Planta
Planta 134, 183-189 (1977)
9 by Springer-Verlag 1977
The Relationship between Leaf Water Potential (~leaf) and the Levels of Abscisic Acid and Ethylene in Excised Wheat Leaves S.T.C. Wright Agricultural Research Council Plant Growth Substance and Systemic Fungicide Unit, Wye College, Near Ashford, Kent TN25 5AH, U.K.
Abstract. The amount of diffusible ethylene from excised wheat leaves (Triticurn aestivurn L. cv. Eclipse) increased when they were subjected to water stress. The quantity of ethylene produced was related to the severity of the stress, reaching a maximum at a leaf water potential (@leaf) of approximately - 1 2 bars. Irrespective of the severity of the stress, the maximum rate of ethylene production usually occurred between 135-270min after applying the stress and then the rate declined. Part of the decline may have been due to an oxygen deficiency in the leaf chambers. In excised water-stressed leaves there was a sigmoid relationship between increasing ethylene and abscisic acid (ABA) levels and decreasing leaf water potential values. The two curves were displaced from each other by approximately 1 bar, with ethylene evolution leading that of ABA accumulation. The maximum rate of increase in ethylene occurred between - 8 and - 9 bars and for ABA between - 9 9and - 10 bars. A significant increase in the levels of these two plant growth regulators was found when the @le,f decreased outside the normal diurnal @leaf range by 1 bar for ethylene and 2 bars for ABA. Because of the sigmoid nature of the curves there was no distinct threshold @leaf value triggering-off an increase in ethylene or ABA, but with ABA the curve became very steep at a @leaf value of - 9.3 bars and this could be looked upon as a kind of " t h r e s h o l d " value. It seems unlikely that the stress-induced ethylene evolution in excised wheat leaves stimulated the accumulation of ABA, because when the leaves were subjected to a substantial water stress (e.g. @leaf= -- 12 bars) ABA increased immediately and at a faster rate than ethylene. 9 ABA=abscisic acid, GLC=gas-liquid chromatography, RWC=relative water content, TLC=thin-layer chromatography, Oleaf=leaf water potential Abbreviations.
Key words: Abscisic acid - Ethylene - Triticum Water potential - Water stress.
Introduction When plants are subjected to drought or waterlogging, major changes occur in hormone levels (see reviews by Livne and Vaadia, 1972 and Hsiao, 1973). The resulting hormonal imbalance is believed to be responsible for the onset of such stress symptoms as growth inhibition, partial or complete stomatal closure, petiole epinasty, increased root permeability, senescence of lower leaves, etc. These physiological changes help to maintain leaf water potential (i.e. a function of cell turgidity in the leaf) and thereby increase the plant's chances of survival. One hormone which is particularly sensitive to changes in leaf turgor is abscisic acid (ABA) (Wright and Hiron, 1969). Data collected by Zabadal (1974) suggested there might be a threshold leaf water potential which triggers-off ABA biosynthesis (and/or accumulation) under water stress conditions. In his experiments Ambrosia plants were exposed to a desiccating environment and throughout a 23 h period the changes in the levels of ABA and @leaf were determined. The results showed that the ABA accumulating system was "switched-on" in the critical - 1 0 to - 1 2 bars range and then ABA accumulated at a steady rate. Threshold values of @leaf below which ABA levels begin to increase have also been given by other workers; this value was - 8 bars for maize and between - 8 and - 10 bars for sorghum (Beardsell and Cohen, 1975). In contrast many of our own results indicate a more gradual increase in ABA levels with the onset of stress (Wright, 1972) and therefore we postulated that the sudden "switch-on" found
184
S.T.C. Wright: Leaf Water Potential, Ethylene, and A B A
by Zabadal could have been due to harsh experimental treatment. For this reason we have re-investigated this phenomenon using a milder experimental approach. Furthermore, since ethylene biosynthesis is known to be promoted by water stress (e.g. Ben-Yehoshua and Aloni, 1974; E1-Beltagy and Hall, 1974) we included ethylene determinations in our experiments to see if its biosynthesis is induced over a similar ~t~af range as ABA.
