On the Inhibition of Mesocotyl Growth by Ethylene

On the Inhibition of Mesocotyl Growth by EthyleneAuthor(s): C. L. MerSource: New Phytologist, Vol. 73, No. 4 (Jul., 1974), pp. 643-651Published by: Wiley on behalf of the New Phytologist TrustStable URL: http://www.jstor.org/stable/2431233 .

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New Phytol. (1974) 73, 643-65 I.

ON THE INHIBITION OF MESOCOTYL GROWTH BY ETHYLENE

BY C. L. MER

Botany Department (ARC Staff), Imperial College of Science and Technology, South Kensington, London, S.W.7

(Received i9 October I973)

SUMMARY

Oat seedlings have been treated with ethylene-generating substances, (z-chloroethyl)phosphonic acid (ethrel) and fl-hydroxyethylhydrazine (B-OH), as well as with gaseous ethylene in an attempt to confirm the claim that mesocotyl growth can be stimulated by ethylene. The factorially arranged experiments included a standard exposure to light to permit evaluation of the light x ethylene interaction.

Ethrel always inhibited growth and caused the malformations associated with ethylene treatment. In contrast, B-OH brought about the 'CO2 effect'; that is, mesocotyl growth was retarded at first but subsequently enhanced due to an increased number of cells having an unchanged final length, while the growth of the coleoptile was reduced. These plants showed no ethylene- type distortions, however. As ethylene gas itself was invariably inhibitory it cannot be assigned any stimulatory properties at the concentrations used.

One treatment combination (B-OH with C02) caused the death, selectively, of the tip cells of the coleoptile. Even without these cells, allegedly an essential part of the growth-regulatory system of the seedling, a promotion of mesocotyl growth was observed.

In the discussion support is deduced for the view that mesocotyl growth is not controlled by the tip of the coleoptile.

INTRODUCTION

The observation that low concentrations of ethylene will stimulate mesocotyl growth in oat seedlings (Suge, I97I, Table i) introduces a contradiction into the proposition that growth control results from the counter-opposing interplay between promotion and inhibition, respectively by auxin and ethylene. In the experiments reported, however, mesocotyl length in 5-day-old untreated seedlings was only 2.3 mm, as compared with 60-75 mm in etiolated plants (Mer, I957; I969), So that exposure to light must have occurred in Suge's experiments. This inference is supported by his description of the way in which the plants were treated with ethylene. Light is known to interact with ethylene to offset partially, but not to reverse, its inhibitory effect (Pratt and Goeschl, I969).

An attempt has therefore been made to confirm the reported stimulation using two ethylene generators, (2-chloroethyl)phosphonic acid (ethrel) and fl-hydroxyethylhydrazine (B-OH); gaseous ethylene was also used.

MATERIALS AND METHODS

Avena sativa (cv. Victory I, Sviilof) was used in all experiments. The de-husked grains, lightly dusted with 'spergon', were planted and the seedlings were raised and handled as previously described (Mer, I953).

643



644 C. L. MER

Ethrel was used from the start of the experiments up to a concentration of 500 ppm in 'pH 4 water'; that is, London tap-water acidified with a few drops of concentrated nitric acid. B-OH was also supplied in 'pH 4 water' up to a concentration of 5000 ppm, likewise from the beginning of the experiments. Carbon dioxide (C) was administered at a concentration of 50 in the continuously flowing, water-saturated air-stream. Ethylene (E) was used at about IOO-200 ppm in different experiments. It was intro- duced into the air-stream (6o 1 h-') by driving ethylene-saturated water into a gas reservoir at a rate of 6 ml h 1 with a Watson-Marlow peristaltic pump. The content of ethylene in the mixed gases was monitored from time to time by gas-chromatography; the contents varied within the range quoted, but constancy was not aimed at given the vagaries of the two independently flowing gas streams. Nitrate (N) was supplied as a o. I % solution of mixed sodium and potassium nitrates in tap-water. This treatment was included in the experiments with gaseous ethylene, and the solution was provided after the seedlings had been illuminated on the second day.

