144,
Planta 9 by Springer-Verlag 1978
The Stimulation of Cell Extension by Ethylene and Auxin in Aquatic Plants Claire Cookson 1 and Daphne J. Osborne Agricultural Research Council, W.R.O. Developmental Botany, 181A Huntingdon Road, Cambridge C B 3 0 D Y , U.K.
Elongation of the shoots of three aquatic plants (Hydrocharis morsus-ranae, Regnellidium diphyllum and Ranunculus sceleratus) is stimulated by treatment with ethylene or IAA. The effects of the two hormones are additive, and experiments with an ethylene biosynthesis inhibitor and silver ions indicate that the mechanisms by which ethylene and IAA stimulate growth may be different. Hydrocharis and Ranunculus leaf discs synthesize [14C]ethylene from [lgC]methionine, but no [l~C]ethylene is formed by Regnellidium, suggesting the existence of an alternative pathway of ethylene biosynthesis in the fern. Key words: Aquatic plants - Auxin - Cell elonga-
tion - Ethylene - Ethylene biosynthesis - Silverions.
Introduction
Many water plants with aerial leaves or flowers exhibit the phenomenon of accommodation to water depth (Sculthorpe, 1967). When these plants are submerged, elongation is greatly stimulated until the laminae of flowers regain the water surface. Depending on the morphology of the plant, the organ which elongates in this manner may be the flower stalk or petiole, e.g. Nymphaea alba (Gessner, 1959), the stem, e.g. Trapa natans (Frank, 1872) or in ferns, the rachis, e.g. Marsilea quadrifolia (Karsten, 1888). Ku et al. (1970) first suggested that the factor controlling elongation might be ethylene. They showed that rice coleoptiles elongate when treated with eth1 Present address: Tate and Lyle Limited, Group Research and Development, Philip Lyle Memorial Research Laboratory, P.O. Box 68, Reading RG6 2BX, U.K. Abbreviations." IAA=Indole-3-acetic acid; RBA = l~2-amino-4-(2'aminoethoxy)-trans-3-butenoic acid.
ylene and proposed that the reported growth of submerged seedlings (Yamada, 1954; Kefford, 1962) might result from the accumulation of ethylene in the tissue. Musgrave et al. (1972), using Callitriche platycarpa, demonstrated that this species also elongates in response to applied ethylene and that enough endogenous ethylene accumulates in the air spaces of submerged plants to account for the enhanced elongation rate. Subsequently ethylene has been shown to stimulate elongation in other species of water plants, including Ranunculus sceleratus and Regnellidium diphyllum (Musgrave and Walters, 1973/1974), Sagittaria pygmaea and Potamogeton distinctus (Suge and Kusanagi, 1975). There have been a few reports of auxin-stimulated elongation in water plants (Yamada, 1954; Jackson, 1971; Funke, 1939), although McComb (1965) found that indol-3yl-acetic acid (IAA) slightly inhibited elongation of Callitriche stagnalis internodes. This present investigation has sought to elucidate further the hormonal regulation of cell growth in water plants. A study has been made of the involvement of auxin and ethylene, together with the possible substrate for ethylene biosynthesis in Ranunculus sceleratus, Regnellidium diphyllum and Hydrocharis morsHs-raHae.
Materials and Methods Plant Material and Treatment Hydrocharis morsus-ranae L. is a floating rosette plant, with a shortened stem producing petiolate leaves and an unbranched root system (Fig. la). Plants were grown from turions supplied by Perry's Hardy Plant Farm, Enfield, Middlesex. Plants were grown during the summer, in porcelain tanks filled with tap water, ig glasshouses with supplementary light. For experiments either whole plants or detached leaves were used. For whole plants, a 1 cm
0032-0935/78/0144/0039/$01.80
40
C. Cookson and D.J. Osborne: Stimulation of Cell Extension by Ethylene and Auxin
Other Chemicals
HYDROCHARIS MORSLI$ - F~ANAE
REGNELLIDIUM DIPHYLLUM
a
h
RANUNCULUS SCELERATUS
c
Fig. 1 a-c. Diagrammatic representation of a Hydrocharis morsus-
ranae b Regnellidium diphyllum e Ranunculus sceleratus
apical segment was marked on the youngest floating leaf at the start of the experiment. Alternatively the petioles and leaves were detached and the petioles were cut to 1 or 2 cm in length. Plants or detached leaves were floated in plastic boxes containing the treatment solution and were sealed in 61 desiccators fitted with subaseals for ethylene treatment. Regnellidium diphyllum Lindm. is a Brazilian species of the Marsileaceae with a horizontal rhizome bearing erect fronds along its length. Fronds consist of an unbranched rachis with two terminal leaflets (Fig. lb). Plants were obtained from the Botanical Gardens, Cambridge, and were grown in trays of wet m u d in glasshouses with supplementary light, and with heating during winter. For most growth experiments fronds between 5 and 10 cm in height, with a small piece of rhizome attached were selected. They were suspended below the leaflets by loops of plastic-covered wire so that only the leaflets were raised above the surface of the treatment solutions. To monitor growth a 2 cm segment below the leaflets was marked at the start of the experiment and the length measured again at the end of the treatment. For experiments with AgNO3, leaflets with the apical 5 cm of rachis were excised and inserted t h r o u g h perforated perspex holders into flasks filled with sufficient treatment solution to submerge the whole rachis. The increase in total rachis length was recorded at intervals. For ethylene treatments the flasks were enclosed in containers fitted with a subaseal and the appropriate volume of ethylene injected. Ranunculus sceleratus L. is a native semi-aquatic plant. Seeds were collected locally and sown in trays of water-saturated Levingtons compost in glasshouses with supplementary light and with heating during winter. Following germination, a rosette of leaves is produced (Fig. lc). Leaves fi'om these y o u n g plants were detached and their petioles were cut to 2 or 3 cm in length. These were either inserted into beakers of treatment solution t h r o u g h a covering of Nescofilm (Fisons Limited) pierced with holes, or into petri dishes of treatment solutions through perforated perspex holders so that the whole of the petiole was submerged. For ethylene treatments samples were contained in 61 desiccators fitted with a subaseal and the appropriate volume of ethylene injected.
