL AND METHODS . . . . . . . . . . . . . . . . . . a. b. c. d. ea f. g. RESULTS 1. Culture of Seedlings for Coleoptiles . . . . . Auxin o 0 Axial Transport TGSt o o o o o o o o o o o o 0 Lateral Transport TBSt o o o o o o o o o o o o Orientation of Tissue Sections for the Transport TGSt o o o o o o o o o o o o o o o 0 Special Coleoptile Centrifuge . . . . . . . . . Assay of RadioaCtiVity o o o o o o o o o o o 0 Effect of Gravity and Centrifugal Force on Axial Auxin Transport . . . . . . . . . . . . . a. b. C. d. e. Basipetal Transport of IAA in Upright and Inverted Coleoptile Sections . . . Basipetal NAA Transport in Upright and Inverted Sections 0 a o o o o o o o o Auxin Transport Rate in Sections After Return From Inverted to Upright Position 9 o a o o o o o o o o o o 0 Effect of Centrifugal Force on Basi- petal NAA Transport 0 o o o o o o o 0 Effect of Gravity and Centrifugal Force on AcrOpetal Movement . . . . . iii 11 11 ll 11 IN 1L» 16 17 20 20 20 25 31 3h 38 2. Time Course of the Effect of Gravity on Lateral NAA Transport . . . . . 3. Oscillation of Auxin Transport DISCUSSION . . . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . . . iv #2 it? 52 59 61 LIST OF TABLES TABLE PAGE 1 Basipetal IAA transport in upright and in- verted sections 0 c o o o c o c o o o c o o o o 22 2 Basipetal NAA transport in upright and in- verted sections 0 o o o o c o c o c o c o o c o 27 3 Comparison of the gravity effects on basipetal IAA and NAA transport 0 o a o o o o o c o c c c 30 h Auxin Transport Rate in Sections After Return From Inverted to Upright Position . . . . . . . 32 5 Basipetal NAA transport in the direction of gravity and centrifugal forces . . . . . . . . 36 6 Basipetal NAA transport against the direction of gravity and centrifugal forces . . . . . . . 37 7 Acropetal IAA movement in and against the direction of the l x g and 5 x g forces . . . . #1 8 Lateral NAA transport in and against the direction 0f gravity 0 o o o c c c o c c c o o “5 9 Basipetal IAA transport in single coleoptile seCtion o o c o c o o c c c o o o c o o c o c o ”9 FIGURE LIST OF FIGURES The Auxin Transport Test . . . . . . . . . . . 13 Orientation of tissue sections for the tranSport teSt o o o c o o o o o c o o o o o o 15 Special coleOptile centrifuge arm with CQleoptile box at the two ends 0 c c c c c o c 17 Basipetal transport of IAA in upright and in- verted coleoptile sections . . . . . . . . . . 24 Basipetal transport of NAA in upright and in- verted coleOptile sections . . . . . . . . . . 29 Auxin transport rate in sections after return form inverted to upright position . . . . . . 33b Lateral NAA Transport . . . . . . . . . . . . 46 OSCillation Of 1AA Transport 0 o c o o o o o o 51 vi INTRODUCTION AND LITERATURE REVIEW a. Auxin Transport. The movement of auxin through coleOptile sections was first studied by Went (1928) and Van Der Weij (1932, 193M), using 'natural' diffusible auxin obtained from coleOptile tips. The auxin in an agar block (donor) was applied to one end of the section and the reappearance of the growth substance in a second agar block (receptor) at the other end of the section was assayed with the A3323 curvature test. Since the work of Goldsmith and Thimann (1962), radioactive indoleacetic acid (1AA) has been used in most cases, allowing easier and more sensitive assays. No difference has been found in the transport preperties of 'natural' auxin and the synthetic IAA, and at least the diffusible auxin was demonstrated to be most likely 1AA (Raadts and Sfiding, 1957). A s she‘s-1n by Van Der Weij (1932, 1931+) and more ()\ +Jo C) rigorously by Goldsmith (19r6, 1967) auxin transport polar, proceeding preferentially from tip to base N. n v.n .-,J- 4-. +nr‘ m r (basipetal); auxin can move bot by active oiansport ani O O ,Vy' afipp m A (-- quv (- «a (‘3' «q‘1r‘fifi “A gfi 1"\ (N m‘ ,- t.) Liliarjluf‘. Cot/s r )n 7 ”‘1 «.L. all male. -upuitibl‘v o 4.’ v) O flfl+1 ? 4" fly" a W“ f r-Q f‘fl '1 D (awn, ’1 A fin'r\ tar ac.i\c transpoit is aster than diffusion, it can p oceel F4 E0 _ a , ,_fi , , ....L. 1, 4... V ,-. . ‘. ' ' 3. .L' . , process i..ol»ing upoaxo, transpo‘t, immobilization and r1- , ,. J-.. .1 ' a ~ ' , ' it ,thic ctztlnlioy ii rot TO_VIT23. Sore ev1dencc e-v-v-s J- 'L'fl I a . \ MJ'f\ — 4- suggested that tne tianepc rt is repeated from cell CO cell (Leopold and Ha11,966;le0pold and De la Fuente , 1967). Electrical coupling between adjacent Avera coleOpt ile parenchy ma cells was not detected and the resistivity of plasmodes :ata appea red to exceed at least 50 times the al resistivity of the cytOplasm. Thi that m m C C 7 (o m c+ m the unimpeded movement of the molecular or ionic form of auxin via plasmodes -mata is unlikely (Goldsmith, 1968). Experi mnx on saturating donor concentrations ant With inhibitors of transport provided evidence that the sensitive and rate limiting step is the exit rather than the uptake of auxin, presumably at the plasmalemma of each cell (Hertel and Leopold, 1963b). Auxin seems to have no covalent interaction in th. . . 