Light (L) was given on the third day after starting the experiments with ethrel and B-OH when the seedlings were exposed to dim orange-red light for 5 min. In Suge's experiments the i- or 2-day-old seedlings were most probably illuminated before being treated with ethylene. As the intensity of light and the duration of the exposure are unknown it is not possible to repeat the experiments accurately. However, the sequence of events was followed in the present experiments with ethylene by giving 2-day-old plants the standard exposure to light, as above, and then beginning the gas flow.

Measurements of length were made, of both mesocotyls and coleoptiles, from the third day to the seventh day after the start of the experiments. The data are quoted as mean values for groups of twenty to twenty-two replicate seedlings. For certain B-OH- treated and control plants the numbers of cells per mean column, and the mean cell length per millimetre along the entire lengths of the structures, were obtained by em- bedding suitable material in wax, sectioning and counting (Mer and Causton, I963).

EXPERIMENTAL RESULTS

The effect of ethrel The data of Fig. i (a, b), from an experiment in which a concentration of 250 ppm

was used, show that ethrel depressed the growth of mesocotyls and coleoptiles. The treated seedlings were waxy to the touch, were very brittle, and had deformed mesocotyls. They were spindly near the grain with the upper part slowly broadening and flattening towards the node. The base of the coleoptile was narrower than the sub-joined mesocotyl and it looked like a short, sharp spike.

The seedlings reacted to light in the usual way; growth of the mesocotyl was inhibited, that of the coleoptile promoted reciprocally. However, there was no growth flexure in the mesocotyl otherwise invariably seen in those of illuminated seedlings (Mer, I959, I973).

The effect of B-OH on growth This substance, at a concentration of 5000 ppm, induced a growth effect quite unlike

that caused by ethrel. Elongation of the mesocotyl was initially held back (Fig. 2a) to be followed by a burst of growth that gave a clear promotion. Coleoptile growth, on the contrary, was depressed during the whole experimental period (Fig. 2b). Again, sensitivity to light was shown by the reciprocal reduction and enhancement, respectively, of



Mesocotyl growth inhibition by ethylene 645

E 30-

I I I I I I . I I~~~~~~~~ol I Ioo

0~~~~~

3 4 5 6 7 3 4 5 6 7

Time (days)

Fig. I. The effects of 250 ppm of ethrel0light on the growth of the mesocotyls (a), and coleoptiles (b) of oat seedlings. Squares, ethrel; circles, control; open symbols, after illumination.

the mesocotyl and coleoptile. However, the stimulation faded to a depression by the end of the experimental period, but the straightening effect of light was very clearly shown by the mesocotyls of these plants.

None of these seedlings displayed the deformed growth associated with ethylene treatment. In some preliminary work, however, plants grown separately in ethrel and B-OH solutions, were enclosed together within a single chamber in the apparatus. All such seedlings had the broadened, and flattened, upper-part to be seen in ethrel-treated

80 -

-(a) (b)/

100? ,,--!X E 0 ; _. |/

E 3

7 -/ 20 -

0 i -- - - 5 6 1 ? | 4 5 607

01- 01~~~~- .

3 4 5 6 7 3 4 5 6 7

Time (days)

Fig. 2. The effects of 5000 ppm of B-OH + Light on the growth of the mesocotyls (a), and coleoptiles (b) of oat seedlings. Triangles, B-OH; circles, controls; open symbols, after illumination.



646 C. L. MER

plants. Evidently, ethrel released ethylene into the atmosphere even from an acidic medium, and simultaneous treatment with B-OH did not prevent the plants from reacting to it.

The effect of B-OH on cell numbers, and size It was obviously of interest to determine if the effect of B-OH on mesocotyl growth

came about by cell-multiplication, or elongation. The relevant data obtained from representative 7-day-old plants (Table i) indicate that the increased length of the mesocotyl can be accounted for in terms of additional cells. The difference in cell length is too little for significance at P, 0.05; the value of t is only I.2.