Materials and Methods Plant Culture Wheat (Triticum aestivum L. cv. Eclipse) grains were soaked in distilled water for 2 h prior to sowing. The grains were sown with their embryos uppermost in rows 1.5 x 1.5 cm on vermiculite contained in plastic seed boxes (26 x 16 x 5.5 cm). They were covered with 1-cm layer o f vermiculite and the surface pressed firm with a board. Seedlings were grown in a growth r o o m under a 14-h photoperiod, 8.6 klx from Atlas 'daylight' fluorescent tubes, at 23 ~ (night temp. 19 ~ and with a m i n i m u m relative humidity of 70%. Primary leaves (12-13 em in length) were excised above t h e coleoptiles in the middle of the 8th light period and trimmed to 11 cm.
Estimation of Ethylene Gas
General Technique
Ethylene was estimated by gas-liquid c h r o m a t o g r a p h y (GLC) using a Pye 104 dual column c h r o m a t o g r a p h with flame ionisation detectors. Gas samples were injected.on to an alumina column (150 cm long; 5 m m diameter) at 112 ~ with a nitrogen flow rate of 40 ml/ min, giving a retention time for ethylene of approximately 2 rain. K n o w n volumes of a standard gas mixture (10.4 vpm) were used to allow conversion of ethylene peak areas on the c h r o m a t o g r a m s to absolute gas values.
Batches of leaves (4 g) were allowed to wilt at r o o m temperature (normally about 20 ~) by spreading them out on sheets of filter
Methods for Measuring Water Stress
-12
i) Leaf Water Potential (tPleaf) values were measured in a pressure b o m b (based on Scholander et al., 1965) using oxygen-free nitrogen as a gas-pressure source. Primary leaves, which had been excised just above the coleoptile and their inner leaves removed, were threaded singly into the leaf clamping device of the pressure chamber so that the cut end protruded to the outside. The pressure of the gas in the chamber was increased at a constant rate to reach a value corresponding to - 6 bars in about 60 s. The pressure when sap first wetted the cut surface of the leaf, as observed through a low-powered binocular microscope, was taken as the end point. A m e a n of 5 readings was obtained for each treatment.
-10
ii) Relative Water Content (RWC) was calculated according to
excised leaves
glass test t u b e
\
r e d u c t ion adapter
l
t
p,ast,c
Fig. 1. Sealed glass test tube used as leaf chamber (x ~)
A
paper a n d when they had reached the desired degree of water stress they were inserted into a glass test tube (150 m m in length, 29 m m diameter) possessing a ground-glass opening. A reductive adapter (Quickfit 24/29; 14/23) was inserted a n d the whole system (vol. 70 ml) sealed with a ' suba-seal' plastic bung (size 30) (Fig. 1). All ground glass surfaces were greased to prevent gas leakage. Controls of non-stressed leaves were included in each e x p e r i m e n t and these usually possessed ~bj~arvalues between - 6 and - 7 bars. The sealed tubes were returned to the growth room and rotated in a klinostat (1 rpm) under subdued lighting. Gas samples (2 ml) were taken from each tube for ethylene analysis by G L C as required. After gas sampling 2 ml of clean air was injected into the 'leaf c h a m b e r s ' to restore the atmospheric pressure. W h e n the final gas sample had been taken the leaves were re-weighed to check for any loss during the period in the klinostat. Five leaves were removed from each batch for leaf water potential (Oleaf) measurements in a pressure chamber, these leaves were returned to the remainder prior to extracting for A B A (i.e. in some experiments).
the formula below. Turgid weight was found by placing the cut ends of three 2 g batches of leaves in small beakers containing water. The beakers were enclosed in a h u m i d atmosphere at room temperature for 6 h. The submerged ends of the leaves were blotted rapidly and thoroughly before weighing. The leaves were then transferred to an oven at 120 ~ for 48 h and their dry weight was determined. The mean R W C value for the three replicates was found.