Hormones Ethylene (C2H4) was obtained in cylinders from Cambrian Chemicals, Croydon, Surrey and was diluted with air to produce appropriate concentrations for treatments. Indol-3yl-acetic acid (IAA) was from Koch-Light Laboratories Limited, Colnbrook, Bucks. and from Sigma Chemical Company. I A A was supplied as an aqueous solution of the sodium salt.
L[U-14C]methionine was from the Radiochemical Centre, Amersham, U.K. (280 mCi/mmol) or from Schwarz/Mann, Orangeburg, New York, U.S.A. (260 mCi/mmol). For scintillation counting 7 g butyl-PBD [2-(4'-tertbutylphenyl)-5-(4"-biphenyl)-1,3,4-oxadiazole] from Hopkin and Williams Limited, were dissolved in 11 toluene. NE260 scintillation fluid was from Nuclear Enterprises Limited, Sighthill, Edinburgh 1 I, Scotland. The ethylene biosynthesis inhibitor, 1,2-amino-4-(T-aminoethoxy)-trans-3-butenoic acid (RBA), an analogue of rhizobitoxine (Owens et al., 1971 ; Lieberman et al., 1975) was a gift from Morris Lieberman, U.S.D.A., Beltsville, Maryland, U.S.A. and from Hoffm a n - L a Roche Inc., New Jersey, U.S.A. Silver nitrate (Analar grade) was from British Drug Houses Chemicals Ltd., Poole, England.
Ethylene Measurements Plant material was sealed in containers of known volume, with subaseal stoppers. At intervals, a 1 ml aliquot of gas was withdrawn from the container with a hypodermic syringe and the ethylene determined by gas-solid chromatography on a Pye U n i c a m "Series 104" gas c h r o m a t o g r a p h fitted with a hydrogen flame ionization detector and a glass column (80 cm x 4 ram) packed with 100-150 mesh silica gel M.F.C. (Hopkin and Williams), (Jackson and Osborne, 1970). All containers were aerated and resealed after each measurement.
Production of [14 C] ethylene .from [14 C] Methionine by Leaf Discs The m e t h o d was modified from that of Yang et al. (1967). The experiments were carried out in glass vials (height 6 cm, diameter 2 cm) with a side a r m and fitted with a subaseal from which a small glass bucket in a fuse wire basket was suspended by hooks. A circle of filter paper was placed in the b o t t o m of each flask and 0.15 mt distilled water added to maintain a humid atmosphere. Four 1 pl drops of c. 0.2 m m o l 1- ~ L[U-14C] methionine (55.5 or 50 gCi/ml) were placed on a round glass coverslip and a leaf disc, 7 m m in diameter, was placed, abaxial surface downwards, on each drop. The coverslip was lowered into the vial, and a piece of filter paper moistened with 0.1 ml 1 tool 1- ~ K O H was inserted into the side arm to absorb carbon dioxide. The vials were incubated for 3 h at 26 ~ C in the light. After 2 h 0.2 ml mercuric perchlorate solution (0.25 mol 1-1 in 2.5 mol 1-1 perchloric acid) was injected into the glass bucket to absorb the ethylene. At the end of the incubation, the glass bucket in its wire basket was transferred to another subaseal which was inserted in a glass scintillation vial. The filter p a p e r + K O H was removed to another scintillation vial for drying and counting. A new K O H paper was inserted in the incubation vial, which was resealed for a further accumulation period. The ethylene was released from the mercuric perchlorate by injection of 0.1 ml 6 mol 1 1 HC1 into the glass bucket and the incubation vial was left overnight at 4 ~ C. 0.3 ml mercuric acetate (0.1 m011- ~ in methanol) was then injected into the b o t t o m of the scintillation vial and left for 1 h at 4 ~ C to reabsorb the ethylene. 10 ml NE 260 scintillation fluid were added to each sample and the radioactivity was determined in a Beckman LS-250 scintillation counter. Preliminary tests showed that non-radioactive ethylene was totally absorbed and released by these procedures. Confirmation that the supplied [14C]methionine was taken up and metabolised by the tissue was obtained from determinations of a) the radioactivity taken up by the discs, b) the radioactivity
C. Cookson and D.J. Osborne: Stimulation of Cell Extension by Ethylene and Auxin present in respired C02 and c) the radioactivity present in the TCA-insoluble fraction of leaf extracts. The methods used were as follows:
a. Uptake of[~4C]methionine: At the end of the incubation, leaf discs were rinsed and washed for 5 rain in 10 ml mmol 1- * methionine. They were boiled in 3 ml 80% ethanol (v/v) until all chlorophyll was extracted. The volume of ethanol was measured and a 0.1 ml aliquot was dried on a Whatman GFA filter paper disc. The radioactivity was determined by liquid scintillation counting. This ethanol-soluble value, together with those for radioactive ethylene, COa and TCA-insoluble material gives an estimate of total uptake. Only 1-2% of the total radioactivity supplied to the discs was recovered in these fractions in all three species, indicating that the amount of [14C]methionine supplied was unlikely to be limiting to ethylene biosynthesis.
41
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8o
b
c. TCA-insoluble material." The boiled leaf discs were ground in a conical glass homogenizer with 10% (w/v) TCA containing 1 mmol 1- 1 methionine at 2 ~ C. The precipitate was collected on GFA filter paper discs and washed sequentially with 20 ml 5% (w/v) TCA containing 1 mmoll ~ methionine and three washes of 20 ml ethanol. The precipitate and filter paper discs were air dried and the radioactivity determined by liquid scintillation counting. Control flasks were set up with 4 gl [~C]methionine alone to check for decomposition of methionine into '4CO2 and [*~C]ethylene. Although 0.05 0.1% of the total radioactivity was found in CO2 after a 6 h incubation, there was no decomposition to [14C]ethylene
Measurement of Cell Numbers In order to determine whether the increase in length of petioles was due to elongation or division of cells, cell numbers were determined in the petioles of leaves which had been treated in water, auxin or ethylene for 24 h. The petioles were first measured, then pooled and macerated in chromic acid (commercial chromium trioxide solution in 85% H2SO4 diluted 1:8 with water) overnight. The cells were separated from each other by vigorous shaking and aliquots from each sample were transferred to a haemocytometer slide and cell numbers were counted. Ten 0.02 mm 3 aliquots were counted for Hydrocharis and twenty 0.2 mm 3 aliquots were counted for Ranunculus.
