Planta (Berl.) 113, 35--46 (1973) 9 by Springer-Verlag 1973
Synthesis and Release of Sucrose by the Aleurone Layer of Barley: Regulation by Gibberellic Acid Maarten J. Chrispeels, Andrea J. Tenner, and Kenneth D. Johnson Department of Biology, University of California/San Diego, La Jolla, California 92037, USA and Botany Department, California State University/San Diego, San Diego, California 92115, USA Received March 27, 1973
Summary. Aleurone layers of barley contain large amounts of a soluble oligosaccharide which was identified as sucrose (3040 ~g/mg fresh weight). Treatment of the layers with gibberellic acid (GAa) causes the release of sucrose from the cells. This release requires the participation of metabolic processes, including protein synthesis. When embryoless half-seeds are incubated sucrose accumulates in the aleurone layers, but when seeds are germinated the sucrose content of the aleurone layers declines. Labeling experiments with radioactive glucose and fructose show that Meurone layers continuously synthesize sucrose and that the release, but not the synthesis of sucrose is enhanced by GAa.
Introduction
The carbohydrate metabolism of germinating cereal grains has been studied quite extensively over the past 75 years. The endosperm contains alcohol-soluble, water-soluble and water-insoluble carbohydrates all of which are utilized during the growth of the seedling (for a review see MaeLeod, 1960). The breakdown of carbohydrates is in part controlled by the embryo and this control is mediated by gibberellins (GAs) (Yomo, 1960; Paleg, 1960) and in part by the build-up of soluble sugars within the endosperm (Armstrong and Jones, i972). The role of the seutellum involves the conversion of the end product of starch breakdown, glucose, into sucrose and transport of this sucrose to the growing axis (Edelman et al., 1959). Evidence is presented here which suggests that the aleurone layer may play a similar role in the conversion of glucose to sucrose. Materials and Methods Seed Preparation and Treatment. Aleurone layers from seeds of Hordeum vulgare L., cv. Himalaya, were prepared from embryo-less halfseeds imbibed for 3 d as described by Chrispeels and Varner (1967). Care was taken to remove all the starch and the layers were kept in a large beaker of cold, sterile water until enough layers had been prepared for the experiment. The layers were then rinsed 3*
36
M . J . Chrispeels et al. :
and incubated in 2 ml of autoclaved medium containing 2 mM acetate buffer (pH 4.8), 20 mM CaC12, 50 ~zg/ml of chloramphenicol, and with or without 2 tzM gibberellic acid (GA3), unless otherwise noted. Incubation routinely took place at 22 ~ I n some experiments half-seeds were incubated as described above and the aleurone layers were isolated from t h e m after the incubation. I n other experiments half-seeds were incubated on sand, and whole seeds were germinated in sterile, moist vermiculite at room temperature. Aleurone layers and endosperm (in the case of hMf-seeds) were isolated at various times after planting. Alcohol Extraction. After t r e a t m e n t the layers were rinsed 3 times with water, 5 ml of 80 % ethanol was added, and tissue was boiled for 4 rain in loosely-stoppered 25 ml flasks. The flasks were allowed to cool, the liquid was decanted, and the layers were rinsed with 2.5 ml of 80% ethanol. The initial extract and the wash were combined. (Alcoholic extraction involving homogenization of the tissue after boiling in ethanol did not result in a greater yield of sucrose). The ethanol was evaporated at 80 ~ under vacuum in a Rotary Evapomix (Buchler Corp., Fort Lee, N.J., U.S.A.) and the residue was t a k e n up in 6.0 ml of water. Starchy endosperms were treated