18 transport process. Experiments With NAA- 0 transport . + Q _ 18 by Hertel and Flory (1966) showed that no 0 was lost during tea Sport. IAA and NAA reappeared as free, un- changed molecules in the receptors (Goldsmith and Thimann, 1962; Hertel, 1962 , Veen, 1967). b. Gravity Effect on Axial and Lateral Auxin“ Tovem ment. It has been shown for over 30 years that the movement of auxin in the coleOptile can be altered by external 3 stimuli, e.g. light or gravity (see Went and Thimann, 1937). Under the influence of gravity there are well documented effects on both lateral displacement and axial transport of auxin. Comparing axial transport, Van Der Weij (1932) inverted £323; coleOptile sections and found about a 10 percent inhibition of IAA basipetal transport. He concluded that inversion has no large effect, if any at all. Recent experiments with radioactive auxin by Hertel (1962) indicated that there is a 10—20 percent inhibition of basipetal 1AA tranSport in inverted 5 mm corn coleoptile sections. Pickard (1969) repeated Hertel's experiment with gygga and corn coleoptile sections and also obtained a decrease of basipetal transport, but the results were very variable. Naqvi and Gordon (1966) observed that gravity caused a slight decrease of transport velocity and transport capacity in two varieties of corn. The most clear-cut effect of inversion on tranSport has been shown by Little and Goldsmith (1967) with 15 mm gzena coleOptile sections. When IAA-luC was transported basipetally for 8 hours, the inhibitory effect was in the range of 40-80 per- cent. The acrOpetal movement was promoted. The inhibitory effect of basipetal transport increased with time and was correlated with inhibition of growth. Although inversion de- creased velocity of auxin movement it had no effect on uptake, destruction or retention of auxin in the tissue. The influence of gravity on lateral auxin movement was first shown by Dolk (1936) in experiments with Avena and corn coleoptile sections. He demonstrated that in Azgng cole0ptile sections a 20 min stimulation in the horizontal position induced asymmetric auxin tranSport which was de- tected when the plants were returned to an upright position. Early experiments with radioactive auxin, in which the source had been applied and removed before horizontal exposure, failed to detect the unequal distribution of radioactivity (Ching and Fang, 1958; Reisener 1957, 1960). However, Dolk's result were later confirmed by Gillespie and Briggs (1961) on corn coleOptile sections. Experiments with 1AA-14 14 C of Gillespie and Thimann (1961) showed that IAA- C held in tissue and transported out into the basal receptors underwent asymmetric distribution after geo- stimulation. In coleOptile halves, the IAA-luc trans- ported out into lateral receptors was more when the lateral movement was in the direction of gravity (hori— zontal position for the coleOptile half) than when the lateral movement was in the normal direction (vertical position for the coleOptile half) or in the direction against gravity (horizontal position for the coleOptile half) (Gillespie and Thimann, 1963; Hertel and LeOpold, 1963a). Goldsmith and Wilkins (1964) provided further con- vincing evidence that the asymmetric distribution reflects lateral movement of auxin within the sections. When IAA—luC was applied asymmetrically to 15mm corn coleOptile sections, the uptake within 2 hours was the same in both horizontal and vertical sections. The distribution of radioactivity in the coleOptile half away from the source was compared. The radioactivity in the lower horizontal half was more than the activity in the vertical half. This in turn was higher than the radioactivity in the upper horizontal half. This distribution of radioactivity corresponded with the curvature develOped. These data confirmed the first statement of the Cholodney—Went Theory (see Went and Thimann, 1937) which reads: "Growth curvatures, whether induced by internal or external factors, are due to an unequal distribution of auxin between the two sides of the curving organ." 0. The Ppoblem of Geosensors and Gravity Transduction. Accepting the theory that asymmetric auxin distri- bution is caused by gravity and responsible for curvature, the question arises as to how a gravitational or centri- fugal force can act on this transport process. This action has the following main characteristics (for review and dis- cussion see Audus, 1962): 1. Some gravity induced stimulatory process must reach a threshold to elicit a response. The minimum time required to reach the threshold is called presentation time. 2. There is an inverse correlation between the force (g) applied and the presentation time (t) whereby g x t = constant within a certain limit of force region and at a given temperature (see also Johnsson, 1965). 3. The sub-threshold stimuli are additive and can reach the threshold level. This implies that the stimulatory process must stop and start virtually simultanously with the application and relaxation of the stimulus to account for the rather precise summation of sub-threshold stimuli. Reversal of this process thus takes place on relaxation of the stimulus, but with a Speed only a fraction of that of the forward reaction. Two main processes can be distinguished in the over- all process of stimulation: 1. The physical mechanism by which plants (cells) ggpgg the applied force (action of the force on a 'geosensor'): 2. The transduction mechanism by means of which the 'geosensor', via an unknown mechanism, acts on the cell membrane of the lower side of the cell to lead to altered biological processes which ultimately result in geotrOpic bending. How do plants perceive gravitational force and trans- late it into biochemical processes leading to geotropic bending? A number of hypotheses have been preposed for the perception mechanism of gravity by plants (see also review Audus, 1962: Pickard and Thimann, 1966). Czapek (1898) proposed the idea that the cell might sense the weight of