Planta
Planta 134,251-256(1977)
9 by Springer-Verlag 1977
Gibberellic-acid-induced Synthesis and Release of Cell-wall-degrading Endoxylanase by Isolated Aleurone Layers of Barley William V. Dashek* and Maarten J. Chrispeels** Department of Biology, University of California, San Diego, La Jolla, CA 92093, USA
Abstract. When aleurone layers of barley (Hordeum vulgate L.) are incubated with gibberellic acid (GA3) xylose and a r a b i n o s e - b o t h as free sugars and bound to larger m o l e c u l e s - a r e released into the medium. Release begins 1 0 - 12 h after the start of incubation and continues for at least 60 h. At the same time there is a GA3-induced breakdown of the cell wall resulting in a loss of 2/3 of the cell-wall pentose during 60 h of incubation. GA3 causes the appearance in the medium of an enzyme (or enzymes) which hydrolyze larchwood xylan and aleurone-layer arabinoxylan. Release of the enzyme(s) into the medium begins 2 8 - 3 2 h after the start of incubation. Enzyme activity does not accumulate to any large extent in the tissue prior to release into the medium, and is present in very low levels only in the absence of G A 3. Xylanase activity is associated with a protein (or proteins) with a molecular weight of 29,000. The hydrolysis o f the xylans is largely caused by endoxylanase activity, indicating the importance of endoglycosidases in the GA3-induced breakdown of the aleurone cell wall. Key words: Aleurone - Cell-wall breakdown - Endosperm - Gibberellin - Hordeum - Xylanase.
Introduction Mobilization of the endosperm reserves accompanies seedling growth in cereal grains. The protein-rich cells of the aleurone synthesize a variety of hydrolytic enzymes which are released into the starch part of the endosperm where they hydrolyze the stored reserves. This process is controlled by the hormone gibberellic * P r e s e n t address." Department of Botany, State University of New York at Syracuse, Syracuse, NY 13210, USA ** To whom reprint requests should be addressed
acid (GA3) which originates in the growing embryo. When GA3 is added to isolated aleurone tissue the cells synthesize and release into the incubation medium c~-amylase, protease, /~-glucanase, ribonuclease and acid phosphatase. Release into the medium is always dependent on the presence of GAa, while synthesis is GA3-dependent for some enzymes such as c~-amylase and protease, but not for others such as acid phosphatase and/?-(1,3)-glucanase (for a recent review see Varner and Ho, 1976). Ashford and Jacobsen (1974) recently showed that acid phosphatase is synthesized and secreted into the periplasmic space whether GA3 is present or not, but that release into the medium depends on the GA3-mediated breakdown of the cell wall. They confirmed earlier ultrastructural observations by van der Eb and Nieuwdorp (1967) and by Jones (1969) that GA3 brings about a dissolution of the aleurone cell wall. These observations indicate that the cell wall and not the plasma membrane constitutes the effective barrier against movement of the hydrolytic enzymes from the aleurone cells to the starchy part of the endosperm, as first suggested by Varner and Mense (1972). Barley aleurone cells walls were recently shown to consist almost exclusively (85%) of an arabinoxylan which has a linear fi-(1,4)-xylan backbone, with 1/3 of the xylosyl residues substituted with single arabinosyl sidechains (McNeil et al., 1975). Arabinoxylans also constitute a major fraction of the hemicellulose of the cell walls of cereal endosperm (Mares and Stone, 1973) and of monocotyledons in general (Burke et al., 1974). Breakdown of aleurone cell walls may be mediated by exo- and/or endoglycosidases capable of hydrolyzing pentosans. In a recent paper Taiz and Honigman (1976) demonstrated that the addition of GA3 to isolated aleurone layers of barley causes the cells to release enzymes with c~-arabinofuranosidase, fl-xylopyranosidase and fi-(1,4)-xylanase activity.
