Planta (Berl.) 92, 73--84 (1970)
Gibberellic Acid, ~-l,3-Glucanase and the Cell Walls of Barley Aleurone Layers* L ~ c o L ~ TAIZ a n d RUSSELL L. JO~CES Department of Botany, University of California, Berkeley Received November 17, 1969 / January 19, 1970
Summary. A glucanase from barley aleurone layers can be assayed using the algal polysaccharide laminarin as substrate. Gibberellic acid (GA3) enhances the release of this enzyme from isolated aleurone layers but has no significant effect on its synthesis. Concentrations of GAs effective in stimulating this release are in the range of 3 • l0 - n -- 3 • 10-~M. The ~ime course of glucanase release was found to be significantly different from that of a-amylase, glucanase release being completed before that of a-amylase. Evidence based on using various histochemical stains suggests that barley aleurone cell walls contain a fl-l,3-1inked polymer. Following treatment of aleurone layers with GA3, digestion of these walls is seen to occur. These observations strongly suggest that the fi-l,3-glucanase produced by aleurone cells is responsible for the observed cell-wall digestion. Introduction Gibberellie acid (GA~) has been shown to induce de-novo synthesis of ~.-amylase a n d a protease in isolated b a r l e y aleurone layers (Filner a n d Varner, 1967; J a c o b s e n a n d Varner, 1967). I n addition, GA~ enhances t h e a c t i v i t y of several o t h e r h y d r o l y t i c e n z y m e s (Briggs, 1963; Chrispeels a n d Varner, 1967); however, the n a t u r e of the e n h a n c e m e n t of these enzymes (de-novo synthesis or activation) is n o t u n d e r s t o o d . Several r e p o r t s h a v e i n d i c a t e d t h a t a /~-l,3-glucanase is p r o d u c e d by b a r l e y half seeds in response to G A 3 (Briggs, 1963 ; M a c L e o d et al., 1964) a n d is p r e s e n t in b a r l e y m a l t (Dillon a n d O'Colla, 1951; Preece a n d n o g g a n , 1956). I n addition, b a r l e y - m a l t p r o d u c t s h a v e been shown to c o n t a i n a water-soluble, l a e v o r o t a t o r y glucan which possesses a p p r o x i m a t e l y equal n u m b e r s of fl-1,3 a n d fi-1,4 linkages (Preece a n d MacKenzie, 1952; Preece et al., 1954; Aspinall a n d Telfcr, 1954), a n d P o l l a r d (1969) has shown r e c e n t l y t h a t b a r l e y half-seeds release a water-soluble glucan containing some fi-l,3 linkages within 5 h of G A s t r e a t m e n t . This p a p e r reports on t h e fl-l,3-glucanase p r o d u c e d b y isolated b a r l e y aleurone layers a n d secreted from t h e m in response to GAa, a n d t h e localization of t h e possible s u b s t r a t e for this enzyme. * Supported by National Science Foundation Grant GB-8332. The skillful technical assistance of Mrs. Janet Price is gratefully acknowledged.
74
L. Taiz and R. L. Jones:
M a t e r i a l a n d Methods Material, Enzyme Preparation and Determination. Aleurone layers obtained from barley hMf-seeds (Hordeum vulgare L., cv. Himalaya, 1967 harvest) were prepared in a manner shnilar to t h a t described previously (Jones and Varner, 1967). Isolated Meurone layers were incubated in 2 ml of a medium containing 2 ~moles acetate buffer (pH 4.8), 20 ~moles CaC12 and the appropriate concentration of GA S. u-Amylase was assayed as described by Jones and Varner (1967). The fl-l,3-glucanase was prepared from the incubation medium and aleurone layer extracts by precipitation with ethanol. 5 aleurone layers were ground in a Ten Broeck glass homogenizer with 2 ml of acetate buffer (pH 4.8), and a further 3 ml of buffer were used for washing. The extract and washings were combined and centrifuged at 1000 • g for 5 min. The glucanase was precipitated from solution by addition of ethanol to give a final concentration of 80% (v/v). The protein precipitate was collected by centrifugation at 13,000 • g at 2 ~ for 5 rain. The supernatant solution was discarded and the pelleted protein re-dissolved in 1 ml of 2 mM acetate buffer (pH 4.8) containing 20 mM CaC12. The enzyme was assayed using laminarin (Nutritional Biochemicals Corp., Cleveland) as substrata. A mixture containing 0.1% Iaminarin, 20 mM CaCl~ and 2 mM acetate buffer was heated for 2 rain to dissolve the laminarin. The solution was prepared freshly for each assay. A suitable aliquot of enzyme (in our experiments 0.3 ml from 1 ml of dissolved protein precipitate) was added to a test tube, the reaction was initiated by the addition of 1 ml of the substrata solution and the tubes were incubated in a water b a t h for 30 min at 37 ~ The reducing sugars formed after incubation of laminarin with the enzyme were determined by the Nelson-Somogyi method. 1 ml of copper reagent was added to the reaction mixture, followed by immersion in boiling water for 20 rain. The tubes were then cooled in an ice b a t h and 1 ml of arsenomolybdate reagent was added with shaking. The colored products formed were diluted with a further 5 ml of water and the absorbance of the solution measured at 540 m~ with a "Spectronic 20" colorimeter. The colorimeter was adjusted to zero absorbance using a laminarin reagent blank (all components except enzyme). Each assay was compared with a zero time control in which the copper reagent was added before the addition of substrata. Enzyme units were expressed as O.I). 30-rainincubation mixture - - O.D. zero-time incubation mixture • 100.
