Ethylene Production during Clinostat Rotation and Effect on Root Geotropism

Botanisches Institut der Universitat Bonn, 5300 Bonn, BRD Department of Botany, The University of Trondheim, 7000 Trondheim, Norway

Ethylene Production during Clinostat Rotation and Effect on Root Geotropism W. HENSEL and T.-H. IVERSEN With 4 figures Received September 27, 1979 . Accepted October 20, 1979

Summary Ethylene production from 24 h old seedlings of Lepidium sativum L. (garden cress) was followed for 20 h both when the roots were growing normally and during rotation on a horizontal clinostat at 2 rpm. While the ethylene production in both groups was only traceable during the first 2 h, the production of ethylene was afterwards found to be higher in the rotated seedlings. By the use of an «internal standard» gas (propane) the absolute amounts of ethylene on a fresh weight basis were estimated to 2.5 and 8.6 nl . g-l f.w. for the control and rotated group, respectively, after 5 h. Parallel to the production of ethylene a decrease in root elongation was observed. After 5 h the root length of the rotated seedlings was only 81.4 % of that of the control roots. The application of exogenous ethylene at 0.2 ppm had a dramatic decreasing effect on the development of the geotropic curvatures when the treatment period extended a threshold limit in the range of 5 to 6 h from the injection of the gas in the plant chamber. The inhibitory effect of ethylene on the curvature was caused by decreased root elongation. Elongation ceased completely after 6 h. The present results are interpreted in terms of the possible interaction of IAA and ethylene and the possible participation of auxin in root getropism.

Key words: Ethylene, clinostat, root geotropism, Lepidium.

Introduction The idea of ethylene as a natural plant product was established by GANE (1934) and the correlation of ethylene to auxin be MORGAN and HALL (1962, 1964), who found that the level of ethylene production could be regulated by the level of auxin in the tissue. The observation that IAA and ethylene inhibited stem elongation by BURG and BURG (1966) suggested that the inhibitory effect of IAA is mediated only by the induction of ethylene production. It has also been suggested that the inhibitory effect on root elongation which occurs in response to applied IAA (CHADWICK and BURG, 1967) is not due to a direct action of the auxin, but to IAA-induced ethylene Z. P/lanzenphysiol. Bd. 97. S. 343-352. 1980.

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production. In contrast to these ideas ANDREAE et a1. (1968) and DUBUCQ et a!. (1978) suggest a more complex regulation. From experiments with various chemicals (ROBERT et aI., 1975, 1976) it can be concluded, that the induction of ethylene production always causes a growth inhibition, but the inhibition of ethylene production does not in every case cause a stimulation of elongation growth. Beside the mere effect on elongation growth, ethylene is suggested to be involved in the geotropic (gravitropic) response. It inhibits the spontaneous curvature of pea stem segments (BURG and BURG, 1966) and seems to be involved in the phytochrome regulated curvature of pea stems (KANG and BURG, 1972). Ethylene has also been found to affect the geotropic response of lateral branches in peanuts (2rv et a!., 1976). Epinasty of sunflower leaves (PALMER, 1973) and lateral roots of pea (LYON, 1972) during rotation on a horizontal clinostat can also be induced by exogenous ethylene. Placing the plants on a clinostat is assumed to eliminate the unidirectional gravitational field. The epinastic leaf curvature of plants on a clinostat has been thought to indicate that the absence of the unilaJteral gravitational force on the leaves disrupted normal auxin transport in the petioles. However, working with tomato plants LEATHER et a1. (1972), concluded that the epinasty of clinostated plants was due to increased ethylene production and not to cancellation of the gravitational pull on the auxin transport in the petiole. The aim of the present study was to determine the endogenous ethylene production of cress seedlings growing in normal direction and of seedlings rotated on a horizontal clinostat. In addition the effect of exogenously applied ethylene on root elongation and geotropic curvature has been examined.

