Planta (1983) 157:71-73 Planta �9 Springer-Verlag 1983

Ethylene-induced growth and proton excretion in the aquatic plant Nymphoides peltata Michael Malone and Irene Ridge Biology Department, The Open University, Milton Keynes, UK

Abstract. In the presence of auxin, ethylene can p romo te growth in petioles o f N. peltata (S.G. Gruel.) O. Kuntze . Acid buffer will also stimulate growth in the tissue and, in abraded petiole seg- ments, e thylene-st imulated growth is accompanied by a marked acidification o f the medium. Auxin stimulates growth in this tissue and, for various auxin and ethylene t reatments , the a m o u n t o f growth is closely correla ted with the degree o f me- dium acidification. The results are consistent with predict ions o f the ' ac id -g rowth ' theory, and provide the first evidence that ethylene acts by an ' ac id-growth ' mechanism.

Key words: Acid growth theory - Auxin and acid growth - Ethylene and acid growth - Nymphoides.

Introduction

Ethylene can p romo te rapid growth in a variety o f semi-aquatic plant tissues; this is thought to account , in part , for the ability o f these plants to accommoda te to change in water depth (Walters and Osborne 1979). The precise mechanism of this e thylene-induced growth is not known, but renewed cell division is not an impor t an t factor in mature tissues (Cookson and Osborne 1978). In one case, an ethylene-induced increase in wall extensibility has been demons t ra ted (Cookson and Osborne 1979).

We have studied the possible mechanism of eth- y lene-promoted growth in petioles o f the fringed water lily, Nymphoides peltata (S.G. Gmel.) O. Kuntze. There is evidence f rom many land-plant tissues that g rowth p romo t ion by a diverse assem- blage o f factors (Tepfer and C M a n d 1979), includ- ing auxins (Rayle and Cleland 1977), is mediated at least par t ly by p r o t o n flux into the cell wall.

Al though not all workers are in agreement (e.g. Stuart and Jones 1978), there is a firm possibility that wall acidification is a p r imary step in mos t or all cases o f p romo t ion o f rapid growth in plants. Consequently, we have investigated the possible involvement o f changes o f cell-wall p H in ethylene- p romo ted growth. In this report , we present evi- dence for such an involvement.

Material and methods

Nymphoidespeltata was collected from a pond in the field (near Clitheroe, Lancashire, UK) and cultured as a clone in a heated greenhouse. Plants were grown in glass tanks containing approx. 30 cm of water above approx. 5 cm of Levington's compost (Fisons Loughborough UK). For experiments, seg- ments were cut from the apical 1 or 2 cm of the petiole of mature floating leaves. In all cases, segments were 'aged' for at least 2 h prior to treatment, by floating them on distilled water; if the material is not aged in this way, it may show an erratic response to ethylene alone, probably because of the presence of residual endogenous auxin transported down from the leaf lamina prior to excision, as is thought to be the case in another semi-aquatic plant, Regnellidium diphyllum (Walters and Osborne 1979). Normally, ethylene cannot promote growth unless auxin is present (see Fig. 1). Experiments (data not shown) indicate that a 2-h aging period is sufficient t 9 overcome this problem. Segments will then remain insensitive to ethylene (unless auxin is also supplied), until approx. 20 h after their excision, at which time they may again show sensitivity to ethyl- ene alone, possibly because endogenous auxin production is resumed with development of a 'physiological tip ', as is thought to occur in excised segments of some other species (e.g. Arena coleoptile, Vesper and Evans/978).

All experiments were carried out at 22-25 ~ C, under fluo- rescent lighting.

For longer-term growth measurements (i.e. 4 h or more), segment length was estimated using a fine rule. For short-term, high-resolution measurement, length was continuously moni- tored from two, independently treated columns, each consisting of four, l-cm segments, by means of displacement transducers with matching conditioner units (Sangamo, North Bersted, Bognor Regis, UK). Segments were held in specially con- structed chambers and fed with liquid medium and air alternate- ly (normally approx. 1 rain. of each). Each apparatus was

72 M. Malone and I. Ridge: Ethylene, protons and growth in Nymphoides

enclosed in a 35-1 glass tank. For ethylene treatment, gas was injected into the tank through a rubber septum to produce the stated level.