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Fig. 2a and b. Elongation of detached Hydrocharis and Ranunculus petioles treated with a) ethylene or b) IAA for 24 h. a Plants enclosed in 61 desiccators, C z H , ~ concentration checked at 2 h and 22 h. C2H4 removed from one desiccator with mercuric perchlorate (0.25 tool 1- ~ in 2 tool 1 ~ perchloric acid). Hydrocharis: initial petiole length i cm. Means of 10 petioles + SEM. Ranuneulus: initial petiole length 2 cm. Means of 20 petioles _+SEM. b Hydrocharis whole plants floated in 1/2 strength Hoaglands solution. Apical 1 cm marked on the petiole of the youngest leaf. Means of 4 petioles • SEM. Ranunculus: initial petiole length 2 cm. Means of i2 petioles _+SEM
Results I n t h e p e t i o l e s o f Hydrocharis a n d Ranunculus s t i m u l a t i o n o f e l o n g a t i o n b y e t h y l e n e is d e t e c t a b l e a t 0.1 gl 1-1, w i t h m a x i m u m s t i m u l a t i o n f o r t h e s e t w o s p e c i e s a t 10 lal 1- 1 Fig. 2a. S i m i l a r r e s u l t s w e r e o b t a i n e d f o r Regnellidium (cf. M u s g r a v e a n d W a l t e r s , 1974). I n c r e a s e s in t h e r a t e o f cell e l o n g a t i o n i n I A A are evident over the range 10- 6--10- 3 m o l 1( F i g . 2b). D e p e n d i n g o n t h e b a t c h o f p l a n t s t h e o p t i m u m c o n c e n t r a t i o n is b e t w e e n 1 0 - 5 1 0 - 4 t o o l 1- 1 f o r
Hydrocharis w h i c h is s i m i l a r t o t h a t f o r Regnellidium (cf. W a l t e r s a n d O s b o r n e , 1978). I n Ranunculus t h e maximum s t i m u l a t i o n o f g r o w t h is g e n e r a l l y a t 10 3 m o l 1-1 I A A ( F i g . 2 b ) b u t i n s o m e b a t c h e s o f p l a n t s , e l o n g a t i o n is i n h i b i t e d a t t h i s c o n c e n t r a t i o n so 10 - 4 t o o l 1-1 I A A w a s r o u t i n e l y u s e d f o r t h e experiments. The results in Table 1 show the extent of stimulat i o n o f e l o n g a t i o n b y e t h y l e n e (10 gl 1- 1) a n d a u x i n (10 - 4 t o o l 1- ~ o r 1 0 - 5 t o o l 1- 1) i n Hydrocharis a n d Ranunculus p e t i o l e s a n d Regnellidium f r o n d s . W h e n
42
C. Cookson and D.J. Osborne: Stimulation of Cell Extension by Ethylene and Auxin
Table 1. Percentage elongation of petioles or rachis treated with C2H4 or I A A for 24 h Treatment
Hydrocharis ~
Regnellidium a
Ranunculus ~
Control C2H4" I A Ab I A A + CzH4
7_+3 32_+6 46_+4 64_+6
6+_ 2 78-+ 8 36_+ 9 117-+ 14
10_+2 27-+5 29-+5 42-+6
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~ e
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.
= Hyrd och aris
2,5-
J
m e a n s -+ SEM a C2H 4 10 gl 1 1 for all b I A A 10 5 mol 1-1 for Hydrocharis and Regnellidium 10 4 mol 1- 1 for Ranunculus r Detached leaves with 1 cm petioles floated in ~/2 strength Hoagland's solution. 10 leaves/treatment d Whole fronds with marked 2 cm apical segment. 6 fronds/treatment Detached leaves with 3 cm petioles. 8 leaves/treatment The CzH4 effect is significant at the 0.1% level in all three species. The I A A effect is significant at the 0.1% level for Hydroeharis and the 1% level for Ranunuelus and Regnellidium. There is no significant interaction between I A A and ethylene, suggesting an additive effect only
o
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IA
IO.
7
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Table 2. Estimates of cell n u m b e r s in petioles of detached leaves treated with C2H4 or I A A for 24 h Treatment
Control C2H4" IAA b IAA+C2H4 a b c a e
Hydrocharis
Ranunculus
% elongation d
No. of cells petiole c x 10 . 4
% elongation ~
No. of cells/ petiole c x 10 3
2+ 2 39_+ 6 31_+ 5 75_+13
86_+6 90-+4 85_+9 73-+5
7+1 44_+8 31_+2 56_+6
87-+4 89_+4 87_+5 80_+4
o
C2H 4 10 lxl 1 1 for all I A A 10- 5 mol 1 1 for Hydrocharis 10 - 4 mol 1-1 for Ramunculus means of different atiquots_+ SEM m e a n s of 5 petioles (initial length 1 cm) _+SEM m e a n s of 8 petioles (initial length 2 cm) +_SEM
Analysis of variance shows that the differences between cell numbers/petiole are nonsignificant
plants are treated simultaneously with ethylene and IAA, much greater elongation occurs than with either hormone alone and the effects are then generally additive. Cell number counts on Hydrocharis and Ranunculus petioles show that IAA and ethylene-stimulated elongation is the result of cell elongation with no concurrent cell division (Table 2). Indeed, there is no increase in cell numbers compared with controls, even when there is a 50-75% increase in petiole length. Similar results have been reported for RegneIlidiurn (Musgrave and Walters, 1974). The additive effects of ethylene and IAA on elongation suggest the possibility that the modes of action
;
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Fig. 3a and b. Effect of I A A (10 . 4 mol 1-1) on the rate of C 2 H 4 production by detached leaves of a) Hydrocharis and b) Ranunculus. a Each leaf with 2 c m petiole sealed in a 3 dram vial + 4 m l of solution. ( e o = E x . 1, 9 zx = E x . 2). 9 9 and 9 1 4 9 transferred to I A A solution at 2 h; o - - o and ~ - - z x in distilled water. Means + r a n g e of duplicates, b C2H4 p r o d u c t i o n - l e a v e s with 2 cm petioles enclosed in 3 d r a m vials, supported by a strip of filter paper (2 x 6 cm) round the petioles. Three leaves per vial + 2 ml solution. 9 - - 9 I A A supplied at 0 h; o - - o in distilled water. Means of 4 vials _+SEM
of the two hormones are different. However, as IAA is known to stimulate ethylene production in many plant tissues (Morgan and Hall, 1964; Burg and Burg, 1968), the possibility that at least part of the IAAstimulated elongation is due to an auxin-induced increase in ethylene production was explored.