similarly, b u t they were not rinsed with water prior to ethanol extraction. Determination o] Reducing Sugars. Reducing sugars present in the alcoholic tissue extract and the incubation medium were determined before and after mild acid hydrolysis (0.1 N HC1 for 10 rain at 100 ~ and/or before and after invertase t r e a t m e n t (0.1 mg/ml of invertase in 5 mM sodium acetate buffer, p H 4.8, incubated at 37 o for 10 min; invertase from Sigma Chemical Corp., St. Louis, Mo., USA) with the Nelson-Somogyi copper reagent as described b y Ashwell (1957). The results are expressed as glucose equivalents. Paper Chromatography. Sugars and oligosaccharides were separated b y descending paper chromatography on W h a t m a n n 3MM paper using either of the following solvents: ethyl acetate: pyridine: water (8: 2 : 1, v/v) r u n for 20 h or n-butanol: pyridine: water (10: 3 : 3, v/v) r u n for 5 days. After drying the sugars were detected by the alkaline silver-oxide reagent as described b y Menzies and Seakins (1969). The locations of the spots were compared with authentic standards. Labeling with Radioactive Sugars. Aleurone layers incubated with or without GA s for 18 h were rinsed and incubated with the same medium containing [14C]D-glucose (1 mCi/mg; :New England Nuclear Corp., Boston, l~ass., USA) or [laC]D-frnctese (1 mCi/1.5 rag; New England Nuclear Corp.) for 3 h. The incubation medium and a 3-ml rinse were pooled, evaporated to dryness and the residue was dissolved in 1.0 ml of water and analysed. The layers were rinsed several more times in ice-cold water, 5.0 ml of 80% ethanol was added, the tissue boiled for 4 min, and homogenized in the alcoholic extract. Homogenization was carried out with a power-driven conical ground-glass homogenizer. Alcohol-soluble and Mcoholinsoluble fractions were separated b y centrifugation (3 000 • g, 5 rain). The residue was extracted 3 times with 5 ml of 80% ethanol and the extracts combined with the initial supernatant. The Mcohol-soluble fraction was evaporated to dryness and then t a k e n up in 1.0 ml of water. Labeling of the alcohol-soluble metabolites in the tissue and of the metabolites in the original incubation medium was determined after their separation b y paper chromatography in the ethylacetate: pyridine: water solvent. The paper chromatograms were cut in 1-cm strips to determine the radioactivity in glucose, fructose, sucrose and metabolites which did not move from the origin (see J o h n s o n and Chrispeels, 1973). Incorporation of label into the alcoholinsoluble macromolecules was determined by mixing a small aliquot of the resuspended cellular material in 10.0 ml of Aquasol (New England Nuclear Corp.) and counting in a Beckman liquid scintillation spectrometer.
Sucrose Release by Aleurone: Regulation by GA3
37
400
< w ..1 W rr rr ,< (.9 D 59 -Z o D a U.I n-
200-
IO0 --
0
~
~'-CONTROL
I
I
I
I
6
12
18
24
HOURS
OF
INCUBATION
Fig. 1. Effect of GA3 on the release of reducing sugars from aleurone layers. Five aleurone layers/treatment were incubated for the times indicated. The media were collected and assayed for reducing sugars
Results and Discussion
The incubation of isolated aleurone layers from which all starch has been carefully removed resulted in a release of reducing sugars into the medium. The presence of GA s in the incubation medium caused a considerable enhancement of this release (Fig. 1). This observation resembles the initial observations of Paleg (1960) and Yomo (1960) made on embryo-less half seeds of barley. I n the presence of GA 3 as much as 400 Fg of reducing sugar (glucose equivalents) was released in a 24-h period. The aleurone layers contained only small amounts of reducing sugars (15-25 ~g/layer), suggesting that they contained other carbohydrates the release and/or the hydrolysis of which was enhanced by GA s . Paper chromatography of the sugars released by aleurone layers treated with GA 3 for 24 h demonstrated the presence of glucose and fructose and relatively smaller quantities of arabinose and xylose (Fig. 2). The medium also contained an ohgosaccharide which co-chromatographed with sucrose. A semi-quantitative estimation of the released reducing sugars based on the size of the chromatographic spots and the intensity of the color reaction showed that approximately equal quantities of glucose and fructose were released (data not shown). Chromatography of the medium 10 h after treatment with GA s showed the presence of reducing sugars (glucose and fructose) and a comparatively larger amount of the ohgosaceharide (Fig. 3). Mild acid hydrolysis (0.1 N tIC1 at 100 ~ for 10 rain) caused the oligosaccharide spot to disappear, with
38
M.J. Chrispeels et al. :
Fig. 2. Paper chromatography of sugars and oligosaccharides released from aleurone layers. The 24-h media (control and GA3) from the experiment described in Fig. 1 were ehromatographed in ethyl acetate: pyridine: water (8 : 2 : 1) together with appropriate standards (arabinose = A R A , xylose = X Y L , glucose = GLU, fructose = F R U , sucrose = SU). Note the difference in intensity of staining of glucose and fructose when an equimolar solution of these sugars was used as a standard. S T D standards
t h e simultaneous a p p e a r a n c e of glucose a n d fructose in e q u i v a l e n t a m o u n t s . P a p e r c h r o m a t o g r a p h y of an alcoholic e x t r a c t of t h e freshly isolated aleurone layers showed t h e presence of t h e same oligosaccharide which u p o n m i l d acid h y d r o l y s i s gave rise to glucose a n d fructose (Fig. 3). T h e following e x p e r i m e n t s were p e r f o r m e d to characterize t h e oligosaccharide: (1) T h e oligosaccharide c o - c h r o m a t o g r a p h e d with sucrose in e t h y l a c e t a t e : p y r i d i n e : w a t e r (8 : 2 : 1) ; u p o n m i l d acid t r e a t m e n t (0.1 N ttC1 for 10 rain a t 100 ~ t h e spot d i s a p p e a r e d w i t h the s i m u l t a n e o u s a p p e a r a n c e of e q u i v a l e n t a m o u n t s of reducing sugars which coc h r o m a t o g r a p h e d with glucose a n d fructose (Fig. 3). Sucrose is k n o w n
Sucrose Release by Aleurone: Regulation by GA3
39
Fig. 3. Effect of mild acid hydrolysis on the chromatographic pattern of the sugars present in the aleurone layers and the incubation medium. Alcoholic extracts of untreated aleurone layers and incubation media obtained after 10 h of incubation were treated as described and the sugars were chromatographed, before and after mild acid hydrolysis, in ethylacetate: pyridinc:water (8:2:1). A) medium after 10 h incubation, B) same medium hydrolysed in 0.1 N HC1 for 10 min. C) alcoholic extract of aleurone layers before incubation, D) same as C, after mild acid hydrolysis. Abbreviations as in Fig. 2
to be quite labile u n d e r m i l d acid conditions (Pazur, 1970). (2) I a c h r o m a t o g r a p h i c s y s t e m which s e p a r a t e s t h e various disaecharides ( n - b u t a n o l : p y r i d i n e : w a t e r 1 0 : 3 : 3 , r u n d e s e e n d i n g l y for 5 days) t h e oligosaceharide c o - e h r o m a t o g r a p h e d w i t h sucrose ( d a t a n o t shown). (3) I n v e r t a s e r a p i d l y h y d r o l y z e d t h e oligosaceharide, again p r o d u c i n g reducing sugars which e o - c h r o m a t o g r a p h e d w i t h glucose a n d fructose. T r e a t m e n t of alcoholic e x t r a c t s of t h e tissue w i t h either i n v e r t a s e or m i l d acid released t h e same a m o u n t of reducing sugar. W h e n t h e m e d i a