its own cytoplasm. Linsbauer (1907) suggested that the cytOplasmic gel can be considered as a ”hanging net". Upon reorientation, distortion could occur under its own weight to modify its biochemical and physiological behavior. StOppel (1923) pointed out that the redistri- buted weight of the protOplast might alter the tension in plasmodesmata and might therefore account for gravi- perception. Brauner (192b) proposed that the purely physical geoelectric effect upon horizontal orientation might cause lateral transport of auxin. However, Grahm (196h) had later found that the develOpment of the geo- electric effect might be a consequence and not the cause of the asymmetric auxin distribution. Particular attention has been received by the Statolith Theory (Haberlandt, 1900: Nemec, 1900). This theory pro- poses that plants perceive geostimulation by means of movement and/or pressure of one or more of their component parts(the statoliths, most likely amyloplast starch grains) that differ in density from their cellular surroundings. The primary action of statoliths could consist in mechani- cal pressure exerted on contact with the plasmalemma to initiate the transduction mechanism. This process may lead to the localized alteration of the membrane which in turn might result in enhancement of auxin exit in each cell. This finally produces a flux of auxin toward the lower. side of the organ (Hertel and LeOpold, l963b)resu1ting in upward bending. Such a postulated transduction mechanism, however, has no direct support. It is unknown whether the post- ulated statoliths exert a physical action via contact pressure or via another, unknown mechanism. Considering diffusion rates, cyclosis, mobility of cell organelles and the sedimentation rate in a gravi- tational field it is obvious that a significant "direct" sedimentation of auxin molecules is impossible, and for fast geotrOpic reactions it is plausible that the amylo- plasts are the geosensors, if the latter consist of moveable particles (calculation and discussion see Audus, 1962). The Starch Statolith Hypothesis has been supported by much correlative evidence (see review by Wilkins, 1966). Hawker (1932), comparing different species could show that the rate of fall of starch grains to the physically lower side showed a high positive correlation with the presentation time. In experiments of Pickard and Thimann (1966) wheat coleoptiles depleted of amyloplast starch still showed geotrOpic curvature, but with a much longer lag period than the controls (about 3 hours). The ratio of curvature to growth rate, however, was the same. This lead to the conclusion that amyloplast starch grains do not play a critical role in the geotrOpic response. The hypothesis was preposed that the difference in hydro- static pressure between the upper and lower sides of the cell might be translated and amplified to yield the geo- response. This difference, however, of about 1 dyne/cm2 across lO‘p cells of a width of ca. 10,n is exceedingly small when compared with the cell's turgor pressure of about 10 atmospheres which equals 107 dynes/cmz. d. Aim of the Work to be Reported. A In view of the similarity of the gravity effect on lateral and axial auxin transport, a study of the gravity effects on the axial auxin movement may be relevant to the problem of geotropism. Therefore the effects of different g forces on axial auxin transport were investi- gated and time course studies were carried out. A study of the effects of centrifugal forces larger than 1 x g might help to decide whether pressure or the mere asymmetric distribution of a possible sensor is important. Such experiments might lead to an approach to the trans- duction mechanism. This, in turn, might help to gain some understanding of the transport mechanism. In order to establish the relative sequence of lateral transport and bending, the very early time course of lateral transport has to be analyzed. The geotropic bending is a quick response which starts in less than 15 min (Brauner and Zipperer, 1961). It has already been shown that a brief period of horizontal exposure is sufficient 10 to cause lateral auxin asymmetry after return to the up- right position (see above, Dolk, 1936: Hager, 1967). These experiments, however, do not clarify whether the trans- port process was rapidly altered in the early period of stimulation since the period of lateral transport ex- tended over 90 min after horizontal exposure. During the studies on the basipetal transport, oscillations of IAA transport with a period length of more than 60 min were observed: this phenomenon was also further investigated. mr-v-wmvvrxrxrw .-\-lA-J’\) a. C'lt”re of neili gs for CCI‘CILlITQ. Corn seeds (2:1 revs L. hybrid WF9; 38, lot M2M° from Bear Hybrid Corn Co., De atur, 111.) were soaked over- night in to: water and planted in a plastic box (covered, with an air inlet) on 6 layers of wet paper tcvels. The seeilings vere ”rcwr in a darkroom ZUOC, °0~GO per‘ent relative humidity for about 5 davs, in (arknoss in- tererted xith 2 hours of red light each night. Coleoptiles of 20-30 my length here used. The sections Heic out wit' the aid of 2 razor blades m nted at a fixed distance (5 or 2 rm) about 2 mm rem the tips. b. fi‘:‘n. When investigatirg axial and lateral transport, IAA and mai;c1y FAA were used. Kid is biologically active, is transported in an active and pola ar manner as IAA, and offers techrical advanta.es over the latter, e.g. it is more stable for a long period of time (See also Hertel and Flory, 1968). 