Planta (Berl.) 123, 399--302 (1975) 9 by Springer-Verlag 1975
Phytochrome Mediated Changes in Extractable Gibberellin Activity in a Cell-free System from Etiolated Wheat Leaves * R. J. Cooke** a n d P. F. Saunders Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA Received 8 February; accepted 14 February 1975 S u m m a r y . Irradiation with red light causes a phytochrome dependent increase in the extractable amount of acidic gibberellin-like activity in plastid preparations from etiolated leaves of Triticum aestivum L.
Introduction I r r a d i a t i o n of sections of etiolated barley or wheat leaves with red light leads to a rapid rise in the level of extractable gibberellin-like (GA-like) substances detected b y bioassay (Reid et al., 1968; Loveys a n d Wareing, 1971). A similar response to red light has been observed in homogenates of barley leaves (Reid et al., 1972). I n the course of further studies on the photocontrol of gibberellin levels in wheat leaves, we have examined the effect of red light on cell-free systems o b t a i n e d from etiolated leaf sections. I n i t i a l experiments with crude homogenates indicated t h a t a n increase in extractable GA-like a c t i v i t y could be o b t a i n e d b y irradiation of a l O00x g fraction o b t a i n e d b y differential centrifugation. This fraction could be expected to consist principally of plastids a n d accordingly it was decided to investigate the effect of red light on a more highly purified prep a r a t i o n of i n t a c t etioplasts.
Materials and Methods Wheat fruits (Tritieum aestivum L. var. "Kolibri") were soaked for 4 h in rtmni~g tap water, sown in moist vermiculite and left in the dark at 20 ~ C for 7 days, with daily watering. An etioplast preparation was obtained from homogenates of 8 cm segments of first leaf tissue by using a loosely packed Sephadex G-50 column as described by Wellburn and Wellburn (1971). Homogenisation was achieved using six 10 see bursts at top speed in an "Ato-mix" blender and the homogenate was filtered through 6 layers of muslin. Otherwise the methods used were exactly as published. All operations were performed under a dim green safe-light. A portion of the eluate from the Sephadex G-50 column was monitored photometrically at 257 nm and the fractions expected on the basis of published evidence to consist predominantly of etioplasts were bulked. A sample of the bulked fractions was examined by phase contrast microscopy: the majority of the plastids had an opaque appearance indicating that they were structurally intact (Kahn and von Wettstein, 1961), and very little contaminating material was apparent. The light sources used for irradiation were as described by Beevers et al. (1970), and van Staden and Wareing (1972). Immediately after irradiation of the plastid preparations, suf* Abbreviation: G A = gibberellin ** Present address: Departmertt of Plant Biology, Universil~y of Newcastle, Newcastle-
upon-Tyne, England.
300
1~. J. Cooke and P. F. Saunders h (dark)
~ k0
Rf
B { red)
1,(~
0
Rf
Fig. 1A and B. Lettuce hypoeotyl assays of thin layer ehromatograms of the acidic, ethyl acetate soluble fractions of etioplast preparations obtained from wheat leaves. The equivalent of 40 g fresh weight was chromatographed in each case. (A) Dark control; (B) irradiated for 20 rain with red light. Shaded portions of the histograms represent significant promotion a~, the 1% level of probability