40
M.J. Chrispeels et I
3
I
al. :
I
I
400
rr W _J
W ._1 w
SO0
2OO
w
'~T I00
0 s u 0
0 0
t
I
I
I
6
12
18
24
HOURS OF INCUBATION
Fig. 4. Time course of the effect of GAs on the release of acid-hydrolyzable oligosaccharide from isolated aleurone layers. Five aleurone layers/treatment were incubated for the times indicated. The media were collected, combined with the first rinse, and reducing sugars were assayed before and after mild acid hydrolysis
were t r e a t e d similarly i n v e r t a s e t r e a t m e n t released only 80% as m u c h as acid t r e a t m e n t . P a p e r c h r o m a t o g r a p h y p r o v e d t h a t acid t r e a t m e n t released some pentoses, suggesting t h a t soluble p e n t o s a n s m a y be released from the aleurone tissue. T a k e n t o g e t h e r these observations suggest t h a t aleurone cells contain sucrose t h e release of which is enh a n c e d b y GA 3. T h e effect of GA 3 on the a p p e a r a n c e in t h e m e d i u m of sucrose is shown in Fig. 4. The d a t a show t h a t GA s affected b o t h t h e r a t e a n d t h e e x t e n t of sucrose release. T h e a p p e a r a n c e of sucrose in t h e m e d i u m was m a t c h e d b y its d i s a p p e a r a n c e from t h e tissue (Fig. 5). I n t h e presence of GA s t h e level of sucrose d r o p p e d from 300-400 ~g/aleurone layer to a new s t e a d y s t a t e of 50-75 ~g/aleurone layer. T h i s happ• whether t h e aleurone layers were i n c u b a t e d as isolated layers (Fig. 5A) or as half-seeds, still a t t a c h e d to t h e s t a r c h y e n d o s p e r m (Fig. 5B). T h e r e was a t r a n s i e n t decline in t h e oligosaccharide c o n t e n t of t h e control tissue. This t r a n s i e n t dip was less p r o n o u n c e d in t h e half-seeds t h a n in t h e isolated aleurone layers a n d m a y in p a r t result from t h e dissection of aleurone layers a n d in p a r t from t h e change in i n c u b a t i o n conditions (the half-seeds were s h a k e n in buffer, b u t prior to this t h e y h a d been i n c u b a t e d on sand). The oligosaccharide content of t h e control tissues r e t u r n e d to a high level at t h e end of a 24-h i n c u b a t i o n period. T h e release of reducing sugars into t h e m e d i u m can p r o b a b l y be a c c o u n t e d for b y t h e slow hydrolysis of this sucrose. This was confirmed b y t h e o b s e r v a t i o n t h a t isolated aleurone cell-wall fractions h y d r o l y s e
Sucrose Release by Aleurone: Regulation by GA3 I
I
I
I
I
I
41 I
I
I 18
I 24
B=HALF-SEEDS
A, ALEURONE LAYERS
400J %. L~
3oo "1" t.)
Synthesis and Release of Sucrose by the Aleurone Layer of Barley: Regulation by Gibberellic Acid Maarten J. Chrispeels, Andrea J. Tenner, and Kenneth D. Johnson Department of Biology, University of California/San Diego, La Jolla, California 92037, USA and Botany Department, California State University/San Diego, San Diego, California 92115, USA Received March 27, 1973
Summary. Aleurone layers of barley contain large amounts of a soluble oligosaccharide which was identified as sucrose (3040 ~g/mg fresh weight). Treatment of the layers with gibberellic acid (GAa) causes the release of sucrose from the cells. This release requires the participation of metabolic processes, including protein synthesis. When embryoless half-seeds are incubated sucrose accumulates in the aleurone layers, but when seeds are germinated the sucrose content of the aleurone layers declines. Labeling experiments with radioactive glucose and fructose show that Meurone layers continuously synthesize sucrose and that the release, but not the synthesis of sucrose is enhanced by GAa.