252
W.V. Dashek and M.J. Chrispeels: GA3 and Cell-wall-degrading Enzymes in Barley Aleurone
Material and Methods
Incorporation of Radioactivity
Aleurone layers from seeds of barley (Hordeum vulgare L. cv. Himalaya; obtained from Washington State University, (Pullman, 1973 harvest) were prepared from embryo-less half seeds imbibed for 3 days on sterile sand. The layers were incubated in 25-ml flasks at 26~ in 3 ml of buffer containing 2 m M acetate, pH 5.0, 50 ~g/ml chloramphenicol, and 10 m M CaC1/ with or without 1 g M GA3 (Chrispeels and Varner, 1967).
Batches of 150 aleurone layers were labeled for 6 h with 2 IxCi of [14C]arabinose (New England NucIear Corp., Boston, Mass., U S A ; 9.78 mCi/mmol) in the absence of GA3. The layers were then rinsed 3 times with 50 m M arabinose and divided over 16 flasks (8 layers per flask), and further incubated in the incubation buffer with 50 m M arabinose and either with or without GA3. Radioactivity in the macromolecules of the cell wall and the cytoplasm was determined as described in J o h n s o n and Chrispeels (1973).
Preparation of Enzyme Extracts and Cell Walls
Results
The incubation media were collected and the aleurone layers rinsed with 2 ml of incubation buffer. The rinses were added to the media which were then clarified by centrifugation at 12,000 x g for 30 min. The resulting supernatant is referred to as " m e d i u m " . The aleurone layers were rinsed again, then homogenized in a mortar and pestle in a total of 4 ml of incubation buffer. The homogenate was centrifuged at 1000 x g for 10 min to remove the cell walls. This supernatant is referred to as " e x t r a c t " . The cell walls were resuspended and resedimented 3 times in 5 ml of homogenization buffer and then used as a source of enzyme or hydrolyzed to determine the pentose content. The media, extracts and cell-wall suspensions were dialyzed overnight against the incubation buffer to remove any pentoses present.
Degradation of the Aleurone Cell Wall
Preparation of Aleurone Layers
Xylanase Assays Colorimetric xylanase assays were performed with larchwood xylan (Sigma Chemical Co., St. Louis, Mo., USA) or barley aleurone cell wall arabinoxylan as substrates. The arabinoxylan was prepared according to the method of McNeil et al. (1975) and 1 ml of the solution contained the arabinoxylan present in 6 - 8 aleurone layers. The larchwood xylan was used as a 0.5% suspension in 25 m M citrate-phosphate buffer, p H 5.5. One ml of substrate was incubated with 0.5 ml of enzyme, usually for 2 h at 37~ in a shaking waterbath a n d the reaction was terminated by the addition of 4 ml of absolute ethanol. The cloudy solution was centrifuged in the cold for 1 5 m i n at 1 0 0 0 x g and an aliquot (0.1 0.5 ml) of the resulting clear supernatant assayed for pentose residues by the orcinol-ferric chloride-HC1 method of Dische (1962). Absorbance was determined at both 560 n m and 670 n m and a correction was made for interfering hexoses whenever the absorbance at 560 n m was more than half the absorbance at 670 nm. One enzyme unit is defined as the a m o u n t of enzyme which causes the release of 10 gg of pentose residues during the standard 2-h incubation. Viscometricxylanase assays were performed with barley aleurone cell wall arabinoxylan as a substrate in a Cannon-Fenske viscometer. Reactions were run at pH 5.5 at 40 ~ Five m1 of substrate were mixed with 5 - 2 5 ~tl of enzyme and the viscosity of the resulting mixture determined at 2 rain intervals. Typical results are shown in the inset in Fig. 6. One enzyme unit is defined as the a m o u n t of enzyme which causes a change of 1.0 in the relative viscosity of the arabinoxylan solution.
Determination of Sugars Cell walls were resuspended, mixed with an equal volume of 4 N trifluoroacetic acid, and hydrolyzed for 1 h at 121 ~ in sealed hydrolysis ampules. Media were cleared by centrifugation at 12,000 x g for 10 min and an aliquot was used for the determination of pentose (Dische, 1962). An aliquot of the medium was hydrolyzed in 2 N trifluoroacetic acid for 1 h at 120~ and the trifluoroacetic acid removed by evaporation. The sugars were derivatized and separated by gas liquid chromatography (Albersheim et al., 1967). Inositol was included as a standard.
