Planta (Berl.) 93, 309--313 (1970) 9 by Springer-Verlag 1970
Polysomal Changes in Developing Wheat Leaves J. A. PEAI~SON a n d P. F. WAltZInG Department of Botany, University College of Wales, Aberystwyth, Wales (U.K) tZeceived May 26 / June 15, 1970
Summary. When leaf sections of 7-day old dark grown wheat leaves were incubated in white light, they unrolled and greened. Gibberellie acid was able to replace the light requirement and abscisic acid (ABA) inhibited the response to light. The percentage of ribosomes occurring as polysomes increased in response to light but not in response to GA3 treatment. Although ABA inhibited the unrolling and greening in light, it did not cause a preferential decrease or inhibition of polysome formation. Introduction I r r a d i a t i o n of e t i o l a t e d leaf tissue w i t h either white light or a short p e r i o d of red light results in m a r k e d morphological a n d biochemical changes. Some of these changes occur even if t h e leaves are p u t b a c k into t o t a l d a r k n e s s i m m e d i a t e l y after t h e light t r e a t m e n t . Changes o b s e r v e d include unrolling of w h e a t leaves (Virgin, 1962), increased s t a r c h d e g r a d a t i o n in maize (Klein et aI., 1963) a n d increased soluble R N A p o l y m e r a s e a c t i v i t y ( S t o u t et al., 1964). F o l l o w i n g i l l u m i n a t i o n w i t h r e d or white light W i l l i a m s et al. (1968) o b s e r v e d a n increase in t h e percentage of ribosomes occurring as polysomes in maize a n d b e a n leaves. I t has been shown t h a t b o t h GA~ a n d k i n e t i n can s u b s t i t u t e for t h e light t r e a t m e n t in t h e unrolling of w h e a t leaves a n d t h a t abseisie acid (ABA) will i n h i b i t t h e light response (Beevers et al., 1970). Since light has been shown to increase p o l y s o m e s (Wiiliams et al., 1968) t h e effects of G A 3 a n d A B A on t h e p o l y s o m e c o n t e n t of e t i o l a t e d w h e a t leaves were i n v e s t i g a t e d to d e t e r m i n e w h e t h e r a similar specific change occurred in response to these compounds.
Materials and Methods Wheat seedlings (var. "Atle") were grown for 7 days in the dark at 25~ as described previously (Beevers et al., 1970). 3 cm leaf segments were excised 1 em from the tip of the leaves and were floated in petri dishes on either water or test solution. The dishes were kept either in the light or dark. Unrolling and chlorophyll contents were measured as described (Beevers et al., 1970). Polysomes were extracted and charaeterised from 5 g of leaf segments as described by Pearson (1969). The percentage of ribosomes occurring as polysomes is expressed as "percentage of polysomes' '.
310
J. A. Pearson and P. F. Wareing: Results and Discussion
W h e n t h e leaf segments were f l o a t e d on w a t e r in continuous white light for 24 hours, t h e r e was a m a r k e d increase in t h e i r width. Also n o t e d was a n increase in t h e chlorophyll c o n t e n t (Table 1).
Table 1. E//ect o] white light on the unrolling o/etiolated wheat lea/sections Treatment
Leaf width (mm • 4)
Chlorophyll (A 665 nm)
Initial Dark 24 hours Light 24 hours
3.80 4.90 7.70
0.10 0.10 0.41
2 replicate treatments of 10 leaf segments were floated on 10 ml of water in the light or dark at 25~ for 24 hours prior to leaf width measurement.