Introduction
The carbohydrate metabolism of germinating cereal grains has been studied quite extensively over the past 75 years. The endosperm contains alcohol-soluble, water-soluble and water-insoluble carbohydrates all of which are utilized during the growth of the seedling (for a review see MaeLeod, 1960). The breakdown of carbohydrates is in part controlled by the embryo and this control is mediated by gibberellins (GAs) (Yomo, 1960; Paleg, 1960) and in part by the build-up of soluble sugars within the endosperm (Armstrong and Jones, i972). The role of the seutellum involves the conversion of the end product of starch breakdown, glucose, into sucrose and transport of this sucrose to the growing axis (Edelman et al., 1959). Evidence is presented here which suggests that the aleurone layer may play a similar role in the conversion of glucose to sucrose. Materials and Methods Seed Preparation and Treatment. Aleurone layers from seeds of Hordeum vulgare L., cv. Himalaya, were prepared from embryo-less halfseeds imbibed for 3 d as described by Chrispeels and Varner (1967). Care was taken to remove all the starch and the layers were kept in a large beaker of cold, sterile water until enough layers had been prepared for the experiment. The layers were then rinsed 3*
36
M . J . Chrispeels et al. :
and incubated in 2 ml of autoclaved medium containing 2 mM acetate buffer (pH 4.8), 20 mM CaC12, 50 ~zg/ml of chloramphenicol, and with or without 2 tzM gibberellic acid (GA3), unless otherwise noted. Incubation routinely took place at 22 ~ I n some experiments half-seeds were incubated as described above and the aleurone layers were isolated from t h e m after the incubation. I n other experiments half-seeds were incubated on sand, and whole seeds were germinated in sterile, moist vermiculite at room temperature. Aleurone layers and endosperm (in the case of hMf-seeds) were isolated at various times after planting. Alcohol Extraction. After t r e a t m e n t the layers were rinsed 3 times with water, 5 ml of 80 % ethanol was added, and tissue was boiled for 4 rain in loosely-stoppered 25 ml flasks. The flasks were allowed to cool, the liquid was decanted, and the layers were rinsed with 2.5 ml of 80% ethanol. The initial extract and the wash were combined. (Alcoholic extraction involving homogenization of the tissue after boiling in ethanol did not result in a greater yield of sucrose). The ethanol was evaporated at 80 ~ under vacuum in a Rotary Evapomix (Buchler Corp., Fort Lee, N.J., U.S.A.) and the residue was t a k e n up in 6.0 ml of water. Starchy endosperms were treated similarly, b u t they were not rinsed with water prior to ethanol extraction. Determination o] Reducing Sugars. Reducing sugars present in the alcoholic tissue extract and the incubation medium were determined before and after mild acid hydrolysis (0.1 N HC1 for 10 rain at 100 ~ and/or before and after invertase t r e a t m e n t (0.1 mg/ml of invertase in 5 mM sodium acetate buffer, p H 4.8, incubated at 37 o for 10 min; invertase from Sigma Chemical Corp., St. Louis, Mo., USA) with the Nelson-Somogyi copper reagent as described b y Ashwell (1957). The results are expressed as glucose equivalents. Paper Chromatography. Sugars and oligosaccharides were separated b y descending paper chromatography on W h a t m a n n 3MM paper using either of the following solvents: ethyl acetate: pyridine: water (8: 2 : 1, v/v) r u n for 20 h or n-butanol: pyridine: water (10: 3 : 3, v/v) r u n for 5 days. After drying the sugars were detected by the alkaline silver-oxide reagent as described b y Menzies and Seakins (1969). The locations of the spots were compared with authentic standards. Labeling with Radioactive Sugars. Aleurone layers incubated with or without GA s for 18 h were rinsed and incubated with the same medium containing [14C]D-glucose (1 mCi/mg; :New England Nuclear Corp., Boston, l~ass., USA) or [laC]D-frnctese (1 mCi/1.5 rag; New England Nuclear Corp.) for 3 h. The incubation medium and a 3-ml rinse were pooled, evaporated to dryness and the residue was dissolved in 1.0 ml of water and analysed. The layers were rinsed several more times in ice-cold water, 5.0 ml of 80% ethanol was added, the tissue boiled for 4 min, and homogenized in the alcoholic extract. Homogenization was carried out with a power-driven conical ground-glass homogenizer. Alcohol-soluble and Mcoholinsoluble fractions were separated b y centrifugation (3 000 • g, 5 rain). The residue was extracted 3 times with 5 ml of 80% ethanol and the extracts combined with the initial supernatant. The Mcohol-soluble fraction was evaporated to dryness and then t a k e n up in 1.0 ml of water. Labeling of the alcohol-soluble metabolites in the tissue and of the metabolites in the original incubation medium was determined after their separation b y paper chromatography in the ethylacetate: pyridine: water solvent. The paper chromatograms were cut in 1-cm strips to determine the radioactivity in glucose, fructose, sucrose and metabolites which did not move from the origin (see J o h n s o n and Chrispeels, 1973). Incorporation of label into the alcoholinsoluble macromolecules was determined by mixing a small aliquot of the resuspended cellular material in 10.0 ml of Aquasol (New England Nuclear Corp.) and counting in a Beckman liquid scintillation spectrometer.