Ethylene Production IAA ( 1 0 - 4 mol 1 - 1 ) does not stimulate the production of ethylene by Hydrocharis leaves (Fig. 3 a) and neither leaflets nor fronds of Regnellidium exhibit enhanced ethylene production with 10- 5 mol-10 4 mol IAA (Waiters and Osborne, 1978). Therefore, in these two species the IAA-induced elongation of petiole or rachis cannot be attributed to increased ethylene production.
C. Cookson and D.J. Osborne : Stimulation of Cell Extension by Ethylene and Auxin
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Fig, 6a and b. Effect of R B A (2.5 x 10- s mol 1- 1) on a) ethylene production of non-submerged Ranunculus plants over 30 h and b) petiole elongation of detached leaves submerged for 24 h. a C2H4 production: as described for Figure 3b. M e a n s of three vials + S E M . b Elongation: initial petiole length 3 cm. Leaves submerged below water in small test tubes containing 15 ml solution. Means of five p e t i o l e s + S E M . Ethylene treatment as in Fig. 5. t-test; contro[/RBA - P < 5 % ; R B A + C 2 H 4 / R B A - P < 5 % ; control/RBA + C2H 4 - N. S.
Q r70
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50
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30
Time h
Fig. 4a and b. Effect of IAA ( 1 0 - 4 m o l l t) and I A A + R B A (2.5 x 10- 5 mol 1 1) on a) total ethylene production over 24 h and b) petiole elongation by detached Ranunculus leaves, a C2H4 production measured as described for Figure 3 b. Means of four vials + S E M . b Elongation. Initial petiole length 3cm. Leaves floated in test t u b e s + 10 ml solution for 24 h. Means of five leaves + SEM. A t-test shows taht R B A has no significant effect on IAA-stimulated elongation
70-
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Time h
,~
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30-
o
5
50 E
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&
Fig. 5a and h. Effect of R B A (2.5 x 10 5 tool I- z) on a) total ethylene production of non-submerged Hydrocharis plants over 48 h and b) petiole elongation of plants submerged in water for 24 h. a CzH4 production: Whole plants sealed in 150 ml conical flasks with 1 0 0 m l 1/2 strength Hoagland's s o l u t i o n + R B A . Means of three p l a n t s + S E M , b Elongation: Young intact plants with the apical l cm marked on the youngest leaf. Means of three petioles • SEM. Plants treated with ethylene by submerging them in water which had been equilibrated with ethylene overnight. The treatment flasks were sealed and ethylene injected into the air space to give a concentration of at least 0.1%. t-test ; c o n t r o l / R B A - - P < 2% ; R B A + C2H4/RBA - P < 0.1% ; control/RBA + C z H ~ - N.S.
o
O
3
6 Time
9
24
o
h
Fig. 7a and b. Effect of R B A (2.5 x 10- 5 mol 1 1) on a) ethylene production by leaflet and rachis of Regnellidium and b) elongation ofrachis with attached leaflets submerged for 24h. a C2H4 production: Paired leaflets (o . . . . o and 9 . . . . o) were enclosed in 10 ml conical flasks containing either 1 ml H 2 0 or RBA. Means of four leaflets+SEM. 5 m m segments ( 9 and o - - e ) cut from apical 2 cm of rachis and randomised between samples. Four segments enclosed in 1 dram vials+0.5 ml H 2 0 or RBA. Means of three vials+ SEM. b Elongation: Y o u n g fronds submerged. Apical 1 cm marked below the leaflets and measured after 24 h. Means of three fronds_+ SEM.
44
C. Cookson and D.J. Osborne: Stimulation of Cell Extension by Ethylene and Auxin
In contrast, the synthesis of ethylene by Ranunculus leaves is stimulated c. 20% by IAA and the rate of ethylene production in 10- 4 mot 1- 1 IAA is shown in Figure 3 b. However, experiments with the ethylene biosynthesis inhibitor RBA show that IAAinduced ethylene production in Ranunculus can be prevented without significantly effecting the extent of IAA-induced elongation (Fig. 4a and b). Therefore, although IAA does stimulate ethylene synthesis in Ranunculus it is presumed that in this species also, IAA does not induce elongation through a stimulation of ethylene production. Experiments with RBA provide further evidence, to support the proposals of Musgrave et al. (1972) and Musgrave and Walters (1973, 1974) that the elongation of submerged aquatic plants to the water surface is due to the accumulation of ethylene in the tissue. When incubated in a solution of RBA (2.5 x 10- 5 mol 1- 1), ethylene production by Hydrocharis and Ranunculus leaves is inhibited by 80% (Fig. 5 a) and 65% (Fig. 6 a) respectively, and the elongation of the petioles of the submerged leaves is reduced by 96 % in Hydrocharis (Fig. 5 b) and by 76 % in Ranunculus (Fig. 6b). This growth inhibition in RBA is not due to toxicity of the chemical, for a normal submergence response can be largely restored by injecting ethylene into the containers in which the plants are submerged (Figs. 5b and 6b). The failure to completely regain the submerged rate of elongation may be due to the difficulty of supplying the plants with sufficient ethylene, for the rate of diffusion from air into solution and then from solution into the plants is probably slow compared with the rate of diffusion into plants treated in air. The results obtained with Regnellidium fronds are very different. Surprisingly, RBA does not reduce the rate of elongation in submerged material (Fig. 7b) nor does treatment of leaflets with RBA alter their production of ethylene (Fig. 7a); the production by rachis tissue being actually stimulated. RBA experiments can not therefore establish that the endogenously produced ethylene stimulates submerged growth in this species. The results obtained however raised the possibility that Regnellidium possesses an RBA-insensitive pathway for the biosynthesis of ethylene which is an alternative to that from methionine.
