Planta
Planta 135, 51-55 (1977)
9 by Springer-Verlag 1977
The Role of Cotyledons in Phototropism of De-etiolated Seedlings J a n e t E . S h u t t l e w o r t h a n d M. Black Department of Biology, Queen Elizabeth College (University of London), Campden Hill Road, London W8 7AH, U.K.
Abstract. S i m u l a t e d " p h o t o t r o p i c " c u r v a t u r e s caused by differential m a s k i n g o f the c o t y l e d o n s o f de-etiol a t e d seedlings e x p o s e d to white light are u n c o n n e c t e d with true p h o t o t r o p i s m . I n C u c u m i s s a t i v u s L. a n d H e l i a n t h u s a n n u u s L. such c u r v a t u r e s result f r o m a r e d - l i g h t - i n d u c e d i n h i b i t i o n c o m i n g f r o m the e x p o s e d c o t y l e d o n . T r u e p h o t o t r o p i c b e n d i n g in these species u n d e r l o n g - t e r m e x p o s u r e to fairly high i r r a d i a n c e s (as in n a t u r e ) i s a r e s p o n s e to blue light. It occurs even w h e n c o t y l e d o n s are c o m p l e t e l y covered. These results show t h a t the c o t y l e d o n s do n o t perceive the p h o t o t r o p i c stimulus a n d need n o t be i l l u m i n a t e d for p h o t o t r o p i s m to occur. K e y words: Blue light -- C o t y l e d o n s - - C u c u m i s H e l i a n t h u s - - P h o t o t r o p i s m -- P h y t o c h r o m e .
Introduction T h e b e n d i n g t o w a r d s u n i l a t e r a l light e x h i b i t e d by green seedlings in n a t u r e is m o s t p r o b a b l y the " s e c o n d p o s i t i v e " c u r v a t u r e o f p h o t o t r o p i s m (Everett, 1974; Steyer, 1967); t h a t is, it is the response to relatively high energy levels o f light received for p r o l o n g e d p e r i o d s o f time. It has been a s s e r t e d t h a t in this p h e n o m e n o n the c o t y l e d o n s or leaves either perceive the u n i l a t e r a l stimulus ( S h i b a o k a a n d Yam a k i , 1959; L a i n a n d L e o p o l d , 1966) o r at least m u s t be i l l u m i n a t e d for the b e n d i n g to occur ( B r u i n s m a et al., 1975). O n the o t h e r h a n d , o t h e r evidence is a g a i n s t such a p e r c e p t i v e role for the c o t y l e d o n s or leaves ( M o h r a n d Peters, 1960; B r e n n a n a n d G u n c k e l , 1976). W e h a v e s h o w n t h a t in d e - e t i o l a t e d C u c u m i s seedlings the c o t y l e d o n s are i n d e e d i n v o l v e d in p h o t o m o r p h o g e n e t i c c o n t r o l o f h y p o c o t y l extension (Black a n d S h u t t l e w o r t h , 1974). It seemed, however, t h a t only
r e d light o p e r a t i n g t h r o u g h the p h y t o c h r o m e system is active a n d t h a t the effects o f blue light are n o t t r a n s m i t t e d f r o m c o t y l e d o n s to h y p o c o t y l . It b e c a m e i m p o r t a n t , therefore, to c o n s i d e r these findings in the c o n t e x t o f the claims m a d e for the i n v o l v e m e n t o f the c o t y l e d o n s in seedling p h o t o t r o p i s m . W e n o w rep o r t investigations o n this p r o b l e m c a r r i e d out on C u c u m i s s a t i v u s (cucumber) a n d H e l i a n t h u s a n n u u s (sunflower).