Materials and Methods Plant material Seeds of garden cress (Lepidium sativum L.) were soaked for 20 min in tap water and placed on moistened filter paper in petri dishes kept in the vertical position in the dark at 25°C. About 24 hand 35 h (for geotropic experiments) after soaking, seedlings with straight roots of equal lengths, were selected and transferred to a gas-tight plant chamber (25 ml) where the roots grew between two 1 mm-thick slices of 1.25 % agar. For the ethylene production experiments, KOH was added as a CO 2-trap, separated from the plants by a plexiglas bar. In the plant chamber for the geotropic experiments KOH was omitted in routine experiments. All manipulations were carried out under dim green safelight (IVERSEN and SIEGEL, 1976). Ethylene production The gas-tight chamber was made from plexiglas with a removable front pane to place the seedlings into the chamber. The chamber was sealed by rubber seals and silicone-grease. Gas samples were taken through a rubber seal with a gas-tight syringe (100,ul) and injected directly into a Hewlett-Packard gas chromatograph (GC, mod. 5730 A) connected with a chart recorder (H.-P.7123 A). The column characteristics were as follows: alumina column

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60/80 mesh; dimensions 1/4' X 5.85'; column temperature 150°C; FID detector; N 2 -carrier 70 mllmin. Propane was used as an internal standard by injecting a known amount of this gas into the plant chamber directly before taking the gas sample from the chamber. The ethylene concentration was estimated using a calibration curve of peak relations (ethylene/propane on the chart recorder) versus concentration relation. Ethylene was identified by the retention time (54.5 ± 1.4 seconds). Absolute amounts of ethylene were measured in the range of 0.01-0.03 nl per injection. Cress seedlings (50-60) were germinated in the plant chamber either in the normal vertical position or placed on a specially constructed clinostat as described by IVERSEN et al. (1971) rotating at 2rpm. Gas samples were taken after 0.5, 2, 5, 15 and 20h. The injection of the internal standard gas prior to the measurement made it impossible to follow the ethylene production in a time course study of a single group of plants. The values of the ethylene concentration at the time of the measurement were therefore estimated from replicated, individual experiments at any given time. Attempts were also made using a Hewlett Packard gas-chromatograph-mass-spectrometer (GC/MS) mod. 5985 to establish a conclusive identification of the endougenous ethylene production. Geotropic experiments

Ten dark-cultivated cress seedlings germinated in petri dishes in darkness for 35 h, were transferred to agar plates and kept in the normal vertical position in the plant chamber for a 2 h-adaptation period. Thereafter ethylene was injected into the chamber to a final concentration of 0.2 ppm. The plants were stimulated horizontally either immediately, 2 or 4 h after the injection. The development of the geotropic curvatures was followed for 2 h in the continous horizontal position, with photographic exposures made every 10 or 20 min. The angles of curvature and root elongation were measured on images of the roots as described by IVERSEN (1973).

Results

Ethylene production The ethylene production from 50 cress seedlings was followed for 20 h and the results presented in Figure 1 are made directly from the chart scans. The peak height was directly proportional to the amount of ethylene in the injected sample ascertained against a standard. As can be seen from the relative values, measurements of the production both in the control and rotated plant groups during the first 2 h showed only traces not able to be quantified. After 5 h the relative ethylene production from the control seedlings was only 55 % of the rotated plants. In the period from 5 to 15 h there was no further increase in the ethylene production neither in the rotated roots or in the roots growing normally. A further increase in the same range in the control and rotated group, was detected 15 to 20 h after the start of the experiments (Figure 1). In preliminary experiments it was found that the use of an «internal standard» gas was the most reliable way of quantifying the low concentrations of ethylene. Propane was selected for this purpose and was used by injecting a known amount into the plant chamber directly before taking the gas sample from the chamber for GC-analysis. When using propane the quantification of ethylene gave the result

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shown in Table 1. As shown the pattern is slightly different from the results presented in Figure 1. After 5 h the rotated seedlings showed an ethylene production three times higher than that of the control group calculated on a fresh weight basis. After 15 and 20 h of the different treatments the absolute amounts of ethylene were in the same range in each treatment, but the ethylene concentration in the rotated chambers was lower than after 5 h of rotation. Table 1: Ethylene production in nl per g freshweight (f.w.) estimated by means of the internal standard procedure. Standard deviation of the means is indicated. Time (hours)

Control Rotated (nl· g-lf.w.)

0.5 2 5 15 20

Traces Traces 2.51±0.5 2.95±0.16 4.89±1.75

Traces Traces 8.61 ± 1.38 2.87±0.94 5.26±1.17

Development of seedlings The fresh weight of the control and rotated seedlings did not vary significantly (5 % level) from each other during the 20 h period (Figure 2). The length of the rotated seedlings was only 81.4 Ofo of that of the control roots. Fifteen and 20 h after the start of the experiments the root-length of the rotated plants was 77.3 Ofo and 78.3 Ofo, respectively, of the control roots (Figure 2). Z. P/lanzenphysiol. Bd. 97. S. 343-352. 1980.