Ethylene Production from [14 C] Methionine Radioactive labelling experiments showed that both Hydrocharis (Fig. 8a) and Ranunculus (Fig. 8b) synthesize radioactive ethylene from supplied [a4C]methionine. Under similar conditions however, no labelled ethylene was detected in three separate exper-
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Planta 9 by Springer-Verlag 1978
The Stimulation of Cell Extension by Ethylene and Auxin in Aquatic Plants Claire Cookson 1 and Daphne J. Osborne Agricultural Research Council, W.R.O. Developmental Botany, 181A Huntingdon Road, Cambridge C B 3 0 D Y , U.K.
Elongation of the shoots of three aquatic plants (Hydrocharis morsus-ranae, Regnellidium diphyllum and Ranunculus sceleratus) is stimulated by treatment with ethylene or IAA. The effects of the two hormones are additive, and experiments with an ethylene biosynthesis inhibitor and silver ions indicate that the mechanisms by which ethylene and IAA stimulate growth may be different. Hydrocharis and Ranunculus leaf discs synthesize [14C]ethylene from [lgC]methionine, but no [l~C]ethylene is formed by Regnellidium, suggesting the existence of an alternative pathway of ethylene biosynthesis in the fern. Key words: Aquatic plants - Auxin - Cell elonga-
tion - Ethylene - Ethylene biosynthesis - Silverions.
Introduction
Many water plants with aerial leaves or flowers exhibit the phenomenon of accommodation to water depth (Sculthorpe, 1967). When these plants are submerged, elongation is greatly stimulated until the laminae of flowers regain the water surface. Depending on the morphology of the plant, the organ which elongates in this manner may be the flower stalk or petiole, e.g. Nymphaea alba (Gessner, 1959), the stem, e.g. Trapa natans (Frank, 1872) or in ferns, the rachis, e.g. Marsilea quadrifolia (Karsten, 1888). Ku et al. (1970) first suggested that the factor controlling elongation might be ethylene. They showed that rice coleoptiles elongate when treated with eth1 Present address: Tate and Lyle Limited, Group Research and Development, Philip Lyle Memorial Research Laboratory, P.O. Box 68, Reading RG6 2BX, U.K. Abbreviations." IAA=Indole-3-acetic acid; RBA = l~2-amino-4-(2'aminoethoxy)-trans-3-butenoic acid.
ylene and proposed that the reported growth of submerged seedlings (Yamada, 1954; Kefford, 1962) might result from the accumulation of ethylene in the tissue. Musgrave et al. (1972), using Callitriche platycarpa, demonstrated that this species also elongates in response to applied ethylene and that enough endogenous ethylene accumulates in the air spaces of submerged plants to account for the enhanced elongation rate. Subsequently ethylene has been shown to stimulate elongation in other species of water plants, including Ranunculus sceleratus and Regnellidium diphyllum (Musgrave and Walters, 1973/1974), Sagittaria pygmaea and Potamogeton distinctus (Suge and Kusanagi, 1975). There have been a few reports of auxin-stimulated elongation in water plants (Yamada, 1954; Jackson, 1971; Funke, 1939), although McComb (1965) found that indol-3yl-acetic acid (IAA) slightly inhibited elongation of Callitriche stagnalis internodes. This present investigation has sought to elucidate further the hormonal regulation of cell growth in water plants. A study has been made of the involvement of auxin and ethylene, together with the possible substrate for ethylene biosynthesis in Ranunculus sceleratus, Regnellidium diphyllum and Hydrocharis morsHs-raHae.
Materials and Methods Plant Material and Treatment Hydrocharis morsus-ranae L. is a floating rosette plant, with a shortened stem producing petiolate leaves and an unbranched root system (Fig. la). Plants were grown from turions supplied by Perry's Hardy Plant Farm, Enfield, Middlesex. Plants were grown during the summer, in porcelain tanks filled with tap water, ig glasshouses with supplementary light. For experiments either whole plants or detached leaves were used. For whole plants, a 1 cm
0032-0935/78/0144/0039/$01.80
40
C. Cookson and D.J. Osborne: Stimulation of Cell Extension by Ethylene and Auxin
Other Chemicals
HYDROCHARIS MORSLI$ - F~ANAE
REGNELLIDIUM DIPHYLLUM
a
h
RANUNCULUS SCELERATUS
c
Fig. 1 a-c. Diagrammatic representation of a Hydrocharis morsus-
ranae b Regnellidium diphyllum e Ranunculus sceleratus
apical segment was marked on the youngest floating leaf at the start of the experiment. Alternatively the petioles and leaves were detached and the petioles were cut to 1 or 2 cm in length. Plants or detached leaves were floated in plastic boxes containing the treatment solution and were sealed in 61 desiccators fitted with subaseals for ethylene treatment. Regnellidium diphyllum Lindm. is a Brazilian species of the Marsileaceae with a horizontal rhizome bearing erect fronds along its length. Fronds consist of an unbranched rachis with two terminal leaflets (Fig. lb). Plants were obtained from the Botanical Gardens, Cambridge, and were grown in trays of wet m u d in glasshouses with supplementary light, and with heating during winter. For most growth experiments fronds between 5 and 10 cm in height, with a small piece of rhizome attached were selected. They were suspended below the leaflets by loops of plastic-covered wire so that only the leaflets were raised above the surface of the treatment solutions. To monitor growth a 2 cm segment below the leaflets was marked at the start of the experiment and the length measured again at the end of the treatment. For experiments with AgNO3, leaflets with the apical 5 cm of rachis were excised and inserted t h r o u g h perforated perspex holders into flasks filled with sufficient treatment solution to submerge the whole rachis. The increase in total rachis length was recorded at intervals. For ethylene treatments the flasks were enclosed in containers fitted with a subaseal and the appropriate volume of ethylene injected. Ranunculus sceleratus L. is a native semi-aquatic plant. Seeds were collected locally and sown in trays of water-saturated Levingtons compost in glasshouses with supplementary light and with heating during winter. Following germination, a rosette of leaves is produced (Fig. lc). Leaves fi'om these y o u n g plants were detached and their petioles were cut to 2 or 3 cm in length. These were either inserted into beakers of treatment solution t h r o u g h a covering of Nescofilm (Fisons Limited) pierced with holes, or into petri dishes of treatment solutions through perforated perspex holders so that the whole of the petiole was submerged. For ethylene treatments samples were contained in 61 desiccators fitted with a subaseal and the appropriate volume of ethylene injected.