The effect of GA3 on the degradation of the aleurone cell wall was determined by measuring the total pentose residues in the wall and in the incubation medium. Accumulation of pentose residues in the med i u m - s h o w n in Figure 1 - b e g a n 1 0 - 1 2 h after the start of the incubation of the layers with GA 3. The orcinol method employed to determine pentose levels estimated not only the free sugars but also the pentose residues in larger molecules. After 64 h of incubation the medium contained 5 0 0 - 6 0 0 gg of pentose per aleurone layer. In the absence of GA3, release of pentose residues did not start until 48 h after the beginning of incubation. The molecular size of the pentose-containing molecules in the medium was estimated by subjecting the medium to gel filtration chromatography on Sephadex G-50. The results (Fig. 2) indicate that 18 h and 25 h after the start of the incubation a large proportion of the pentose residues was present in molecules excluded from the gel. The presence of free arabinose and xylose in the incubation medium was confirmed by paper chromatography (see Fig. 2 in Chrispeels et al., 1973). Analysis by gas liquid chromatography of the sugar residues in these released molecules indicated that xylose accounted for 60% of the total pentose residues and arabinose for 40% (data not shown). The medium also contained large amounts of free sucrose, glucose and fructose but these were not quantitated. The presence of these sugars in the medium is the result of the GA3-enhanced release of sucrose from the aleurone cells (Chrispeels et al., 1973). Since the cell walls of wheat endosperm are known to be rich in arabinoxylan (Mares and Stone, 1973) we examined the possibility that the small endosperm fragments, which invariably cling to isolated aleurone layers, were the source of the pentose residues in the medium. Using aleurone layers which contained much less and much more than the usual amount of starchy endosperm we observed no differences in the amount of pentose residues released into the medium. Cell-wall breakdown was demonstrated directly
W.V. Dashek and M.J. Chrispeels : GA~ and Cell-wall-degrading Enzymes in Barley Aleurone
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Fig. 2. Gel filtration on Sephadex G-50 of the media from l0 aleurone layers collected after 18 h and 25 h of incubation. The eluate was assayed for pentose by the orcinol method. Blue dextran eluted in tube Nr, 11 and the position of the small molecules was determined with a xylose standard
Fig. 4. Metabolic stability of [l*C]arabinose labeled macromolecules in the cytoplasm and the cell wall of aleurone layers. The layers were labeled with [14C]arabinose for 6 h, rinsed, and further incubated with 50 mM arabinose either with or without GA3. Samples were harvested at the times indicated and the radioactivity in the cytoplasmic macromolecules and the cell wall determined. Open symbols: control closed symbols: GA3
by measuring the total pentose residues in the isolated cell walls after hydrolysis of the walls in 2 N trifluoroacetic acid. Each aleurone layer lost about 400 gg of cell wall pentose during a 64-h incubation (Fig. 3). This number agrees reasonably well with the amount of pentose which accumulated in the medium. In the absence of GA3 cell-wall pentose remained unchanged for up to 48 h of incubation. It was not possible to determine exactly at which time GA 3induced cell-wall dissolution began because of the variability between the samples. The loss of hexoses from the cell wall was not determined.
Aleurone layers readily convert [14C]arabinose into [l*C]xylose and incorporate both pentoses into cellular macromolecules (Johnson and Chrispeels, 1973). The metabolic stability of these newly synthesized macromolecules was tested in the presence and absence of GA3. Aleurone layers were allowed to incorporate [14C]arabinose for 6 h. The layers were then rinsed and further incubated with 50 mM unlabeled arabinose and in the presence or absence of GA3. The radioactivity in cytoplasmic and cell wall polymers was determined. The results (Fig. 4) indicate that the radioactivity in cytoplasmic polymers was
254
w.v. Dashek and M.J. Chrispeels: GA3 and Cell-wall-degradingEnzymesin Barley Aleurone
secreted and/or metabolically labile, while the radioactivity in cell-wall polymers was metabolically stable. The newly synthesized cell-wall arabinoxylans were apparently not degraded in the presence of GA3 and differed in this respect from the bulk of the arabinoxylans in the wall.
