Planta 9 by Springer-Verlag I977
Planta 134, 119-125 (1977)
Studies on the Movement and Distribution of Ethylene in Vicia faba L. M. Zeroni 1, P.H. Jerie 2, and M.A. Hall Department of Botany and Microbiology, The University College of Wales, Penglais, Aberystwyth SY23 3DA, U.K.
Abstract. In Vicia f a b a ethylene does not appear to
move between different parts of the plant in physiologically significant amounts. The 'resistance' to longitudinal m o v e m e n t is such that lateral emanation effectively isolates different parts of the plant from each other. When emanation is prevented, ethylene can be channelled to any part of the plant. Exposure of one section of a plant to 14C-labelled ethylene (up to 200 lal/1) increased the internal concentration in other parts with ethylene that did not originate from the feeding chamber. A basipetal gradient of endogenous ethylene concentration was found in the lacuna of intact plants, the source of ethylene being the stem tissue. The permeability of stem tissue to ethylene decreases with age. The concentration of ethylene in tissues surrounding the lacuna is always higher than that in the lacuna and it is argued that ' c o m p a r t m e n t a t i o n ' of ethylene occurs within these tissues. Key words: C o m p a r t m e n t a t i o n - Ethylene Viciafaba.
Move-
ment of ethylene -
tion of ethylene in roots and aerial parts of plants increases when the root system is waterlogged or is treated with ethylene (El Beltagy and Hall, 1974; Smith and Jackson, 1974). These findings are in accord with other work, using several species, indicating that gases may diffuse through the plant as though in a system of continuous air spaces. Such effects have been shown with oxygen (Evans and Ebert, 1960; Greenwood, 1967a and b; Healy and Armstrong, 1972), and hydrogen and nitrogen (Brown, 1947). The possibility that ethylene m a y move from its site(s) of synthesis to other parts of the plant raises a number of questions not only in relation to the role of the gas in plant growth and development but also because such m o v e m e n t might affect estimates of rates of ethylene biosynthesis in a particular location. The aim of the present work, therefore, was to determine the significance of the movement and distribution of ethylene in Vicia faba.
Materials and Methods Introduction
A number of studies have suggested that movement of ethylene may occur between different parts of the plant. Thus, Jackson and Campbell (1975) demonstrated that, in tomato, labelled ethylene moves from the roots to the stem and petioles, apparently providing an explanation for the results of Z i m m e r m a n et al. (1931), who showed that exposure of t o m a t o roots to ethylene results in epinasty of the petioles. Similarly, it has been shown that the internal concentra1
Permanentaddress: Department of Botany, Tel-Aviv Univer-
sity, Tel-Aviv, Israel 2
Permanent address." Horticultural Research Station, Tatura,
Victoria, Australia
Plant Culture
Plants of Vicia faba var. Aquadulce were grown under a 16 h photoperiod either in a heated glasshouse or in a growth chamber at 20+_2~ Depending on which tissues were required the plants were grown in Levington compost or in fine gravel in 10 cm pots or in deeper containers of vermiculite. Ethylene movement within the root system was determined using two week old plants (Table 6). In other experiments the plants were 5-6 weeks old (Tables 1-5, 7) with the exception of data in Table 8 where 4-month-old field grown plants were used. Plant Manipulation
Plant tissues were enclosed in gas-tight chambers made from plastic syringes or plastic tubes fitted with silicone rubber septa. The
120 plant part was supported in the chamber with the rubber head from the syringe plunger which incorporated a suitable hole and was split on one side. A quick-setting silicone rubber (Silicoset 101, ICI Ltd.) was used to seal the chambers and wherever possible water was added to a height of 1 cm inside the chamber. For studies on movement to and from the roots the fine gravel or vermiculite in which the plants were growing was gently removed with water. The chamber used to enclose roots always contained a small a m o u n t of water. The remaining open part of the root system was held in running tap water to prevent desiccation. Plant segments were always cut under water a n d the basal end stood in water throughout the experiment, Intact plants were embedded in 2% agar by drawing them into a plastic tube 2.6 cm in diameter into which agar at 36-37~ was then poured. This procedure did not appear to h a r m the plants as the roots continued to grow in agar. The lacuna was divided into sections by injecting silicone grease. Control plants were fitted with chambers identical to those on the treated plants. In some experiments ' b l a n k ' plants without chambers were included to check whether the chambers affected the results. U p w a r d and downward diffusion of ethylene in plastic tubes (0.4 em diameter) closed at one end and of similar length to the lacunae of the bean plants used was investigated. At zero time a wide-bore glass tap from a feeding chamber containing ethylene was opened. At the end of each experimental period the tap was closed and the tube quickly divided with two clamps into three sections. For ethylene determination the gas in each section was withdrawn under a m m o n i u m sulphate. Labelled ethylene (1~C2H,, A m e r s h a m , 95 m Ci/mmol) was injected into the feeding chambers. In experiments where [lr ylene was diluted with unlabelled ethylene the specific activity was determined in each feeding chamber just prior to dissection. The radiochemical purity of 114C]ethylene as supplied and after movement through and extraction from the plant was determined by radiogaschromatography (Panax Radiogas Detector System) and found to be 99.42% and 99.74% respectively.
Sampling and Analysis Unless otherwise stated experiments were terminated 200 rain after ethylene application. Plants were dissected under a saturated solution of a m m o n i u m sulphate. The gas from lacuna sections was drawn into plastic syringes. The internal concentration of ethylene within tissues was determined using a modification of the method of Beyer and Morgan (1970). The tissue was placed under saturated a m m o n i u m sulphate inside an inverted 50 ml syringe fitted on the needle taper with a piece of closed thick-walled rubber tube. A volume of air (2.00 ml) was injected into each syringe and a vacuum of 30 cm Hg was applied for 3 rain to the syringes in a v a c u u m desiccator. After samples were taken for ethylene determination the volume of gas withdrawn from the tissue by the vacuum treatment was measured (_+0.01 ml). Ethylene was measured using a Pye U n i c a m 104 gas chromatograph with a glass column (210 x 0.4 cm) packed with alumina (85-100 mesh). The oven temperature was 120~ and the nitrogen flow rate 60 ml/min. Labelled ethylene was determined by injecting 1 ml gaseous samples into a 3.8 ml glass vial containing 0.3 ml a m m o n i u m sulphate and 3 ml of toluene scintillant (5 g/1 PPO). The samples were counted at an efficiency of 88-91% in a Beckman L S - 2 0 0 B scintillation spectrometer. The quantity of Iabelled ethylene was generally calculated as a percentage of the ethylene determined by gas chromatography. As a small piece of tissue did not liberate a sufficient volume of gas under v a c u u m extraction to enable the concentration to be calculated accurately, such pieces were placed directly in the vials described above and counted with an efficiency of 82-88%.
M. Zeroni et al. : Movement and Distribution of Ethylene
Description of Plant Parts In the succeeding experiments the ' apical region' is taken to consist of the upper part of the stem from the apex down to the node of the oldest folded leaf. Leaves were counted basipetally starting from the oldest folded leaf. Upper, middle and lower sections of lacunae were of equal length. The 'transition z o n e ' included tissue from just below the position of the cotyledons to a point on the solid stem about 1.5 cm above the cotyledons. The ' u p p e r ' and 'lower' root segments were 2 cm long sections of the main root removed sequentially from the root below the transition zone and above the root tip. The ' r o o t tip' encompassed the terminal 4-6 cm of the main root.