Hormones Ethylene (C2H4) was obtained in cylinders from Cambrian Chemicals, Croydon, Surrey and was diluted with air to produce appropriate concentrations for treatments. Indol-3yl-acetic acid (IAA) was from Koch-Light Laboratories Limited, Colnbrook, Bucks. and from Sigma Chemical Company. I A A was supplied as an aqueous solution of the sodium salt.
L[U-14C]methionine was from the Radiochemical Centre, Amersham, U.K. (280 mCi/mmol) or from Schwarz/Mann, Orangeburg, New York, U.S.A. (260 mCi/mmol). For scintillation counting 7 g butyl-PBD [2-(4'-tertbutylphenyl)-5-(4"-biphenyl)-1,3,4-oxadiazole] from Hopkin and Williams Limited, were dissolved in 11 toluene. NE260 scintillation fluid was from Nuclear Enterprises Limited, Sighthill, Edinburgh 1 I, Scotland. The ethylene biosynthesis inhibitor, 1,2-amino-4-(T-aminoethoxy)-trans-3-butenoic acid (RBA), an analogue of rhizobitoxine (Owens et al., 1971 ; Lieberman et al., 1975) was a gift from Morris Lieberman, U.S.D.A., Beltsville, Maryland, U.S.A. and from Hoffm a n - L a Roche Inc., New Jersey, U.S.A. Silver nitrate (Analar grade) was from British Drug Houses Chemicals Ltd., Poole, England.
Ethylene Measurements Plant material was sealed in containers of known volume, with subaseal stoppers. At intervals, a 1 ml aliquot of gas was withdrawn from the container with a hypodermic syringe and the ethylene determined by gas-solid chromatography on a Pye U n i c a m "Series 104" gas c h r o m a t o g r a p h fitted with a hydrogen flame ionization detector and a glass column (80 cm x 4 ram) packed with 100-150 mesh silica gel M.F.C. (Hopkin and Williams), (Jackson and Osborne, 1970). All containers were aerated and resealed after each measurement.
Production of [14 C] ethylene .from [14 C] Methionine by Leaf Discs The m e t h o d was modified from that of Yang et al. (1967). The experiments were carried out in glass vials (height 6 cm, diameter 2 cm) with a side a r m and fitted with a subaseal from which a small glass bucket in a fuse wire basket was suspended by hooks. A circle of filter paper was placed in the b o t t o m of each flask and 0.15 mt distilled water added to maintain a humid atmosphere. Four 1 pl drops of c. 0.2 m m o l 1- ~ L[U-14C] methionine (55.5 or 50 gCi/ml) were placed on a round glass coverslip and a leaf disc, 7 m m in diameter, was placed, abaxial surface downwards, on each drop. The coverslip was lowered into the vial, and a piece of filter paper moistened with 0.1 ml 1 tool 1- ~ K O H was inserted into the side arm to absorb carbon dioxide. The vials were incubated for 3 h at 26 ~ C in the light. After 2 h 0.2 ml mercuric perchlorate solution (0.25 mol 1-1 in 2.5 mol 1-1 perchloric acid) was injected into the glass bucket to absorb the ethylene. At the end of the incubation, the glass bucket in its wire basket was transferred to another subaseal which was inserted in a glass scintillation vial. The filter p a p e r + K O H was removed to another scintillation vial for drying and counting. A new K O H paper was inserted in the incubation vial, which was resealed for a further accumulation period. The ethylene was released from the mercuric perchlorate by injection of 0.1 ml 6 mol 1 1 HC1 into the glass bucket and the incubation vial was left overnight at 4 ~ C. 0.3 ml mercuric acetate (0.1 m011- ~ in methanol) was then injected into the b o t t o m of the scintillation vial and left for 1 h at 4 ~ C to reabsorb the ethylene. 10 ml NE 260 scintillation fluid were added to each sample and the radioactivity was determined in a Beckman LS-250 scintillation counter. Preliminary tests showed that non-radioactive ethylene was totally absorbed and released by these procedures. Confirmation that the supplied [14C]methionine was taken up and metabolised by the tissue was obtained from determinations of a) the radioactivity taken up by the discs, b) the radioactivity
C. Cookson and D.J. Osborne: Stimulation of Cell Extension by Ethylene and Auxin present in respired C02 and c) the radioactivity present in the TCA-insoluble fraction of leaf extracts. The methods used were as follows:
a. Uptake of[~4C]methionine: At the end of the incubation, leaf discs were rinsed and washed for 5 rain in 10 ml mmol 1- * methionine. They were boiled in 3 ml 80% ethanol (v/v) until all chlorophyll was extracted. The volume of ethanol was measured and a 0.1 ml aliquot was dried on a Whatman GFA filter paper disc. The radioactivity was determined by liquid scintillation counting. This ethanol-soluble value, together with those for radioactive ethylene, COa and TCA-insoluble material gives an estimate of total uptake. Only 1-2% of the total radioactivity supplied to the discs was recovered in these fractions in all three species, indicating that the amount of [14C]methionine supplied was unlikely to be limiting to ethylene biosynthesis.
41
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8o
b
c. TCA-insoluble material." The boiled leaf discs were ground in a conical glass homogenizer with 10% (w/v) TCA containing 1 mmol 1- 1 methionine at 2 ~ C. The precipitate was collected on GFA filter paper discs and washed sequentially with 20 ml 5% (w/v) TCA containing 1 mmoll ~ methionine and three washes of 20 ml ethanol. The precipitate and filter paper discs were air dried and the radioactivity determined by liquid scintillation counting. Control flasks were set up with 4 gl [~C]methionine alone to check for decomposition of methionine into '4CO2 and [*~C]ethylene. Although 0.05 0.1% of the total radioactivity was found in CO2 after a 6 h incubation, there was no decomposition to [14C]ethylene
Measurement of Cell Numbers In order to determine whether the increase in length of petioles was due to elongation or division of cells, cell numbers were determined in the petioles of leaves which had been treated in water, auxin or ethylene for 24 h. The petioles were first measured, then pooled and macerated in chromic acid (commercial chromium trioxide solution in 85% H2SO4 diluted 1:8 with water) overnight. The cells were separated from each other by vigorous shaking and aliquots from each sample were transferred to a haemocytometer slide and cell numbers were counted. Ten 0.02 mm 3 aliquots were counted for Hydrocharis and twenty 0.2 mm 3 aliquots were counted for Ranunculus.