ficient ice-cold methanol to give a final concentration of 80 % was added. The resultant extracts were stirred for 1 h in a cold room at 5 ~ C and then the methanol was removed on a rotary evaporator at 35 ~ C in vacuo. A n acidic, ethyl acetate-soluble fraction was obtained from each of the aqueous residues using standard techniques (see, for example, Loveys and Wareing, 1971) and ehromatographed on 0.25 mm layers of Kieselgel GF~ 4 in chloroform/ethyl acetate/ acetic acid (50:50:1; v/v/v). After being thoroughly dried, chromatograms were divided into 10 l~f zones and each zone was tested using a lettuce hypocotyl bioassay (Frarddand and Wareing 1960). Results Fig. 1 illustrates t h e effect of 20 m i n of r e d light on t h e levei of e x t r a c t a b l e acidic gibberellin-like substances p r e s e n t in a p r e p a r a t i o n o b t a i n e d from 40 g of leaf sections. T h e n a t u r e of this e x p e r i m e n t p r e c l u d e d s i m u l t a n e o u s r e p l i c a t i o n ; it has, however, been r e p e a t e d on n u m e r o u s s e p a r a t e occasions with essentially i d e n t i c a l results. Clearly, r e d light brings a b o u t a consistent increase in t h e level of t w o c h r o m a t o g r a p h i c a l l y s e p a r a b l e p e a k s of OA-like a c t i v i t y . The R I of these peaks, on t h i n l a y e r a n d on p a p e r c h r o m a t o g r a m s is similar to t h e Rf of t h e GAlike c o m p o u n d s which a p p e a r following red i r r a d i a t i o n of i n t a c t leaf sections. F u r t h e r e x p e r i m e n t s showed t h a t 5 m i n of r e d light, followed b y 15 m i n i n c u b a t i o n in t h e d a r k , has a similar p r o m o t i v e effect on t h e e x t r a c t a b l e G A levels. If, however, t h e 5 min r e d light t r e a t m e n t is followed b y 15 min of far-red i r r a d i a t i o n , t h e n t h e level of GA-like s u b s t a n c e s as m e a s u r e d b y b i o a s s a y is m a r k e d l y r e d u c e d while f a r - r e d light given alone has no a p p r e c i a b l e effect (Fig. 2). On t h e basis of this degree of r e d / f a r - r e d r e v e r s i b i l i t y it is reasonable to suppose t h a t p h y t o c h r o m e is t h e p h o t o r e c e p t o r involved. I n view of these o b s e r v a t i o n s it is p r o p o s e d t h a t t h e e t i o p l a s t is t h e site of t h e r e d light i n d u c e d increase in e x t r a c t a b l e gibberellin a c t i v i t y which has been observed in w h e a t leaf sections. O t h e r workers h a v e d e m o n s t r a t e d a n association b e t w e e n GA-like substances a n d chloroplasts ( S t o d d a r t , 1968; 1969; R a i l t o n a n d W a r e i n g , 1973; F r y d m a n a n d W a r e i n g , 1973; R a i l t o n a n d Reid, 1974). F u r t h e r m o r e , t h e r e is evidence from o t h e r sources to suggest t h a t p h y t o c h r o m e is associated with p l a s t i d s (e.g. W e l l b u r n a n d W e l l b u r n , 1973; P r a t t a n d Colem a n , 1974). T h e relevance of t h e p r e s e n t w o r k to t h e d e v e l o p m e n t a l changes i n d u c e d b y r e d light in w h e a t leaves will be discussed in a further, m o r e d e t a i l e d
Phytochrome Mediated Changes in Gibberellin A (dark)
301
B(5R, 15D)
E
v
Rf
-~3
1.0
C(5R, 15FR)
()
Rf
D (15 FR)
o
[3. >.
:x2
I
Fig. 2A--D. As Fig. 1: (A) Dark control; (B) 5 min red light + 15 rain dark; (C) 5 rain red light + 15 min far red light; (D) 15 min far red light
publication. Regardless of their physiological significance the experiments described here are of considerable i m p o r t a n c e in t h a t t h e y d e m o n s t r a t e a rapid p h y t o c h r o m e m e d i a t e d response in a well-defined cell free system. As such, t h e y could open the way to detailed studies on the biochemistry of p b y t o c h r o m e action. R. J. C. acknowledges the receipt of a Research Studentship from the Science Research Council.