Table i. The effect of B-OH on cell numbers and size Control B-OH

Mesocotyl length (mm) 68 I00 Number of cells per mean column I30 197 Cell length 5I4.7+ I3.7 (62) 537.2?+I.7 (99)

Mean length of cells in mm x io with standard errors. The number of observations is in brackets.

The interaction between B-OH and carbon dioxide Experiments have been carried out in which carbon dioxide treatment was com-

bined in turn with each of these substances. The results of those with ethrel will not be presented as no unexpected features were observed. The promotive effect of carbon dioxide on mesocotyl elongation was largely eliminated in the presence of ethrel, but the

120 (a) 80 (b)

9, -0 /

60 - 40 -~~ -

90 2 60 ,

O / 1, I I I I O_1

3 4 5 6 7 3 4 5 6 7 Time (days)

Fig. 3. The effects of I00o ppm of B-OH +C02 on the growth of the mesocotyls (a), and coleoptiles (b) of oat seedlings. Triangles, B-OH; circles, controls; open symbols, with carbon dioxide.



Mesocotyl growth inhibition by ethylene 647 mesocotyls were just perceptibly longer than those of plants grown in ethrel alone; consequently, the activity of carbon dioxide had not been wholly suppressed by ethylene.

With B-OH, however, the concentration of 5000 ppm hitherto used proved to be so inhibitory in combination with carbon dioxide that the majority of the plants succumbed. The amount of B-OH was progressively reduced to IOO ppm at which low level mesocotyl length was slightly increased beyond that of the controls (Fig. 3a; compare a and v). In combination with carbon dioxide they grew very much longer just surpassing those of the plants grown in CO2 alone (Fig. 3a; compare o and v). The coleoptiles became successively shorter in the sequence of treatments B-OH, C02, and B-OH + CO2 (Fig. 3b). Two measurements for the length of the coleoptile on the fourth day (Fig. 3b; o and v) were unaccountably longer than required by the flow of the growth curve.