Light and Electron Microscopy. Thin sections of barley aleurone layers were prepared for light and electron microscopy by methods previously described (Jones, 1969a, b). For light microscopy, aleurone pieces were fixed in 3 % glutaraldehyde and embedded in Epon. Sections were cut at 0.2--0.4 ~z and stained with Aniline blue-black for protein and b y the periodic acid-Schiff's reagent (PAS) for carbohydrates. Sections of fresh, unfixed aleurone layers were cut at approximately 5 [x for staining with aniline blue and lacmoid. Fresh sections were placed on a microscope slide and covered with 2 drops of 0.05 % water-soluble aniline blue or lacmoid dye in 0.01 M K2HPO4--KaPO4 buffer (pH 10). After 10--20 rain, the sections were rinsed with distilled water and viewed in the microscope with visible and ultra violet light. Photomicrographs were obtained in the U.V. using Kodak high speed Ektachrome film. For electron microscopy, cell wall materials were visualized after staining sections with the periodic acid-Schiff's-silver stain (Rambonrg, 1967). Ultra-thin sections of glutaraldehyde-fixed barley aleurone ceils were mounted on gold grids, oxidized for 20 min on a 1% solution of periodic acid and stained with the silver-
Gibberellic Acid and Cell Walls of Barley Aleurone
75
metheuamine stain for two periods of 30 rain. The stained sections were washed in a 5 % solution of sodium thiosulfate for 5 rain, followed by rinsing in water and observecl in a Zeiss EM9A electron microscope. Results
Conditions o] Enzyme Assay. Several e x p e r i m e n t s were c o n d u c t e d to d e t e r m i n e o p t i m a l conditions for the a s s a y of t h e fi-],3-glueanase obt a i n e d f r o m b a r l e y aleurone layers. L a m i n a r i n was used as s u b s t r a t e since it is the only commercially available fl-l,3-glucan. In addition, laminarin is the preferred substrate for fl-l,3-glucanases isolated from several plant (Huotari et al., 1968; Dillon and O'Colla, 1951 ; Preece and Garg, 1961) and animal (Epel et al., 1969) sources. Using 0.1% laminarin as substrate, the pll optimum for the barley glueanase was found to be in the range of pll 4.75--5.5. Since pH 4.8 has been found optimal for the assay of other barley hydrolases, this pll was also chosen for the assay of barley /~-l,3-glucanase. A time-course for the hydrolysis of laminarin was also determined. For a 30-rain incubation period, the amount of reducing sugar produced in the reaction mixture was linear. Table 1. Development o/fi-l,3-glucanase activity in barley seeds imbibed on water Enzyme sourcea
Glucanase activity
Dry seed 24 h imbibed 48 h imbibed 96 h imbibed
9.0 24.0 87.0 225.0
a Enzyme from 10 dry seeds or 5 imbibed seeds.
Physiology o/ Barley fl-l,3-Glucanase. The fi-l,3-glucanase of b a r l e y aleurone layers is p r e s e n t in b o t h d r y a n d i m b i b e d seeds. H o w e v e r , t h e level of e x t r a c t a b l e e n z y m e increases w i t h t h e d u r a t i o n of i m b i b i t i o n of t h e half-seeds on moistened, sterile s a n d (Table l). T r e a t m e n t of isolated aleurone layers w i t h G A 3 results in t h e release of/~-l,3-glucanase into t h e i n c u b a t i o n m e d i u m while t h e level of e x t r a c t a b l e glucanase decreases (Fig. 1 A). The release of t h e e n z y m e begins 4 h after GA a t r e a t m e n t a n d is essentially complete a t 1 6 - - 1 8 h (Fig. 1 A). Control seeds do n o t release a p p r e c i a b l e a m o u n t s of t h e enzyme (Fig. 1 B). The effect of v a r y i n g concentrations of GA 3 on glucanase release was also examined. W h e n aleurone layers were t r e a t e d w i t h v a r y i n g conc e n t r a t i o n s of G A 3 for 12 h, e n z y m e release was i n i t i a t e d a t 5 • 10 -l~ M a n d was s a t u r a t e d a t 5 • 10 -7 M (Fig. 2A). I t o w e v e r , when t h e incubation p e r i o d was e x t e n d e d to 18 h, t h e release of glucanase was i n i t i a t e d
76
L. Taiz and R. L. Jones:
B
A 200
2OO
. / ~
I~o
.~ 160
9
/~o~.
o//
120
g |
6o
:-7:
I
0
4
8
12
16
20
24
0
4
12
8
6
20
214
Time in hours
Fig. l A and B. Time course for the production of fl-l,3-glucanase by 5 aleurone layers. A. Enzyme from GAs (5 • 10-; M) treated aleurone layers. B. Enzyme from water controls, o - 9 enzyme released into medium; |174 enzyme extracted from aleurone layers; =--., total enzyme (released ~ extracted) zoo 160
120
f/ ._~
5,
B 160
11"
j~
"~o --9 - o ~ ' ' ' ' e ~
120
~
/\ ~176
o (_9
40
40
0
-II
-I0
-9
-S
-7
-6
GA3
0
-I1
-I10
-9
M x 5
Fig. 2A and B. Concentration range of GAa effective in stimulating fi-l,3-glucanase release from isolated barley aleurone layers. A. Sampled at 12 h; B. at 18h. o-- 9 released enzyme; Q--Q, extracted enzyme; 9 total enzyme a t lower GAa c o n c e n t r a t i o n s (Fig. 2 B). A t 12 h, GA 3 concentrations of 5 • 10-9 to 5 • 10-6 M caused a n increase i n total glucanase, while a t 18 h there was no significant increase i n glueanase levels relative to water controls (Fig. 2). The time course of glneanase release was compared with s - a m y l a s e secretion from GAa-treated aleurone layers b y p l o t t i n g the percent of t o t a l e n z y m e secreted with time (Fig. 3). I t is clear from Fig. 3 t h a t glucanase release is completed before t h a t of ~-amylase.
Gibberellie Acid and Cell Walls of Barley Aleurone
77
(::: 8o
"~
60
9
o
40
4
8
i6
I
I
I
I
i
20
24
28
32
36
40
Time in hours
Fig. 3. Time course of fl-l,3-glucanase and e-amylase release from GA 3 (5 • 10-7 M) treated aleurone layers. Enzyme units expressed as % of total enzyme released over 42 h time period. 9 9 released glucanase. 9 released e-amylase Table 2. A comparison o/ the staining properties and glycosidic linkages o/ various
carbohydrate polymers Carbohydrate polymer
Sieve-tube callose Yeast-wall glucan Laminarin Barley-Meurone wall Cellulose Barley-seed Starch
Staining characteristics of polymer Aniline blue UV fluo- Visible rescence
Lacmoid Visible
Nature of glycosidic linkage in polymer
Reference
yellowgreen yellowgreen yellowgreen yellowgreen none none
blue
blue
fl-l,3
1, 2, 3, 4, 7
blue
blue
fl-l,3
1, 3, 5, 7
blue
blue
fl-l,3
4, 6, 7
blue
blue
none none
none none
f l 1,4 e- 1,4
4
References: 1, Currier (1957); 2, Kessler (1958); 3, Frey-Wysling and 1V[fihlethMer (1965); 4, Parker (1964); 5, Northeote and ttorne (1952); 6, Meeuse (1962); 7, Clarke and Stone (1963).