References Beevers, L., Loveys, B.R., Pearson, J.A., Wareing, P . F . : Phytochrome and hormonal control of expansion and greening of etiolated wheat leaves. Planta (Berl.) 90, 286 294 (1970) Frankland, B., Wareing, P . F . : Effect of gibberellic acid on hypocotyl growth of lettuce seedlings. Nature (Lond.) 185, 255-256 (1960) Frydman, V. M., Wareing, P. F. : Phase change in Hedera helix L. I. Gibberellin-likes:lbstances in the growth phase. J. exp. Bot. 24, 1131-1138 (1973) Kahn, A., Wettstein, D. von: Macromolecular physiology of plastids. II. Structure of isolated spinach chloroplasts. J. Ultrastruct. Res. 5, 557-574 (1961) Loveys, B. R., Wareing, P. F. : The red light controlled production of gibberellin in etiolated wheat leaves. Planta (Berl.) 98, 109 116 (1971) Pratt, H. H., Coleman, H. A. : Phytochrome distribution in etiolated grass seedlings as assayed by an indirect antibody-labellingmethod. Amer. J. Bot. 61, 195 202 (1974) l~ailton, I. D., Reid, D.M.: Studies on gibberellins in shoots of light-grown peas. I. A reevaluation of the data. P1, Sci. Lett. 2,157-163 (1974) Railton, I. D., Wareing, P. F. : Effects of daylength on endogenous gibberellins in Solanum andigena. I. Changes in levels of free acidic gibberellin-likesubstances in the leaves. Physiol. Plant. (Cph.) 28, 88-94 (1973) Reid, D.M., Clements, J.B., Carr, D . J . : Red light induction of gibberellin synthesis in leaves. Nature (Lond.) 217, 580-582 (1968) Reid, D. M., Tuing, M. S., Durley, 1~. C., Railton, I. D. : Red light enhanced conversion of tritiated gibberellin A9 into other gibberellin-like substances in homogenates of etiolated barley leaves. Planta (Berl.) 108, 67-75 (1972)
302
R . J . Cooke and P. F. Saunders
Staden, J. van, Wareing, P. F. : The effect of light on endogenous cytokinin levels in seeds of R u m e x obtusi/olius. Planta (Berl.) 104, 126-133 (1972) Stoddart, J. L. : The association of gibberellin-like activity with the chloroplast fraction of leaf homogenates. Planta (Berl.) 81,106-112 (1968) Stoddart, J. L. : Incorporation of kaurenoic acid into gibberellins by chloroplast preparations of Brassica oleracea. Phytochem. 8, 831-837 (1969) Wellburn, A. R., Wcllburn, A. F. M. : A new method for the isolation of etioplasts with intact membranes. J. exp. Bot. 23, 972-979 (1971) Wellburn, F. A. M., Wellburn, A. R. : l~esponse of etioplasts in situ and in isolated suspensions to pre-illumination with various combinations of red, far-red and blue light. New Phytol. 72, 55-60 (1973)
Phytochrome Mediated Changes in Extractable Gibberellin Activity in a Cell-free System from Etiolated Wheat Leaves * R. J. Cooke** a n d P. F. Saunders Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA Received 8 February; accepted 14 February 1975 S u m m a r y . Irradiation with red light causes a phytochrome dependent increase in the extractable amount of acidic gibberellin-like activity in plastid preparations from etiolated leaves of Triticum aestivum L.
Introduction I r r a d i a t i o n of sections of etiolated barley or wheat leaves with red light leads to a rapid rise in the level of extractable gibberellin-like (GA-like) substances detected b y bioassay (Reid et al., 1968; Loveys a n d Wareing, 1971). A similar response to red light has been observed in homogenates of barley leaves (Reid et al., 1972). I n the course of further studies on the photocontrol of gibberellin levels in wheat leaves, we have examined the effect of red light on cell-free systems o b t a i n e d from etiolated leaf sections. I n i t i a l experiments with crude homogenates indicated t h a t a n increase in extractable GA-like a c t i v i t y could be o b t a i n e d b y irradiation of a l O00x g fraction o b t a i n e d b y differential centrifugation. This fraction could be expected to consist principally of plastids a n d accordingly it was decided to investigate the effect of red light on a more highly purified prep a r a t i o n of i n t a c t etioplasts.
Materials and Methods Wheat fruits (Tritieum aestivum L. var. "Kolibri") were soaked for 4 h in rtmni~g tap water, sown in moist vermiculite and left in the dark at 20 ~ C for 7 days, with daily watering. An etioplast preparation was obtained from homogenates of 8 cm segments of first leaf tissue by using a loosely packed Sephadex G-50 column as described by Wellburn and Wellburn (1971). Homogenisation was achieved using six 10 see bursts at top speed in an "Ato-mix" blender and the homogenate was filtered through 6 layers of muslin. Otherwise the methods used were exactly as published. All operations were performed under a dim green safe-light. A portion of the eluate from the Sephadex G-50 column was monitored photometrically at 257 nm and the fractions expected on the basis of published evidence to consist predominantly of etioplasts were bulked. A sample of the bulked fractions was examined by phase contrast microscopy: the majority of the plastids had an opaque appearance indicating that they were structurally intact (Kahn and von Wettstein, 1961), and very little contaminating material was apparent. The light sources used for irradiation were as described by Beevers et al. (1970), and van Staden and Wareing (1972). Immediately after irradiation of the plastid preparations, suf* Abbreviation: G A = gibberellin ** Present address: Departmertt of Plant Biology, Universil~y of Newcastle, Newcastle-
upon-Tyne, England.