Materials and Methods Cucumber seeds (Cucumis sativus L. cv. Ridge Long Green) were obtained from Suttons Seeds, Reading, U.K. They were imbibed for 4 h in distilled water, rinsed 3 times and sown in wet vermiculite to germinate and grow in darkness for 31/2 days at 26~177176 Etiolated seedlings 3.5 cm high were selected and transferred to fresh vermiculite. They were exposed to white fluorescent light (5.0 W m -2) at 26~177176 for 30 h when they had become characteristically de-etiolated with expanded cotyledons but no visible growth of the primary leaves. Sunflower seeds (Helianthus annuus L. cv. Tall Single) were from Dobie's Seed Company, Chester, U.K. They were imbibed in distilled water for 24 h, rinsed 3 times and sown in wet vermiculite. They were germinated and grown in darkness at 26~177176 for 3 days when they reached 4.0 to 5.0 cm high. The coats were removed and the seedlings transplanted in fresh, wet vermiculite and de-etiolated at 26~176 for 2 days under a daily regime of 14 h white fluorescent light (5.0 W m -2) and 10 h darkness. Seedlings then had expanded cotyledons and no visible primary leaf growth. In the experiments described below cucumber seedlings were used immediately following the 30 h de-etiolating light treatment; sunflower seedlings were used 5 h after the end of the second dark period. For experiments with unilateral light de-etiolated seedlings (5 per container) were transplanted in a line along the long edge of a small plastic tray (13.0 x 8.5 cm and 6.0 cm deep) containing wet vermiculite. Unilateral light was obtained through an opening (7.0 cm wide and 38.0 cm long) in one side of an otherwise lighttight box lined with non-reflecting black cloth. Two trays were held in here with seedlings aligned such that the mid-veins of the cotyledons wereat right angles to the incident light, rendering one cotyledon of each pair closer to the light source than the
52
J.E. Shuttleworth and M. Black: Role of Cotyledons in Phototropism
other. Unilateral white light was obtained from one Atlas "White" 20-watt fluorescent tube placed horizontally in front of the box opening. The system was maintained at 26~ 0.1~ and 60% relative humidity in a controlled environment chamber. Unilateral red and blue light were obtained using a similar system held in cabinets with constant temperature (26~176 but with no controlled relative humidity. Here, a 15-watt Atlas "Grolux" fluorescent tube covered with 2 layers of Cinemoid filter no. 14 (ruby red) transmitting from 600 to 700 nm, peaking at 660 nm (half-band width: approx. 25 nm) was used horizontally for unilateral red light. For unilateral blue a 15-watt Atlas "Tropical Daylight" fluorescent tube covered with 2 layers of Cinemoid filter no. 20 (deep blue) transmitting from 400-510 nm (peak 440 nm; half-band width: approx. 45 nm) was used. Where necessary, cotyledons were completely shielded from irradiation with aluminium foil envelopes. Curvatures were determined by measuring the angle between the upper and lower regions
of the hypocotyl with a protractor. This was done after removing the seedling from the vermiculite in cases where a single measurement was to be made or in situ as in time-course experiments. Experimental conditions for seedlings held in diffuse (nonunilateral) light were as described above except that light-tight boxes were not used and irradiation was from above. Each experiment never consisted of less than 10 seedlings, and was repeated several times.
Experimental Results and Discussion Curvature Induced by a Cotyledon It has been shown that covering one c o t y l e d o n of Helianthus annuus seedlings, p r e s u m a b l y to simulate the differential i l l u m i n a t i o n in u n i l a t e r a l light, causes b e n d i n g of the hypocotyl away f r o m the d a r k e n e d side ( L a m a n d Leopold, 1966). C u r v a t u r e s of the hypocotyl can also be i n d u c e d in Cucumis by the same t e c h n i q u e (Fig. 1). This result was expected in view of o u r previous finding that a n i n h i b i t o r y effect of white light is t r a n s m i t t e d from the cotyledons to the h y p o c o t y l (Black a n d Shuttleworth, 1974). I n the present e x p e r i m e n t this i n h i b i t o r y influence is restricted to one side of the h y p o c o t y l - t h e side b e n e a t h the i l l u m i n a t e d cotyledon. T h e i n h i b i t i o n is due entirely to red light, for even at high energy levels of the blue w a v e b a n d n o h y p o c o t y l b e n d i n g is caused by decreased g r o w t h b e n e a t h the o n e lighted c o t y l e d o n (Fig. 2). O n the contrary, however, a c o t y l e d o n irradiated with blue light somewhat e n h a n c e s hypocotyl g r o w t h o n that side. Helianthus seedlings with one c o t y l e d o n exposed exhibit similar b e h a v i o u r towards
Fig. 1. Curvature induced in cucumber seedlings by darkening one cotyledon with aluminium foil- in continuous non-unilateral white light for 8 h
28
Ci
2A
!/t
oo 16 -~
c
-
~ 16-
/
~. i
~ - 8
8
3 -'~ o
"~
c
,rrodi . . . .
p'o
1s'.o
1:0 5:0 Irmdiance (Wm2/
16.0
~-A.
8 -" 0:5 1:o
0:5
3 -8: o
'
'
'
,60
-,2 i*/i
(WRY)
-4
-4.
-16
-8
-8
-20
l\#._.___,l" r T /'I ,*Ti/T.
l
i'll" l
Fig. 2. Dose-response relationship for curvature induced in cucumber seedlings by continuous non-unilateral irradiation (one cotyledon darkened) a white light, b red light, e blue light (bars represent +S.E.)