Cytological Observations on Barley Aleurone Cell Walls. T h e walls of aleurone cells were localized using various histochemical stains. These walls stain markedly with PAS, a general carbohydrate stain (Fig. 4). In addition, aleurone layers produce a marked fluorescence with aniline blue when observed under ultraviolet light (Fig. 4a). This stain has
78
L. Taiz and R. L. Jones:
Fig. 4 a - - ~
Gibberellic Acid and Cell Walls of Barley Aleurone
79
been shown to be specific for carbohydrates containing fl-l,3-1inked glucose residues (Currier, 1957, and Table 2). The cells of the starchy endosperm and the seed coat do not fluoresce with aniline blue although they stain strongly with PAS (Fig. 4). Similar results were obtained using laemoid dye, which selectively stains aleurone cell walls a deep blue. This stain has also been shown to be specific for glucans containing /~-l,3-1inkages (Parker, 1964). The wails of aleurone cells are only weakly birefringent while those of the starchy endosperm and of the seed coat are highly birefringent when observed in polarized light. When thin sections of aleurone layers isolated from seeds imbibed on water for 3 days are examined after PAS staining, the walls are uniformly stained (Fig. 4b). However, with increased exposure of these cells to GA a, regions of the wall become PAS-negative (Fig. 4c--f). This histochemical evidence suggest that the wall is digested resulting in the removal of the PAS-positive carbohydrate. I n addition, the observations indicate that this digestion predominates in the region of the cell wall closest to the starchy endosperm (see Fig. 4 c--f). This localized digestion is apparent in all layers of the aleurone, although it is most marked in the cell layer which is adjacent to the seed coat (Fig. r The digestion of the cell wall and the localized nature of this digestion has been confirmed at the electron-microscope level. Using the PASsilver stain, the deposit of silver was found to be markedly reduced in the region of the cell wall closest to the starchy endosperm (Figs. 5, 6). In the walls of the cells adjacent to the seed coat digestion is restricted to the wall oriented away from the coat and towards the starchy endosperm and, at least in the time periods used, does not proceed beyond the region of the middle lamella (Fig. 5). Likewise, in the cells which lie adjacent to the starchy endosperm, cell-wall digestion occurs in the ceil-wall region closest to the endosperm cells (Fig. 6). Discussion A glucanase produced by isolated barley aleurone cells can be readily assayed using the algal fi-l,3-1inked glucan laminarin as substrate. Since this enzyme can use laminarin substrate it must be specific to at least a fi-],3-1inked glucose polymer. Fig. 4. a UV photomicrograph of aniline-blue-stained barley half seed. Section cut at 5 ~. Prominent yellow-green fluorescence is confined to the walls of the aleurone cells. X 440. b Section of PAS-stained control aleurone layer. Note uniform staining of the cell wall. • 920. c---f Sections of aleurone layers stained with PAS after varying times of exposure to GAs . Note increased breakdown of wall in that region of the wall closest to the starchy endosperm, c GA3 for 16 h; • d Ga3 for 22 h; x960. e GA3 for 22 h; • f GA3 for 32 h; X920
80
L. Taiz and 1~. L. Jones:
Fig. 5. Electronmicrograph of aleurone cell stained with PAS-silver. Cell treated with GA s for 18 h. Note the absence of silver grains (arrows) in the region of the wall closest to the starchy endosperm. The digestion of the wall in this region does not progress beyond the middle lamella to the wall of the adjacent cell. x4,860
Gibberellic Acid and Cell Walls Of Barley Aleurone
81
Fig. 6. View of cell adjacent to t h a t shown in Fig. 5. Again, digestion is seen in t h a t region of the wall oriented away from the seed coat and toward the starchy endosperm. Note t h a t the lateral walls are relatively intact. PAS-silver stain. • 4,950 6 Planta (BerI.), Bd. 92
82
L. Taiz and R. L. Jones:
GA 3 does not significantly affect the synthesis of fi-l,3-glucanase in barley aleurone layers. The synthesis of this enzyme begins soon after imbibition on water and it accumulates within the cells. This contrasts markedly with ~-amylase and protease which do not accumulate within aleurone cells, but whose synthesis is caused by GA 3 (Jacobsen and Varner, 1967; Filner and Varner, 1967). GA 3 stimulates the release of fi-l,3-glucanase from isolated aleurone layers; glucanase release from water controls is directly proportional to the time of imbibition of the half-seeds. Thus, as with the synthesis and secretion of s-amylase and ribonuclease, GA 3 does not exert an "all-or-none" effect on glucanase secretion. The level of GA 3 required to initiate the release of glucanase was found to v a r y with the time of exposure of aleurone layers to GA 3 (Fig. 2). Incubation time in GAa also affects the total enzyme levels relative to water controls. Thus, up to 12 h, GAa stimulates total enzyme synthesis while after 16 h, the level of glucanase obtained from GAatreated and water-control aleurone layers is equal (Figs. 1, 2). I t is possible t h a t the small initial enhancement of synthesis by GA 8 is an indirect result of GAa-stimulated secretion since at 16 h there is no quantitative difference in total enzyme levels. The concentration level of GA a which enhances glncanase release is similar to that required for ribonuclease formation and release (Chrispeels and Varner, 1967) but is lower than t h a t level required to initiate ~-amylase and protease synthesis and release (Chrispeels and Varner, 1967; Jacobsen and Varner, 1967). I t is significant that the effect of GA a on ribonuclease at low concentrations is primarily on release rather than synthesis (Chrispeels and Varner, 1967). The time course of glucanase release was examined and compared with the release of ~-amylase (Fig. 3). To facilitate such a comparison between glucanase and ~-amylase, results were expressed as a percent of total secreted enzyme per unit time. GAa-stimulated glucanase release begins within 4 h and is completed at 16--18 h whereas ~-amylase release begins at 4 h and continues for 42 h. The shorter duration of glucanase secretion m a y reflect an early cessation of synthesis relative to ~-amylase. Cytological studies of the aleurone layer were conducted with partieular emphasis on the nature of the cell wall. Aleurone cell wails stain markedly with PAS (Fig. 4) ; however, this is a non-specific carbohydrate stain. Aleurone cell walls also stain blue with laemoid and aniline blue, and exhibit fluorescence in UV light after exposure to aniline blue. Currier (1957) has shown t h a t sieve-tube eallose and yeast cell walls fluoresce in UV light after aniline-blue staining. These sources of aniline blue fluorescence are also known to contain carbohydrates with fi-l,3-1inkages
Gibberellic Acid and Cell Walls of Barley Aleurone
83
(Table 2). Parker (1964) has shown that sieve-plate callose and laminarin stain deeply with lacmoid whereas cellulose does not. Laminarin is also known to contain/~-1,3 linkages (Meeuse, 1962 ; Table 2). On the basis of the staining properties of aleurone cell walls in lacmoid and aniline blue, and the observations presented in Table 2 on the substrate for these stains, it is suggested that aleurone cell walls posses fi-l,3-1inkages. This cytological observation on the nature of the linkages in barley aleurone cell walls is supported by observations that barley malt, as mentioned before, contains a water-soluble, laevo-rotatory glucan possessing fi-l,3 and fl-l,4-1inkages and the release by barley half-seeds of a glucan containing fl-l,3-1inkages after 5 h of GAa treatment. The cell walls of imbibed aleurone cells stain uniformly with PAS (Fig. 4b). However, with increased exposure of these cells to GAs, regions of the cell wall become PAS negative and appear colorless (Fig. 4e--f). This confirms the observations of Jones (1969c) suggesting that cell wall digestion is occurring. This digestion of aleurone cell walls appears to occur in a localized manner (Fig. 4e--f). Within 14--16 h of GAa addition, digestion predominates in the region of the wall which lies relatively nearest to the starchy endosperm. Localization of digestion occurs in this region of the cell wall irrespective of the position of the cell in the aleurone layer (Fig. 4 c--f). This localized nature of cell-wall breakdown has been confirmed at the electron microscope level using the PAS-silver stain (Figs. 5, 6). The reasons for the polarized nature of cell wall breakdown need further study. No morphological polarization within the aleurone cells, which could account for this digestion pattern, has been noted, nor is there so far any histochemieal evidence, either at the light or the eleetronmieroscope level, for substrate differences within the wall of these cells.