300
1~. J. Cooke and P. F. Saunders h (dark)
~ k0
Rf
B { red)
1,(~
0
Rf
Fig. 1A and B. Lettuce hypoeotyl assays of thin layer ehromatograms of the acidic, ethyl acetate soluble fractions of etioplast preparations obtained from wheat leaves. The equivalent of 40 g fresh weight was chromatographed in each case. (A) Dark control; (B) irradiated for 20 rain with red light. Shaded portions of the histograms represent significant promotion a~, the 1% level of probability
ficient ice-cold methanol to give a final concentration of 80 % was added. The resultant extracts were stirred for 1 h in a cold room at 5 ~ C and then the methanol was removed on a rotary evaporator at 35 ~ C in vacuo. A n acidic, ethyl acetate-soluble fraction was obtained from each of the aqueous residues using standard techniques (see, for example, Loveys and Wareing, 1971) and ehromatographed on 0.25 mm layers of Kieselgel GF~ 4 in chloroform/ethyl acetate/ acetic acid (50:50:1; v/v/v). After being thoroughly dried, chromatograms were divided into 10 l~f zones and each zone was tested using a lettuce hypocotyl bioassay (Frarddand and Wareing 1960). Results Fig. 1 illustrates t h e effect of 20 m i n of r e d light on t h e levei of e x t r a c t a b l e acidic gibberellin-like substances p r e s e n t in a p r e p a r a t i o n o b t a i n e d from 40 g of leaf sections. T h e n a t u r e of this e x p e r i m e n t p r e c l u d e d s i m u l t a n e o u s r e p l i c a t i o n ; it has, however, been r e p e a t e d on n u m e r o u s s e p a r a t e occasions with essentially i d e n t i c a l results. Clearly, r e d light brings a b o u t a consistent increase in t h e level of t w o c h r o m a t o g r a p h i c a l l y s e p a r a b l e p e a k s of OA-like a c t i v i t y . The R I of these peaks, on t h i n l a y e r a n d on p a p e r c h r o m a t o g r a m s is similar to t h e Rf of t h e GAlike c o m p o u n d s which a p p e a r following red i r r a d i a t i o n of i n t a c t leaf sections. F u r t h e r e x p e r i m e n t s showed t h a t 5 m i n of r e d light, followed b y 15 m i n i n c u b a t i o n in t h e d a r k , has a similar p r o m o t i v e effect on t h e e x t r a c t a b l e G A levels. If, however, t h e 5 min r e d light t r e a t m e n t is followed b y 15 min of far-red i r r a d i a t i o n , t h e n t h e level of GA-like s u b s t a n c e s as m e a s u r e d b y b i o a s s a y is m a r k e d l y r e d u c e d while f a r - r e d light given alone has no a p p r e c i a b l e effect (Fig. 2). On t h e basis of this degree of r e d / f a r - r e d r e v e r s i b i l i t y it is reasonable to suppose t h a t p h y t o c h r o m e is t h e p h o t o r e c e p t o r involved. I n view of these o b s e r v a t i o n s it is p r o p o s e d t h a t t h e e t i o p l a s t is t h e site of t h e r e d light i n d u c e d increase in e x t r a c t a b l e gibberellin a c t i v i t y which has been observed in w h e a t leaf sections. O t h e r workers h a v e d e m o n s t r a t e d a n association b e t w e e n GA-like substances a n d chloroplasts ( S t o d d a r t , 1968; 1969; R a i l t o n a n d W a r e i n g , 1973; F r y d m a n a n d W a r e i n g , 1973; R a i l t o n a n d Reid, 1974). F u r t h e r m o r e , t h e r e is evidence from o t h e r sources to suggest t h a t p h y t o c h r o m e is associated with p l a s t i d s (e.g. W e l l b u r n a n d W e l l b u r n , 1973; P r a t t a n d Colem a n , 1974). T h e relevance of t h e p r e s e n t w o r k to t h e d e v e l o p m e n t a l changes i n d u c e d b y r e d light in w h e a t leaves will be discussed in a further, m o r e d e t a i l e d
Phytochrome Mediated Changes in Gibberellin A (dark)
301
B(5R, 15D)
E
v
Rf
-~3
1.0
C(5R, 15FR)
()
Rf
D (15 FR)
o
[3. >.