(a) A (b) 70 - - -

40 -

~~~~~~~~~. ..... -4

E 10-- -

E~~~

-C

-~100 (c) ,A (d)

50 _ /S,;+2/ _ o"40

3 4 5 6 7 3 4 5 6 7

Time (days)

Fig. 4. The effects of nitrates, and ethylene on the growth of the mesocotyls (a, b), and coleoptiles (c, d) in combination with + light on the second day after the start of the experiment. Squares, nitrates and light; triangles, nitrates; diamonds, light; circles, controls; open symbols, with ethylene.



648 C. L. MER

In experiments with carbon dioxide and B-OH (ioOO ppm), some interesting plants were obtained (Plate i). Almost half the seedlings were killed by the dual treatment but those which survived had very distorted plumules in the early stages. Also, there was a circular patch of blackened, dead, cells at the coleoptile tip from which a zone of senescing cells extended down a lateral flank. The leaves tended to force their way through this weak point to give a bifid aspect to the seedling. When 7 days old the plants had mesocotyls with a mean length of 96 mm, only I3 mm less than those of plants grown in carbon dioxide alone (Fig. 3a). The coleoptiles, however, reached a final length of I7.5 mm, the least in all the experiments carried out with these substances.

The effect of gaseous ethylene In this experiment, for which ordinary tap-water was used as the basic medium,

nitrate supply (N), light (L), and ethylene (E) were combined in a 23 factorial arrange- ment. Control conditions obtained for 2 days when, having illuminated half the seedlings, they were all transferred in darkness to compartmented dishes containing, separately, the nitrate solution and fresh tap-water to complete the statistical design.

The data (Fig. 4: a and b, mesocotyls; c and d, coleoptiles) have been plotted with the measurements of the ethylene-treated plants in open symbols to display its effect directly. Mesocotyl growth was inhibited; a stimulatory effect after illumination was entirely absent. However, the positive light/ethylene interaction (Table 2; L x E) arose because the mesocotyls of the illuminated plants grew to a uniform final length irrespective of treatment with ethylene (Fig. 4, a, b; compare v and v). In Table 2, further, a positive ethylene/nitrate interaction (E x N) is also recorded; the seedlings supplied with the extra nutrients were less susceptible to ethylene (compare the separation between the o and A curves, with that between the A and *). The provision of nutrients gave a slight boost to mesocotyl growth after illumination, but this was not maintained suffi- ciently to influence the overall result (Fig. 4a, b; compare n and m), nor to constitute an effect that could be called a promotion with any conviction.

Although coleoptile growth was sharply reduced by gaseous ethylene (Fig. 4c, d) it was not thereby prevented from making the usual positive response to illumination, and likewise to nitrate supply, but both reactions were on a small scale. A light/ethylene interaction again reached significant levels, but it changed with time from negative to positive (Table 2).

These results indicated the possibility that seedlings might be made less sensitive to

Table 2. The effects of ethylene (E), light (L), and nitrate supply (N), and their mutual interactions, on the growth of oat seedling plumules

Mesocotyls Coleoptiles Sampling day

3 4 5 6 7 3 4 5 6 7 Variant

E I I55 2I 9 49 3IO 1573 ii8o I82I 762 N I 38 7 I9 7 6 8 IIO 2I2 I63 ExN I I 23 6 I5 4 I 26 7 I L 546 846 585 IOI4 752 I98 2I3 76 I I LxN I 7 I I2 I3 I I I I I L x E 6i 82 83 3I 53 9 29 207 8 I7 LxNxE I 5 I 6 I I I I I I

F-ratios are quoted: positive effects are in ordinary type while negative ones are in italics. With i and I 50 degrees of freedom an F-ratio larger than 3.9I indicates significance at P, 0.05; a value larger than 6.8i is needed for significance at P, o.oi. Ratios less than 3.9I, however slightly, are marked with i.



THE NEW PHYTOLOGIST, 73, 4 PLATE I

Four-day-old seedlings grown in B-OH (iooo ppm) and air containing carbon dioxide at a concentration Of 5%. The distorted growth of the plumule is evident, also the black tip of the coleoptile. A zone of senescing cells extending down the flank of the coleoptile is also visible.

C. L. MER-MESOCOTYL GROWTH INHIBITION BY ETHYLENVE (faci-ng page 648)



Mesocotyl growth inhibition by ethylene 649 ethylene, immune even, if they were treated earlier with nutrients. A further experiment was therefore conducted in which the appropriate seedlings were given a larger dose of nitrates (o. Is5%) from its beginning, with the onset of ethylene flow again following the exposure to light on the second day. The results, and the significant interactions, were virtually identical with those already presented and so they need not be detailed any further. Sensitivity to ethylene remained.

The interaction between gaseous ethylene and B-OH In this experiment the stimulatory effect of B-OH was set against the inhibitory one