References Aspinall, G. 0., Telfer, R. G. J.: Cereal gums. Part I. The methylation of barley glucosans. J. chem. Soc. 1954, 3519--3522. Briggs, D. E.: Biochemistry of barley germination: Action of gibberellic acid on barley endosperm. J. Inst. Brewing 69, 13--19 (1963). Chrispeels, M. J., Varner, J. E. : Gibbereltic acid-enhanced synthesis and release of e-amylase and ribonuelease by isolated barley aleurone layers. Plant Physiol. 42, 398--406 (1967). Clarke, A. E., Stone, B. A. : Chemistry and biochemist1T of fi-l,3-glucans. Rev. Pure App1. Chem. 13, 134 (i963). Currier, I-I. B. : Callose substance in plant cells. Amer. J. Bot. 44, 478--488 (1957). Dillon, T., O'Colla, P. : The enzymic hydrolysis of 1 : 3-1inked polyglucosans. Chem. and Ind. 1951, 111. Epel, D., Weaver, A.M., Muchmore, A.V., Schimke, R.T.: fl-l,3-glucanase of sea urchin eggs: Release from particles at fertilization. Science 163, 294--296 (1969). 6*
84
L. Taiz and R. L. Jones: Gibberellic Acid and Cell Walls of Barley Aleurone
Filner, P., Varner, J. E. : A simple and unequivocal test for de novo synthesis of enzymes: Density labelling of barley s-amylase with H20 TM. Proc. nat. Acad. Sci. (Wash.) 58, 1520-1526 (1967). Frey-Wyssling, A., •uhlethaler, K. : Ultrastructural plant cytology, p. 290. Amsterdam: Elsevier 1965. Houtari, F. I., Nelson, T . E . , Smith, ~., Kirkwood, S. : Purification of an exofl-D-(1-3)-glucanase from basidiomycete species QM806. J. biol. Chem. 248, 952--956 (1968). Jacobsen, J. V., Varner, J. E.: Gibberellic acid-induced synthesis of protease by isolated aleurone layers of barley. Plant Physiol. 42, 1596--1600 (1967). Jones, R. L. : The fine structure of barley aleurone cells. Planta (Berl.) 85, 359--375 (1969a). - - Gibberellic acid and the fine structure of barley aleurone cells. I. Changes during the lag phase of co-amylase synthesis. Planta (Berl.) 87, 119--133 (1969b). - - Gibberellic acid and the fine structure of barley aleurone cells. II. Changes during the synthesis and secretion of u-amylase. Planta (Berl.) 88, 73--86 ( 1969 c). Varner, J. E. : The bioassay of gibberellins. Planta (Berl.) 72, 53--59 (1967). Kessler, G. : Zur Charakterisierung der SiebrShrenkallose. Ber. schweiz, bot. Ges. 68, 5 4 2 (1968). MacLeod, A. M., Duffus, J. H., Johnston, C. S. : Development of hydrolytic enzymes in germinating grain. J. Inst. Brewing 70, 521--528 (1964). Meeuse, B. J . D . : Storage products. In: Physiology and biochemistry of algae (R. A. Lewin, ed.), p. 289--313. New York: Acad. Press 1962. Northcote, D. H., Home, R. W. : Chemical composition and structure of the yeast cell wall. Biochem. J. 51, 232--236 (1952). Parker, B.: Chemical nature of sieve tube callus in macrocystis. Phycologia 4, 2 7 4 2 (1964). Pollard, C. J. : A survey of the sequences of some effects of gibberellic acid in the metabolism of cereal grains. Plant Physiol. 44, 1227--1232 (1969). Preece, I . A . , Aitken, ]%. A., Dick, J. A. : Non-starchy polysaccharides of cereal grains. VI. Preliminary study of the enzymolysis of barley fl-glucosan. J. Inst. Brewing 60, 497--507 (1954). - - Garg, N. K. : Enzymatic degradation of cereal hemicelluloses. IV. Quantitative aspects of fl-glucan degradation. J. Inst. Brewing 67, 267--273 (1961). - - Hoggan, J. : Enzymic degradation of cereal hemicelluloses. I. Observations on the fl-glucanase system and its development during malting. J. Inst. Brewing 62, 4 8 6 4 9 6 (1956). - - macKenzie, K. G. : Non-starchy polysaccharides of cereal grains. I. Fractionation of the barley gums. J. Inst. Brewing 58, 353--363 (1952). Rambourg, A. : An improved silver methenamine technique for the detection of periodic acid-reactive complex carbohydrates with electron microscope. J. Histochem. Cytoehem. 15, 409~412 (1967). Varner, J. E. : Gibberellic acid controlled synthesis of u-amylase in barley endosperm. Plant Physiol. 89, 4 1 3 ~ 1 5 (1964). -