Ethylene production and root geotropism

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Ethylene and geotropic response

Treatment with 0.2 ppm ethylene estimated from the mean amount of endogenous ethylene produced after rotation, produced a dramatic decrease in the geotropic

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Fig. 3: Geotropic curvature in cress roots treated with 0.2 ppm ethylene applied exogenously into the plant chamber. The treatment times which also include a 120 min stimulation period are as follows: 0 h; control (e); 2 h (D); 4 h (*); 6 h (0). Standard deviations are indicated. Z. PJlanzenphysiol. Rd. 97. S. 343-352. 1980.

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curvature of the cress seedlings when the treatment period extended a threshold limit (Figure 3). Only occasionally did a few plants in each chamber lose their geotropic responsiveness totally after 4 h of ethylene treatment while the rest of the plant group showed normal curvature development. After 5 h - not shown in Figure 3 the responsiveness of the roots was generally decreased. When the total ethylene treatment period was 6 h only a slight curvature had developed in the roots after 2 h of horizontal stimulation (Figure 3). At the same time the roots appeared thin and translucent indicating that destructive processes had been initiated in the tissue as a result of the ethylene treatment. The inhibitory effect of ethylene on the curvature was caused by decreased root elongation. While only a small decrease in growth can be observed after 2 and 4 h the roots stop elongating after 6 h of ethylene treatment (Figure 4).

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Discussion Identification and quantification of ethylene

One of the problems in working with ethylene is to obtain conclusive identification of the gas. It is normally not sufficient to use retention time data from only one GC-colomn to assign an ethylene identity. As stressed by WARD et al. (1978) co-chromatography on two columns whose separation is based on different properties adds confidence but a conclusive identification can only be established by a GC-MS-procedure. To obtain sufficient material for this type of analysis concentration by condensation of ethylene is necessary. In the present study attempts were made to identify the volatiles produced in the plant chambers using GC-MS. Preliminary results have shown that the volatile component is ethylene although it should be added that nitrogen with a mass number in the same range as ethylene demands a mass-spectrometer with high resolution for conclusive identification.

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Ethane is difficult to separate from ethylene but in the present study an alumina column was selected because this has the advantage of giving a larger difference in retention time between ethane and ethylene than other types of columns (WARD et aI., 1978). Another problem in the study was the quantification of the ethylene production. Preliminary experiments with low ethylene concentrations showed, that it was not possible to use a direct quantification by means of a calibration-curve with an ethylene standard. Injection of a second gas of known concentration as an «internal standard» gave more reproducible and reliable results, providing that the chamber was small enough to ensure a complete mixing of the two gases. In this connection another factor may influence the ethylene production; oxygen may become limiting in such a small-volume chamber, the tissues will then respire anaerobically and affect both the aerobic ethylene-producing system and the other physiological processes under observation. In the present study no precautions were taken to ensure aeration of the plant chamber but it is not thought that anaerobiosis will occur during the time period used for the cress experiments. The quantification of the ethylene measurements after 15 and 20 h (Table 1) shows that there is some diffusion of ethylene out of the chamber. A small loss of ethylene due to leakage from the chamber may explain the underestimation of the concentration calculated by means of the internal standard. For comparison of the treatments it is therefore necessary to abandon the absolute quantification and only use the relative amounts of produced ethylene (Figure 1). Ethylene production and growth-response

In most of the previous studies IAA was used to induce ethylene production (e. g. BURG and BURG, 1966; CHADWICK and BURG, 1970). Only little information is given about endogenous ethylene production. ROBERT et aI. (1975) showed that cress roots-segments grown in nutrient solution produced ethylene up to 4 h without any further increase up to 8 h. Cress roots grown on agar-plates in moist air, show generally the same pattern of ethylene production: an increase from 2 to 5 h followed by a period of no further ethylene production from 5 to 15 h (Figure 1). Another increase is measurable after 20 h. Rotating the seedlings on a horizontal clinostat with their root-axis parallel to the rotation-axis increases the ethylene production compared to the controls. It was shown by LARSEN (1953), that clinostat rotation acts as a continuous geotropic stimulation on the treated plants. So the increased ethylene production after rotation could be due to this geotropic stimulation on the treated plants. It is interesting to note, that this stimulation leads only to an initial ethylene response. The production rate ceases at the 5 h-level and increases after 15 h of rotation thus following the same general pattern as the control (Figure 1). Comparable results were given by LEATHER et aI. (1972), who rotated intact tomato plants. Z. P/lanzenphysiol. Bd. 97. S. 343-352. 1980.