GA3-induced Xylanase Activity We tested for the presence of xylanase activity by incubating aliquots of tissue extracts or incubation medium with commercially available larchwood xylan or with arabinoxylan prepared from aleurone layers, and measuring the total pentose residues in the alcoholic supernatant of the reaction mixture. Similar results were generally obtained with either substrate. Preliminary experiments indicated that xylanase activity was present in the incubation medium of aleurone layers incubated in the presence of GA3 for 48 h, and could be measured quantitatively by using sufficient enzyme to produce an absorbance of 0 . 1 - 0 . 6 units under the standard assay conditions (see Material and Methods). The release of alcohol-soluble products from the alcohol-insoluble larchwood xylan or aleurone-layer arabinoxylan could be caused by the activity of both exo- and endopeptosanases. Both types of enzymes are present in barley malt (Preece and MacDougal, 1958) and produced by aleurone layers incubated with GA3 (Taiz and Honigman, 1976). The extent to which both types of enzymes contributed to the formation of reaction products was determined by measuring the molecular size of the alcoholsoluble reaction products on a Sephadex G-50 column. The results (Fig. 5)
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Fig. 6. Time course of the appearance of endoxylanase activity in the extract and the incubation medium of aleurone layers incubated with GA 3. No detectable xylanase activity was found in the absence of GA3. The inset shows the raw data on the decrease in running time of the reaction mixture in the viscometer. The reaction mixture consisted of 4 ml of cell wall arabinoxylan and 5 ml or 10 ~tl of enzyme from a 48-h medium
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Fig. 7. Determination of the molecular weight of xylanase by gel filtration on Sephadex G-100. Bovine serum albumin (BSA), ovalbumin, e-chymotrypsinogen and ribonuclease (RNase) were used as standards
The results presented in this paper demonstrate that GA3 induces the breakdown of the walls of aleurone cells of barley. During a 64-h incubation the aleurone cells lost a/3 of their cell wall pentose (about 400 gg per layer) while free pentoses and pentose-containing macromolecules accumulated in the medium (about 500 lag per layer). Since arabinoxylan makes up 80% of the aleurone cell wall (McNeil, 1975) this finding constitutes an analytical confirmation of ultrastructural (Jones, 1969; van der Eb and Nieuwdorp, 1967) and cytochemical (Taiz and Jones, 1970) evidence concerning the GA 3-enhanced breakdown of the wall. Our results also confirm the finding of Taiz and Honigman (1976) that GA3 induces the formation and release of an enzyme or enzymes with xylanase activity. Barley malt contains several enzymes which degrade pentosans (Preece and MacDougal, 1958) and GA3 induces the formation and the release of both exo- and endopeptosanases in aleurone cells. The enzyme(s) detected by our assays degraded commercial larchwood xylan as well as cell-wall arabinoxylan; degradation was followed colorimetrically through the formation of alcohol-soluble products, or viscometrically. Since the products of the reaction were larger than individual sugars we concluded that the enzyme measured by both procedures-colorimetric and viscometric- was an endoxylanase. The possibility that exoxylanase contributed to the formation of reaction product could not be excluded. Xylanase activity appeared in the medium after 26-30 h and release from the tissue continued for 12-16 h. Tissue extracts contained small amounts of endoxylanase (after 24 h of incubation) but there was no accumulation in the tissue of a large amount of endoxylanase prior