-
R. L. Jones Department of Botany University of California Berkeley, California 94720, U.S.A.
Gibberellic Acid, ~-l,3-Glucanase and the Cell Walls of Barley Aleurone Layers* L ~ c o L ~ TAIZ a n d RUSSELL L. JO~CES Department of Botany, University of California, Berkeley Received November 17, 1969 / January 19, 1970
Summary. A glucanase from barley aleurone layers can be assayed using the algal polysaccharide laminarin as substrate. Gibberellic acid (GA3) enhances the release of this enzyme from isolated aleurone layers but has no significant effect on its synthesis. Concentrations of GAs effective in stimulating this release are in the range of 3 • l0 - n -- 3 • 10-~M. The ~ime course of glucanase release was found to be significantly different from that of a-amylase, glucanase release being completed before that of a-amylase. Evidence based on using various histochemical stains suggests that barley aleurone cell walls contain a fl-l,3-1inked polymer. Following treatment of aleurone layers with GA3, digestion of these walls is seen to occur. These observations strongly suggest that the fi-l,3-glucanase produced by aleurone cells is responsible for the observed cell-wall digestion. Introduction Gibberellie acid (GA~) has been shown to induce de-novo synthesis of ~.-amylase a n d a protease in isolated b a r l e y aleurone layers (Filner a n d Varner, 1967; J a c o b s e n a n d Varner, 1967). I n addition, GA~ enhances t h e a c t i v i t y of several o t h e r h y d r o l y t i c e n z y m e s (Briggs, 1963; Chrispeels a n d Varner, 1967); however, the n a t u r e of the e n h a n c e m e n t of these enzymes (de-novo synthesis or activation) is n o t u n d e r s t o o d . Several r e p o r t s h a v e i n d i c a t e d t h a t a /~-l,3-glucanase is p r o d u c e d by b a r l e y half seeds in response to G A 3 (Briggs, 1963 ; M a c L e o d et al., 1964) a n d is p r e s e n t in b a r l e y m a l t (Dillon a n d O'Colla, 1951; Preece a n d n o g g a n , 1956). I n addition, b a r l e y - m a l t p r o d u c t s h a v e been shown to c o n t a i n a water-soluble, l a e v o r o t a t o r y glucan which possesses a p p r o x i m a t e l y equal n u m b e r s of fl-1,3 a n d fi-1,4 linkages (Preece a n d MacKenzie, 1952; Preece et al., 1954; Aspinall a n d Telfcr, 1954), a n d P o l l a r d (1969) has shown r e c e n t l y t h a t b a r l e y half-seeds release a water-soluble glucan containing some fi-l,3 linkages within 5 h of G A s t r e a t m e n t . This p a p e r reports on t h e fl-l,3-glucanase p r o d u c e d b y isolated b a r l e y aleurone layers a n d secreted from t h e m in response to GAa, a n d t h e localization of t h e possible s u b s t r a t e for this enzyme. * Supported by National Science Foundation Grant GB-8332. The skillful technical assistance of Mrs. Janet Price is gratefully acknowledged.
74
L. Taiz and R. L. Jones:
M a t e r i a l a n d Methods Material, Enzyme Preparation and Determination. Aleurone layers obtained from barley hMf-seeds (Hordeum vulgare L., cv. Himalaya, 1967 harvest) were prepared in a manner shnilar to t h a t described previously (Jones and Varner, 1967). Isolated Meurone layers were incubated in 2 ml of a medium containing 2 ~moles acetate buffer (pH 4.8), 20 ~moles CaC12 and the appropriate concentration of GA S. u-Amylase was assayed as described by Jones and Varner (1967). The fl-l,3-glucanase was prepared from the incubation medium and aleurone layer extracts by precipitation with ethanol. 5 aleurone layers were ground in a Ten Broeck glass homogenizer with 2 ml of acetate buffer (pH 4.8), and a further 3 ml of buffer were used for washing. The extract and washings were combined and centrifuged at 1000 • g for 5 min. The glucanase was precipitated from solution by addition of ethanol to give a final concentration of 80% (v/v). The protein precipitate was collected by centrifugation at 13,000 • g at 2 ~ for 5 rain. The supernatant solution was discarded and the pelleted protein re-dissolved in 1 ml of 2 mM acetate buffer (pH 4.8) containing 20 mM CaC12. The enzyme was assayed using laminarin (Nutritional Biochemicals Corp., Cleveland) as substrata. A mixture containing 0.1% Iaminarin, 20 mM CaCl~ and 2 mM acetate buffer was heated for 2 rain to dissolve the laminarin. The solution was prepared freshly for each assay. A suitable aliquot of enzyme (in our experiments 0.3 ml from 1 ml of dissolved protein precipitate) was added to a test tube, the reaction was initiated by the addition of 1 ml of the substrata solution and the tubes were incubated in a water b a t h for 30 min at 37 ~ The reducing sugars formed after incubation of laminarin with the enzyme were determined by the Nelson-Somogyi method. 1 ml of copper reagent was added to the reaction mixture, followed by immersion in boiling water for 20 rain. The tubes were then cooled in an ice b a t h and 1 ml of arsenomolybdate reagent was added with shaking. The colored products formed were diluted with a further 5 ml of water and the absorbance of the solution measured at 540 m~ with a "Spectronic 20" colorimeter. The colorimeter was adjusted to zero absorbance using a laminarin reagent blank (all components except enzyme). Each assay was compared with a zero time control in which the copper reagent was added before the addition of substrata. Enzyme units were expressed as O.I). 30-rainincubation mixture - - O.D. zero-time incubation mixture • 100.