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Fig. 2a and b. Elongation of detached Hydrocharis and Ranunculus petioles treated with a) ethylene or b) IAA for 24 h. a Plants enclosed in 61 desiccators, C z H , ~ concentration checked at 2 h and 22 h. C2H4 removed from one desiccator with mercuric perchlorate (0.25 tool 1- ~ in 2 tool 1 ~ perchloric acid). Hydrocharis: initial petiole length i cm. Means of 10 petioles + SEM. Ranuneulus: initial petiole length 2 cm. Means of 20 petioles _+SEM. b Hydrocharis whole plants floated in 1/2 strength Hoaglands solution. Apical 1 cm marked on the petiole of the youngest leaf. Means of 4 petioles • SEM. Ranunculus: initial petiole length 2 cm. Means of i2 petioles _+SEM
Results I n t h e p e t i o l e s o f Hydrocharis a n d Ranunculus s t i m u l a t i o n o f e l o n g a t i o n b y e t h y l e n e is d e t e c t a b l e a t 0.1 gl 1-1, w i t h m a x i m u m s t i m u l a t i o n f o r t h e s e t w o s p e c i e s a t 10 lal 1- 1 Fig. 2a. S i m i l a r r e s u l t s w e r e o b t a i n e d f o r Regnellidium (cf. M u s g r a v e a n d W a l t e r s , 1974). I n c r e a s e s in t h e r a t e o f cell e l o n g a t i o n i n I A A are evident over the range 10- 6--10- 3 m o l 1( F i g . 2b). D e p e n d i n g o n t h e b a t c h o f p l a n t s t h e o p t i m u m c o n c e n t r a t i o n is b e t w e e n 1 0 - 5 1 0 - 4 t o o l 1- 1 f o r
Hydrocharis w h i c h is s i m i l a r t o t h a t f o r Regnellidium (cf. W a l t e r s a n d O s b o r n e , 1978). I n Ranunculus t h e maximum s t i m u l a t i o n o f g r o w t h is g e n e r a l l y a t 10 3 m o l 1-1 I A A ( F i g . 2 b ) b u t i n s o m e b a t c h e s o f p l a n t s , e l o n g a t i o n is i n h i b i t e d a t t h i s c o n c e n t r a t i o n so 10 - 4 t o o l 1-1 I A A w a s r o u t i n e l y u s e d f o r t h e experiments. The results in Table 1 show the extent of stimulat i o n o f e l o n g a t i o n b y e t h y l e n e (10 gl 1- 1) a n d a u x i n (10 - 4 t o o l 1- ~ o r 1 0 - 5 t o o l 1- 1) i n Hydrocharis a n d Ranunculus p e t i o l e s a n d Regnellidium f r o n d s . W h e n
42
C. Cookson and D.J. Osborne: Stimulation of Cell Extension by Ethylene and Auxin
Table 1. Percentage elongation of petioles or rachis treated with C2H4 or I A A for 24 h Treatment
Hydrocharis ~
Regnellidium a
Ranunculus ~
Control C2H4" I A Ab I A A + CzH4
7_+3 32_+6 46_+4 64_+6
6+_ 2 78-+ 8 36_+ 9 117-+ 14
10_+2 27-+5 29-+5 42-+6
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m e a n s -+ SEM a C2H 4 10 gl 1 1 for all b I A A 10 5 mol 1-1 for Hydrocharis and Regnellidium 10 4 mol 1- 1 for Ranunculus r Detached leaves with 1 cm petioles floated in ~/2 strength Hoagland's solution. 10 leaves/treatment d Whole fronds with marked 2 cm apical segment. 6 fronds/treatment Detached leaves with 3 cm petioles. 8 leaves/treatment The CzH4 effect is significant at the 0.1% level in all three species. The I A A effect is significant at the 0.1% level for Hydroeharis and the 1% level for Ranunuelus and Regnellidium. There is no significant interaction between I A A and ethylene, suggesting an additive effect only
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Table 2. Estimates of cell n u m b e r s in petioles of detached leaves treated with C2H4 or I A A for 24 h Treatment
Control C2H4" IAA b IAA+C2H4 a b c a e
Hydrocharis
Ranunculus
% elongation d
No. of cells petiole c x 10 . 4
% elongation ~
No. of cells/ petiole c x 10 3
2+ 2 39_+ 6 31_+ 5 75_+13
86_+6 90-+4 85_+9 73-+5
7+1 44_+8 31_+2 56_+6
87-+4 89_+4 87_+5 80_+4
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C2H 4 10 lxl 1 1 for all I A A 10- 5 mol 1 1 for Hydrocharis 10 - 4 mol 1-1 for Ramunculus means of different atiquots_+ SEM m e a n s of 5 petioles (initial length 1 cm) _+SEM m e a n s of 8 petioles (initial length 2 cm) +_SEM
Analysis of variance shows that the differences between cell numbers/petiole are nonsignificant
plants are treated simultaneously with ethylene and IAA, much greater elongation occurs than with either hormone alone and the effects are then generally additive. Cell number counts on Hydrocharis and Ranunculus petioles show that IAA and ethylene-stimulated elongation is the result of cell elongation with no concurrent cell division (Table 2). Indeed, there is no increase in cell numbers compared with controls, even when there is a 50-75% increase in petiole length. Similar results have been reported for RegneIlidiurn (Musgrave and Walters, 1974). The additive effects of ethylene and IAA on elongation suggest the possibility that the modes of action
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Fig. 3a and b. Effect of I A A (10 . 4 mol 1-1) on the rate of C 2 H 4 production by detached leaves of a) Hydrocharis and b) Ranunculus. a Each leaf with 2 c m petiole sealed in a 3 dram vial + 4 m l of solution. ( e o = E x . 1, 9 zx = E x . 2). 9 9 and 9 1 4 9 transferred to I A A solution at 2 h; o - - o and ~ - - z x in distilled water. Means + r a n g e of duplicates, b C2H4 p r o d u c t i o n - l e a v e s with 2 cm petioles enclosed in 3 d r a m vials, supported by a strip of filter paper (2 x 6 cm) round the petioles. Three leaves per vial + 2 ml solution. 9 - - 9 I A A supplied at 0 h; o - - o in distilled water. Means of 4 vials _+SEM
of the two hormones are different. However, as IAA is known to stimulate ethylene production in many plant tissues (Morgan and Hall, 1964; Burg and Burg, 1968), the possibility that at least part of the IAAstimulated elongation is due to an auxin-induced increase in ethylene production was explored.