The increase in w i d t h is similar to t h a t r e p o r t e d b y Beevers et al. (1970), when t h e leaf segments were i l l u m i n a t e d for 10 m i n with red light followed b y darkness. Comparison of t h e p o l y s o m a l profiles, on sucrose d e n s i t y gradients, from leaf tissue i n c u b a t e d in t h e light or t h e d a r k reveals a larger p e r c e n t a g e of polysomes in t h e light t r e a t e d m a t e r i a l (Fig. 1). The " p e r centage of p o l y s o m e s " increases from 22 to 66%. This m a y r e p r e s e n t t h e synthesis of new R N A species or some c o m p o n e n t necessary for p o l y s o m e assembly. Similar increases in response to light h a v e been o b s e r v e d b y Clark et al. (1964) a n d W i l l i a m s et al. (1968). As can be seen from Table 2, i n c u b a t i o n of t h e leaf sections in light on A B A solutions resulted in a n i n h i b i t i o n of unrolling a n d of chlorophyll synthesis. Despite this i n h i b i t i o n of unrolling, t h e " p e r c e n t a g e of polysomes" from light control a n d A B A t r e a t e d leaf segments was essentially Table 2. E//ect o/ abscisie acid on the unrolling o/ etiolated wheat leaves in response to white light ABA concentration (M)
Leaf width (mm • 4)
Chlorophyll (A 665 nm)
0 1.9 • 10-5 3.8 • l0 -5 7.6 • 10 5
8.40 7.34 5.34 4.90
0.41 0.32 0.30 0.27
2 replicate treatments of 10 leaf sections were incubated on the appropriate solution for 24 hours in white light at 25~
Polysomal Changes in Wheat Leaves
311
1.0[
&254nmI
051
/
0
t
Bottom
--Sedimentation
Top
Fig. 1. Sucrose density gradient profile of wheat leaf polysomes. Tissue was incubated on water at 25~C in the light or dark for 24 hours after which time polysomes were extracted, light treated; ...... dark treated
the same (Fig. 2). This indicates t h a t although there was a decrease in the yield of ribosomes from the A B A treated material, there was no differential effect on polysome formation. I t would seem unlikely then t h a t A B A inhibits the unrolling of wheat leaves b y a preferential inhibition of m-I~NA synthesis as seen b y a decrease in "percentage of polysome". W h e n the leaf sections were incubated for 24 hours in total darkness on GA 3 solutions t h e y unrolled (Table 3). Analysis of the polysomal component of the tissue, however, revealed no preferential increase in polysomes (Fig. 3). Thus, it appears t h a t a large preferential increase in the synthesis of m - R N A is not required for the wheat leaf unrolling in response to GA s . Nucleic acid and protein synthesis appears to be involved in the unrolling response as is seen b y the inhibition of the process b y Aetinomyein D, 6-methylpurine and eycloheximide (unpublished results). However, the specificity of these inhibitors is open to discussion (Ellis et al., 1969). F r o m the above results it would appear t h a t although the unrolling of
J. A. Pearson and P. F. Wareing:
312
1.0
A25hnrn
0.5
0
Bottom
--,-Sedimentotion
Top
Fig. 2. Sucrose density gradient profile of wheat leaf polysomes. Tissue was incubated for 24 hours at 25 ~C. - dark control; ....... light incubated on water ; ..... light incubated on 3.8 x 10-s M ABA
Table 3. Effect o/ GA 3 on the unrolling o/ wheat lea] sections in total darkness GA a concentration (M)
Leaf width (ram x 4)
Chlorophyll (A 665 nm)
0 2.9 x 10 -7 2.9 • 10 s 1.45 x 10-5 2.9 x 10-5
6.35 7.40 7.90 8.40 7.80
0.11 0.10 0.10 0.11 0.11
2 replicate treatments of 10 segments were incubated on 10 ml of appropriate solution in total darkness at 25~ for 24 hours prior to measurement of width.
w h e a t l e a v e s i n r e s p o n s e t o l i g h t is a s s o c i a t e d w i t h a n i n c r e a s e i n p o l y s o m e s , n o s u c h i n c r e a s e is s e e n i n t h e d a r k i n r e s p o n s e t o G A 3. T h e u n r o l l i n g r e s p o n s e m a y n o t r e q u i r e t h e s y n t h e s i s of m - R N A . H o w e v e r , i t is p o s s i b l e t h a t o n l y a v e r y s m a l l p r o p o r t i o n of m-1%NA s y n t h e s i s is a l t e r e d a n d t h a t t h i s c h a n g e is o u t s i d e t h e s e n s i t i v i t y of d e t e c t i o n b y sucrose density gradient analysis.