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Parallel to the increased ethylene production is a reduced elongation of the rotated seedlings compared to the controls. This starts at 5 h and continues almost at the same level during the measuring period (Figure 2). These results may explain the controversial observations of growth reactions of roots grown on horizontal clinostats. If gas-tight chambers were used (e. g. LARSEN, 1953) the ethylene production lead to a reduced root-growth. Roots grown in chambers which allowed gas exchange show no difference in growth compared to controls (unpublished results). Increased root growth during rotation (VEEN, 1964) may be explained by the growth-stimulating effect of ethylene, if the rotation-induced ethylene-production does not exceed a certain threshold (KONINGS and JACKSON, 1979).

Ethylene and geotropic response As shown in Figures 3 and 4 exogenous ethylene results in a decrease in the development of the geotropic root curvature by inhibiting the root elongation. Ethylene applied exogenously might interfere with the normal auxin metabolism e. g. by mediating the generally accepted inhibitory action of IAA on root elongation. As recently stressed by KONINGS and JACKSON (1979), a certain amount of endogenous ethylene is necessary to maintain normal root growth. Increasing the concentration of exogenous ethylene leads to increased growth up to a certain threshold-concentration. Further increase reduces growth. In the present study the applied ethylene in the range of 0.2 ppm together with the endogenously produced gas in the gas-tight plant chamber seems to exceed this threshold and causes reduction of growth. It is interesting to note, that a period of 2 h normal growth followed by 2 h horizontal exposition (each in an ethylene atmosphere) leads to an initial increase of graviresponse although the overall growth of the roots is reduced. After 6 h of ethylene treatment, the gas has an absolute destructive and toxic effect on the root tissue. An effect of ethylene on endoplasmic reticulum membranes has been demonstrated by SARGENT and OSBORNE (1975) in expanding cells of etiolated shoots. They proposed that the changes in rough endoplasmic membranes resulted from a «stabilisation» of the membranes brought about by a reduced rate of phospholipid turnover in the presence of ethylene. An effect on endoplasmic membrane metabolism may generally be extended to also include the lysosomal membranes which after ethylene treatment might release hydrolyzing enzymes and initiate the destructive processes in the root cells. In conclusion it can be said that the present results show that the endogenous ethylene production by cress seedlings is increased by rotating the plants on a horizontal clinostat thus reducing the overall growth. The influence of exogenous ethylene on the endogenous ethylene equilibrium of the roots (KONINGS and JACKSON, 1979) and the complete cessation of root growth in the Z. Pflanzenphysiol. Bd. 97. S. 343-352. 1980.

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present gravitropic experiments (Figures 3 and 4) suggest at least a participation of ethylene in the overall growth of the roots. The involvement of the IAA/ethylene mediating system in the differential growth response after gravitropic stimulation as proposed by CHADWICK and BURG (1970) cannot completely be ruled out but it is not possible from such experiments to give conclusive evidence on IAA/ethylene regulated graviresponse. Acknowledgement We would like to thank the «Studienstiftung des Deutschen Volkes» for the student grant awarded to W. HENSEL. Thanks are also due to Professor A. SIEVERS for helpful criticism of the manuscript.

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ROBERT, M. L., H. F. TAYLOR, and R. L. WAIN: Ethylene production by cress roots and excised cress root segments and its inhibition by 3,5-diiodo-4-hydroxybenzoic acid. Planta 126, 273-284 (1975). - - - The effects of 3,5-diiodo-4-hydroxybenzoic acid on the oxidation of IAA and auxin-induced ethylene production by cress root segments. Planta 129,53-57 (1976). SARGENT, J. A. and D. J. OSBORNE: An effect of ethylene on the endoplasmic reticulum of expanding cells of etiolated shoots of Pisum sativum L. Planta 124, 199-205 (1975). VEEN, B. W.: Increased growth of roots of Vicia faba L. on a horizontal clinostat. Act. Bot. Neerl. 13, 91-96 (1964). WARD, T. M., M. WRIGHT, J. A. ROBERTS, R. SELF, and D. J. OSBORNE: Analytical procedures for the assay and identification of ethylene. In: J. R. HILLMAN (Ed.): Isolation of plant growth substances. Soc. for Exp. BioI. Seminar Series. New York, 4, 135-151 (1978). Zrv, M., D. KOLLER, and A. H. HALEVY: Ethylene and the geotropic response of lateral branches in peanuts (Arachis hypogaea L.) Plant Cell PhysioI. 17, 333-339 (1976). Professor TOR-HENNING IVERSEN, Department of Botany, The University of Trondheim, 7000 Trondheim, Norway.

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