of ethylene, at the respective concentrations of iooo ppm and 150 ppm. The data for mesocotyl growth, which have been extracted from a larger factorial experiment, show that ethylene had the more powerful effect.

80 _

- --- -

E~~~~~~

20 El I I I I I 3 4 5 6 7

Time (days)

Fig. 5. The effect of ethylene (about 150 ppm) on the stimulation of mesocotyl growth by B-OH (ioOO ppm). Squares, B-OH; circles, control; open symbols, with ethylene.

DISCUSSION

All efforts to confirm Suge's observation that ethylene will promote the growth of oat seedling mesocotyls have been unsuccessful; the gas acted as an inhibitor. These opposite conclusions arise from the different final lengths of the mesocotyls of the control seedlings; they were 2.3 mm in Suge's experiments and 60-70 mm in the present ones. Although Suge did not mention illumination as a factor in his experimentation the final mesocotyl length of 2.3 mm, effectively no growth at all, indicates that a substantial over-exposure to light must have been administered at an early stage of development, almost certainly before the second day. Evidently, this occurred when the plants were taken from the darkroom and put into the special glass vessels in which they were to be treated with ethylene.

However, with a supposedly uniform exposure to light the different lengths of the mesocotyls of the control and treated seedlings cannot be explained; some additional



650 C. L. MER

factor must be invoked. A possible suggestion can be obtained from Goodwin's (1941) observation that oat seedlings are more sensitive to light when 2 days old, than when they are i day old. If the plants used by Suge were of different ages when exposed, differential growth would take place subsequently in darkness.

In addition to this possible extra variable in Suge's work a lingering doubt remains about the efficacy of the treatment with ethylene. The various concentrations were made up by injecting ethylene, with a micro-pipette, into a soo-ml glass vessel fitted with a serum cap. The strongest concentration (i0oo ppm) would then require only o.s ml of ethylene. The vessel was thereafter placed in a darkroom and left undisturbed until the plants were measured. But, the mere introduction of the active gas into the still, closed, atmosphere would not give the selected concentration uniformly without efficient mixing; on this important detail nothing was said.

Although attention has been focused on Suge's data with oat seedlings, the points at issue being readily apparent, stimulatory effects of ethylene have been reported using rice (Ku et al., I970; Suge, Katsura and Inada, I971). All these data require caution as regards interpretation because of the inadvertent exposure to light during transfer of the seedlings to the glass vessel before the start of treatment with ethylene. Further, Ku et al. made some measurements under 'dim green light' without remarking on its possible activity, morphogenetically speaking. Etiolated oat seedlings are very sensitive to green light (Mer, i966), and if rice plants happen to be equally so the absence of all mention of the mesocotyl in these papers might be explained. If the mesocotyl did not grow at all, then the plants must have been exposed excessively to light. The reported effect of ethylene would then be an interaction with illumination. If it grew only slightly it was wholly disregarded, because measurements of coleoptiles were alone published. Whatever the circumstances the growth of the coleoptile cannot be properly understood if that of the mesocotyl is ignored.

Ethrel and B-OH have been hitherto regarded as equivalents for ethylene generation, but the data presented here show that with etiolated oat seedlings they brought about quite different responses. Ethrel always depressed growth, of the coleoptile more so than of the mesocotyl. In contrast, B-OH evoked what is usually referred to as 'the CO2 effect' (Mer and Richards, I950; Causton and Mer, i966); that is, after an initial reduction of the mesocotyl it elongated rapidly to give an ultimate growth promotion and, at the same time, the growth of the coleoptile was very restricted. As with carbon dioxide (Mer and Causton, i963), B-OH increased cell number without varying cell size (Table i). Ethrel, moreover, distorted the plumular members, B-OH did not: the mesocotyls of ethrel-treated plants showed no sign, apart from shortness, of having been exposed to light whereas the straightening response was shown very obviously by those which had been grown in a B-OH solution. Seemingly, B-OH was not metabolized by these plants to ethylene. This conclusion is supported by their susceptibility to gaseous ethylene (Fig. 5) and also to something inhibitory, presumably ethylene, released from ethrel solutions (see page 644).