Results
Ethylene Movement Only a small proportion of the ethylene supplied in high concentration to one region of the plant could be detected in other parts of the plant. The largest amount of ethylene moved between the apical region and the upper section of the lacuna and stem (Table 1). In the lower lacuna ethylene movement resulted in a 4.3-fold increase above the control level of ethylene after 200 rain. The gradient between the lower lacuna and the feeding chamber was greater than 600: 1. In the lower region of the stem the ethylene concentration increased as a result of feeding the apical region but only a small proportion (17.6%) of the increase could be accounted for as labelled ethylene. Although in plants fed through the apical region the upper lacuna and stem contained substantial activity, in adjacent leaves the ethylene concentration increased by only 1-5% over the endogenous concentration in control leaves, representing about 0.01%
Table 1. Movement of ethylene (specific activity 2820 cpm/nl) from the apical region of Vicia faba. Each column shows data from a single plant representative of 12 blank, 8 control and 8 treatment plants. Total length of the stem was 42 to 48 cm
Blank
Control
Treatment
C2H4 (nl/ml)
C2H4 (nl/ml)
Total CzH 4 (nl/ml)
14C2H 4 (nl/ml)
14CzH 4 as a % of total ethylene
0.08
191.30
191.30
100
Feeding chamber Lacuna upper middle lower
0.17 0.10 0.08
0.10 0.06 0.07
3.90 0.44 0.30
3.99 0.41 0.21
102 91.8 71.6
Stem upper middle lower
0.64 0.30 0.53
0.32 0.39 0.32
3.71 0.64 0.83
2.50 0.22 0.09
67.3 33.8 10.9
M. Zeroni et al. : Movement and Distribution of Ethylene
121
Table 2. The effects of feeding ethylene (specific activity 1380 cpm/nl) to the apical region or to a lower leaf on the internal concentration of ethylene in other aerial parts of the plant. Figures are the mean of the number of plants indicated in brackets _+standard deviation Recipient organs a
Feeding Organ Apical Region
Leaf (4th or 5th)
Control C~H4 (nl/ml)
Treatment Total C2H4 (nl/ml)
Apical Region Lacuna Stem 2nd leaf(2.5) 3rd leaf (9) 4th leaf 09) 5th leaf(31)
0.14_+0.08(13) 0.64-+0.32(13) 0.46_+0.21(9) 0.55+0.29(13) 0.69+-0.62(11) 0.71 (3)
Treatment
Control C2H 4 (nl/ml) 1 4 C 2 H 4 as a
Total
percentage of total ethylene
C2H4 (nl/ml)
14C),H4 as a percentage of total ethylene
1.3 _+0.8 (4) 0.19_+0.10(7) 0.84_+0.60(7) 0.16 (2) 0.61_+0.56(4) 0.36_+0.12(4) 0.64 (2)
15.8_+11.6 20.7_+24.1 35.0_+18.6 0.0 0.0 0.0 7.8
0.53_+0.23(4) 0.14+0.04(7) 0.65+_0.20(7) 0.26 (2) 0.47_+0.32(4) 0.35_+0.28(4) 0.25+-0.03(4)
1.80_+1.40(10) 95.4 _ + 1 4 . 0 2.0 +_1.8 (10) 45.5 _ + 1 7 . 9 0.93_+0.44(6) 2.4 _+ 4.2 0.91_+1.02(11) 0.25_+ 0 . 6 1 0.40_+0.23(10) 2.7 +_ 4.8 0.39+_0.18(5) 0.50-+ 0 . 8 7
Figures in brackets denote average distance from apical region (cm) Table 3. The effect of feeding ethylene to the apical region on ethylene emanation from and internal concentration of a leaf and vice versa. Leaf feeding chamber 160 nl CzH4/ml, apical region feeding chamber 300 nl C2HJml, specific activity 833 cpm/nl. Plants were analysed after 180 min. Figures are the mean of the number of plants indicated in brackets_+ standard deviation. The distance between the apical region and the second leaf was 2-3 cm Emanating tissue
Apical region 2nd leaf
Control
Treatment
C2H4 emanation C 2 H 4 internM (nl/organ/h) (nl/ml)
C2H 4 internal C2H 4 emanation 14C2H 4 as a concentration (nl/organ/h) percentage of total emanation (nl/ml)
14C2H4 as a percentage of total internal ethylene
1.79_+1.90(4) 1.71 _+1.69(10)
1.75_+1.40 (6) 1.77_+ 1.60(18)
2.9 2.4
0.53_+0.23(4) 0.46_+0.21(9)
o f the c o n c e n t r a t i o n in the feeding c h a m b e r (Table 2). F e e d i n g the f o u r t h a n d fifth leaf with ethylene increased the ethylene c o n c e n t r a t i o n in the l a c u n a a n d stem by only 0 - 5 0 % , a m u c h smaller increase t h a n t h a t o b s e r v e d w h e n the apical r e g i o n was fed. N o activity c o u l d be d e t e c t e d in o t h e r leaves b u t the apical r e g i o n was e n r i c h e d by u p to 16% w i t h labelled ethylene. A s no e m a n a t i o n o f r a d i o a c t i v i t y c o u l d be d e t e c t e d (Table 3) we believe t h a t the figures given for i n t e r n a l c o n c e n t r a t i o n r e p r e s e n t all the ethylene that moved. T h e v a r i a b i l i t y o f the results in T a b l e s 2 a n d 3 is high. A p a r t i a l e x p l a n a t i o n for this is d e m o n s t r a t e d by the results in T a b l e 4. W h e n e m a n a t i o n f r o m a p e t i o l e was c o m p l e t e l y p r e v e n t e d , ethylene m o v e m e n t to the l a c u n a i n c r e a s e d eleven fold. A s the enclosure o f a n y leaf involves at least the p a r t i a l covering o f the petiole, the a m o u n t o f ethylene arriving at the' distal e n d w o u l d be largely influenced by the length o f the r e m a i n i n g o p e n petiole. This was, o f necessity, a v a r i a b l e in the e x p e r i m e n t s d e s c r i b e d here. H o w ever, it also seems likely t h a t p a r t o f the v a r i a t i o n
0.0 0.0
1.28_+0.81(4) 0.93 _+0.44(6)
is due to o t h e r u n c o n t r o l l e d factors or to i n d i v i d u a l v a r i a t i o n b e t w e e n plants. M o v e m e n t o f ethylene f r o m the aerial p a r t s to the r o o t was i n v e s t i g a t e d in u n d i s t u r b e d p l a n t s growing in gravel. A t the end o f the e x p e r i m e n t a l p e r i o d the r o o t s were w a s h e d out u n d e r a m m o n i u m s u l p h a t e a n d were v a c u u m extracted. W h e n the a p i c a l region was fed with 150 gl/1 [14C]ethylene n o activity c o u l d be d e t e c t e d in the r o o t system, Even w h e n the c o n c e n t r a t i o n o f l a b e l l e d ethylene in the t r a n s i t i o n z o n e was increased by feeding directly into the l a c u n a cut 12 c m Table 4. The effect of preventing emanation from the petiole (1.6 cm
long) on the movement of ethylene (specific activity 8075 cpm/nl) from a fed leaf to the lacuna. Figures in brackets are standard deviations. The experiment was carried out in triplicate C2H r (nl/ml)
Leaf chamber Lacuna
Open petiole
Sealed petiole
72.8 (7.43) 0.019 (0.009)
65.40 (0.650) 0.21 (0.028)
122
M. Zeroni et al. : Movement and Distribution of Ethylene
Table 5. Movement of ethylene (specific activity 6960 cpm/nl) from the lacuna to the root. Plants were cut 12 cm above the transition zone with the feeding chamber in direct contact with the open lacuna. Each column shows data obtained from a single plant which was representative of 10 plants Control
Stem Transition zone Root
Treatment
C2H 4
C2H 4
14C2H 4 as
(nl/ml)
(nl/ml)
percentage of total
0.38 1.40 0.61
4.78 1.26 0.60
109.0 36.0 3.9
Table 6. The movement of ethylene (700 nl/ml; specific activity 608cpm/nl) from a fed root tip to a 6 c m section of root 1 cm above it and emanation therefrom. The volume of ethylene inside the fed root segment was 1.55 nl. The experiment was performed in seven plants and the results shown are from one plant representative of all Time
60 190 270
CzH4
Vol. of
14C2H 4 as
Vol. of
emanation (hi/section/h)
14C2H 4
a % of total
emanated (nl) emanation
14C2H ~ inside emanating root section (nl)
5.07 6.55
2.34 6.26 8.28
38.2 36.5 32.1
0.013
above the gravel, no activity could be detected in the root on most occasions (Table 5). When ethylene was fed to the root tip, emanation of labelled ethylene from a 6 cm segment starting 25.0 10.0
0~. Ok. ~ ~ ~ ~
t ~ - - D
Q---I O--O 1--1
1 cm above the feeding chamber occurred rapidly such that after 270 min the total volume of labelled ethylene in the emanation chamber was 640 times the volume within that segment (Table 6). The movement of labelled ethylene through whole plants in both directions is shown in Figure 1. The activity in successive segments of the plant declines rapidly and approached zero at the distal end. When plants were embedded in agar the volume of labelled ethylene always exceeded that in the open plants.