Light and Electron Microscopy. Thin sections of barley aleurone layers were prepared for light and electron microscopy by methods previously described (Jones, 1969a, b). For light microscopy, aleurone pieces were fixed in 3 % glutaraldehyde and embedded in Epon. Sections were cut at 0.2--0.4 ~z and stained with Aniline blue-black for protein and b y the periodic acid-Schiff's reagent (PAS) for carbohydrates. Sections of fresh, unfixed aleurone layers were cut at approximately 5 [x for staining with aniline blue and lacmoid. Fresh sections were placed on a microscope slide and covered with 2 drops of 0.05 % water-soluble aniline blue or lacmoid dye in 0.01 M K2HPO4--KaPO4 buffer (pH 10). After 10--20 rain, the sections were rinsed with distilled water and viewed in the microscope with visible and ultra violet light. Photomicrographs were obtained in the U.V. using Kodak high speed Ektachrome film. For electron microscopy, cell wall materials were visualized after staining sections with the periodic acid-Schiff's-silver stain (Rambonrg, 1967). Ultra-thin sections of glutaraldehyde-fixed barley aleurone ceils were mounted on gold grids, oxidized for 20 min on a 1% solution of periodic acid and stained with the silver-
Gibberellic Acid and Cell Walls of Barley Aleurone
75
metheuamine stain for two periods of 30 rain. The stained sections were washed in a 5 % solution of sodium thiosulfate for 5 rain, followed by rinsing in water and observecl in a Zeiss EM9A electron microscope. Results
Conditions o] Enzyme Assay. Several e x p e r i m e n t s were c o n d u c t e d to d e t e r m i n e o p t i m a l conditions for the a s s a y of t h e fi-],3-glueanase obt a i n e d f r o m b a r l e y aleurone layers. L a m i n a r i n was used as s u b s t r a t e since it is the only commercially available fl-l,3-glucan. In addition, laminarin is the preferred substrate for fl-l,3-glucanases isolated from several plant (Huotari et al., 1968; Dillon and O'Colla, 1951 ; Preece and Garg, 1961) and animal (Epel et al., 1969) sources. Using 0.1% laminarin as substrate, the pll optimum for the barley glueanase was found to be in the range of pll 4.75--5.5. Since pH 4.8 has been found optimal for the assay of other barley hydrolases, this pll was also chosen for the assay of barley /~-l,3-glucanase. A time-course for the hydrolysis of laminarin was also determined. For a 30-rain incubation period, the amount of reducing sugar produced in the reaction mixture was linear. Table 1. Development o/fi-l,3-glucanase activity in barley seeds imbibed on water Enzyme sourcea
Glucanase activity
Dry seed 24 h imbibed 48 h imbibed 96 h imbibed
9.0 24.0 87.0 225.0
a Enzyme from 10 dry seeds or 5 imbibed seeds.
Physiology o/ Barley fl-l,3-Glucanase. The fi-l,3-glucanase of b a r l e y aleurone layers is p r e s e n t in b o t h d r y a n d i m b i b e d seeds. H o w e v e r , t h e level of e x t r a c t a b l e e n z y m e increases w i t h t h e d u r a t i o n of i m b i b i t i o n of t h e half-seeds on moistened, sterile s a n d (Table l). T r e a t m e n t of isolated aleurone layers w i t h G A 3 results in t h e release of/~-l,3-glucanase into t h e i n c u b a t i o n m e d i u m while t h e level of e x t r a c t a b l e glucanase decreases (Fig. 1 A). The release of t h e e n z y m e begins 4 h after GA a t r e a t m e n t a n d is essentially complete a t 1 6 - - 1 8 h (Fig. 1 A). Control seeds do n o t release a p p r e c i a b l e a m o u n t s of t h e enzyme (Fig. 1 B). The effect of v a r y i n g concentrations of GA 3 on glucanase release was also examined. W h e n aleurone layers were t r e a t e d w i t h v a r y i n g conc e n t r a t i o n s of G A 3 for 12 h, e n z y m e release was i n i t i a t e d a t 5 • 10 -l~ M a n d was s a t u r a t e d a t 5 • 10 -7 M (Fig. 2A). I t o w e v e r , when t h e incubation p e r i o d was e x t e n d e d to 18 h, t h e release of glucanase was i n i t i a t e d
76
L. Taiz and R. L. Jones:
B
A 200
2OO
. / ~
I~o
.~ 160
9
/~o~.
o//
120
g |
6o
:-7:
I
0
4
8
12
16
20
24
0
4
12
8
6
20
214
Time in hours
Fig. l A and B. Time course for the production of fl-l,3-glucanase by 5 aleurone layers. A. Enzyme from GAs (5 • 10-; M) treated aleurone layers. B. Enzyme from water controls, o - 9 enzyme released into medium; |174 enzyme extracted from aleurone layers; =--., total enzyme (released ~ extracted) zoo 160
120
f/ ._~
5,
B 160
11"
j~
"~o --9 - o ~ ' ' ' ' e ~
120
~
/\ ~176
o (_9
40
40
0
-II
-I0
-9
-S
-7
-6
GA3
0
-I1
-I10
-9
M x 5
Fig. 2A and B. Concentration range of GAa effective in stimulating fi-l,3-glucanase release from isolated barley aleurone layers. A. Sampled at 12 h; B. at 18h. o-- 9 released enzyme; Q--Q, extracted enzyme; 9 total enzyme a t lower GAa c o n c e n t r a t i o n s (Fig. 2 B). A t 12 h, GA 3 concentrations of 5 • 10-9 to 5 • 10-6 M caused a n increase i n total glucanase, while a t 18 h there was no significant increase i n glueanase levels relative to water controls (Fig. 2). The time course of glneanase release was compared with s - a m y l a s e secretion from GAa-treated aleurone layers b y p l o t t i n g the percent of t o t a l e n z y m e secreted with time (Fig. 3). I t is clear from Fig. 3 t h a t glucanase release is completed before t h a t of ~-amylase.
Gibberellie Acid and Cell Walls of Barley Aleurone
77
(::: 8o
"~
60
9
o
40
4
8
i6
I
I
I
I
i
20
24
28
32
36
40
Time in hours
Fig. 3. Time course of fl-l,3-glucanase and e-amylase release from GA 3 (5 • 10-7 M) treated aleurone layers. Enzyme units expressed as % of total enzyme released over 42 h time period. 9 9 released glucanase. 9 released e-amylase Table 2. A comparison o/ the staining properties and glycosidic linkages o/ various
carbohydrate polymers Carbohydrate polymer
Sieve-tube callose Yeast-wall glucan Laminarin Barley-Meurone wall Cellulose Barley-seed Starch
Staining characteristics of polymer Aniline blue UV fluo- Visible rescence
Lacmoid Visible
Nature of glycosidic linkage in polymer
Reference
yellowgreen yellowgreen yellowgreen yellowgreen none none
blue
blue
fl-l,3
1, 2, 3, 4, 7
blue
blue
fl-l,3
1, 3, 5, 7
blue
blue
fl-l,3
4, 6, 7
blue
blue
none none
none none
f l 1,4 e- 1,4
4
References: 1, Currier (1957); 2, Kessler (1958); 3, Frey-Wysling and 1V[fihlethMer (1965); 4, Parker (1964); 5, Northeote and ttorne (1952); 6, Meeuse (1962); 7, Clarke and Stone (1963).