20
e White --x I
Planta 135, 51-55 (1977)
9 by Springer-Verlag 1977
The Role of Cotyledons in Phototropism of De-etiolated Seedlings J a n e t E . S h u t t l e w o r t h a n d M. Black Department of Biology, Queen Elizabeth College (University of London), Campden Hill Road, London W8 7AH, U.K.
Abstract. S i m u l a t e d " p h o t o t r o p i c " c u r v a t u r e s caused by differential m a s k i n g o f the c o t y l e d o n s o f de-etiol a t e d seedlings e x p o s e d to white light are u n c o n n e c t e d with true p h o t o t r o p i s m . I n C u c u m i s s a t i v u s L. a n d H e l i a n t h u s a n n u u s L. such c u r v a t u r e s result f r o m a r e d - l i g h t - i n d u c e d i n h i b i t i o n c o m i n g f r o m the e x p o s e d c o t y l e d o n . T r u e p h o t o t r o p i c b e n d i n g in these species u n d e r l o n g - t e r m e x p o s u r e to fairly high i r r a d i a n c e s (as in n a t u r e ) i s a r e s p o n s e to blue light. It occurs even w h e n c o t y l e d o n s are c o m p l e t e l y covered. These results show t h a t the c o t y l e d o n s do n o t perceive the p h o t o t r o p i c stimulus a n d need n o t be i l l u m i n a t e d for p h o t o t r o p i s m to occur. K e y words: Blue light -- C o t y l e d o n s - - C u c u m i s H e l i a n t h u s - - P h o t o t r o p i s m -- P h y t o c h r o m e .
Introduction T h e b e n d i n g t o w a r d s u n i l a t e r a l light e x h i b i t e d by green seedlings in n a t u r e is m o s t p r o b a b l y the " s e c o n d p o s i t i v e " c u r v a t u r e o f p h o t o t r o p i s m (Everett, 1974; Steyer, 1967); t h a t is, it is the response to relatively high energy levels o f light received for p r o l o n g e d p e r i o d s o f time. It has been a s s e r t e d t h a t in this p h e n o m e n o n the c o t y l e d o n s or leaves either perceive the u n i l a t e r a l stimulus ( S h i b a o k a a n d Yam a k i , 1959; L a i n a n d L e o p o l d , 1966) o r at least m u s t be i l l u m i n a t e d for the b e n d i n g to occur ( B r u i n s m a et al., 1975). O n the o t h e r h a n d , o t h e r evidence is a g a i n s t such a p e r c e p t i v e role for the c o t y l e d o n s or leaves ( M o h r a n d Peters, 1960; B r e n n a n a n d G u n c k e l , 1976). W e h a v e s h o w n t h a t in d e - e t i o l a t e d C u c u m i s seedlings the c o t y l e d o n s are i n d e e d i n v o l v e d in p h o t o m o r p h o g e n e t i c c o n t r o l o f h y p o c o t y l extension (Black a n d S h u t t l e w o r t h , 1974). It seemed, however, t h a t only
r e d light o p e r a t i n g t h r o u g h the p h y t o c h r o m e system is active a n d t h a t the effects o f blue light are n o t t r a n s m i t t e d f r o m c o t y l e d o n s to h y p o c o t y l . It b e c a m e i m p o r t a n t , therefore, to c o n s i d e r these findings in the c o n t e x t o f the claims m a d e for the i n v o l v e m e n t o f the c o t y l e d o n s in seedling p h o t o t r o p i s m . W e n o w rep o r t investigations o n this p r o b l e m c a r r i e d out on C u c u m i s s a t i v u s (cucumber) a n d H e l i a n t h u s a n n u u s (sunflower).