256
W.V. Dashek and M.J. Chrispeels: GA3 and Ce11-wall-degrading Enzymes in Barley Aleurone
to its release into the medium as is the case with ribonuclease (Chrispeels and Varner, 1967) and fl(1,3)-glucanase (Jones, 1971). Taiz and Honigman (1976) found equivalent amounts of endoxylanase in the tissue and in the medium at the time of maximum enzyme release (48 h). The discrepancy between these two observations may be due to differences in the seeds. The relationship between cell-wall breakdown and enzymatic activity remains unresolved. The accumulation of free pentoses and macromolecular pentose in the medium indicates that both exo- and endopentosanases are involved in cell-wall breakdown. Ultrastructural and cytochemical experiments indicate that cell-wall breakdown has progressed considerably by 24 h (Taiz and Jones, 1970; van der Eb and Nieuwdorp, 1967). Our own analyses indicate that breakdown starts after 12-16 h and that less than 20% of the cell-wall pentosan has been hydrolyzed at 24 h. The secretion of exoxylanase and exoarabanase begins 1 2 - 16 h after the start of incubation (Taiz and Honigman, 1976), while endoxylanase is not secreted until 12 h later. It is difficult to see at the moment how the activity of these exoglycosydases can account for the early release of macromolecular pentose in the medium. The resolution of this problem may have to await the characterization of these pentose-containing macromolecules. It is remarkable that the massive degradation of the aleurone cell wall cannot be followed by determining the metabolic lability of the newly synthesized cell-wall pentosans. Secretion of labeled macromolecules is a rapid process which is completed in 3 0 - 6 0 rain (Chrispeels, 1976) and most of the decrease in cytoplasmic radioactivity can therefore be attributed to metabolic lability of macromolecules. The results indicate that the cell wall contains two arabinoxylan components: one which is readily degraded when the tissue is incubated with GA3, and one which is relatively stable. Taiz and Jones (1973) made a: similar observation when they treated aleurone layers with a mixture of hydrolytic enzymes. They found that the inner layer of the wall was quite resistant to degradation by fungal enzymes which removed all of the outer portion of the wall. We suggest that the newly synthesized pentosans are added to this resistant cell wall layer. Dr. K.D. Johnson of San Diego State University carried out the experiment described in Figure 4. We thank him for allowing us to incorporate this experiment in our paper. The work was supported by the US. Energy Research and Development Administration (ERDA) contract E(04-3)-34/159.
References A1bersheim, P., Nevins, D.J., English, P.D., Karr, A.: A method for the analysis of sugars in plant cell wall polysaccharides by gas liquid chromatography. Carbohyd. Res. 5, 340-345 (1967) Ashford, A.E., Jacobsen, LV. : Cytochemical localization of phosphatase in barley aleurone cells. The pathway of gibberellic acid-induced enzyme release. Planta [Bed.] 120, 80-105 (1974) Burke, D., Kaufman, P., McNeil, M., Albersheim, P.: The structure of plant cell walls. VI. A survey of the walls of suspensioncultured monocots. Plant Physiol. 54, 109-115 (1974) Chrispeels, M.J. : Biosynthesis, intracellular transport, and secretin of extracellular macromolecules. Ann. Rev. Plant Physiol. 27, 1 9 - 3 8 (1976) Chrispeels, M.J., Tenner, A.J., Johnson, K.D.: Synthesis and release of sucrose by the aleurone layer of barley: Regulation by gibberellic acid. Planta [Berl.] 113, 3 5 - 4 6 (1973) Chrispeels, M.J., Varner, J.E. : Gibberellic acid-enhanced synthesis and release of a-amylase and ribonuclease by isolated barley aleurone layers. Plant Physiol. 