:x2
I
Fig. 2A--D. As Fig. 1: (A) Dark control; (B) 5 min red light + 15 rain dark; (C) 5 rain red light + 15 min far red light; (D) 15 min far red light
publication. Regardless of their physiological significance the experiments described here are of considerable i m p o r t a n c e in t h a t t h e y d e m o n s t r a t e a rapid p h y t o c h r o m e m e d i a t e d response in a well-defined cell free system. As such, t h e y could open the way to detailed studies on the biochemistry of p b y t o c h r o m e action. R. J. C. acknowledges the receipt of a Research Studentship from the Science Research Council.
References Beevers, L., Loveys, B.R., Pearson, J.A., Wareing, P . F . : Phytochrome and hormonal control of expansion and greening of etiolated wheat leaves. Planta (Berl.) 90, 286 294 (1970) Frankland, B., Wareing, P . F . : Effect of gibberellic acid on hypocotyl growth of lettuce seedlings. Nature (Lond.) 185, 255-256 (1960) Frydman, V. M., Wareing, P. F. : Phase change in Hedera helix L. I. Gibberellin-likes:lbstances in the growth phase. J. exp. Bot. 24, 1131-1138 (1973) Kahn, A., Wettstein, D. von: Macromolecular physiology of plastids. II. Structure of isolated spinach chloroplasts. J. Ultrastruct. Res. 5, 557-574 (1961) Loveys, B. R., Wareing, P. F. : The red light controlled production of gibberellin in etiolated wheat leaves. Planta (Berl.) 98, 109 116 (1971) Pratt, H. H., Coleman, H. A. : Phytochrome distribution in etiolated grass seedlings as assayed by an indirect antibody-labellingmethod. Amer. J. Bot. 61, 195 202 (1974) l~ailton, I. D., Reid, D.M.: Studies on gibberellins in shoots of light-grown peas. I. A reevaluation of the data. P1, Sci. Lett. 2,157-163 (1974) Railton, I. D., Wareing, P. F. : Effects of daylength on endogenous gibberellins in Solanum andigena. I. Changes in levels of free acidic gibberellin-likesubstances in the leaves. Physiol. Plant. (Cph.) 28, 88-94 (1973) Reid, D.M., Clements, J.B., Carr, D . J . : Red light induction of gibberellin synthesis in leaves. Nature (Lond.) 217, 580-582 (1968) Reid, D. M., Tuing, M. S., Durley, 1~. C., Railton, I. D. : Red light enhanced conversion of tritiated gibberellin A9 into other gibberellin-like substances in homogenates of etiolated barley leaves. Planta (Berl.) 108, 67-75 (1972)
302
R . J . Cooke and P. F. Saunders
Staden, J. van, Wareing, P. F. : The effect of light on endogenous cytokinin levels in seeds of R u m e x obtusi/olius. Planta (Berl.) 104, 126-133 (1972) Stoddart, J. L. : The association of gibberellin-like activity with the chloroplast fraction of leaf homogenates. Planta (Berl.) 81,106-112 (1968) Stoddart, J. L. : Incorporation of kaurenoic acid into gibberellins by chloroplast preparations of Brassica oleracea. Phytochem. 8, 831-837 (1969) Wellburn, A. R., Wcllburn, A. F. M. : A new method for the isolation of etioplasts with intact membranes. J. exp. Bot. 23, 972-979 (1971) Wellburn, F. A. M., Wellburn, A. R. : l~esponse of etioplasts in situ and in isolated suspensions to pre-illumination with various combinations of red, far-red and blue light. New Phytol. 72, 55-60 (1973)