Materials and Methods Cucumber seeds (Cucumis sativus L. cv. Ridge Long Green) were obtained from Suttons Seeds, Reading, U.K. They were imbibed for 4 h in distilled water, rinsed 3 times and sown in wet vermiculite to germinate and grow in darkness for 31/2 days at 26~177176 Etiolated seedlings 3.5 cm high were selected and transferred to fresh vermiculite. They were exposed to white fluorescent light (5.0 W m -2) at 26~177176 for 30 h when they had become characteristically de-etiolated with expanded cotyledons but no visible growth of the primary leaves. Sunflower seeds (Helianthus annuus L. cv. Tall Single) were from Dobie's Seed Company, Chester, U.K. They were imbibed in distilled water for 24 h, rinsed 3 times and sown in wet vermiculite. They were germinated and grown in darkness at 26~177176 for 3 days when they reached 4.0 to 5.0 cm high. The coats were removed and the seedlings transplanted in fresh, wet vermiculite and de-etiolated at 26~176 for 2 days under a daily regime of 14 h white fluorescent light (5.0 W m -2) and 10 h darkness. Seedlings then had expanded cotyledons and no visible primary leaf growth. In the experiments described below cucumber seedlings were used immediately following the 30 h de-etiolating light treatment; sunflower seedlings were used 5 h after the end of the second dark period. For experiments with unilateral light de-etiolated seedlings (5 per container) were transplanted in a line along the long edge of a small plastic tray (13.0 x 8.5 cm and 6.0 cm deep) containing wet vermiculite. Unilateral light was obtained through an opening (7.0 cm wide and 38.0 cm long) in one side of an otherwise lighttight box lined with non-reflecting black cloth. Two trays were held in here with seedlings aligned such that the mid-veins of the cotyledons wereat right angles to the incident light, rendering one cotyledon of each pair closer to the light source than the
52
J.E. Shuttleworth and M. Black: Role of Cotyledons in Phototropism
other. Unilateral white light was obtained from one Atlas "White" 20-watt fluorescent tube placed horizontally in front of the box opening. The system was maintained at 26~ 0.1~ and 60% relative humidity in a controlled environment chamber. Unilateral red and blue light were obtained using a similar system held in cabinets with constant temperature (26~176 but with no controlled relative humidity. Here, a 15-watt Atlas "Grolux" fluorescent tube covered with 2 layers of Cinemoid filter no. 14 (ruby red) transmitting from 600 to 700 nm, peaking at 660 nm (half-band width: approx. 25 nm) was used horizontally for unilateral red light. For unilateral blue a 15-watt Atlas "Tropical Daylight" fluorescent tube covered with 2 layers of Cinemoid filter no. 20 (deep blue) transmitting from 400-510 nm (peak 440 nm; half-band width: approx. 45 nm) was used. Where necessary, cotyledons were completely shielded from irradiation with aluminium foil envelopes. Curvatures were determined by measuring the angle between the upper and lower regions
of the hypocotyl with a protractor. This was done after removing the seedling from the vermiculite in cases where a single measurement was to be made or in situ as in time-course experiments. Experimental conditions for seedlings held in diffuse (nonunilateral) light were as described above except that light-tight boxes were not used and irradiation was from above. Each experiment never consisted of less than 10 seedlings, and was repeated several times.
Experimental Results and Discussion Curvature Induced by a Cotyledon It has been shown that covering one c o t y l e d o n of Helianthus annuus seedlings, p r e s u m a b l y to simulate the differential i l l u m i n a t i o n in u n i l a t e r a l light, causes b e n d i n g of the hypocotyl away f r o m the d a r k e n e d side ( L a m a n d Leopold, 1966). C u r v a t u r e s of the hypocotyl can also be i n d u c e d in Cucumis by the same t e c h n i q u e (Fig. 1). This result was expected in view of o u r previous finding that a n i n h i b i t o r y effect of white light is t r a n s m i t t e d from the cotyledons to the h y p o c o t y l (Black a n d Shuttleworth, 1974). I n the present e x p e r i m e n t this i n h i b i t o r y influence is restricted to one side of the h y p o c o t y l - t h e side b e n e a t h the i l l u m i n a t e d cotyledon. T h e i n h i b i t i o n is due entirely to red light, for even at high energy levels of the blue w a v e b a n d n o h y p o c o t y l b e n d i n g is caused by decreased g r o w t h b e n e a t h the o n e lighted c o t y l e d o n (Fig. 2). O n the contrary, however, a c o t y l e d o n irradiated with blue light somewhat e n h a n c e s hypocotyl g r o w t h o n that side. Helianthus seedlings with one c o t y l e d o n exposed exhibit similar b e h a v i o u r towards
Fig. 1. Curvature induced in cucumber seedlings by darkening one cotyledon with aluminium foil- in continuous non-unilateral white light for 8 h
28
Ci
2A
!/t
oo 16 -~
c
-
~ 16-
/
~. i
~ - 8
8
3 -'~ o
"~
c
,rrodi . . . .
p'o
1s'.o
1:0 5:0 Irmdiance (Wm2/
16.0
~-A.
8 -" 0:5 1:o
0:5
3 -8: o
'
'
'
,60
-,2 i*/i
(WRY)
-4
-4.
-16
-8
-8
-20
l\#._.___,l" r T /'I ,*Ti/T.
l
i'll" l
Fig. 2. Dose-response relationship for curvature induced in cucumber seedlings by continuous non-unilateral irradiation (one cotyledon darkened) a white light, b red light, e blue light (bars represent +S.E.)
20
e White --x I