Ethylene Production IAA ( 1 0 - 4 mol 1 - 1 ) does not stimulate the production of ethylene by Hydrocharis leaves (Fig. 3 a) and neither leaflets nor fronds of Regnellidium exhibit enhanced ethylene production with 10- 5 mol-10 4 mol IAA (Waiters and Osborne, 1978). Therefore, in these two species the IAA-induced elongation of petiole or rachis cannot be attributed to increased ethylene production.
C. Cookson and D.J. Osborne : Stimulation of Cell Extension by Ethylene and Auxin
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Fig, 6a and b. Effect of R B A (2.5 x 10- s mol 1- 1) on a) ethylene production of non-submerged Ranunculus plants over 30 h and b) petiole elongation of detached leaves submerged for 24 h. a C2H4 production: as described for Figure 3b. M e a n s of three vials + S E M . b Elongation: initial petiole length 3 cm. Leaves submerged below water in small test tubes containing 15 ml solution. Means of five p e t i o l e s + S E M . Ethylene treatment as in Fig. 5. t-test; contro[/RBA - P < 5 % ; R B A + C 2 H 4 / R B A - P < 5 % ; control/RBA + C2H 4 - N. S.
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Fig. 4a and b. Effect of IAA ( 1 0 - 4 m o l l t) and I A A + R B A (2.5 x 10- 5 mol 1 1) on a) total ethylene production over 24 h and b) petiole elongation by detached Ranunculus leaves, a C2H4 production measured as described for Figure 3 b. Means of four vials + S E M . b Elongation. Initial petiole length 3cm. Leaves floated in test t u b e s + 10 ml solution for 24 h. Means of five leaves + SEM. A t-test shows taht R B A has no significant effect on IAA-stimulated elongation
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Fig. 5a and h. Effect of R B A (2.5 x 10 5 tool I- z) on a) total ethylene production of non-submerged Hydrocharis plants over 48 h and b) petiole elongation of plants submerged in water for 24 h. a CzH4 production: Whole plants sealed in 150 ml conical flasks with 1 0 0 m l 1/2 strength Hoagland's s o l u t i o n + R B A . Means of three p l a n t s + S E M , b Elongation: Young intact plants with the apical l cm marked on the youngest leaf. Means of three petioles • SEM. Plants treated with ethylene by submerging them in water which had been equilibrated with ethylene overnight. The treatment flasks were sealed and ethylene injected into the air space to give a concentration of at least 0.1%. t-test ; c o n t r o l / R B A - - P < 2% ; R B A + C2H4/RBA - P < 0.1% ; control/RBA + C z H ~ - N.S.
o
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3
6 Time
9
24
o
h
Fig. 7a and b. Effect of R B A (2.5 x 10- 5 mol 1 1) on a) ethylene production by leaflet and rachis of Regnellidium and b) elongation ofrachis with attached leaflets submerged for 24h. a C2H4 production: Paired leaflets (o . . . . o and 9 . . . . o) were enclosed in 10 ml conical flasks containing either 1 ml H 2 0 or RBA. Means of four leaflets+SEM. 5 m m segments ( 9 and o - - e ) cut from apical 2 cm of rachis and randomised between samples. Four segments enclosed in 1 dram vials+0.5 ml H 2 0 or RBA. Means of three vials+ SEM. b Elongation: Y o u n g fronds submerged. Apical 1 cm marked below the leaflets and measured after 24 h. Means of three fronds_+ SEM.
44
C. Cookson and D.J. Osborne: Stimulation of Cell Extension by Ethylene and Auxin
In contrast, the synthesis of ethylene by Ranunculus leaves is stimulated c. 20% by IAA and the rate of ethylene production in 10- 4 mot 1- 1 IAA is shown in Figure 3 b. However, experiments with the ethylene biosynthesis inhibitor RBA show that IAAinduced ethylene production in Ranunculus can be prevented without significantly effecting the extent of IAA-induced elongation (Fig. 4a and b). Therefore, although IAA does stimulate ethylene synthesis in Ranunculus it is presumed that in this species also, IAA does not induce elongation through a stimulation of ethylene production. Experiments with RBA provide further evidence, to support the proposals of Musgrave et al. (1972) and Musgrave and Walters (1973, 1974) that the elongation of submerged aquatic plants to the water surface is due to the accumulation of ethylene in the tissue. When incubated in a solution of RBA (2.5 x 10- 5 mol 1- 1), ethylene production by Hydrocharis and Ranunculus leaves is inhibited by 80% (Fig. 5 a) and 65% (Fig. 6 a) respectively, and the elongation of the petioles of the submerged leaves is reduced by 96 % in Hydrocharis (Fig. 5 b) and by 76 % in Ranunculus (Fig. 6b). This growth inhibition in RBA is not due to toxicity of the chemical, for a normal submergence response can be largely restored by injecting ethylene into the containers in which the plants are submerged (Figs. 5b and 6b). The failure to completely regain the submerged rate of elongation may be due to the difficulty of supplying the plants with sufficient ethylene, for the rate of diffusion from air into solution and then from solution into the plants is probably slow compared with the rate of diffusion into plants treated in air. The results obtained with Regnellidium fronds are very different. Surprisingly, RBA does not reduce the rate of elongation in submerged material (Fig. 7b) nor does treatment of leaflets with RBA alter their production of ethylene (Fig. 7a); the production by rachis tissue being actually stimulated. RBA experiments can not therefore establish that the endogenously produced ethylene stimulates submerged growth in this species. The results obtained however raised the possibility that Regnellidium possesses an RBA-insensitive pathway for the biosynthesis of ethylene which is an alternative to that from methionine.
Ethylene Production from [14 C] Methionine Radioactive labelling experiments showed that both Hydrocharis (Fig. 8a) and Ranunculus (Fig. 8b) synthesize radioactive ethylene from supplied [a4C]methionine. Under similar conditions however, no labelled ethylene was detected in three separate exper-
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