The data further show that the response of etiolated oat seedlings to ethylene can be modified by their nutritional status. The effect of an increase in the nitrogen supply (as nitrate) has alone been investigated in this connexion. The nutrients acted to reduce the severity of the inhibition caused by ethylene; considerably with mesocotyl growth (Fig. 4a, b), somewhat less so with the coleoptiles (Fig. 4c, d). Any discussion of the role of ethylene as a natural growth regulator either by itself, or in combination/antagonism with other growth-controlling substances ought, therefore, take account of the nutritional



Mesocotyl growth inhibition by ethylene 65I status of the experimental material. Similar considerations apply to light, which also tended to diminish the efficacy of ethylene as an inhibitor: experimental results obtained while using variable, uncontrolled, lighting conditions may be difficult, if not impossible, to interpret.

The selective toxicity of one treatment, B-OH with C02, for the tip cells of the coleoptile (see page 646 and Plate i) provides a discussion point in a different direction; namely, the idea that these particular cells control the elongation of the mesocotyl. This hypothesis (Went, I928; Huisinga, I964) was thought to have been shown untenable (Mer, 195I), but a re-examination of the situation vis-a-vis regeneration prompted the view that it might be impossible to find out if the mesocotyl can indeed grow under the unattainable condition of the complete absence of coleoptilar auxin (Mer, I972). The seedlings showing destruction of the tip cells were in a fourth-day sample; it would not be unreasonable to suppose that these cells were metabolically dislocated from the onset of germination. No experiments have so far been undertaken to determine whether, or no, regeneration did occur. All that can be said positively to suggest a lack of auxin is that the coleoptiles grew very feebly. Hence it may be alleged that the cell-division and -extension processes in the mesocotyl which later resulted in its growth to 96 mm were taking place when the tip cells of the coleoptile were not functioning normally. The argu- ment supports the view that the coleoptile tip does not control the elongation of the mesocotyl.

ACKNOWLEDGMENTS

Thanks are due to Mrs Jean Eller for her excellent technical assistance, and to Dr J. W. Millbank for the estimations of ethylene content in the gas mixtures.

REFERENCES CAUSTON, R. D. & MER, C. L. (I966). Analytical studies of the growth of the etiolated seedling of Avena

sativa. I. Meristematic activity in the mesocotyl with special reference to the effect of carbon dioxide. New Phytol., 65, 87.

GOODWIN, R. H. (I94I) On the inhibition of the first internode of Avena by light. Am. J3. Bot., 28, 325. HuISINGA, B. (I964). Influence of light on growth, geotropism and guttation of Avena seedlings in total

darkness. Acta bot. neerl., 13, 445. Ku, H-S., SUGE, H., RAPPAPORT, L. & PRATT, H. K. (I970). Stimulation of rice coleoptile growth by ethyl-

ene. Planta, 90, 333. MER, C. L. (I95i). A critical study of the auxin theory of growth regulation in the mesocotyl of Avena

sativa. Ann. Bot., N.S. 15, I79. MER, C. L. (I953) An examination of the factors affecting variability in the growth of the mesocotyl and

coleoptile of etiolated Avena seedlings. Ann. Bot., N.S. 17, 569. MER, C. L. (I957). Further observations on the effect of carbon dioxide on the growth of etiolated oat

seedlings. Ann. Bot., N.S. 21, I3. MER, C. L. (I959). A study of the growth and photoperceptivity of etiolated oat seedlings. J. exp. Bot., Io,

220. MER, C. L. (I966). The inhibition of cell division in the mesocotyl of etiolated oat plants by light of

different frequencies. Ann. Bot., N.S. 30, I7. MER, C. L. (I969). Plant growth in relation to endogenous auxin, with special reference to cereal seedlings.

New Phytol., 68, 275. MER, C. L. (I972). Aspects of growth control in cereal seedlings. In: Hormonal Regulation in Plant Growth

and Development (Ed. by H. Kaldewey & Y. Vardar), p. I07. Chemie Verlag. MER, C. L. (I973). On the mechanism of the 'photo-geo' response in the mesocotyl of oat seedlings. Planta,

III, 179.

MER, C. L. & CAUSTON, D. R. (I963). Carbon dioxide: a factor influencing cell division. Natuire, Lond., 199, 360.

MER, C. L. & RICHARDS, F. J. (I950). Carbon dioxide and the extension growth of etiolated oat seedlings. Nature, Lond., 165, I79.

PRATT, H. K. & GOESCHL, J. D. (I969). Physiological roles of ethylene in plants. A. Rev. Pl. Physiol., 20, 54I. SUGE, H. (I97I). Stimulation of oat and rice mesocotyl growth by ethylene. Pl. Cell Physiol., 12, 83I. SUGE, H., KATSURA, N. & INADA, K. (I97I) Ethylene-light relationships in the growth of the rice coleoptile.

Planta, IOI, 365. WENT, F. W. (I928). Wuchsstoff und Wachstum. Recl Trav. bot. Neerl., 25, I.



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On the Inhibition of Mesocotyl Growth by Ethylene