2 -8 o
-6
-4
100
95
90
85
RWC
'
I
I
I
1%
2
4
6
8
10 wt loss
(i.e,~mid l i g h t period)
Fig. 2. Relationship between leaf water potential values and (i) relative water content (RWC), and (ii) % fresh wt loss, in excised wheat leaves taken from plants growing under our conditions
fresh
RWC =
Fresh or stressed w t - d r y wt Turgid w t - d r y wt
iii) Percentage Fresh Weight Loss was used as a quick and convenient m e t h o d of estimating the degree of water stress in excised leaves when setting up an experiment (see General technique). At the end of the experiment Olo~f values were measured and
S.T.C. Wright: Leaf Water Potential, Ethylene, and ABA these values are given with the % flesh weight losses in some experiments. The relationship between 0~f values and the other two methods of estimating water stress under our conditions are shown in Figure 2. Control leaves (i.e. 0% fresh wt loss) usually possessed a 0~,af value of about -6.5 bars which corresponds to a RWC of 96%.
Extraction, Purification and Bioassay Procedures for EndogenousAbscisic Acid (ABA) The leaves from two ethylene replicates (i.e. equivalent to 8 g of original leaves) were cut into 3 mm sections and placed in 200 ml ether for 24 h at 5~ The ether was decanted into a separating funnel and any aqueous phase discarded. The ether extract was shaken with three 20 ml vols of 2 per cent sodium bicarbonate solution. The bulked bicarbonate extract was acidified with N HC1 to pH 2.5 and shaken with two volumes of 200 ml ether. The bulked ether extract was dried with anhydrous sodium sulphate and taken to dryness. The extract containing 'free' ABA was stored at 2~. The extracts were further purified on 0.25 mm thick fluorescent silica-gel coated TLC plates developed in a mixture of n-butanol, n-propanol, 0.88 ammonia and water (2:6:1:2 v/v) and the zone on each plate corresponding to ABA marker spots (i.e. quenched spots under UV) was scraped off and eluted with methanol. A drop of plant extract was added to each marker spot since the Rf value of ABA was reduced by certain components in the extracts. Each eluate was reapplied to another TLC plate which was developed 6 times in a mixture of chloroform, benzene, and acetic acid (100:100:1.5 v/v). The zone in the ABA region was eluted again. The eluate was then applied to a paper chromatogram (Whatman No. 1) which was developed in an ascending manner in a mixture of isopropanol, 0.88 ammonia and water (10:1:1 v/v) allowed to run 20.5 cm from the origin. After drying overnight the chromatograms were divided into 21 horizontal sections, but only those sections corresponding to the ABA zone (Re0.4 0.7) were bioassayed. Each section was placed in a plastic dish containing 2 ml distilled water and bioassayed in the wheat coleoptile straight growth test (Wright, 1969). The growth inhibiting activity per segment was converted into gg equivalents of ABA using a dosage response curve of (+)-ABA.
Results
Ethylene Evolution in Relation to Leaf Water Potential (Oleaf) of Excised Wheat Leaves Duplicate 4 g batches of excised wheat leaves were allowed to wilt u n t i l they had lost 2 to 8% of their fresh weight a n d t h e n inserted into leaf c h a m b e r s (Fig. 1). T h e sealed c h a m b e r s were r e t u r n e d to the g r o w t h r o o m a n d r o t a t e d in a klinostat (1 rpm). T w o c o n t r o l tubes c o n t a i n i n g non-stressed leaves were also included. G a s samples (2 ml) were t a k e n for ethylene analysis every 135 m i n for the first 675 rain a n d then a final s a m p l i n g at 24 h. Ethylene biosynthesis was greatest in the severely stressed leaves (i.e. 8% fresh wt l o s s - e q u i v a l e n t to a 0~eaf of -- 1 1.5 bars) (Fig. 3), a n d repeat experiments
185 200
I.-~
8%
150
100
so
9
-
:L
= 2% o%
0'
' 135
2 7' 0
' 405
5 4' 0
' 675
II
' 1440 min
Fig. 3. The effect of different levels of water stress-shown as percentage fresh weight loss-on ethylene diffusion from excised wheat leaves
have s h o w n a Ole,f o f a b o u t - 12 bars is o p t i m a l for ethylene biosynthesis in excised wheat leaves. After a period of a b o u t 11 h (i.e. 675 m i n in Fig. 3) the ethylene c o n c e n t r a t i o n in the leaf c h a m b e r s levelled off. Since a n a d e q u a t e supply o f oxygen is essential for ethylene biosynthesis (e.g. Burg a n d T h i m a n n , 1959) i t was possible that the levelling off was due to a r e d u c t i o n in the oxygen levels in the leaf chambers. This hypothesis was tested in the following experiment.