Cytological Observations on Barley Aleurone Cell Walls. T h e walls of aleurone cells were localized using various histochemical stains. These walls stain markedly with PAS, a general carbohydrate stain (Fig. 4). In addition, aleurone layers produce a marked fluorescence with aniline blue when observed under ultraviolet light (Fig. 4a). This stain has
78
L. Taiz and R. L. Jones:
Fig. 4 a - - ~
Gibberellic Acid and Cell Walls of Barley Aleurone
79
been shown to be specific for carbohydrates containing fl-l,3-1inked glucose residues (Currier, 1957, and Table 2). The cells of the starchy endosperm and the seed coat do not fluoresce with aniline blue although they stain strongly with PAS (Fig. 4). Similar results were obtained using laemoid dye, which selectively stains aleurone cell walls a deep blue. This stain has also been shown to be specific for glucans containing /~-l,3-1inkages (Parker, 1964). The wails of aleurone cells are only weakly birefringent while those of the starchy endosperm and of the seed coat are highly birefringent when observed in polarized light. When thin sections of aleurone layers isolated from seeds imbibed on water for 3 days are examined after PAS staining, the walls are uniformly stained (Fig. 4b). However, with increased exposure of these cells to GA a, regions of the wall become PAS-negative (Fig. 4c--f). This histochemical evidence suggest that the wall is digested resulting in the removal of the PAS-positive carbohydrate. I n addition, the observations indicate that this digestion predominates in the region of the cell wall closest to the starchy endosperm (see Fig. 4 c--f). This localized digestion is apparent in all layers of the aleurone, although it is most marked in the cell layer which is adjacent to the seed coat (Fig. r The digestion of the cell wall and the localized nature of this digestion has been confirmed at the electron-microscope level. Using the PASsilver stain, the deposit of silver was found to be markedly reduced in the region of the cell wall closest to the starchy endosperm (Figs. 5, 6). In the walls of the cells adjacent to the seed coat digestion is restricted to the wall oriented away from the coat and towards the starchy endosperm and, at least in the time periods used, does not proceed beyond the region of the middle lamella (Fig. 5). Likewise, in the cells which lie adjacent to the starchy endosperm, cell-wall digestion occurs in the ceil-wall region closest to the endosperm cells (Fig. 6). Discussion A glucanase produced by isolated barley aleurone cells can be readily assayed using the algal fi-l,3-1inked glucan laminarin as substrate. Since this enzyme can use laminarin substrate it must be specific to at least a fi-],3-1inked glucose polymer. Fig. 4. a UV photomicrograph of aniline-blue-stained barley half seed. Section cut at 5 ~. Prominent yellow-green fluorescence is confined to the walls of the aleurone cells. X 440. b Section of PAS-stained control aleurone layer. Note uniform staining of the cell wall. • 920. c---f Sections of aleurone layers stained with PAS after varying times of exposure to GAs . Note increased breakdown of wall in that region of the wall closest to the starchy endosperm, c GA3 for 16 h; • d Ga3 for 22 h; x960. e GA3 for 22 h; • f GA3 for 32 h; X920
80
L. Taiz and 1~. L. Jones:
Fig. 5. Electronmicrograph of aleurone cell stained with PAS-silver. Cell treated with GA s for 18 h. Note the absence of silver grains (arrows) in the region of the wall closest to the starchy endosperm. The digestion of the wall in this region does not progress beyond the middle lamella to the wall of the adjacent cell. x4,860
Gibberellic Acid and Cell Walls Of Barley Aleurone
81
Fig. 6. View of cell adjacent to t h a t shown in Fig. 5. Again, digestion is seen in t h a t region of the wall oriented away from the seed coat and toward the starchy endosperm. Note t h a t the lateral walls are relatively intact. PAS-silver stain. • 4,950 6 Planta (BerI.), Bd. 92
82
L. Taiz and R. L. Jones:
GA 3 does not significantly affect the synthesis of fi-l,3-glucanase in barley aleurone layers. The synthesis of this enzyme begins soon after imbibition on water and it accumulates within the cells. This contrasts markedly with ~-amylase and protease which do not accumulate within aleurone cells, but whose synthesis is caused by GA 3 (Jacobsen and Varner, 1967; Filner and Varner, 1967). GA 3 stimulates the release of fi-l,3-glucanase from isolated aleurone layers; glucanase release from water controls is directly proportional to the time of imbibition of the half-seeds. Thus, as with the synthesis and secretion of s-amylase and ribonuclease, GA 3 does not exert an "all-or-none" effect on glucanase secretion. The level of GA 3 required to initiate the release of glucanase was found to v a r y with the time of exposure of aleurone layers to GA 3 (Fig. 2). Incubation time in GAa also affects the total enzyme levels relative to water controls. Thus, up to 12 h, GAa stimulates total enzyme synthesis while after 16 h, the level of glucanase obtained from GAatreated and water-control aleurone layers is equal (Figs. 1, 2). I t is possible t h a t the small initial enhancement of synthesis by GA 8 is an indirect result of GAa-stimulated secretion since at 16 h there is no quantitative difference in total enzyme levels. The concentration level of GA a which enhances glncanase release is similar to that required for ribonuclease formation and release (Chrispeels and Varner, 1967) but is lower than t h a t level required to initiate ~-amylase and protease synthesis and release (Chrispeels and Varner, 1967; Jacobsen and Varner, 1967). I t is significant that the effect of GA a on ribonuclease at low concentrations is primarily on release rather than synthesis (Chrispeels and Varner, 1967). The time course of glucanase release was examined and compared with the release of ~-amylase (Fig. 3). To facilitate such a comparison between glucanase and ~-amylase, results were expressed as a percent of total secreted enzyme per unit time. GAa-stimulated glucanase release begins within 4 h and is completed at 16--18 h whereas ~-amylase release begins at 4 h and continues for 42 h. The shorter duration of glucanase secretion m a y reflect an early cessation of synthesis relative to ~-amylase. Cytological studies of the aleurone layer were conducted with partieular emphasis on the nature of the cell wall. Aleurone cell wails stain markedly with PAS (Fig. 4) ; however, this is a non-specific carbohydrate stain. Aleurone cell walls also stain blue with laemoid and aniline blue, and exhibit fluorescence in UV light after exposure to aniline blue. Currier (1957) has shown t h a t sieve-tube eallose and yeast cell walls fluoresce in UV light after aniline-blue staining. These sources of aniline blue fluorescence are also known to contain carbohydrates with fi-l,3-1inkages
Gibberellic Acid and Cell Walls of Barley Aleurone
83
(Table 2). Parker (1964) has shown that sieve-plate callose and laminarin stain deeply with lacmoid whereas cellulose does not. Laminarin is also known to contain/~-1,3 linkages (Meeuse, 1962 ; Table 2). On the basis of the staining properties of aleurone cell walls in lacmoid and aniline blue, and the observations presented in Table 2 on the substrate for these stains, it is suggested that aleurone cell walls posses fi-l,3-1inkages. This cytological observation on the nature of the linkages in barley aleurone cell walls is supported by observations that barley malt, as mentioned before, contains a water-soluble, laevo-rotatory glucan possessing fi-l,3 and fl-l,4-1inkages and the release by barley half-seeds of a glucan containing fl-l,3-1inkages after 5 h of GAa treatment. The cell walls of imbibed aleurone cells stain uniformly with PAS (Fig. 4b). However, with increased exposure of these cells to GAs, regions of the cell wall become PAS negative and appear colorless (Fig. 4e--f). This confirms the observations of Jones (1969c) suggesting that cell wall digestion is occurring. This digestion of aleurone cell walls appears to occur in a localized manner (Fig. 4e--f). Within 14--16 h of GAa addition, digestion predominates in the region of the wall which lies relatively nearest to the starchy endosperm. Localization of digestion occurs in this region of the cell wall irrespective of the position of the cell in the aleurone layer (Fig. 4 c--f). This localized nature of cell-wall breakdown has been confirmed at the electron microscope level using the PAS-silver stain (Figs. 5, 6). The reasons for the polarized nature of cell wall breakdown need further study. No morphological polarization within the aleurone cells, which could account for this digestion pattern, has been noted, nor is there so far any histochemieal evidence, either at the light or the eleetronmieroscope level, for substrate differences within the wall of these cells.