Distribution of Ethylene in Stem Tissue and in the Lacuna A gradient of ethylene concentration was found in the lacuna. The concentration of ethylene was always highest in the upper section while in the lower section it was usually less than in the middle, but varied between about 50% and 150% of the middle (Tables 1 and 8). In plants fed through the apical region the gradient was steeper (Tables 1 and 9). The gradient of ethylene concentration down the lacuna was not reflected in the stem tissues. Ethylene diffusion in closed plastic tubes shows a rapid equilibration such that after three hours the ratio of ethylene concentration between the sections was 1:0.9:0.86 (Fig. 2, Table 9) and at 65 h no gradient could be measured9 The patterns of upward and downward diffusion were identical (Fig. 2). It is possible that changes in the permeability of the stem with age would affect the ethylene gradient
Apical Region Fed, Enclosed in Agar Apical Region Fed Open Root Tip Fed, Enclosed in Agar Root Tip Fed, Open
17 / / I1
1.0
o
D E
.IO
"~ 9 o
\
Ii,/
I/ 9
it
i ~
Z
/
.oi
/
\o ..-'"ix.....
l i /
I
i l
\
I" 9001 D~
90001
Apical Region
Stem
Transition Zone
Upper Root Segments
PLANT SEGMENTS
Lower Root Segments
Root Tip
Fig. 1. Distribution of ethylene within plants supplied with the gas (100 nl/ml; specific activity 8075 cpm/ml) through the root or the apical region. Plants were 55 to 65 cm long. The experiment was carried out in triplicate. The position of plant segments on the lower axis is proportional to the distances involved
M. Zeroni et al. : Movement and Distribution of Ethylene
'~ 100
Table 9. The ratios of ethylene concentration in the three sections of plastic tubes and in sections of the lacuna of blank, control, and apically fed plants
_.~._...o--/z--2-~o
1 O1 c-
_ // ~
80
0
123
.~9 I/ -"o~ / "
Sections /)//
"/
6c
/
,,~/
o
dt -
/
4O
/
o/
o Proximal ] Fed ,', Middle { downwards 13 DisLa[ J
/"
-/
/ / m// /~ /"
O
-, 20 z / / , . /
13
0 ~
" 60
-r"
9 Proximal ] Fed 9 Middle I upwards 9
Distal
L 120 Time (min)
i // 180
65brs
Plastic tube (Fig. 2, 180 min) Blank (Table 1) Control (Table 1) Apically fed (Table 1) Control (Table 8) Blocked lacuna (Table 8) Apicalty fed, blocked lacuna (Table 8)
Upper Middle
Lower
1 1 1 1 1 1 1
0.86 0.47 0.70 0.08 0.37 0.32 0.02
0.90 0.59 0.60 0.11 0.39 0.40 0.04
Fig. 2. Downward and upward diffusion of ethylene (15 nl/ml) in plastic tubes. Each section of tube was 15 cm long
Table 7. Mean permeability of stem sections of different ages to ethylene (195 nl/ml) fed to the lacuna. The experiment was carried out in triplicate Time (min)
30 90 180
b
Ethylene emanation (nl/cm 2 stem surface/h) Upper stem
Middle stem
Lower stem
28.4 41.2 46.4 a
6.24 7.60 6.47 b
4.61 6.84 6.54 c
Range 27.8-82.0 Range 6.12-6.67 Range 4.72-7.83
9).
Table 8. The effect of blocking the lacuna on the concentration of ethylene in sections of the lacuna and stem and on the movement of ethylene fed to the apical region of field grown plants. The experiment was carried out in triplicate and repeated twice with similar results. The lacunae were blocked 90 h and ethylene added 3.3h before dissection. The plants were 100 to 120cm tall. Ethylene concentration in feeding chamber 225nl/ml, specific activity 559 cpm/nl Control Blocked C2H 4 lacuna, (nl/ml) no ethylene added Call 4 (nl/ml)
BlOcked lacuna, ethylene added C2H 4 (nl/ml)
14C2H ~ as a percentage of total ethylene
2.68
2.86
152.1
90.7
Lacuna Upper Middle Lower
0.46 0.18 0.17
0.53 0.21 0.17
4.25 0.16 0.09
94.9 14.2 0.0
Stem Upper Middle Lower
3.33 4.11 1.85
3.04 3.74 4.66
5.89 4.71 2.55
47.8 8.37 3.17
Apical Region
in the lacuna. Sections of young upper stem were 6-7 times more permeable to ethylene than middle and lower sections (Table 7). The increase in emanation with time is possibly due to incomplete equilibration between the feeding chamber and the lacuna. Blocking the lacuna with silicone grease for 40 or 90 h did not alter the concentration of ethylene in the lacuna sections or in the adjacent stem sections (Table 8). In plants fed through the apical region the block between upper and middle sections dramatically reduced ethylene movement past that point which resulted in a much steeper gradient than in apically fed plants with an open lacuna (see Tables 1 and In all experiments the concentration of endogenous ethylene in the lacuna was always at least three times lower than that in any of the tissues surrounding it, i.e. apical region, stem and transition zone.