The Effect on Total Ethylene Evolution of Transferring Water-stressed Leaves to Leaf Chambers Containing Clean Air After Each Ethylene Sampling (i.e. Every 135 min) Batches of leaves (4 g) were stressed to a ~bleaf of - 1 2 bars (approx. 9 % fresh wt loss) a n d placed in leaf chambers. Three tubes were left sealed a n d ethylene samples t a k e n every 135 m i n as in the previous e x p e r i m e n t (Fig. 4, curve A). A n o t h e r set of 3 leaf c h a m b e r s were o p e n e d after each ethylene s a m p l i n g a n d the leaves quickly spread o u t o n a sheet of glass to release a n y t r a p p e d ethylene a n d t h e n r e t u r n e d i m m e d i a t e l y to clean leaf c h a m b e r s (Fig. 4, histog r a m B1). I n F i g u r e 4, curve B2 is a s u m m a t i o n curve m a d e u p f r o m levels f o u n d in B1. F o r the first 405 m i n (curves A a n d B2) there was n o difference between the treatments, b u t b e y o n d this p o i n t there was a suggestion ( a n d this was b o r n e out in repeat experiments), that m o r e ethylene was p r o d u c e d by the leaves transferred to clean air every 135 min. This c o u l d have been due to the extra available oxygen, b u t equally m a y have been caused by a slight increase
186
S.T.C. Wright: Leaf Water Potential, Ethylene, and ABA
in @leafimposed inadvertently during leaf transfer every 135 min. Some of the increase would certainly be accounted for from ethylene trapped in the internal leaf spaces diffusing into the leaf chamber. As a precautionary measure leaves were transferred to clean tubes every 135 min when experiments were to run longer than 405 rain. At this stag e we were ready to carry out the next experiment which was the main objective of this study.
200 B2
15o
~> loo
~o
t~
Planta 134, 183-189 (1977)
9 by Springer-Verlag 1977
The Relationship between Leaf Water Potential (~leaf) and the Levels of Abscisic Acid and Ethylene in Excised Wheat Leaves S.T.C. Wright Agricultural Research Council Plant Growth Substance and Systemic Fungicide Unit, Wye College, Near Ashford, Kent TN25 5AH, U.K.
Abstract. The amount of diffusible ethylene from excised wheat leaves (Triticurn aestivurn L. cv. Eclipse) increased when they were subjected to water stress. The quantity of ethylene produced was related to the severity of the stress, reaching a maximum at a leaf water potential (@leaf) of approximately - 1 2 bars. Irrespective of the severity of the stress, the maximum rate of ethylene production usually occurred between 135-270min after applying the stress and then the rate declined. Part of the decline may have been due to an oxygen deficiency in the leaf chambers. In excised water-stressed leaves there was a sigmoid relationship between increasing ethylene and abscisic acid (ABA) levels and decreasing leaf water potential values. The two curves were displaced from each other by approximately 1 bar, with ethylene evolution leading that of ABA accumulation. The maximum rate of increase in ethylene occurred between - 8 and - 9 bars and for ABA between - 9 9and - 10 bars. A significant increase in the levels of these two plant growth regulators was found when the @le,f decreased outside the normal diurnal @leaf range by 1 bar for ethylene and 2 bars for ABA. Because of the sigmoid nature of the curves there was no distinct threshold @leaf value triggering-off an increase in ethylene or ABA, but with ABA the curve became very steep at a @leaf value of - 9.3 bars and this could be looked upon as a kind of " t h r e s h o l d " value. It seems unlikely that the stress-induced ethylene evolution in excised wheat leaves stimulated the accumulation of ABA, because when the leaves were subjected to a substantial water stress (e.g. @leaf= -- 12 bars) ABA increased immediately and at a faster rate than ethylene. 9 ABA=abscisic acid, GLC=gas-liquid chromatography, RWC=relative water content, TLC=thin-layer chromatography, Oleaf=leaf water potential Abbreviations.