Polysomal Changes in Wheat Leaves 2.75
313 A
1.01
f 'A254nm i i
0.5~11
I
-~-Sedimentation
TopBottom ~Sedimentation
.Top
Fig. 3. Sucrose density gradient profile of wheat leaf polysomes. Tissue was incubated in total darkness for 24 hours at 25~C. A water control; B incubated on 1.45 • 10-5 M GA3
References Beevers, L., Loveys, B., Pearson, J. A., Wareing, P. F. : Pytochrome and hormonal control of expansion and greening of etiolated wheat leaves. Planta (Berl.) 90, 286--294 (1970). Clark, M. F., Matthews, R. E. F., Ralph, R. K. : Ribosomes and polyribosomes in Brassica pelcinensis. Biochim. biophys. Acta (Amst.) 91, 284 (1964). Klein, W.H., Price, L., Mitrakos, K.: Light stimulated starch degradation in plastids and leaf morphogenesis. Photochem. and Photobiol. 2, 233--240 (1963). MacDonald, I. R., Ellis, R. J. : Does cycloheximide inhibit protein synthesis specifically in plant tissues ? Nature (Lond.) 222, 791 (1969). Pearson, J. A. : Isolation and characterisation of polysomes and polysomal RNA from radish leaves. Exp. Cell Res. ~7, 235--239 (1969). Stout, E. R., Parenti, R., Mans, 1%. J.: An increase in RNA polymerase activity after illumination of dark grown maize seedlings. Biochem. biophys. Res. Commun. 29, 322 (1967). Virgin, H. I. : Light induced unfolding of the grass leaf. Physiol. Plant. (Copenh.) 15, 380--389 (1962). Williams, G. R., Novelli, G. D. : Ribosome changes following illumination of darkgrown plants. Biochim. biophys. Acta (Amst.) 155, 183--192 (1968). Dr. J. A. Pearson Department of Botany University College of Wales Aberystwyth, Wales, U.K.
Polysomal Changes in Developing Wheat Leaves J. A. PEAI~SON a n d P. F. WAltZInG Department of Botany, University College of Wales, Aberystwyth, Wales (U.K) tZeceived May 26 / June 15, 1970
Summary. When leaf sections of 7-day old dark grown wheat leaves were incubated in white light, they unrolled and greened. Gibberellie acid was able to replace the light requirement and abscisic acid (ABA) inhibited the response to light. The percentage of ribosomes occurring as polysomes increased in response to light but not in response to GA3 treatment. Although ABA inhibited the unrolling and greening in light, it did not cause a preferential decrease or inhibition of polysome formation. Introduction I r r a d i a t i o n of e t i o l a t e d leaf tissue w i t h either white light or a short p e r i o d of red light results in m a r k e d morphological a n d biochemical changes. Some of these changes occur even if t h e leaves are p u t b a c k into t o t a l d a r k n e s s i m m e d i a t e l y after t h e light t r e a t m e n t . Changes o b s e r v e d include unrolling of w h e a t leaves (Virgin, 1962), increased s t a r c h d e g r a d a t i o n in maize (Klein et aI., 1963) a n d increased soluble R N A p o l y m e r a s e a c t i v i t y ( S t o u t et al., 1964). F o l l o w i n g i l l u m i n a t i o n w i t h r e d or white light W i l l i a m s et al. (1968) o b s e r v e d a n increase in t h e percentage of ribosomes occurring as polysomes in maize a n d b e a n leaves. I t has been shown t h a t b o t h GA~ a n d k i n e t i n can s u b s t i t u t e for t h e light t r e a t m e n t in t h e unrolling of w h e a t leaves a n d t h a t abseisie acid (ABA) will i n h i b i t t h e light response (Beevers et al., 1970). Since light has been shown to increase p o l y s o m e s (Wiiliams et al., 1968) t h e effects of G A 3 a n d A B A on t h e p o l y s o m e c o n t e n t of e t i o l a t e d w h e a t leaves were i n v e s t i g a t e d to d e t e r m i n e w h e t h e r a similar specific change occurred in response to these compounds.