3-lndoleacetic acid a (=NAA) were used in 11Ln C cio Carboxyl—labeled 1AA- I! 1.»! 1 carboxyl-labeled NAA- ‘C were both purch good from c. Axial Transport Test To prepare agar blo (:IAA) and l-naphthalene acetic both unlabeled and labeled form. 33 mC/mH) and 5C.6 rC/m ) (specific activity (Specific activity ’—’ " 4 clear Chicago Corp. cks, Difco Br actc- -agar 1. 5 percent 11 was melted and poured into steel molds on microscope slides chilled from underneath with ice, and cut flat. The blocks were 23 mm in diameter and 1 mm thick. Re- ceptor blocks contained only agar; donor blocks were made by adding auxin to the molten agar before pouring it into molds (Fig. l). The original agar blocks as described were divided into smaller blocklets for each transport test, 3 blocks for 8-12 sections, 4 blocks for 4-6 sections, and 8 blocks for 1-2 sections. After cutting, coleOptile sections were transferred to receptor blocks placed on microscope slides. Four plasticine columns slightly higher than the sections were placed at the four corners of the slide. The slide with the donor block was placed down on the plasticine until all sections were in good contact with the donor (Fig. 1). If not otherwise specified, Upright coleOptile sections were pretreated with cold IAA and NAA for 60 min prior to application of the IAA—lQC or NAA-luC donor in order to avoid the disturbance at set-up steady state. If inverted sections were to be used, they were inverted immediately after application of the donor by inverting the assemblies. The assemblies were kept in a plastic box (covered, with an air inlet) lined with 2 layers of wet paper towels. The manipulation was done under dim green light from a flourescent lamp covered with u layers of 13 9‘ Lg (2:2 c g t . V Figure 1. The auxin transport te‘st. 14 yellow and blue ce110phane (see Fig. 2a, for tissue orientation). d. Lateral Transporthest. Donor blocks were 3 mm wide and ca. 15 mm long, re- ceptor blocks were 5 mm wide and ca. 20 mm long. 5 mm long coleOptile sections out about 2 mm below the coleoptile tips were depleted of endogenous auxin by transport between two plain agar blocks for 60 min and subsequencely were cut into halves. Each half was placed on a separate 4 mm of the tissue ./ lateral receptor block so that about 2. was on agar. The donor block was placed on the apical end of the sections. Each test was run with 5 coleOptile halves and for the same uptake and transport period. The sections in the assembly were kept in the vertical position and changed to a horizontal position for different trans- port intervals. Transport in the direction of gravity and against gravity was compared (Fig. 2b). e. Orientation of Tissue Sections for the Transpprt Test. To test auxin movement, auxin is applied to the tissue by a dgggp agar block and its reappearance is measured in the receptor block. 531a; movement is studied when the donor-receptor connection, i.e. the movement, is along the long axis of the coleOptile. Axial movement is basipetal when the donor is applied at the morphologically apical cut end of a section; the movement is towards the basal end (Fig. 2a). 15 a. Axial Transport Test. Basipetal Basipetal Acropetal Acropetal Upright Inverted Upright Inverted I‘ll m Donor [:::] Receptor Direction of Gravitationl ‘ Force b. Lateral Transport Test Figgre 2. Orientation of tissue sections for the transport test. 16 Acropetal movement is in the Opposite direction. The sections can be oriented morphologically upright or inverted. In the case of basipetal movement (no centrifugal force involved) the transport in upright sections proceeds in the direction of gravity while in inverted sections it proceeds against the direction of gravity. The reverse is true for acropetal movement (Fig. 2a). Lateral movement is assayed with sections that are sliced in half lengthwise, the donor is applied at the apical end of the half-section as in basipetal transport, but in this case the auxin allowed to exit from the longitudinal cut surface into a lateral receptor (Fig. 2b). When centrifugal forces are applied, the movement in the direction of centrifugal force is tested when the cut surface touching the receptor faces away from the centrifuge center. f. §pecial Coleoptile Centrifuge. For experiments with centrifugal forces, a Special coleOptile centrifuge, designed by R. Hertel and R. Geyer, (see Fig. 3) was used. The centrifuge axis was vertical, thus centrifugal forces were applied in the horizontal direction. The centrifuge arm can hold objects at the two ends with the distances from the central axis to the middle of the coleOptile boxes being 0.36m (=r). The assemblies 17 [ON . I /;~ / j“ \ '/ [1 ----------- <—-> Coleoptile-box Central axis ‘ Orientation of coleoptile sections in the cole0ptile box IO x g Resultant force Figurgji. a. Special coleoptile centrifuge arm with colo0pti1e box at the two ends. b.' The vector sum (resultant force on coleoptile sections) of centrifugal force and gravitational force. 