Key words: Abscisic acid - Ethylene - Triticum Water potential - Water stress.
Introduction When plants are subjected to drought or waterlogging, major changes occur in hormone levels (see reviews by Livne and Vaadia, 1972 and Hsiao, 1973). The resulting hormonal imbalance is believed to be responsible for the onset of such stress symptoms as growth inhibition, partial or complete stomatal closure, petiole epinasty, increased root permeability, senescence of lower leaves, etc. These physiological changes help to maintain leaf water potential (i.e. a function of cell turgidity in the leaf) and thereby increase the plant's chances of survival. One hormone which is particularly sensitive to changes in leaf turgor is abscisic acid (ABA) (Wright and Hiron, 1969). Data collected by Zabadal (1974) suggested there might be a threshold leaf water potential which triggers-off ABA biosynthesis (and/or accumulation) under water stress conditions. In his experiments Ambrosia plants were exposed to a desiccating environment and throughout a 23 h period the changes in the levels of ABA and @leaf were determined. The results showed that the ABA accumulating system was "switched-on" in the critical - 1 0 to - 1 2 bars range and then ABA accumulated at a steady rate. Threshold values of @leaf below which ABA levels begin to increase have also been given by other workers; this value was - 8 bars for maize and between - 8 and - 10 bars for sorghum (Beardsell and Cohen, 1975). In contrast many of our own results indicate a more gradual increase in ABA levels with the onset of stress (Wright, 1972) and therefore we postulated that the sudden "switch-on" found
184
S.T.C. Wright: Leaf Water Potential, Ethylene, and A B A
by Zabadal could have been due to harsh experimental treatment. For this reason we have re-investigated this phenomenon using a milder experimental approach. Furthermore, since ethylene biosynthesis is known to be promoted by water stress (e.g. Ben-Yehoshua and Aloni, 1974; E1-Beltagy and Hall, 1974) we included ethylene determinations in our experiments to see if its biosynthesis is induced over a similar ~t~af range as ABA.
Materials and Methods Plant Culture Wheat (Triticum aestivum L. cv. Eclipse) grains were soaked in distilled water for 2 h prior to sowing. The grains were sown with their embryos uppermost in rows 1.5 x 1.5 cm on vermiculite contained in plastic seed boxes (26 x 16 x 5.5 cm). They were covered with 1-cm layer o f vermiculite and the surface pressed firm with a board. Seedlings were grown in a growth r o o m under a 14-h photoperiod, 8.6 klx from Atlas 'daylight' fluorescent tubes, at 23 ~ (night temp. 19 ~ and with a m i n i m u m relative humidity of 70%. Primary leaves (12-13 em in length) were excised above t h e coleoptiles in the middle of the 8th light period and trimmed to 11 cm.
Estimation of Ethylene Gas
General Technique
Ethylene was estimated by gas-liquid c h r o m a t o g r a p h y (GLC) using a Pye 104 dual column c h r o m a t o g r a p h with flame ionisation detectors. Gas samples were injected.on to an alumina column (150 cm long; 5 m m diameter) at 112 ~ with a nitrogen flow rate of 40 ml/ min, giving a retention time for ethylene of approximately 2 rain. K n o w n volumes of a standard gas mixture (10.4 vpm) were used to allow conversion of ethylene peak areas on the c h r o m a t o g r a m s to absolute gas values.
Batches of leaves (4 g) were allowed to wilt at r o o m temperature (normally about 20 ~) by spreading them out on sheets of filter
Methods for Measuring Water Stress
-12
i) Leaf Water Potential (tPleaf) values were measured in a pressure b o m b (based on Scholander et al., 1965) using oxygen-free nitrogen as a gas-pressure source. Primary leaves, which had been excised just above the coleoptile and their inner leaves removed, were threaded singly into the leaf clamping device of the pressure chamber so that the cut end protruded to the outside. The pressure of the gas in the chamber was increased at a constant rate to reach a value corresponding to - 6 bars in about 60 s. The pressure when sap first wetted the cut surface of the leaf, as observed through a low-powered binocular microscope, was taken as the end point. A m e a n of 5 readings was obtained for each treatment.