Levels of ethylene were checked by gas-solid chromatogra- phy using a Pye GCD gas chromatograph (Pye-Unicam, Cam- bridge, UK) with an alumina column as described by Ward et al. (1978).

Medium acidification was monitored using a 'micro-'pH electrode, with reference electrode (Microelectrodes Inc., Lon- donderry, N.H., USA), to sample from 10-ml beakers contain- ing 26, l-cm, abraded segments; together with 4ml of 1 mM K+-citrate/phosphate buffer, pH 6.3, plus 0.1 mM CaC1 z. Oxygenation was continued throughout, and rapid stir- ring of the solutions was done during pH measurement. Ethy- lene gas was slowly but continuously, bubbled through the solu- tions where appropriate.

Segments were abraded by gently rubbing them with 120-mesh carborundum powder (The Carborundum Co., Man- chester, UK). Excess powder was rinsed off with distilled water.

Auxin (indole-3-acetic acid) was purchased from the Sigma Chemical Co., Poole, Dorset, UK, and applied as the K + salt. Ethylene (99.8% purity, BDH Ltd., Poole, Dorset, UK) where used, was always applied at saturating levels (i.e. 100 ixl 1-1 or more), to ensure a full response.

Results and discussion

Figure 1 shows that in aged segments o f the Nym- phoides petiole: a )e thy lene alone will not induce growth; b) ethylene promotes growth in the pres- ence o f auxin; c) auxin alone induces a lesser pro- mot ion than auxin plus ethylene. In all these re- spects, Nymphoides appears to be similar to several o ther water-plant species (Imaseki and Pjon 1970; Samarakoon e ta l . 1980; Walters and Osborne 1979). Other results (not shown) demons t ra te tha t increasing the IAA concent ra t ion beyond approx. 10 -6 M in the absence o f ethylene causes no fur- ther increase in growth rate; however at all levels o f IAA tested (10- 8 _ 10 - 3 M), applicat ion o f eth- ylene approximate ly doubles the max imum rate of growth achieved. Thus IAA alone cannot saturate the growth response in this mater ial ; and the pro- mot ion o f long-term growth by ethylene reflects a real s t imulation of growth rate, and not, for example, a p ro longa t ion o f the dura t ion o f auxin- induced growth.

A possible involvement o f p ro tons in growth p romot ion in Nymphoides petiole segments is indi- cated by the data in Fig. 2, which show that an acid pH stimulates growth in this tissue, part icular- ly if the outer cuticle is disrupted by abrading.

Tha t ethylene induces medium acidification f rom abraded Nymphoides petiole segments when it p romotes their growth (i.e. in the presence of auxin), is shown in Fig. 3. This acidification was not due to increased CO2 product ion , since it could not be reversed by bubbling ni trogen th rough the medium (data not shown). N o r was it due to leak-

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Ethylene IAA// ' -t//

i t

Time 1 h

Fig. 1. Effect of ethylene on auxin(IAA)-induced elongation in columns of four, 1-cm, aged Nymphoides segments. Length was monitored using a transducer. Both hormones were supplied at saturating levels (i.e. 10-5M IAA, 100 gl 1 -x ethylene). Ethylene was added at the first arrow (upper curve only), and IAA at the second arrow (both curves)

A

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10 LU

0 -,,y I

pH of treatment medium

Fig. 2. Effect of medium pH on growth of abraded (o), and non-abraded (o) Nyrnphoides segments. One-cm segments were incubated in 10 mM K+-citrate/phosphate buffer for 4 h. Each point is a mean value for eight segments, _+SE

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6.6

KEY: Eth.