18 were arranged so that the coleOptiles were in a horizontal position; thus the centrifugal force was applied in the axial direction of the section. (The overall direction of the force exerted on coleOptile sections was not strictly parallel to the axis but was the vector sum of the centrifugal force and the gravitational force, see Fig. 3). The centrifugal force is calculated from the equation 2 = 1.115 x 10‘3x n2 x r where f = centrifugal acceleration (m/secz) g = gravitational acceleration = 9.81 m/sec2 Z = g = centrifugal force expressed in multiples L) o f the gravitational force g n = revolutions per min (rpm) if r is constant, Z is preportional to n2 The centrifuge forces (Z) used in these experiments were 5 x g (at n = 111.6 rpm), 6 x g (at n = 122.3 rpm) and 10 x g (at n = 157.8 rpm). Since the centrifuge had a very strong drive (3/u horse power) the arm could be accelerated to the required g-force within seconds. Braking was achieved within 30 seconds, with the help of manually applied pressure. e. Assav of Radioactivity. After the end of a transport period, agar receptors and tiss e were transferred to counting vials containing 5 ml of Bray's solution (fig PPO and 0.2e POPOP, both from C) 19 Packard Instr. Co., Downers Grove, 111.; 60g naphtalene, 100 m1 methanol, diluted to 1000 ml with p-dioxane) and were subsequencely allowed to extract for at least 15 min before counting at room temperature. A Beckman liquid 11%,, U .T scintillation counter Lodel OPE-100 (with 90 percent counting efficiency with window fully Opened) was used. ’11 ihe values (cpm) given in the results section are corrected for background, which was at ca. 30 epm in all experiments. RESULTS 1. Effect of Gravity and Centrifugal Force on Axial Auxin Transport. a. Basipetal Transport of IAA in Upright and Inverted Colegptile Sections. The effect of gravity on axial auxin movement has been documented by comparing basipetal IAA transport in upright and inverted tissue (see Introduction). To con- firm this basic finding, such tests were performed using donors containing 2 x lO-6M IAA-luC and 2 mm long coleOp- tile sections. For each transport test, sections were cut from a single coleOptile (h sections) from about 2-10 mm be- low the tip. The donors and receptors were changed every 30 min throughout the M hours of the eXperimental period. The results are shown in Table 1. In 4 tests, the transport in inverted sections was inhibited on the average by ca. 30 percent in the first 30 min; after this initial period the inhibitory effect seems to be constant in the range of 10-20 percent (Table 1, also Fig. 4a, b). The radioactivity in the tissue sections was higher in the inverted sections than the upright sections, but the total uptake (=Cpm in receptor + cpm held in tissue) might have been slightly less at the end of the 4 hours transporting period. Because the counts were rather variable, the in- hibitory effect in the first 30 min of transport was evaluated statistically by the Student 't' Test (Hodges 20 21 and Lehmann, 1964) from the equation: z_o—i where U transported from the upright sections; II o *d 3‘ U = average of the U replicates = 119 cpm. I = cpm transported from the inverted sections; I = average of the I replicates = 88.5 Cpm. 1' = Standard error of the mean of the difference of each replicate = 11.27 Cpm. -9 z = LEE—1577i = 2.7o> 2.58 The Z value shows that the difference between U and I is significant at the 1 percent level. Therefore, the effect of inversion on basipetal 1AA transport was mani- fested at 30 min after reorientation. Differences appearing within 15 min seen in some other experiments were suggestive but not significant. 22 «n.oa mo.mH ma.ma mo.n~ accuuooa on.m om.-n mo.- aw.aa na.u~ nu.wa mo.¢~ wouno>GH :« coaugpwscw R oo.mu¢ me.cHH sm.¢~¢ en.¢o as.no w~.~n mH.ow «N.m¢ mm.mm n~.n¢ um.w~ uuuouuo.vuapo¢um Ibm~m Nana ween «mm «Hm man «on one nee omm aw owauo>< ohms owed mmou son was «on use mom mom com me acme owed moau Hue ace one owe mmm Hon own um Haem aHON wmmm own was mac oae «we Nee emm moH aweo meH meme ooh men use ecm Hum «Ho nae mma wouuo>cH no.m~n wo.nn em.~me an.oa wH.mo Hc.¢o mo.m¢ mm.¢¢ me.mw s~.no «N.H~ uuuouuo unavaoum chum fiwmfi meme o~m com «no one man men fine aHH owmuo>< ommm mnofi wfimm wfio 5mm wmm ofio one Nae can an mane monfi oflcn Haw wen mom men man nwo mum ooH wumq mama dean con mme who ewe nme Nme mmm mm whee mfima ccan mmo mww Nmn mun 5mm Ham mwm fine . unwwuma oeNnoHN cfimuowa omfiuoma onauowa omfinoo couco oo-om omnc sausage a“ mafia oxauo: enema» cu uncunooou :33 95395» 5 sun muouoooou 5 Sou Eau Emu mo 63m Awooa as my asowuuua wouuo>a« can uswauo: cw uncoonmnu <a« cg :.9Il::1-!.: acuuapwsaa N mn.wwm oo.m¢~ ac.maa nm.n¢ w~.m¢ ac.nu mu.¢m mo.mn «n.0u mn.o~ un.~ Humouno vuavuaum wmam «fine ¢wo~ mfin «on aH¢ mum nqm dwm «on m owauo><. «mow xfi¢o «new anq mac nwm mme o¢m omm ao~ H fifimm some #onu mnn mum qu men «an own muH H woaw noon Howu n¢¢ ~¢¢ wen mam m¢~ Hmm on m omood nmwo swam owe awn anq Ho¢ moq ¢nm nmu o wouno>au ~a.om~ ca.mw mm.n- mo.~m -.nm ww.o~ nm.ma mm.- ao.oH m~.~a hm.n M uouuo vuavuuum mmom «man ooad ooh ohm mac awn mom onq mmm ma umuum>< omum noon mcwm fine NNB mom ¢mm own woq Cum 5 ¢amm Nana noo¢ nae woo oao umm onm non «wm o omwo «Nam ommq mqm new coo mwm son Ho¢ nae um Huaofi «sum o¢m¢ oqw mam new own man ooq can em uxwfiump ceuuofim Camuowfi owanoma anfiuoua omauoa canoe oouom cane. nuances cu o8«9.. oxoua: oaoaau :« auounouuu fiauou mcacgasuu ca Eco muouaooou :« Baa Emu Emu «0 85m Awaofi BE my macauouu wouuu>:« can uzwauas ca uncomcauu «<2 Hauoawuam .N «Haas Figure 5. Basipetal trenSport of NAA-luc in upright and inverted coleoptile sections (data from Table 2) a. The amount of radioactivity delivered for every 30 min of transport (Table 2) b. The total radioactivity transported until the time indicated in the abscissa. o Hue Hmo use .330 Aawuv . .350 3:5 30 Table 3. Comparison of the gravity effects on basipetal IAA and NAA transport Sum of cpm epm epm in receptors cpm in remaining