-10
ii) Relative Water Content (RWC) was calculated according to
excised leaves
glass test t u b e
\
r e d u c t ion adapter
l
t
p,ast,c
Fig. 1. Sealed glass test tube used as leaf chamber (x ~)
A
paper a n d when they had reached the desired degree of water stress they were inserted into a glass test tube (150 m m in length, 29 m m diameter) possessing a ground-glass opening. A reductive adapter (Quickfit 24/29; 14/23) was inserted a n d the whole system (vol. 70 ml) sealed with a ' suba-seal' plastic bung (size 30) (Fig. 1). All ground glass surfaces were greased to prevent gas leakage. Controls of non-stressed leaves were included in each e x p e r i m e n t and these usually possessed ~bj~arvalues between - 6 and - 7 bars. The sealed tubes were returned to the growth room and rotated in a klinostat (1 rpm) under subdued lighting. Gas samples (2 ml) were taken from each tube for ethylene analysis by G L C as required. After gas sampling 2 ml of clean air was injected into the 'leaf c h a m b e r s ' to restore the atmospheric pressure. W h e n the final gas sample had been taken the leaves were re-weighed to check for any loss during the period in the klinostat. Five leaves were removed from each batch for leaf water potential (Oleaf) measurements in a pressure chamber, these leaves were returned to the remainder prior to extracting for A B A (i.e. in some experiments).
the formula below. Turgid weight was found by placing the cut ends of three 2 g batches of leaves in small beakers containing water. The beakers were enclosed in a h u m i d atmosphere at room temperature for 6 h. The submerged ends of the leaves were blotted rapidly and thoroughly before weighing. The leaves were then transferred to an oven at 120 ~ for 48 h and their dry weight was determined. The mean R W C value for the three replicates was found.
2 -8 o
-6
-4
100
95
90
85
RWC
'
I
I
I
1%
2
4
6
8
10 wt loss
(i.e,~mid l i g h t period)
Fig. 2. Relationship between leaf water potential values and (i) relative water content (RWC), and (ii) % fresh wt loss, in excised wheat leaves taken from plants growing under our conditions
fresh
RWC =
Fresh or stressed w t - d r y wt Turgid w t - d r y wt
iii) Percentage Fresh Weight Loss was used as a quick and convenient m e t h o d of estimating the degree of water stress in excised leaves when setting up an experiment (see General technique). At the end of the experiment Olo~f values were measured and
S.T.C. Wright: Leaf Water Potential, Ethylene, and ABA these values are given with the % flesh weight losses in some experiments. The relationship between 0~f values and the other two methods of estimating water stress under our conditions are shown in Figure 2. Control leaves (i.e. 0% fresh wt loss) usually possessed a 0~,af value of about -6.5 bars which corresponds to a RWC of 96%.
Extraction, Purification and Bioassay Procedures for EndogenousAbscisic Acid (ABA) The leaves from two ethylene replicates (i.e. equivalent to 8 g of original leaves) were cut into 3 mm sections and placed in 200 ml ether for 24 h at 5~ The ether was decanted into a separating funnel and any aqueous phase discarded. The ether extract was shaken with three 20 ml vols of 2 per cent sodium bicarbonate solution. The bulked bicarbonate extract was acidified with N HC1 to pH 2.5 and shaken with two volumes of 200 ml ether. The bulked ether extract was dried with anhydrous sodium sulphate and taken to dryness. The extract containing 'free' ABA was stored at 2~. The extracts were further purified on 0.25 mm thick fluorescent silica-gel coated TLC plates developed in a mixture of n-butanol, n-propanol, 0.88 ammonia and water (2:6:1:2 v/v) and the zone on each plate corresponding to ABA marker spots (i.e. quenched spots under UV) was scraped off and eluted with methanol. A drop of plant extract was added to each marker spot since the Rf value of ABA was reduced by certain components in the extracts. Each eluate was reapplied to another TLC plate which was developed 6 times in a mixture of chloroform, benzene, and acetic acid (100:100:1.5 v/v). The zone in the ABA region was eluted again. The eluate was then applied to a paper chromatogram (Whatman No. 1) which was developed in an ascending manner in a mixture of isopropanol, 0.88 ammonia and water (10:1:1 v/v) allowed to run 20.5 cm from the origin. After drying overnight the chromatograms were divided into 21 horizontal sections, but only those sections corresponding to the ABA zone (Re0.4 0.7) were bioassayed. Each section was placed in a plastic dish containing 2 ml distilled water and bioassayed in the wheat coleoptile straight growth test (Wright, 1969). The growth inhibiting activity per segment was converted into gg equivalents of ABA using a dosage response curve of (+)-ABA.