References Aspinall, G. 0., Telfer, R. G. J.: Cereal gums. Part I. The methylation of barley glucosans. J. chem. Soc. 1954, 3519--3522. Briggs, D. E.: Biochemistry of barley germination: Action of gibberellic acid on barley endosperm. J. Inst. Brewing 69, 13--19 (1963). Chrispeels, M. J., Varner, J. E. : Gibbereltic acid-enhanced synthesis and release of e-amylase and ribonuelease by isolated barley aleurone layers. Plant Physiol. 42, 398--406 (1967). Clarke, A. E., Stone, B. A. : Chemistry and biochemist1T of fi-l,3-glucans. Rev. Pure App1. Chem. 13, 134 (i963). Currier, I-I. B. : Callose substance in plant cells. Amer. J. Bot. 44, 478--488 (1957). Dillon, T., O'Colla, P. : The enzymic hydrolysis of 1 : 3-1inked polyglucosans. Chem. and Ind. 1951, 111. Epel, D., Weaver, A.M., Muchmore, A.V., Schimke, R.T.: fl-l,3-glucanase of sea urchin eggs: Release from particles at fertilization. Science 163, 294--296 (1969). 6*
84
L. Taiz and R. L. Jones: Gibberellic Acid and Cell Walls of Barley Aleurone
Filner, P., Varner, J. E. : A simple and unequivocal test for de novo synthesis of enzymes: Density labelling of barley s-amylase with H20 TM. Proc. nat. Acad. Sci. (Wash.) 58, 1520-1526 (1967). Frey-Wyssling, A., •uhlethaler, K. : Ultrastructural plant cytology, p. 290. Amsterdam: Elsevier 1965. Houtari, F. I., Nelson, T . E . , Smith, ~., Kirkwood, S. : Purification of an exofl-D-(1-3)-glucanase from basidiomycete species QM806. J. biol. Chem. 248, 952--956 (1968). Jacobsen, J. V., Varner, J. E.: Gibberellic acid-induced synthesis of protease by isolated aleurone layers of barley. Plant Physiol. 42, 1596--1600 (1967). Jones, R. L. : The fine structure of barley aleurone cells. Planta (Berl.) 85, 359--375 (1969a). - - Gibberellic acid and the fine structure of barley aleurone cells. I. Changes during the lag phase of co-amylase synthesis. Planta (Berl.) 87, 119--133 (1969b). - - Gibberellic acid and the fine structure of barley aleurone cells. II. Changes during the synthesis and secretion of u-amylase. Planta (Berl.) 88, 73--86 ( 1969 c). Varner, J. E. : The bioassay of gibberellins. Planta (Berl.) 72, 53--59 (1967). Kessler, G. : Zur Charakterisierung der SiebrShrenkallose. Ber. schweiz, bot. Ges. 68, 5 4 2 (1968). MacLeod, A. M., Duffus, J. H., Johnston, C. S. : Development of hydrolytic enzymes in germinating grain. J. Inst. Brewing 70, 521--528 (1964). Meeuse, B. J . D . : Storage products. In: Physiology and biochemistry of algae (R. A. Lewin, ed.), p. 289--313. New York: Acad. Press 1962. Northcote, D. H., Home, R. W. : Chemical composition and structure of the yeast cell wall. Biochem. J. 51, 232--236 (1952). Parker, B.: Chemical nature of sieve tube callus in macrocystis. Phycologia 4, 2 7 4 2 (1964). Pollard, C. J. : A survey of the sequences of some effects of gibberellic acid in the metabolism of cereal grains. Plant Physiol. 44, 1227--1232 (1969). Preece, I . A . , Aitken, ]%. A., Dick, J. A. : Non-starchy polysaccharides of cereal grains. VI. Preliminary study of the enzymolysis of barley fl-glucosan. J. Inst. Brewing 60, 497--507 (1954). - - Garg, N. K. : Enzymatic degradation of cereal hemicelluloses. IV. Quantitative aspects of fl-glucan degradation. J. Inst. Brewing 67, 267--273 (1961). - - Hoggan, J. : Enzymic degradation of cereal hemicelluloses. I. Observations on the fl-glucanase system and its development during malting. J. Inst. Brewing 62, 4 8 6 4 9 6 (1956). - - macKenzie, K. G. : Non-starchy polysaccharides of cereal grains. I. Fractionation of the barley gums. J. Inst. Brewing 58, 353--363 (1952). Rambourg, A. : An improved silver methenamine technique for the detection of periodic acid-reactive complex carbohydrates with electron microscope. J. Histochem. Cytoehem. 15, 409~412 (1967). Varner, J. E. : Gibberellic acid controlled synthesis of u-amylase in barley endosperm. Plant Physiol. 89, 4 1 3 ~ 1 5 (1964). -
-
R. L. Jones Department of Botany University of California Berkeley, California 94720, U.S.A.