total receptors in tissue uptake Time in minutes 0-30 30-60 60-90 Experiment I Upright 362 822 1057 2241 1117 3358 297 761 996 2054 1033 3087 IAA 330 792 1027 2149 1075 3224 Inverted 247 727 910 1884 ' 1157 3041 333 779 910 2052 1205 3257 290 753 925 1968 1187 3149 Z inhibition _12.12 4.92 9.93 8.42 -9.86 2.32 Upright 111 700 750 1561 2476 4037 99 770 880 1749 2687 4436 NAA 105 735 815 1655 2582 4236 Inverted 56 487 525 1068 2839 3907 56 547 452 1055 2543 3598 56 517 489 1062 2691 3753 Z inhibition 46.67 29.66 40.00 35.83 -4.22 11.42 Experiment 11 Upright 306 745 775 1826 932 2758 274 680 713 1667 1038 2705 IAA 290 713 744 1747 .985 2732 ' . Inverted 243 626 633 1493 ;977 2470 163 455 ._§l§_ 1236 1064 _j!flfll_ 199 501 626 1365 1021 2385 2 inhibition 31.38 29.73 15.86 21.87 -3.65 12.70 Upright 64 543 658 1265 3353 4618 56 533 632 1221 3422 4643 NAA 60 538 645 1243 3386 4631 Inverted 23 219 310 552 3987 4539- 27 234 358 619 3184 3803 25 227 334 586 3586 4171 2 inhibition 58.33 57.81 52.86 -5.91 9.93 48.86 31 c. Auxin Transport Rate in Sections After Return From Inverted to Upright Position. In order to test decrease of the transport by in- version and to further examine the time course of the gravity response, inverted coleOptile sections when transporting NAA at a steady rate were returned to an up- right position. 5 mm coleOptile sections were pretreated 6 M unlabeled NAA for 60 min. Four sections for 6 each transport test were used. 10' M NAA-luc was applied with 10‘ in the donors and the receptors were changed and collected every 10 min. The tissue sections were allowed to transport NAA-luC for 30 min in the upright position; then they were inverted and NAA-luC movement continued in this position for 60 min. After this period the tissue sections were erected and allowed to transport for another 60 min. The control assemblies transported in the upright position throughout the transport period. Table 4 and Figure 6 show that the decrease in trans- port rate of the inverted sections appeared very early, perhaps within 10 min. After 30 min- the transport rate of the inverted sections seems to reach the low steady state compared to controls. Reorientation of tissue sections to the upright position increased the transport rate observably after 10 min. After 20-30 min in the up- right position the transport rate seemed to have returned to the control level, perhaps with an overshoot. 32 (I ‘I‘ull'..r n.00 0.m~ 0.5 0 0.5 0.0 0.HH m.0~ 0.HH n.n m.u .0.» 0.0 0.~ 5.0 0.~ .w uouuo vuswsaum Ilsa @ mulm H“. H H fimflflfimfl.mmmfl Hoow uwa mam sou 00H NHH H0 mm mm. mm mm mm 00 ma 5 0 -0m mam 00m sou 00H ONH mag odd ~0H 00 mag nod nu 0H 0 0~ usoaumoua 0.n~ 5.0 0.H~ 0.0a n.0fi n.n 0.HH n.0~ 0.5 m.~ 0.~ m.n 0.mH 0.0 m.0 0.N fl uouuo . . . .0stcwum Ilsa NM mm. mm. m WM. m mm. H a H PM. H. M w. m. 3.36.2. ~50m 05g wmfi 00H 05H 00H 00H 00H 00H nma 0m~ 0mH no «N 0 N uswwuenv mficm 55H 05H «0a wna Hag 00H omfi 0MH N¢H mmH n 0 Houusoo ~NH no ma osmmgu onetoea 0¢Hucn~ 0mH10N~ 0N~a0~a 0a~n00~ 00H100 00:00 00:05 ounce 00-00 00:00 oeuom 00-0w 0~10~ 0H:0 mouscwe ca 800 a“ mafia mMOunooou sH Emu scuuwmom unwaua: ou vouuo>sH Bosh annuom Houm¢ msoauoom an apex uuoamsuue cha< .0 ofipme 33 Figure 6. Auxin transport rate in sections after return from inverted to upright position (data from Table “)0 33 b cpm in RECEPTQR/lo min Jk ~00 ill. 044 H WA 0 4 HH NCO .r I. II HH I. u H H00 .. H~0 .. 0 we .r . w I 0030:.” . w a U , .. l Hag/«manna _n.l.uJ-£ 0 . 0 0 v4 0 0 a a 8 N0 #0 00 m0 H00 H~0 H00 H00 8H3.” AB»: 0 34 d. Effect of Centrifugal Force on Basipetal NAA transport. After an effect of gravity (1 x g) on basipetal auxin transport in upright and inverted sections had been shown above, further experiments were done to investigate the basipetal transport in or against the direction of centri- fugal forces higher than 1 x g. Table 5 shows the basipetal transport along the direction of different g forces. The experiments were done with 5 mm sections; 6 sections for each transport test were used. The tissue was depleted for 60 min prior to the application of a 10'6M NAA-lac donor. The ratio of radioactivity in the receptor over the total uptake (= cpm in tissue + cpm in receptor) was calculated and taken as a corrected measure of transport. As shown in Table 5a, after 60 min of transport, basipetal auxin movement appeared to be increased in the direction of higher centrifugal force when compared to the l x g control. With the technique described artifacts may be possible; e.g. little pieces of the donor or water droplets containing NAA-14C could be driven towards the receptor when transport was in the direction of high centrifugal forces. To eliminate such possibilities and to further test the effect of centrifugal force, the pulse technique of Goldsmith (1967) was used. A donor with lO-6M NAA-lac was applied for 30 min at l x g to all sections; it was 35 then changed to plain agar (Table 5b). The NAA-14C within the sections was allowed to be transported basipetally for a further 30 min at l x g, 5 x g and 10 x g. The re- sults show that the ratio of average cpm transported over uptake is more at 5 x g and 10 x g than at l x g. Thus the basipetal transport is indeed increased over the l x g control. The increase in basipetal transport along the direction of centrifugal force, then, is a genuine effect. The pulse technique of Goldsmith (1967) was also used to verify whether an increased force against the direction of transport will result in a greater inhibitory effect than 1 x g. Two experiments were performed in which the 6 tissue sections were pretreated with unlabeled 2 x 10‘ M IAA and lO-5M NAA, respectively. A donor with 10-6M NAA was applied for 30 min at l x g for all of the assemblies. After this uptake period the sections were transferred to plain agar receptors and donors and allowed to transport in the direction against the force in 10 x g and l x g. A set of coleOptiles for tranSport in the direction of force at l x g served asthe control. The ratios of radioactivity exported into the receptors over total uptake were compared. It can be seen from Table 6 that the transport against the direction of force was inhibited and this inhibitory effect increased with an increase in the force applied. 