Discussion
Even when the natural gradients of ethylene concentration that could be observed in the plants used here were exceeded by more than 100 fold the contribution of movement of labelled ethylene to the ethylene measured in other regions of the plant was small. This leads to the conclusion that, given natural gradients, ethylene does not diffuse between plant parts in physiologically significant amounts. Ethylene moved more readily between the apical region and upper lacuna and stem than between other parts of the plant, possibly due to the longitudinally oriented air spaces in the apparently solid stem above the lacuna (Hunter, 1915). The two main plant factors which appear to limit ethylene movement are resistance in the longitudinal direction and the loss of ethylene by emanation from the surface of the plant. There is a complex system
124
of air spaces in all parts of Viciafaba (Hunter, 1915) and studies on oxygen diffusion have shown that the air spaces are continuous, at least between the leaves and all but the youngest root tissue (Evans and Ebert, 1960; Greenwood, 1967b; Healy and Armstrong, 1972) and that the resistance to gas movement lies largely within the system of air spaces (Coult and Vallance, 1958). When plants covered with agar were fed with [14C]ethylene through the apical region, the level of labelled ethylene declined steadily through the plant as far as the lowest root segment and then declined rapidly in the root tip. When ethylene was fed through the root tip a similar decline was observed between the feeding chamber and the lower root segments with an almost constant level in the rest of the plant (Fig. 1). This may result from a much higher longitudinal resistance in the young root than elsewhere in the plant as well as from the interruption in the connection between the lacuna and the ring of lysogenic cavities found in the cortex of the lower stem and upper root (Hunter, 1915). While the mode of movement of ethylene is similar to the mode of oxygen diffusion it is not possible to determine from our data whether the ethylene concentration in the root of apicallyfed plants increases at the same rapid rate as reported for oxygen (Greenwood, 1967a). Hall (1976) gives results which show that ethylene movement from the surrounding atmosphere into leaves of Viciafaba is less than expected by simple diffusion and so the possibility remains that the plant offers a greater resistance to the diffusion of ethylene compared to oxygen. Emanation from the surface of the plant further limits longitudinal ethylene movement by more than 10-fold (Fig. 1, Table 4). Thus ethylene in the leaves cannot move effectively to the rest of the plant because of emanation from the petiole (Table 4), or from one point to another in the root system for the same reason as shown by the ratio of the volume emanated to internal volume of ethylene (Table 6), or even in the open air space of the lacuna because of emanation through the stem as shown by the difference in ethylene gradients between plastic tubes and lacunae (Table 9). Similarly, Healy and Armstrong (1972) showed that the amount of oxygen diffusing along the root is greatly reduced if emanation is not prevented. It follows that when emanation is prevented ethylene can be channelled to distant parts of the plant. In waterlogged soil channelling occurs in the underwater parts of the plant but it is uncertain what proportion of the ethylene reported in aerial parts originated from below the waterline. The concentration of ethylene increases in waterlogged soil (Smith and
M. Z e r o n i et al. : M o v e m e n t a n d D i s t r i b u t i o n of E t h y l e n e
Russell, 1969), and in the roots (Smith and Jackson, 1974) and aerial parts of waterlogged plants (E1-Beltagy and Hall, 1974; Jackson and Campbell, 1975). Jackson and Campbell (1975) also demonstrated movement of labelled ethylene from the roots of waterlogged tomato plants to the stem and petioles. However the total movement was small and it is difficult to see how the amount moved could have greatly increased the internal concentration. In several of our experiments the movement of labelled ethylene could not account for the consequent increase in the internal concentration of a distant tissue. This was shown in the apical regions, leaves and lower stems (Tables 1 and 3). The mechanism responsible for this effect is unclear, but it seems unlikely that sufficient ethylene moved to stimulate autocatalytic synthesis. It seems possible that leaf epinasty and the increase in ethylene concentration in stems and petioles of root-fed tomato plants (Jackson and Campbell, 1975) may be due to this phenomenon rather than to movement of ethylene from the roots. Steam girdling of the stem, which reduced epinasty and lessened the increase in internal concentration of ethylene, could well be affecting other processes, particularly as a direct effect of this treatment on the movement of labelled ethylene from the roots was not reported. A comparison of the ethylene concentrations in lacunae and surrounding tissues presented several problems. The difference between the patterns of the gradients of ethylene established in plastic tubes and those found in the lacunae of blank and control plants suggests that downward diffusion is not the major controlling factor in the maintenance of the gradient in the latter. If the apical region were the source of ethylene in the lacuna then lateral emanation, given the high permeability of young stem tissues (Table 7), could establish a downward gradient but this cannot be the explanation because the gradient remains when ethylene movement through the whole lacuna is prevented (Table 8). Figure 2 shows that there is no significant flotation effect of ethylene in air. Further, it was invariably found that the concentration of ethylene in the gas extracted from the stem, apical region, and transition zone was at least three times greater than that in the lacuna, which raises the problem of why the lacuna does not appear to equilibrate with its surrounding tissue. The relation between lacuna and stem concentration would also appear to exclude movement from the lacuna to the outside but such a movement is indicated by the ethylene gradient found in the lacuna. We suggest that ethylene is held at a higher, albeit variable concentration in the cell compared to the air spaces. This view is supported by
M. Zeroni et al. : Movement and Distribution of Ethylene
the lack of a direct relation between emanation and internal concentration and the fact that in some circumstances labelled ethylene moves into a tissue but no emanation can be detected (Table 3). We can only speculate as to whether this' compartmentation' consists of ethylene dissolved in or otherwise attached to certain parts of the cell or whether a ' c o m p a r t m e n t ' is formed by an anatomical structure such as the vascular bundles isolated by the closely packed cells of the bundle sheath. Thus, it is argued that the ethylene concentration in the tissue air spaces is at least three times lower than that shown by vacuum extraction and that the lacuna is in fact equilibrated with the adjacent air spaces. The endogenous ethylene in the lacuna originates from the stem tissue with the younger tissues producing more ethylene than the older ones (Abeles, 1973). Presumably ethylene in the top of the lacuna diffuses down the concentration gradient in the lacuna but quickly reaches a zone where the concentration in the air spaces of the stem is lower than that further down the lacuna and is emanated to the outside. That the stem is sufficiently permeable to support this view is shown by the steeper gradients in the lacunae of apically fed plants. We are grateful to the SRC for a grant towards some of this work.
125 Beyer, E.M. Jr., Morgan, P.W.: A method for determining the concentration of ethylene in gas phase of vegetative plant tissue. Plant Physiol. 46, 352-354 (1970) Brown, R. : The gaseous exchange between the root and the shoot of the seedling of Cucurbita pepo. Ann. Bot. 11, 417-437 (1947) Coult, D.A., Vallance, K.B. : Observation on the gaseous exchanges which take place between Menyanthes trifoliata L and its environment. II. J. exp. Bot. 9, 384-402 (1958) E1-Beltagy, A.S., Hall, M.A. : Effect of water stress upon endogeous ethylene levels in Vicia faba. New Phytol. 73, 47-60 (1974) Evans, N.T., Ebert, M.: Radioactive oxygen in the study of gas transport down the root of Viciafaba. J. exp. Bot. 11, 246-257 (1960) Greenwood, D.J.: Studies on the transport of oxygen through the stems and roots of vegetable seedlings. New Phytol. 66, 337-347 (1967a) Greenwood, D.J.: Studies on oxygen transport through mustard seedlings (Sinapis alba L.). New Phytol. 66, 597 606 (1967b) Hall, M.A.: Ethylene involvement in senescence processes. Ann. appl. Biol. In press (1976) Healy, M.T., Armstrong, W. : The effectiveness of internal oxygen transport in a mesophyte (Pisum sativum L.). Planta (Berl.) 103, 302-309 (1972) Hunter, C.: The aerating system of Vicia faba. Ann. Bot. 29, 627 634 (1915) Jackson, M.B., Campbell, D.J. : Movement of ethylene from roots to shoots, a factor in the responses of tomato plants to waterlogged soil conditions. New Phytol. 74, 397-406 (1975) Smith, K.A., Jackson, M.B. : Ethylene, waterlogging and plant growth. Rep. Agric. Res. Coun. Letcombe Lab. 1973, 60 75 (1974) Smith, K.A., Russell, R.S. : Occurrence of ethylene and its significance in anaerobic soil. Nature (Lond.) 222, 769-771 (1969) Zimmerman, P.W., Hitchcock, A.E., Crocker, W. : The movement of gases into and through plants. Contr. Boyce Thompson Inst. P1. Res. 3, 313-320 (1931)
References Abeles, F.B.: Ethylene in plant biology. New York: Academic Press 1973
Received 6 September; accepted 22 November 1976
Planta 134, 119-125 (1977)
Studies on the Movement and Distribution of Ethylene in Vicia faba L. M. Zeroni 1, P.H. Jerie 2, and M.A. Hall Department of Botany and Microbiology, The University College of Wales, Penglais, Aberystwyth SY23 3DA, U.K.