Results
Ethylene Evolution in Relation to Leaf Water Potential (Oleaf) of Excised Wheat Leaves Duplicate 4 g batches of excised wheat leaves were allowed to wilt u n t i l they had lost 2 to 8% of their fresh weight a n d t h e n inserted into leaf c h a m b e r s (Fig. 1). T h e sealed c h a m b e r s were r e t u r n e d to the g r o w t h r o o m a n d r o t a t e d in a klinostat (1 rpm). T w o c o n t r o l tubes c o n t a i n i n g non-stressed leaves were also included. G a s samples (2 ml) were t a k e n for ethylene analysis every 135 m i n for the first 675 rain a n d then a final s a m p l i n g at 24 h. Ethylene biosynthesis was greatest in the severely stressed leaves (i.e. 8% fresh wt l o s s - e q u i v a l e n t to a 0~eaf of -- 1 1.5 bars) (Fig. 3), a n d repeat experiments
185 200
I.-~
8%
150
100
so
9
-
:L
= 2% o%
0'
' 135
2 7' 0
' 405
5 4' 0
' 675
II
' 1440 min
Fig. 3. The effect of different levels of water stress-shown as percentage fresh weight loss-on ethylene diffusion from excised wheat leaves
have s h o w n a Ole,f o f a b o u t - 12 bars is o p t i m a l for ethylene biosynthesis in excised wheat leaves. After a period of a b o u t 11 h (i.e. 675 m i n in Fig. 3) the ethylene c o n c e n t r a t i o n in the leaf c h a m b e r s levelled off. Since a n a d e q u a t e supply o f oxygen is essential for ethylene biosynthesis (e.g. Burg a n d T h i m a n n , 1959) i t was possible that the levelling off was due to a r e d u c t i o n in the oxygen levels in the leaf chambers. This hypothesis was tested in the following experiment.
The Effect on Total Ethylene Evolution of Transferring Water-stressed Leaves to Leaf Chambers Containing Clean Air After Each Ethylene Sampling (i.e. Every 135 min) Batches of leaves (4 g) were stressed to a ~bleaf of - 1 2 bars (approx. 9 % fresh wt loss) a n d placed in leaf chambers. Three tubes were left sealed a n d ethylene samples t a k e n every 135 m i n as in the previous e x p e r i m e n t (Fig. 4, curve A). A n o t h e r set of 3 leaf c h a m b e r s were o p e n e d after each ethylene s a m p l i n g a n d the leaves quickly spread o u t o n a sheet of glass to release a n y t r a p p e d ethylene a n d t h e n r e t u r n e d i m m e d i a t e l y to clean leaf c h a m b e r s (Fig. 4, histog r a m B1). I n F i g u r e 4, curve B2 is a s u m m a t i o n curve m a d e u p f r o m levels f o u n d in B1. F o r the first 405 m i n (curves A a n d B2) there was n o difference between the treatments, b u t b e y o n d this p o i n t there was a suggestion ( a n d this was b o r n e out in repeat experiments), that m o r e ethylene was p r o d u c e d by the leaves transferred to clean air every 135 min. This c o u l d have been due to the extra available oxygen, b u t equally m a y have been caused by a slight increase
186
S.T.C. Wright: Leaf Water Potential, Ethylene, and ABA
in @leafimposed inadvertently during leaf transfer every 135 min. Some of the increase would certainly be accounted for from ethylene trapped in the internal leaf spaces diffusing into the leaf chamber. As a precautionary measure leaves were transferred to clean tubes every 135 min when experiments were to run longer than 405 rain. At this stag e we were ready to carry out the next experiment which was the main objective of this study.
200 B2
15o
~> loo
~o
t~