Materials and Methods Wheat seedlings (var. "Atle") were grown for 7 days in the dark at 25~ as described previously (Beevers et al., 1970). 3 cm leaf segments were excised 1 em from the tip of the leaves and were floated in petri dishes on either water or test solution. The dishes were kept either in the light or dark. Unrolling and chlorophyll contents were measured as described (Beevers et al., 1970). Polysomes were extracted and charaeterised from 5 g of leaf segments as described by Pearson (1969). The percentage of ribosomes occurring as polysomes is expressed as "percentage of polysomes' '.
310
J. A. Pearson and P. F. Wareing: Results and Discussion
W h e n t h e leaf segments were f l o a t e d on w a t e r in continuous white light for 24 hours, t h e r e was a m a r k e d increase in t h e i r width. Also n o t e d was a n increase in t h e chlorophyll c o n t e n t (Table 1).
Table 1. E//ect o] white light on the unrolling o/etiolated wheat lea/sections Treatment
Leaf width (mm • 4)
Chlorophyll (A 665 nm)
Initial Dark 24 hours Light 24 hours
3.80 4.90 7.70
0.10 0.10 0.41
2 replicate treatments of 10 leaf segments were floated on 10 ml of water in the light or dark at 25~ for 24 hours prior to leaf width measurement.
The increase in w i d t h is similar to t h a t r e p o r t e d b y Beevers et al. (1970), when t h e leaf segments were i l l u m i n a t e d for 10 m i n with red light followed b y darkness. Comparison of t h e p o l y s o m a l profiles, on sucrose d e n s i t y gradients, from leaf tissue i n c u b a t e d in t h e light or t h e d a r k reveals a larger p e r c e n t a g e of polysomes in t h e light t r e a t e d m a t e r i a l (Fig. 1). The " p e r centage of p o l y s o m e s " increases from 22 to 66%. This m a y r e p r e s e n t t h e synthesis of new R N A species or some c o m p o n e n t necessary for p o l y s o m e assembly. Similar increases in response to light h a v e been o b s e r v e d b y Clark et al. (1964) a n d W i l l i a m s et al. (1968). As can be seen from Table 2, i n c u b a t i o n of t h e leaf sections in light on A B A solutions resulted in a n i n h i b i t i o n of unrolling a n d of chlorophyll synthesis. Despite this i n h i b i t i o n of unrolling, t h e " p e r c e n t a g e of polysomes" from light control a n d A B A t r e a t e d leaf segments was essentially Table 2. E//ect o/ abscisie acid on the unrolling o/ etiolated wheat leaves in response to white light ABA concentration (M)
Leaf width (mm • 4)
Chlorophyll (A 665 nm)
0 1.9 • 10-5 3.8 • l0 -5 7.6 • 10 5
8.40 7.34 5.34 4.90
0.41 0.32 0.30 0.27
2 replicate treatments of 10 leaf sections were incubated on the appropriate solution for 24 hours in white light at 25~
Polysomal Changes in Wheat Leaves
311
1.0[
&254nmI
051
/
0
t
Bottom
--Sedimentation
Top
Fig. 1. Sucrose density gradient profile of wheat leaf polysomes. Tissue was incubated on water at 25~C in the light or dark for 24 hours after which time polysomes were extracted, light treated; ...... dark treated