42, 398-406 (1967) Dische, Z. : Color reaction of carbohydrates. In : Methods in Carbohydrate Chemistry, vol. I, pp. 477 - 512, Whistler, R.L., Wolfram, M.L., eds. New York: Acad. Press 1962 Johnson, K.D., Chrispeels, M.J.: Regulation of pentosan biosynthesis in barley aleurone tissue by gibberellic acid. Planta [Berl.] 111, 353-364 (1973) Jones, R.L. : Gibberellic acid and the fine structure of barley aleutone cells. II. Changes during the synthesis and secretion of a-amylase, Planta (Berl.) 88, 7 3 - 86 (1969) Jones, R.L.: Gibberellic acid-enhanced release of fl-(1,3)-glucanase from glucanase from barley aleurone cells. Plant Physiol. 47, 412-416 (1971) Mares, D.J.o Stone, B.A. : Studies on wheat endosperm. I. Chemical composition and ultrastructure of the ceil walls. Aust. J. biol. Sci. 26, 793-812 (1973) McNeil, M., Albersheim, P., Taiz, L., Jones, R.L. : The structure of plant cell walls. VII. Barley aleurone cells. Plant Physiol. 55, 6 4 - 6 8 (1975) Preece, I.A., MacDougal, M.: Enzymatic degradation of cereal hemicelluloses. II. Pattern of pentosan degradation. J. Inst. Brewing 64, 489-500 (1958) Taiz, L., Honigman, W.A.: Production of celI wall hydrolyzing enzymes by barley aleurone layers in response to gibberellic acid. Plant Physiol. 58, 380-386 (1976) Taiz, L., Jones, R . L : Gibberellic acid, fl-(1,3)-glucanase and the cell walls of barley aleurone layers. Planta (Bed.) 92, 73--84 (1970) Taiz, L., Jones, R.L. : Plasmodesmata and an associated cell wail component in barley aleurone tissue. Amer. J. Bot. 60, 6 7 - 7 5 (1973) Van der Eb, A.A., Nieuwdorp, P.J. : Electron microscopic structure of the aleurone cells of barley during germination. Acta bot. need. 15, 690-699 (1967) Varner, J.E., Ho, D.T.: The role of hormones in the integration of seedling growth. 34th Syrup., Soc. Devel. Biol. (in press). New York: Acad. Press (1976) Varner, J.E., Mense, R.: Characteristics of the process of enzyme release from secretory plant cells. Plant Physiol. 49, 187-189 (1972)
Received 12 October 1976; accepted 17 January 1977
Planta 134,251-256(1977)
9 by Springer-Verlag 1977
Gibberellic-acid-induced Synthesis and Release of Cell-wall-degrading Endoxylanase by Isolated Aleurone Layers of Barley William V. Dashek* and Maarten J. Chrispeels** Department of Biology, University of California, San Diego, La Jolla, CA 92093, USA
Abstract. When aleurone layers of barley (Hordeum vulgate L.) are incubated with gibberellic acid (GA3) xylose and a r a b i n o s e - b o t h as free sugars and bound to larger m o l e c u l e s - a r e released into the medium. Release begins 1 0 - 12 h after the start of incubation and continues for at least 60 h. At the same time there is a GA3-induced breakdown of the cell wall resulting in a loss of 2/3 of the cell-wall pentose during 60 h of incubation. GA3 causes the appearance in the medium of an enzyme (or enzymes) which hydrolyze larchwood xylan and aleurone-layer arabinoxylan. Release of the enzyme(s) into the medium begins 2 8 - 3 2 h after the start of incubation. Enzyme activity does not accumulate to any large extent in the tissue prior to release into the medium, and is present in very low levels only in the absence of G A 3. Xylanase activity is associated with a protein (or proteins) with a molecular weight of 29,000. The hydrolysis o f the xylans is largely caused by endoxylanase activity, indicating the importance of endoglycosidases in the GA3-induced breakdown of the aleurone cell wall. Key words: Aleurone - Cell-wall breakdown - Endosperm - Gibberellin - Hordeum - Xylanase.