Abstract. In Vicia f a b a ethylene does not appear to
move between different parts of the plant in physiologically significant amounts. The 'resistance' to longitudinal m o v e m e n t is such that lateral emanation effectively isolates different parts of the plant from each other. When emanation is prevented, ethylene can be channelled to any part of the plant. Exposure of one section of a plant to 14C-labelled ethylene (up to 200 lal/1) increased the internal concentration in other parts with ethylene that did not originate from the feeding chamber. A basipetal gradient of endogenous ethylene concentration was found in the lacuna of intact plants, the source of ethylene being the stem tissue. The permeability of stem tissue to ethylene decreases with age. The concentration of ethylene in tissues surrounding the lacuna is always higher than that in the lacuna and it is argued that ' c o m p a r t m e n t a t i o n ' of ethylene occurs within these tissues. Key words: C o m p a r t m e n t a t i o n - Ethylene Viciafaba.
Move-
ment of ethylene -
tion of ethylene in roots and aerial parts of plants increases when the root system is waterlogged or is treated with ethylene (El Beltagy and Hall, 1974; Smith and Jackson, 1974). These findings are in accord with other work, using several species, indicating that gases may diffuse through the plant as though in a system of continuous air spaces. Such effects have been shown with oxygen (Evans and Ebert, 1960; Greenwood, 1967a and b; Healy and Armstrong, 1972), and hydrogen and nitrogen (Brown, 1947). The possibility that ethylene m a y move from its site(s) of synthesis to other parts of the plant raises a number of questions not only in relation to the role of the gas in plant growth and development but also because such m o v e m e n t might affect estimates of rates of ethylene biosynthesis in a particular location. The aim of the present work, therefore, was to determine the significance of the movement and distribution of ethylene in Vicia faba.
Materials and Methods Introduction
A number of studies have suggested that movement of ethylene may occur between different parts of the plant. Thus, Jackson and Campbell (1975) demonstrated that, in tomato, labelled ethylene moves from the roots to the stem and petioles, apparently providing an explanation for the results of Z i m m e r m a n et al. (1931), who showed that exposure of t o m a t o roots to ethylene results in epinasty of the petioles. Similarly, it has been shown that the internal concentra1
Permanentaddress: Department of Botany, Tel-Aviv Univer-
sity, Tel-Aviv, Israel 2
Permanent address." Horticultural Research Station, Tatura,
Victoria, Australia
Plant Culture
Plants of Vicia faba var. Aquadulce were grown under a 16 h photoperiod either in a heated glasshouse or in a growth chamber at 20+_2~ Depending on which tissues were required the plants were grown in Levington compost or in fine gravel in 10 cm pots or in deeper containers of vermiculite. Ethylene movement within the root system was determined using two week old plants (Table 6). In other experiments the plants were 5-6 weeks old (Tables 1-5, 7) with the exception of data in Table 8 where 4-month-old field grown plants were used. Plant Manipulation
Plant tissues were enclosed in gas-tight chambers made from plastic syringes or plastic tubes fitted with silicone rubber septa. The
120 plant part was supported in the chamber with the rubber head from the syringe plunger which incorporated a suitable hole and was split on one side. A quick-setting silicone rubber (Silicoset 101, ICI Ltd.) was used to seal the chambers and wherever possible water was added to a height of 1 cm inside the chamber. For studies on movement to and from the roots the fine gravel or vermiculite in which the plants were growing was gently removed with water. The chamber used to enclose roots always contained a small a m o u n t of water. The remaining open part of the root system was held in running tap water to prevent desiccation. Plant segments were always cut under water a n d the basal end stood in water throughout the experiment, Intact plants were embedded in 2% agar by drawing them into a plastic tube 2.6 cm in diameter into which agar at 36-37~ was then poured. This procedure did not appear to h a r m the plants as the roots continued to grow in agar. The lacuna was divided into sections by injecting silicone grease. Control plants were fitted with chambers identical to those on the treated plants. In some experiments ' b l a n k ' plants without chambers were included to check whether the chambers affected the results. U p w a r d and downward diffusion of ethylene in plastic tubes (0.4 em diameter) closed at one end and of similar length to the lacunae of the bean plants used was investigated. At zero time a wide-bore glass tap from a feeding chamber containing ethylene was opened. At the end of each experimental period the tap was closed and the tube quickly divided with two clamps into three sections. For ethylene determination the gas in each section was withdrawn under a m m o n i u m sulphate. Labelled ethylene (1~C2H,, A m e r s h a m , 95 m Ci/mmol) was injected into the feeding chambers. In experiments where [lr ylene was diluted with unlabelled ethylene the specific activity was determined in each feeding chamber just prior to dissection. The radiochemical purity of 114C]ethylene as supplied and after movement through and extraction from the plant was determined by radiogaschromatography (Panax Radiogas Detector System) and found to be 99.42% and 99.74% respectively.
Sampling and Analysis Unless otherwise stated experiments were terminated 200 rain after ethylene application. Plants were dissected under a saturated solution of a m m o n i u m sulphate. The gas from lacuna sections was drawn into plastic syringes. The internal concentration of ethylene within tissues was determined using a modification of the method of Beyer and Morgan (1970). The tissue was placed under saturated a m m o n i u m sulphate inside an inverted 50 ml syringe fitted on the needle taper with a piece of closed thick-walled rubber tube. A volume of air (2.00 ml) was injected into each syringe and a vacuum of 30 cm Hg was applied for 3 rain to the syringes in a v a c u u m desiccator. After samples were taken for ethylene determination the volume of gas withdrawn from the tissue by the vacuum treatment was measured (_+0.01 ml). Ethylene was measured using a Pye U n i c a m 104 gas chromatograph with a glass column (210 x 0.4 cm) packed with alumina (85-100 mesh). The oven temperature was 120~ and the nitrogen flow rate 60 ml/min. Labelled ethylene was determined by injecting 1 ml gaseous samples into a 3.8 ml glass vial containing 0.3 ml a m m o n i u m sulphate and 3 ml of toluene scintillant (5 g/1 PPO). The samples were counted at an efficiency of 88-91% in a Beckman L S - 2 0 0 B scintillation spectrometer. The quantity of Iabelled ethylene was generally calculated as a percentage of the ethylene determined by gas chromatography. As a small piece of tissue did not liberate a sufficient volume of gas under v a c u u m extraction to enable the concentration to be calculated accurately, such pieces were placed directly in the vials described above and counted with an efficiency of 82-88%.
M. Zeroni et al. : Movement and Distribution of Ethylene
Description of Plant Parts In the succeeding experiments the ' apical region' is taken to consist of the upper part of the stem from the apex down to the node of the oldest folded leaf. Leaves were counted basipetally starting from the oldest folded leaf. Upper, middle and lower sections of lacunae were of equal length. The 'transition z o n e ' included tissue from just below the position of the cotyledons to a point on the solid stem about 1.5 cm above the cotyledons. The ' u p p e r ' and 'lower' root segments were 2 cm long sections of the main root removed sequentially from the root below the transition zone and above the root tip. The ' r o o t tip' encompassed the terminal 4-6 cm of the main root.