36 eH.en oHn.o sooH «he eHe.o new awe an.en me~.o oeHH use eoe.o who see oe.m mee.o wee ewm eme.o . tee nee Ne.mH hoe.o sew nee Hue.o “he coo . co.ee ee~.o see “an eem.e.. ewe ewe m x as. ea.e amm.o NwHH ewm wnm.o eeHH she he.n~ oom.o enoH see Hem.o eNoH not w x n .e. nn.me m-.o Heou Noe sum.o eme was NH.ee seo.o «Hon Ne "no.0 «Hon NoH w x oH oe.H mw~.o oomH eHn ee~.o soeH «an oo.on o3~.o emNH can ms~.o woeH. one u x e um.H- eom.o oAMH awn oom.o menH nee w.w N .a L 4 800 Emu oxsuaa oxmuns noouou HsuoH oamuHH Houaouom Hauoa enmmHB nouaooom eunuch HawsMHuuaou nous: noumouom acumooom omsouoaH N Houuaoo as nouuom Aw x H0 huH>¢uw nova: uuoamssua HowSmHuusou m=OHHu> nova: uuommsmua .0ouucua as? assausuuu Haw:MHuu:oo osu ouomen m x H us noussHa on now uaoaoua one: ouosov HHs “ooHsa 00 u<oz .0 H .00Huon uncoossuu waHuav usomoun “0:00 .0 .uouuou HawauHuucoo was huH>¢uw no aoHuuoqu one s« uuoamssuu «<2 HsuonHusm .m oHnsH 37 n I. liliuililil | I i l l 0n.0H 00.0n 0H0.0 mum 0N0 n00.0 0M0 Hun . .00~.0 mmHH III. .II: III. .II: HHO mm0.0 000 000 Hmm.0 000 000 0n~.0 uwHH nm0 00m.0 000 00m mom.0 ~50 0mm . HmN.0 000H 00m .0 SJ 8.8 3.06 Wm We...» 3.; m m . 026 $2 own «00.0 new 5mm 000.0 «mm nms mHm.0 00HH «mm 0H0.0 n00 000 wm0.0 ~00 mun 00m.0 mam . mum .- Eno Emu 800 m x H m N 0H _ oxsuns oxsuap . oxmuas muH>auw :uHa Houoe osuoHH noueooom Hsuoe osmoHH neunooom Hsuoa osmoHH nounooom :oHanHnsH N neumouom neumwoom noummoom aHouu:00v Am x Hv huH>suw «0 Am a Hv Am a 0H0 moouom aoHuuouHc one sH uuoamsuua huH>0uw uosHsms uuoausuua HawsmHuucoo uocstu uuoamssua .<uuw mo :oHuooqu are uasHaws uuoamssuu <42 HauoaHuam .0 oHnsH p.) [—4 ’d O w e. Effect of Gravity and Centrifrg ce on_Acropeta1 Movement. From the results on the basipetal auxin transport, one might eXpect that acrOpetal auxin movement is also higher when in the direction of gravitational force than when against it, regardless of the mechanism of the move- ment, active transport or diffusion. Although obtaining only relatively smallamountscd‘acrOpetal auxin exit, Little and Goldsmith (1967) could verify this prediction. In order to confirm the gravity effect on acropetal movement, the inversion effect was tested in preliminary experimen s using 8 sections (2 mm long; four sections from each of 2 coleoptiles) for one transport test. The results show that there were no receptor counts significantly 14 above the background when 10'6M IAA- C was tranSported 6M NAA-*uC up to 240 min. When 6. l — the IAA-1L0 concentration was increased to 5 X 10 M, a for up to 160 min and 10- significant fraction of auxin was delivered into the apical receptors after 90 min of transport. (The absence of counts in receptors during transport of 10—6M radioactive auxin transport indicates that there is no leakage of auxin via external moisture into the receptors.) Further experiments were performed to test the effect of the direction of gravity and centrifugal force on acro- petal auxin movement using relatively high donor concentra- tions. The radioactivity in the apical receptors of up- 39 right and inverted sections was assayed after 90 min of transport.‘ The data (Table 7) are highly variable, but taken together the experiments provide suggestive evidence that the acrOpetal movement in the inverted sections was enhanced over that in the upright sections. Also, when a 5 x g centrifugal force was applied (Table 7d) the movement in the direction of the force seemed to be higher than that against it. 40 Table 7. Acropetal movement at 1 x g and 5 x g of IAA-lac for 90 min after 60 min pretreatment with 5 x 10-6M cold IAA. All donors contained 5 x 10-6M IAA-1a C; the final concentration was made by adding appropriate amount of unlabeled IAA. In a, b and c gravity (1 x g) was the applied force, while in d movement with and against centrifugal force (5 x g) was tested. Each transport test contained 12 sections (2 mm long) cutting from 3 coleOptiles. 41 Upright Inverted Donor Total Total Concentration Receptor Tissue Uptake Receptor Tissue Uptake cpm cpm a. 278 2984 3262 38 3487 3525 177 3313 3490 859 4415 5274 - 30 3903 3933 37 3651 3688 10 M 263 3458 3721 312 3418 3730 283 3304 3581 608 3148 3756 582 3273 3855 45 3227 3272 Average 269373 3373 3642 3171142 3557 3874 b. 442 1797 2239 840 2264 3104 77 2089 2166 29 2068 2097 -5 76 1871 1947 19 2029 2048 1.5 x 10 M 377 2183 2560 57 1862 1919 88 1944 2032 - 278 1783 2061 801 2263 3064 Average 223267 1945 2168 3491193 2105 2454 c. 16 1540 1556 38 1900 1938 38 1732 1770 180 2208 2388 -5 30 1944 1974 64 1883 1947 5 x 10 M 81 1588 1669 15 2276 2291 79 2111 2190 170 1930 2100 49 1664 1713 39 2035 2074 Average 49211 1763 1812 84229 2039 2123 t I Centrifugal Force 5 x g In ‘he Direction of Force Against the Directon of . Force _ Total Total Receptor Tissue Uptake