M i_l+ 6.3

6,0 \

5.7 ~ ~ I*

5.4

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Elongation (% of initial length)

Fig. 3. Medium acidification plotted against growth; abraded Nymphoides petiole segments, were treated with various levels of auxin (IAA), with and without ethylene, over 4 h. The curve shown is a calculated best fit. Elongations are means for 12 seg- ments, + SE

M. Malone and I. Ridge : Ethylene, protons and growth in Nymphoides 73

age of acidic substances from the d a m a g e d , abraded surface of the segments, since controls showed no such acidification, although they too were abraded (Fig. 3). The medium acidification probably reflects wall acidification caused by some ion transfer at the plasmalemma, as is thought to occur in many other systems (Rayle and Cleland 1977).

A clear relationship is evident between the amount of growth and the degree of medium acidi- fication, for various combinations of auxin and ethylene (Fig. 3). It is particularly noteworthy that ethylene induces mediun acidification when it pro- motes growth (i.e. in the presence of auxin), but not when there is no growth promotion (i.e. in the absence of auxin).

From these data, it can be seen t ha t the two major criteria indicating an acid-growth mecha- nism (CMand 1980), are fulfilled when ethylene (and auxin) promotes growth in Nymphoides, namely: 1) acidic solutions promote growth; 2) the growth promoters also stimulate medium-, and therefore presumably wall-, acidification. These findings provide further support for the acid- growth theory, and argue for its wide applicability. The results also indicate that despite additivity be- tween their effects, auxin's and ethylene's promo- tions of growth involve the same mechanism of wall loosening; one would thus predict that the steady-state wall pH reached during auxin-induced growth would not be as low as that induced when auxin plus ethylene are promoting growth.

In view of differences between their effects on growth (e.g. the dependence of ethylene on the presence of auxin, and the greater promotion pos- sible when ethylene is present), there must be dif- ferences in the pathways leading to auxin-induced, as opposed to ethylene-induced, H+-excretion. What these might be is not known.

Further data on auxin-, H +-, and ethylene-in- duced growth in water plants, together with a wider discussion of the implications will be pre- sented in a future report.

The award of an S.R.C. studentship to M.M. is gratefully ac- knowledged.

References

CMand, R.E. (1980) Auxin and H § excretion; the state of our knowledge. In: Plant growth substances 1979, pp. 71- 78, Skoog, F., ed. Springer, Berlin Heidelberg New York

Cookson, C., Osborne, D.J. (1978) The stimulation of cell ex- tension by ethylene and auxin in aquatic plants. Planta 144, 39-47

Cookson, C., Osborne, D.J. (1979) The effect of ethylene and attxin on cell wall extensibility of the semi-aquatic fern, Reg- nellidium diphyllum. Planta 146, 303-307

Imaseki, H., Pjon, C.J. (1970) The effect of ethylene on auxin induced growth of excised rice coleoptile segments. Plant Cell Physiol. 11, 827-829

Rayle, D.L., Cleland, R. (1977) Control of plant cell enlarge- ment by hydrogen ions. Curr. Top. Dev. Biol. 11, 187-214

Samarakoon, A., Boesel, I., Horton, R. (1980) The control of petiole elongation in Ranunculus sceleratus (abstr.). Plant Physiol. 65, Suppl., 42

Stuart, D.A., Jones, R.L. (1978) The role of acidification in gibberetlic acid-, and fusicoccin-induced elongation growth of lettuce hypocotyl sections. Planta 142, 135-145

Tepfer, M., Cleland, R.E. (1979) A comparison of acid-induced cell wall loosening in Valonia ventricosa and in oat coleop- tiles. Plant Physiol. 63, 898-902

Vesper, M.J., Evans, M.L. (1978) Time-dependent changes in the auxin sensitivity of coleoptile segments. Plant Physiol. 61, 204-208

Walters, J., Osborne, D.J. (1979) Ethylene and auxin-induced cell growth in relation to auxin transport and metabolism and ethylene production in the semi-aquatic plant, Regnelli- dium diplyllum. Planta 146, 309-317

Ward, T.M., Wright, M., Roberts, J.A., Self, R., Osborne, D.J. (1978) Analytical procedures for the assay and identification of ethylene. In: Isolation of plant growth substances, pp. 135-151, Hillman, J.R., ed. Cambridge University Press, Cambridge

Received 20 July; accepted 2 November 1982