Results
Ethylene Movement Only a small proportion of the ethylene supplied in high concentration to one region of the plant could be detected in other parts of the plant. The largest amount of ethylene moved between the apical region and the upper section of the lacuna and stem (Table 1). In the lower lacuna ethylene movement resulted in a 4.3-fold increase above the control level of ethylene after 200 rain. The gradient between the lower lacuna and the feeding chamber was greater than 600: 1. In the lower region of the stem the ethylene concentration increased as a result of feeding the apical region but only a small proportion (17.6%) of the increase could be accounted for as labelled ethylene. Although in plants fed through the apical region the upper lacuna and stem contained substantial activity, in adjacent leaves the ethylene concentration increased by only 1-5% over the endogenous concentration in control leaves, representing about 0.01%
Table 1. Movement of ethylene (specific activity 2820 cpm/nl) from the apical region of Vicia faba. Each column shows data from a single plant representative of 12 blank, 8 control and 8 treatment plants. Total length of the stem was 42 to 48 cm
Blank
Control
Treatment
C2H4 (nl/ml)
C2H4 (nl/ml)
Total CzH 4 (nl/ml)
14C2H 4 (nl/ml)
14CzH 4 as a % of total ethylene
0.08
191.30
191.30
100
Feeding chamber Lacuna upper middle lower
0.17 0.10 0.08
0.10 0.06 0.07
3.90 0.44 0.30
3.99 0.41 0.21
102 91.8 71.6
Stem upper middle lower
0.64 0.30 0.53
0.32 0.39 0.32
3.71 0.64 0.83
2.50 0.22 0.09
67.3 33.8 10.9
M. Zeroni et al. : Movement and Distribution of Ethylene
121
Table 2. The effects of feeding ethylene (specific activity 1380 cpm/nl) to the apical region or to a lower leaf on the internal concentration of ethylene in other aerial parts of the plant. Figures are the mean of the number of plants indicated in brackets _+standard deviation Recipient organs a
Feeding Organ Apical Region
Leaf (4th or 5th)
Control C~H4 (nl/ml)
Treatment Total C2H4 (nl/ml)
Apical Region Lacuna Stem 2nd leaf(2.5) 3rd leaf (9) 4th leaf 09) 5th leaf(31)
0.14_+0.08(13) 0.64-+0.32(13) 0.46_+0.21(9) 0.55+0.29(13) 0.69+-0.62(11) 0.71 (3)
Treatment
Control C2H 4 (nl/ml) 1 4 C 2 H 4 as a
Total
percentage of total ethylene
C2H4 (nl/ml)
14C),H4 as a percentage of total ethylene
1.3 _+0.8 (4) 0.19_+0.10(7) 0.84_+0.60(7) 0.16 (2) 0.61_+0.56(4) 0.36_+0.12(4) 0.64 (2)
15.8_+11.6 20.7_+24.1 35.0_+18.6 0.0 0.0 0.0 7.8
0.53_+0.23(4) 0.14+0.04(7) 0.65+_0.20(7) 0.26 (2) 0.47_+0.32(4) 0.35_+0.28(4) 0.25+-0.03(4)
1.80_+1.40(10) 95.4 _ + 1 4 . 0 2.0 +_1.8 (10) 45.5 _ + 1 7 . 9 0.93_+0.44(6) 2.4 _+ 4.2 0.91_+1.02(11) 0.25_+ 0 . 6 1 0.40_+0.23(10) 2.7 +_ 4.8 0.39+_0.18(5) 0.50-+ 0 . 8 7
Figures in brackets denote average distance from apical region (cm) Table 3. The effect of feeding ethylene to the apical region on ethylene emanation from and internal concentration of a leaf and vice versa. Leaf feeding chamber 160 nl CzH4/ml, apical region feeding chamber 300 nl C2HJml, specific activity 833 cpm/nl. Plants were analysed after 180 min. Figures are the mean of the number of plants indicated in brackets_+ standard deviation. The distance between the apical region and the second leaf was 2-3 cm Emanating tissue
Apical region 2nd leaf
Control
Treatment
C2H4 emanation C 2 H 4 internM (nl/organ/h) (nl/ml)
C2H 4 internal C2H 4 emanation 14C2H 4 as a concentration (nl/organ/h) percentage of total emanation (nl/ml)
14C2H4 as a percentage of total internal ethylene
1.79_+1.90(4) 1.71 _+1.69(10)
1.75_+1.40 (6) 1.77_+ 1.60(18)
2.9 2.4
0.53_+0.23(4) 0.46_+0.21(9)
o f the c o n c e n t r a t i o n in the feeding c h a m b e r (Table 2). F e e d i n g the f o u r t h a n d fifth leaf with ethylene increased the ethylene c o n c e n t r a t i o n in the l a c u n a a n d stem by only 0 - 5 0 % , a m u c h smaller increase t h a n t h a t o b s e r v e d w h e n the apical r e g i o n was fed. N o activity c o u l d be d e t e c t e d in o t h e r leaves b u t the apical r e g i o n was e n r i c h e d by u p to 16% w i t h labelled ethylene. A s no e m a n a t i o n o f r a d i o a c t i v i t y c o u l d be d e t e c t e d (Table 3) we believe t h a t the figures given for i n t e r n a l c o n c e n t r a t i o n r e p r e s e n t all the ethylene that moved. T h e v a r i a b i l i t y o f the results in T a b l e s 2 a n d 3 is high. A p a r t i a l e x p l a n a t i o n for this is d e m o n s t r a t e d by the results in T a b l e 4. W h e n e m a n a t i o n f r o m a p e t i o l e was c o m p l e t e l y p r e v e n t e d , ethylene m o v e m e n t to the l a c u n a i n c r e a s e d eleven fold. A s the enclosure o f a n y leaf involves at least the p a r t i a l covering o f the petiole, the a m o u n t o f ethylene arriving at the' distal e n d w o u l d be largely influenced by the length o f the r e m a i n i n g o p e n petiole. This was, o f necessity, a v a r i a b l e in the e x p e r i m e n t s d e s c r i b e d here. H o w ever, it also seems likely t h a t p a r t o f the v a r i a t i o n
0.0 0.0
1.28_+0.81(4) 0.93 _+0.44(6)
is due to o t h e r u n c o n t r o l l e d factors or to i n d i v i d u a l v a r i a t i o n b e t w e e n plants. M o v e m e n t o f ethylene f r o m the aerial p a r t s to the r o o t was i n v e s t i g a t e d in u n d i s t u r b e d p l a n t s growing in gravel. A t the end o f the e x p e r i m e n t a l p e r i o d the r o o t s were w a s h e d out u n d e r a m m o n i u m s u l p h a t e a n d were v a c u u m extracted. W h e n the a p i c a l region was fed with 150 gl/1 [14C]ethylene n o activity c o u l d be d e t e c t e d in the r o o t system, Even w h e n the c o n c e n t r a t i o n o f l a b e l l e d ethylene in the t r a n s i t i o n z o n e was increased by feeding directly into the l a c u n a cut 12 c m Table 4. The effect of preventing emanation from the petiole (1.6 cm
long) on the movement of ethylene (specific activity 8075 cpm/nl) from a fed leaf to the lacuna. Figures in brackets are standard deviations. The experiment was carried out in triplicate C2H r (nl/ml)
Leaf chamber Lacuna
Open petiole
Sealed petiole
72.8 (7.43) 0.019 (0.009)
65.40 (0.650) 0.21 (0.028)
122
M. Zeroni et al. : Movement and Distribution of Ethylene
Table 5. Movement of ethylene (specific activity 6960 cpm/nl) from the lacuna to the root. Plants were cut 12 cm above the transition zone with the feeding chamber in direct contact with the open lacuna. Each column shows data obtained from a single plant which was representative of 10 plants Control
Stem Transition zone Root
Treatment
C2H 4
C2H 4
14C2H 4 as
(nl/ml)
(nl/ml)
percentage of total
0.38 1.40 0.61
4.78 1.26 0.60
109.0 36.0 3.9
Table 6. The movement of ethylene (700 nl/ml; specific activity 608cpm/nl) from a fed root tip to a 6 c m section of root 1 cm above it and emanation therefrom. The volume of ethylene inside the fed root segment was 1.55 nl. The experiment was performed in seven plants and the results shown are from one plant representative of all Time
60 190 270
CzH4
Vol. of
14C2H 4 as
Vol. of
emanation (hi/section/h)
14C2H 4
a % of total
emanated (nl) emanation
14C2H ~ inside emanating root section (nl)
5.07 6.55
2.34 6.26 8.28
38.2 36.5 32.1
0.013
above the gravel, no activity could be detected in the root on most occasions (Table 5). When ethylene was fed to the root tip, emanation of labelled ethylene from a 6 cm segment starting 25.0 10.0
0~. Ok. ~ ~ ~ ~
t ~ - - D
Q---I O--O 1--1
1 cm above the feeding chamber occurred rapidly such that after 270 min the total volume of labelled ethylene in the emanation chamber was 640 times the volume within that segment (Table 6). The movement of labelled ethylene through whole plants in both directions is shown in Figure 1. The activity in successive segments of the plant declines rapidly and approached zero at the distal end. When plants were embedded in agar the volume of labelled ethylene always exceeded that in the open plants.