Receptor Tissue Uptake cpm cpm d. 92 5148 5240 80 4817 4897 -5 21 4419 4440 180 4588 4696 S x 10 M 20 4993 5013 31 4463 4494 16 5261 5277 202 5205 5407 Average 37218 4955 4992 105i36 4768 4873 “ 42 2. Time Course of the Effect of Gravity on Lateral NAA Transport. After it had been shown that both basipetal and acro- petal auxin movement are affected by gravity and centrifugal forces and that this effect is a rapid one, the gravity effect on lateral auxin transport and its time course were examined since this transport is thought to play an important part in the geotrOpic response. The lateral transport in the physically upward and downward direction was compared using coleOptile halves. NAA was used since its axial movement had shown more pronounced gravity effects than did the IAA transport; and since NAA can mediate a geotrOpic bending reaction (Anker, 1962), but its lateral displacement underifimainfluence of gravity had not yet been documented. A test consisted of two transport assemblies (see Material and Methods, Fig. 2b), each with 5 coleOptile halves. Donors with 10‘5M NAA-lac were applied after 60 min of depletion. The sections in the assemblies were left in the upright position for different time periods (tV = 0, 10, 15, 20, 30 min) and then changed to the horizontal position to allow transport in the downward (Td) or upward (Tu) direction for t - (BO-tv) min. A11 counts arriving in the lateral receptor during the total 30 min were registered, including those that were exported during the upright exposure. In Fig. 7 the time of horizontal exposure (t) is shown on the abscissa. The lateral trans- port was evaluated in each test as the percent difference of downward minus upward tranSport over the sum of trans- port in both assemblies. _r'n NAA asymmetry (%) = Td ‘u x 100 Td+Tu The percent of NAA asymmetry was averaged from many experiments performed at different times. Its mean value and the standard error of the mean were calculated and the results are shown in Fig. 7. It can be seen that a difference could be observed within 10 min after reorienting the tissue. Table 8. Lateral transport of 5 coleoptile halves (5 mm long) from donor containing lO-SM NAA-14C in the downward direction (Td) (with gravity) and upward direction (Tu) (against gravity) after different times in horizontal position (see text, Material and Methods). 45 cpm in receptor ' e * T1 ' T2 Time in Horizontal T1 T2 -———-——- X 100 Exposure T1o+ T2 0 min 27 31 -16** 25 36 ‘ -18 72 40 29 _2 .2. 13 Average 88 - 82 8t12.1 * cpm transported in vertical position ** the mean value of T1- T2 is corrected to be zero T - T '1d Tu d “ x 100 Td + Tu 10 min 17 8 36 47 11 62 41 29 , 17 31 14 38 29 21 16 82 44 30 _z .9. -13 Average {88 12 2728.8 15 min 80 36 38 26 13 - 33 32 12 45 49 24 _ 34 1.9. .1 45 Average '8; 18 3932.7 { 20 min 17 9. 31 i 46 9 67 ’ 98 42 40 95 20 65 120 19 . 73 55 25 38 17 13 13 26 9 . 49 87 24 67 .12. .2 ..___.__.3o Average 88 18 4716.4. 30 min '45 16 48 55 22. 43 79 14 ' 70 82 12 74 31 5 72 Average 88 14 6126.6 46 1H o o s '55 40.. /i > I o 60 s ‘o‘ p '4 20" 9 ~+ : ; IO 20 30 J. Time (min) Figgre 7. The percent difference in lateral transport in the downward direction along gravity as compared to the upward direction against gravity as a function of time in horizontal position (see Table 8, text and Material and Methods). 47 3. Oscillation of Auxin Transport. In many experiments on basipetal IAA-lac transport (e.g. Table l) in which the receptors were changed at intervals up to 30 min, the rate of delivery of radio- activity showed a more or less pronounced oscillatory pattern with a period length of ca. 60-100 min. The data in Table 9, Fig. 8, document a particularly clear case of transport oscillation. In this experiment, the basipetal 6M IAA-1” transport of 5 X 10- C in a single 2 mm coleoptile section was tested. Prior to the start of the experiment the seedlings were kept in 27°C and 90 percent relative humidity for about 30 hours. The transport test, however, was performed at the standard temperature of 24° . After cutting, the sections were either directly (that is immediately) placed in a transport assembly, or were first subjected to a depletion period of 40 min. Donors and receptors were changed every 25 min. The transport in both series was compared. For each series, 4 sections from one coleoptile were used which all show a similar transport pattern (Table 9; example in Fig. 8a and b; in Fig. 8c, the average of 4 sections is given). In sections used immediately after cutting a minimum of trans- port was reached at about 140 min after cutting and donor application (e.g..Fig. 8a and c). In tissue depleted for 40 min the minimum was reached at about 100 min (e.g., Fig. 8b and c) after donor application, that is also 140 min 48 after cutting. This may indicate that the oscillation had been started at the moment of cutting the sections (see also Kirk and Jacobs, 1968). 49 I‘ .III acunooou sH Emu mflmmn «wean 0n00~ onenH 0HO0H NHHHOm 0~w00m 0HH~0 uwsuo>< «an me «Nu me NwH mmu Hmu 0m 0 00m 00m w0~ 55H 00H mwm 00m 00 m 000 can Hmu wnH «NH mHm sum 50 N 00m 00m 5mm an mom 00m 00m 00 H.oz soHuoom soHuoHaov .55 00 enema anowH whoeH wooe~ mHueoN euumew HHueHN Noam ewtuo>< 5mm 05H 0mH new 00m NNN NHN mm 0 0mm «NH mmH Hum new wHN 00m mm m nwm 00H .00H mmu ohm wNN 00H mm N mom H0~ 0NH 00w 0mm mam 00m mm H.oz :oHuuom wsHuuso nouns uncensoua maHuonH 05H1m0H m0H-0NH 0~H-mm menon onum0 m0-0~ 0~-0 moussHa sH weHH A wsoH SEN v coHuoou uHHuaooHoo onsHm nH unoamcuuu <