the same (Fig. 2). This indicates t h a t although there was a decrease in the yield of ribosomes from the A B A treated material, there was no differential effect on polysome formation. I t would seem unlikely then t h a t A B A inhibits the unrolling of wheat leaves b y a preferential inhibition of m-I~NA synthesis as seen b y a decrease in "percentage of polysome". W h e n the leaf sections were incubated for 24 hours in total darkness on GA 3 solutions t h e y unrolled (Table 3). Analysis of the polysomal component of the tissue, however, revealed no preferential increase in polysomes (Fig. 3). Thus, it appears t h a t a large preferential increase in the synthesis of m - R N A is not required for the wheat leaf unrolling in response to GA s . Nucleic acid and protein synthesis appears to be involved in the unrolling response as is seen b y the inhibition of the process b y Aetinomyein D, 6-methylpurine and eycloheximide (unpublished results). However, the specificity of these inhibitors is open to discussion (Ellis et al., 1969). F r o m the above results it would appear t h a t although the unrolling of
J. A. Pearson and P. F. Wareing:
312
1.0
A25hnrn
0.5
0
Bottom
--,-Sedimentotion
Top
Fig. 2. Sucrose density gradient profile of wheat leaf polysomes. Tissue was incubated for 24 hours at 25 ~C. - dark control; ....... light incubated on water ; ..... light incubated on 3.8 x 10-s M ABA
Table 3. Effect o/ GA 3 on the unrolling o/ wheat lea] sections in total darkness GA a concentration (M)
Leaf width (ram x 4)
Chlorophyll (A 665 nm)
0 2.9 x 10 -7 2.9 • 10 s 1.45 x 10-5 2.9 x 10-5
6.35 7.40 7.90 8.40 7.80
0.11 0.10 0.10 0.11 0.11
2 replicate treatments of 10 segments were incubated on 10 ml of appropriate solution in total darkness at 25~ for 24 hours prior to measurement of width.
w h e a t l e a v e s i n r e s p o n s e t o l i g h t is a s s o c i a t e d w i t h a n i n c r e a s e i n p o l y s o m e s , n o s u c h i n c r e a s e is s e e n i n t h e d a r k i n r e s p o n s e t o G A 3. T h e u n r o l l i n g r e s p o n s e m a y n o t r e q u i r e t h e s y n t h e s i s of m - R N A . H o w e v e r , i t is p o s s i b l e t h a t o n l y a v e r y s m a l l p r o p o r t i o n of m-1%NA s y n t h e s i s is a l t e r e d a n d t h a t t h i s c h a n g e is o u t s i d e t h e s e n s i t i v i t y of d e t e c t i o n b y sucrose density gradient analysis.
Polysomal Changes in Wheat Leaves 2.75
313 A
1.01
f 'A254nm i i
0.5~11
I
-~-Sedimentation
TopBottom ~Sedimentation
.Top
Fig. 3. Sucrose density gradient profile of wheat leaf polysomes. Tissue was incubated in total darkness for 24 hours at 25~C. A water control; B incubated on 1.45 • 10-5 M GA3
References Beevers, L., Loveys, B., Pearson, J. A., Wareing, P. F. : Pytochrome and hormonal control of expansion and greening of etiolated wheat leaves. Planta (Berl.) 90, 286--294 (1970). Clark, M. F., Matthews, R. E. F., Ralph, R. K. : Ribosomes and polyribosomes in Brassica pelcinensis. Biochim. biophys. Acta (Amst.) 91, 284 (1964). Klein, W.H., Price, L., Mitrakos, K.: Light stimulated starch degradation in plastids and leaf morphogenesis. Photochem. and Photobiol. 2, 233--240 (1963). MacDonald, I. R., Ellis, R. J. : Does cycloheximide inhibit protein synthesis specifically in plant tissues ? Nature (Lond.) 222, 791 (1969). Pearson, J. A. : Isolation and characterisation of polysomes and polysomal RNA from radish leaves. Exp. Cell Res. ~7, 235--239 (1969). Stout, E. R., Parenti, R., Mans, 1%. J.: An increase in RNA polymerase activity after illumination of dark grown maize seedlings. Biochem. biophys. Res. Commun. 29, 322 (1967). Virgin, H. I. : Light induced unfolding of the grass leaf. Physiol. Plant. (Copenh.) 15, 380--389 (1962). Williams, G. R., Novelli, G. D. : Ribosome changes following illumination of darkgrown plants. Biochim. biophys. Acta (Amst.) 155, 183--192 (1968). Dr. J. A. Pearson Department of Botany University College of Wales Aberystwyth, Wales, U.K.