Distribution of Ethylene in Stem Tissue and in the Lacuna A gradient of ethylene concentration was found in the lacuna. The concentration of ethylene was always highest in the upper section while in the lower section it was usually less than in the middle, but varied between about 50% and 150% of the middle (Tables 1 and 8). In plants fed through the apical region the gradient was steeper (Tables 1 and 9). The gradient of ethylene concentration down the lacuna was not reflected in the stem tissues. Ethylene diffusion in closed plastic tubes shows a rapid equilibration such that after three hours the ratio of ethylene concentration between the sections was 1:0.9:0.86 (Fig. 2, Table 9) and at 65 h no gradient could be measured9 The patterns of upward and downward diffusion were identical (Fig. 2). It is possible that changes in the permeability of the stem with age would affect the ethylene gradient
Apical Region Fed, Enclosed in Agar Apical Region Fed Open Root Tip Fed, Enclosed in Agar Root Tip Fed, Open
17 / / I1
1.0
o
D E
.IO
"~ 9 o
\
Ii,/
I/ 9
it
i ~
Z
/
.oi
/
\o ..-'"ix.....
l i /
I
i l
\
I" 9001 D~
90001
Apical Region
Stem
Transition Zone
Upper Root Segments
PLANT SEGMENTS
Lower Root Segments
Root Tip
Fig. 1. Distribution of ethylene within plants supplied with the gas (100 nl/ml; specific activity 8075 cpm/ml) through the root or the apical region. Plants were 55 to 65 cm long. The experiment was carried out in triplicate. The position of plant segments on the lower axis is proportional to the distances involved
M. Zeroni et al. : Movement and Distribution of Ethylene
'~ 100
Table 9. The ratios of ethylene concentration in the three sections of plastic tubes and in sections of the lacuna of blank, control, and apically fed plants
_.~._...o--/z--2-~o
1 O1 c-
_ // ~
80
0
123
.~9 I/ -"o~ / "
Sections /)//
"/
6c
/
,,~/
o
dt -
/
4O
/
o/
o Proximal ] Fed ,', Middle { downwards 13 DisLa[ J
/"
-/
/ / m// /~ /"
O
-, 20 z / / , . /
13
0 ~
" 60
-r"
9 Proximal ] Fed 9 Middle I upwards 9
Distal
L 120 Time (min)
i // 180
65brs
Plastic tube (Fig. 2, 180 min) Blank (Table 1) Control (Table 1) Apically fed (Table 1) Control (Table 8) Blocked lacuna (Table 8) Apicalty fed, blocked lacuna (Table 8)
Upper Middle
Lower
1 1 1 1 1 1 1
0.86 0.47 0.70 0.08 0.37 0.32 0.02
0.90 0.59 0.60 0.11 0.39 0.40 0.04
Fig. 2. Downward and upward diffusion of ethylene (15 nl/ml) in plastic tubes. Each section of tube was 15 cm long
Table 7. Mean permeability of stem sections of different ages to ethylene (195 nl/ml) fed to the lacuna. The experiment was carried out in triplicate Time (min)
30 90 180
b
Ethylene emanation (nl/cm 2 stem surface/h) Upper stem
Middle stem
Lower stem
28.4 41.2 46.4 a
6.24 7.60 6.47 b
4.61 6.84 6.54 c
Range 27.8-82.0 Range 6.12-6.67 Range 4.72-7.83
9).
Table 8. The effect of blocking the lacuna on the concentration of ethylene in sections of the lacuna and stem and on the movement of ethylene fed to the apical region of field grown plants. The experiment was carried out in triplicate and repeated twice with similar results. The lacunae were blocked 90 h and ethylene added 3.3h before dissection. The plants were 100 to 120cm tall. Ethylene concentration in feeding chamber 225nl/ml, specific activity 559 cpm/nl Control Blocked C2H 4 lacuna, (nl/ml) no ethylene added Call 4 (nl/ml)
BlOcked lacuna, ethylene added C2H 4 (nl/ml)
14C2H ~ as a percentage of total ethylene
2.68
2.86
152.1
90.7
Lacuna Upper Middle Lower
0.46 0.18 0.17
0.53 0.21 0.17
4.25 0.16 0.09
94.9 14.2 0.0
Stem Upper Middle Lower
3.33 4.11 1.85
3.04 3.74 4.66
5.89 4.71 2.55
47.8 8.37 3.17
Apical Region
in the lacuna. Sections of young upper stem were 6-7 times more permeable to ethylene than middle and lower sections (Table 7). The increase in emanation with time is possibly due to incomplete equilibration between the feeding chamber and the lacuna. Blocking the lacuna with silicone grease for 40 or 90 h did not alter the concentration of ethylene in the lacuna sections or in the adjacent stem sections (Table 8). In plants fed through the apical region the block between upper and middle sections dramatically reduced ethylene movement past that point which resulted in a much steeper gradient than in apically fed plants with an open lacuna (see Tables 1 and In all experiments the concentration of endogenous ethylene in the lacuna was always at least three times lower than that in any of the tissues surrounding it, i.e. apical region, stem and transition zone.