Sucrose Release by Aleurone: Regulation by GA3
37
400
< w ..1 W rr rr ,< (.9 D 59 -Z o D a U.I n-
200-
IO0 --
0
~
~'-CONTROL
I
I
I
I
6
12
18
24
HOURS
OF
INCUBATION
Fig. 1. Effect of GA3 on the release of reducing sugars from aleurone layers. Five aleurone layers/treatment were incubated for the times indicated. The media were collected and assayed for reducing sugars
Results and Discussion
The incubation of isolated aleurone layers from which all starch has been carefully removed resulted in a release of reducing sugars into the medium. The presence of GA s in the incubation medium caused a considerable enhancement of this release (Fig. 1). This observation resembles the initial observations of Paleg (1960) and Yomo (1960) made on embryo-less half seeds of barley. I n the presence of GA 3 as much as 400 Fg of reducing sugar (glucose equivalents) was released in a 24-h period. The aleurone layers contained only small amounts of reducing sugars (15-25 ~g/layer), suggesting that they contained other carbohydrates the release and/or the hydrolysis of which was enhanced by GA s . Paper chromatography of the sugars released by aleurone layers treated with GA 3 for 24 h demonstrated the presence of glucose and fructose and relatively smaller quantities of arabinose and xylose (Fig. 2). The medium also contained an ohgosaccharide which co-chromatographed with sucrose. A semi-quantitative estimation of the released reducing sugars based on the size of the chromatographic spots and the intensity of the color reaction showed that approximately equal quantities of glucose and fructose were released (data not shown). Chromatography of the medium 10 h after treatment with GA s showed the presence of reducing sugars (glucose and fructose) and a comparatively larger amount of the ohgosaceharide (Fig. 3). Mild acid hydrolysis (0.1 N tIC1 at 100 ~ for 10 rain) caused the oligosaccharide spot to disappear, with
38
M.J. Chrispeels et al. :
Fig. 2. Paper chromatography of sugars and oligosaccharides released from aleurone layers. The 24-h media (control and GA3) from the experiment described in Fig. 1 were ehromatographed in ethyl acetate: pyridine: water (8 : 2 : 1) together with appropriate standards (arabinose = A R A , xylose = X Y L , glucose = GLU, fructose = F R U , sucrose = SU). Note the difference in intensity of staining of glucose and fructose when an equimolar solution of these sugars was used as a standard. S T D standards
t h e simultaneous a p p e a r a n c e of glucose a n d fructose in e q u i v a l e n t a m o u n t s . P a p e r c h r o m a t o g r a p h y of an alcoholic e x t r a c t of t h e freshly isolated aleurone layers showed t h e presence of t h e same oligosaccharide which u p o n m i l d acid h y d r o l y s i s gave rise to glucose a n d fructose (Fig. 3). T h e following e x p e r i m e n t s were p e r f o r m e d to characterize t h e oligosaccharide: (1) T h e oligosaccharide c o - c h r o m a t o g r a p h e d with sucrose in e t h y l a c e t a t e : p y r i d i n e : w a t e r (8 : 2 : 1) ; u p o n m i l d acid t r e a t m e n t (0.1 N ttC1 for 10 rain a t 100 ~ t h e spot d i s a p p e a r e d w i t h the s i m u l t a n e o u s a p p e a r a n c e of e q u i v a l e n t a m o u n t s of reducing sugars which coc h r o m a t o g r a p h e d with glucose a n d fructose (Fig. 3). Sucrose is k n o w n
Sucrose Release by Aleurone: Regulation by GA3
39