Discussion
Even when the natural gradients of ethylene concentration that could be observed in the plants used here were exceeded by more than 100 fold the contribution of movement of labelled ethylene to the ethylene measured in other regions of the plant was small. This leads to the conclusion that, given natural gradients, ethylene does not diffuse between plant parts in physiologically significant amounts. Ethylene moved more readily between the apical region and upper lacuna and stem than between other parts of the plant, possibly due to the longitudinally oriented air spaces in the apparently solid stem above the lacuna (Hunter, 1915). The two main plant factors which appear to limit ethylene movement are resistance in the longitudinal direction and the loss of ethylene by emanation from the surface of the plant. There is a complex system
124
of air spaces in all parts of Viciafaba (Hunter, 1915) and studies on oxygen diffusion have shown that the air spaces are continuous, at least between the leaves and all but the youngest root tissue (Evans and Ebert, 1960; Greenwood, 1967b; Healy and Armstrong, 1972) and that the resistance to gas movement lies largely within the system of air spaces (Coult and Vallance, 1958). When plants covered with agar were fed with [14C]ethylene through the apical region, the level of labelled ethylene declined steadily through the plant as far as the lowest root segment and then declined rapidly in the root tip. When ethylene was fed through the root tip a similar decline was observed between the feeding chamber and the lower root segments with an almost constant level in the rest of the plant (Fig. 1). This may result from a much higher longitudinal resistance in the young root than elsewhere in the plant as well as from the interruption in the connection between the lacuna and the ring of lysogenic cavities found in the cortex of the lower stem and upper root (Hunter, 1915). While the mode of movement of ethylene is similar to the mode of oxygen diffusion it is not possible to determine from our data whether the ethylene concentration in the root of apicallyfed plants increases at the same rapid rate as reported for oxygen (Greenwood, 1967a). Hall (1976) gives results which show that ethylene movement from the surrounding atmosphere into leaves of Viciafaba is less than expected by simple diffusion and so the possibility remains that the plant offers a greater resistance to the diffusion of ethylene compared to oxygen. Emanation from the surface of the plant further limits longitudinal ethylene movement by more than 10-fold (Fig. 1, Table 4). Thus ethylene in the leaves cannot move effectively to the rest of the plant because of emanation from the petiole (Table 4), or from one point to another in the root system for the same reason as shown by the ratio of the volume emanated to internal volume of ethylene (Table 6), or even in the open air space of the lacuna because of emanation through the stem as shown by the difference in ethylene gradients between plastic tubes and lacunae (Table 9). Similarly, Healy and Armstrong (1972) showed that the amount of oxygen diffusing along the root is greatly reduced if emanation is not prevented. It follows that when emanation is prevented ethylene can be channelled to distant parts of the plant. In waterlogged soil channelling occurs in the underwater parts of the plant but it is uncertain what proportion of the ethylene reported in aerial parts originated from below the waterline. The concentration of ethylene increases in waterlogged soil (Smith and
M. Z e r o n i et al. : M o v e m e n t a n d D i s t r i b u t i o n of E t h y l e n e
Russell, 1969), and in the roots (Smith and Jackson, 1974) and aerial parts of waterlogged plants (E1-Beltagy and Hall, 1974; Jackson and Campbell, 1975). Jackson and Campbell (1975) also demonstrated movement of labelled ethylene from the roots of waterlogged tomato plants to the stem and petioles. However the total movement was small and it is difficult to see how the amount moved could have greatly increased the internal concentration. In several of our experiments the movement of labelled ethylene could not account for the consequent increase in the internal concentration of a distant tissue. This was shown in the apical regions, leaves and lower stems (Tables 1 and 3). The mechanism responsible for this effect is unclear, but it seems unlikely that sufficient ethylene moved to stimulate autocatalytic synthesis. It seems possible that leaf epinasty and the increase in ethylene concentration in stems and petioles of root-fed tomato plants (Jackson and Campbell, 1975) may be due to this phenomenon rather than to movement of ethylene from the roots. Steam girdling of the stem, which reduced epinasty and lessened the increase in internal concentration of ethylene, could well be affecting other processes, particularly as a direct effect of this treatment on the movement of labelled ethylene from the roots was not reported. A comparison of the ethylene concentrations in lacunae and surrounding tissues presented several problems. The difference between the patterns of the gradients of ethylene established in plastic tubes and those found in the lacunae of blank and control plants suggests that downward diffusion is not the major controlling factor in the maintenance of the gradient in the latter. If the apical region were the source of ethylene in the lacuna then lateral emanation, given the high permeability of young stem tissues (Table 7), could establish a downward gradient but this cannot be the explanation because the gradient remains when ethylene movement through the whole lacuna is prevented (Table 8). Figure 2 shows that there is no significant flotation effect of ethylene in air. Further, it was invariably found that the concentration of ethylene in the gas extracted from the stem, apical region, and transition zone was at least three times greater than that in the lacuna, which raises the problem of why the lacuna does not appear to equilibrate with its surrounding tissue. The relation between lacuna and stem concentration would also appear to exclude movement from the lacuna to the outside but such a movement is indicated by the ethylene gradient found in the lacuna. We suggest that ethylene is held at a higher, albeit variable concentration in the cell compared to the air spaces. This view is supported by
M. Zeroni et al. : Movement and Distribution of Ethylene
the lack of a direct relation between emanation and internal concentration and the fact that in some circumstances labelled ethylene moves into a tissue but no emanation can be detected (Table 3). We can only speculate as to whether this' compartmentation' consists of ethylene dissolved in or otherwise attached to certain parts of the cell or whether a ' c o m p a r t m e n t ' is formed by an anatomical structure such as the vascular bundles isolated by the closely packed cells of the bundle sheath. Thus, it is argued that the ethylene concentration in the tissue air spaces is at least three times lower than that shown by vacuum extraction and that the lacuna is in fact equilibrated with the adjacent air spaces. The endogenous ethylene in the lacuna originates from the stem tissue with the younger tissues producing more ethylene than the older ones (Abeles, 1973). Presumably ethylene in the top of the lacuna diffuses down the concentration gradient in the lacuna but quickly reaches a zone where the concentration in the air spaces of the stem is lower than that further down the lacuna and is emanated to the outside. That the stem is sufficiently permeable to support this view is shown by the steeper gradients in the lacunae of apically fed plants. We are grateful to the SRC for a grant towards some of this work.
125 Beyer, E.M. Jr., Morgan, P.W.: A method for determining the concentration of ethylene in gas phase of vegetative plant tissue. Plant Physiol. 46, 352-354 (1970) Brown, R. : The gaseous exchange between the root and the shoot of the seedling of Cucurbita pepo. Ann. Bot. 11, 417-437 (1947) Coult, D.A., Vallance, K.B. : Observation on the gaseous exchanges which take place between Menyanthes trifoliata L and its environment. II. J. exp. Bot. 9, 384-402 (1958) E1-Beltagy, A.S., Hall, M.A. : Effect of water stress upon endogeous ethylene levels in Vicia faba. New Phytol. 73, 47-60 (1974) Evans, N.T., Ebert, M.: Radioactive oxygen in the study of gas transport down the root of Viciafaba. J. exp. Bot. 11, 246-257 (1960) Greenwood, D.J.: Studies on the transport of oxygen through the stems and roots of vegetable seedlings. New Phytol. 66, 337-347 (1967a) Greenwood, D.J.: Studies on oxygen transport through mustard seedlings (Sinapis alba L.). New Phytol. 66, 597 606 (1967b) Hall, M.A.: Ethylene involvement in senescence processes. Ann. appl. Biol. In press (1976) Healy, M.T., Armstrong, W. : The effectiveness of internal oxygen transport in a mesophyte (Pisum sativum L.). Planta (Berl.) 103, 302-309 (1972) Hunter, C.: The aerating system of Vicia faba. Ann. Bot. 29, 627 634 (1915) Jackson, M.B., Campbell, D.J. : Movement of ethylene from roots to shoots, a factor in the responses of tomato plants to waterlogged soil conditions. New Phytol. 74, 397-406 (1975) Smith, K.A., Jackson, M.B. : Ethylene, waterlogging and plant growth. Rep. Agric. Res. Coun. Letcombe Lab. 1973, 60 75 (1974) Smith, K.A., Russell, R.S. : Occurrence of ethylene and its significance in anaerobic soil. Nature (Lond.) 222, 769-771 (1969) Zimmerman, P.W., Hitchcock, A.E., Crocker, W. : The movement of gases into and through plants. Contr. Boyce Thompson Inst. P1. Res. 3, 313-320 (1931)
References Abeles, F.B.: Ethylene in plant biology. New York: Academic Press 1973
Received 6 September; accepted 22 November 1976