Fig. 3. Effect of mild acid hydrolysis on the chromatographic pattern of the sugars present in the aleurone layers and the incubation medium. Alcoholic extracts of untreated aleurone layers and incubation media obtained after 10 h of incubation were treated as described and the sugars were chromatographed, before and after mild acid hydrolysis, in ethylacetate: pyridinc:water (8:2:1). A) medium after 10 h incubation, B) same medium hydrolysed in 0.1 N HC1 for 10 min. C) alcoholic extract of aleurone layers before incubation, D) same as C, after mild acid hydrolysis. Abbreviations as in Fig. 2
to be quite labile u n d e r m i l d acid conditions (Pazur, 1970). (2) I a c h r o m a t o g r a p h i c s y s t e m which s e p a r a t e s t h e various disaecharides ( n - b u t a n o l : p y r i d i n e : w a t e r 1 0 : 3 : 3 , r u n d e s e e n d i n g l y for 5 days) t h e oligosaceharide c o - e h r o m a t o g r a p h e d w i t h sucrose ( d a t a n o t shown). (3) I n v e r t a s e r a p i d l y h y d r o l y z e d t h e oligosaceharide, again p r o d u c i n g reducing sugars which e o - c h r o m a t o g r a p h e d w i t h glucose a n d fructose. T r e a t m e n t of alcoholic e x t r a c t s of t h e tissue w i t h either i n v e r t a s e or m i l d acid released t h e same a m o u n t of reducing sugar. W h e n t h e m e d i a
40
M.J. Chrispeels et I
3
I
al. :
I
I
400
rr W _J
W ._1 w
SO0
2OO
w
'~T I00
0 s u 0
0 0
t
I
I
I
6
12
18
24
HOURS OF INCUBATION
Fig. 4. Time course of the effect of GAs on the release of acid-hydrolyzable oligosaccharide from isolated aleurone layers. Five aleurone layers/treatment were incubated for the times indicated. The media were collected, combined with the first rinse, and reducing sugars were assayed before and after mild acid hydrolysis
were t r e a t e d similarly i n v e r t a s e t r e a t m e n t released only 80% as m u c h as acid t r e a t m e n t . P a p e r c h r o m a t o g r a p h y p r o v e d t h a t acid t r e a t m e n t released some pentoses, suggesting t h a t soluble p e n t o s a n s m a y be released from the aleurone tissue. T a k e n t o g e t h e r these observations suggest t h a t aleurone cells contain sucrose t h e release of which is enh a n c e d b y GA 3. T h e effect of GA 3 on the a p p e a r a n c e in t h e m e d i u m of sucrose is shown in Fig. 4. The d a t a show t h a t GA s affected b o t h t h e r a t e a n d t h e e x t e n t of sucrose release. T h e a p p e a r a n c e of sucrose in t h e m e d i u m was m a t c h e d b y its d i s a p p e a r a n c e from t h e tissue (Fig. 5). I n t h e presence of GA s t h e level of sucrose d r o p p e d from 300-400 ~g/aleurone layer to a new s t e a d y s t a t e of 50-75 ~g/aleurone layer. T h i s happ• whether t h e aleurone layers were i n c u b a t e d as isolated layers (Fig. 5A) or as half-seeds, still a t t a c h e d to t h e s t a r c h y e n d o s p e r m (Fig. 5B). T h e r e was a t r a n s i e n t decline in t h e oligosaccharide c o n t e n t of t h e control tissue. This t r a n s i e n t dip was less p r o n o u n c e d in t h e half-seeds t h a n in t h e isolated aleurone layers a n d m a y in p a r t result from t h e dissection of aleurone layers a n d in p a r t from t h e change in i n c u b a t i o n conditions (the half-seeds were s h a k e n in buffer, b u t prior to this t h e y h a d been i n c u b a t e d on sand). The oligosaccharide content of t h e control tissues r e t u r n e d to a high level at t h e end of a 24-h i n c u b a t i o n period. T h e release of reducing sugars into t h e m e d i u m can p r o b a b l y be a c c o u n t e d for b y t h e slow hydrolysis of this sucrose. This was confirmed b y t h e o b s e r v a t i o n t h a t isolated aleurone cell-wall fractions h y d r o l y s e
Sucrose Release by Aleurone: Regulation by GA3 I
I
I
I
I
I
41 I
I
I 18
I 24
B=HALF-SEEDS
A, ALEURONE LAYERS
400J %. L~
3oo "1" t.)