Introduction Mobilization of the endosperm reserves accompanies seedling growth in cereal grains. The protein-rich cells of the aleurone synthesize a variety of hydrolytic enzymes which are released into the starch part of the endosperm where they hydrolyze the stored reserves. This process is controlled by the hormone gibberellic * P r e s e n t address." Department of Botany, State University of New York at Syracuse, Syracuse, NY 13210, USA ** To whom reprint requests should be addressed
acid (GA3) which originates in the growing embryo. When GA3 is added to isolated aleurone tissue the cells synthesize and release into the incubation medium c~-amylase, protease, /~-glucanase, ribonuclease and acid phosphatase. Release into the medium is always dependent on the presence of GAa, while synthesis is GA3-dependent for some enzymes such as c~-amylase and protease, but not for others such as acid phosphatase and/?-(1,3)-glucanase (for a recent review see Varner and Ho, 1976). Ashford and Jacobsen (1974) recently showed that acid phosphatase is synthesized and secreted into the periplasmic space whether GA3 is present or not, but that release into the medium depends on the GA3-mediated breakdown of the cell wall. They confirmed earlier ultrastructural observations by van der Eb and Nieuwdorp (1967) and by Jones (1969) that GA3 brings about a dissolution of the aleurone cell wall. These observations indicate that the cell wall and not the plasma membrane constitutes the effective barrier against movement of the hydrolytic enzymes from the aleurone cells to the starchy part of the endosperm, as first suggested by Varner and Mense (1972). Barley aleurone cells walls were recently shown to consist almost exclusively (85%) of an arabinoxylan which has a linear fi-(1,4)-xylan backbone, with 1/3 of the xylosyl residues substituted with single arabinosyl sidechains (McNeil et al., 1975). Arabinoxylans also constitute a major fraction of the hemicellulose of the cell walls of cereal endosperm (Mares and Stone, 1973) and of monocotyledons in general (Burke et al., 1974). Breakdown of aleurone cell walls may be mediated by exo- and/or endoglycosidases capable of hydrolyzing pentosans. In a recent paper Taiz and Honigman (1976) demonstrated that the addition of GA3 to isolated aleurone layers of barley causes the cells to release enzymes with c~-arabinofuranosidase, fl-xylopyranosidase and fi-(1,4)-xylanase activity.
252
W.V. Dashek and M.J. Chrispeels: GA3 and Cell-wall-degrading Enzymes in Barley Aleurone
Material and Methods
Incorporation of Radioactivity
Aleurone layers from seeds of barley (Hordeum vulgare L. cv. Himalaya; obtained from Washington State University, (Pullman, 1973 harvest) were prepared from embryo-less half seeds imbibed for 3 days on sterile sand. The layers were incubated in 25-ml flasks at 26~ in 3 ml of buffer containing 2 m M acetate, pH 5.0, 50 ~g/ml chloramphenicol, and 10 m M CaC1/ with or without 1 g M GA3 (Chrispeels and Varner, 1967).
Batches of 150 aleurone layers were labeled for 6 h with 2 IxCi of [14C]arabinose (New England NucIear Corp., Boston, Mass., U S A ; 9.78 mCi/mmol) in the absence of GA3. The layers were then rinsed 3 times with 50 m M arabinose and divided over 16 flasks (8 layers per flask), and further incubated in the incubation buffer with 50 m M arabinose and either with or without GA3. Radioactivity in the macromolecules of the cell wall and the cytoplasm was determined as described in J o h n s o n and Chrispeels (1973).
Preparation of Enzyme Extracts and Cell Walls
Results
The incubation media were collected and the aleurone layers rinsed with 2 ml of incubation buffer. The rinses were added to the media which were then clarified by centrifugation at 12,000 x g for 30 min. The resulting supernatant is referred to as " m e d i u m " . The aleurone layers were rinsed again, then homogenized in a mortar and pestle in a total of 4 ml of incubation buffer. The homogenate was centrifuged at 1000 x g for 10 min to remove the cell walls. This supernatant is referred to as " e x t r a c t " . The cell walls were resuspended and resedimented 3 times in 5 ml of homogenization buffer and then used as a source of enzyme or hydrolyzed to determine the pentose content. The media, extracts and cell-wall suspensions were dialyzed overnight against the incubation buffer to remove any pentoses present.
Degradation of the Aleurone Cell Wall
Preparation of Aleurone Layers
Xylanase Assays Colorimetric xylanase assays were performed with larchwood xylan (Sigma Chemical Co., St. Louis, Mo., USA) or barley aleurone cell wall arabinoxylan as substrates. The arabinoxylan was prepared according to the method of McNeil et al. (1975) and 1 ml of the solution contained the arabinoxylan present in 6 - 8 aleurone layers. The larchwood xylan was used as a 0.5% suspension in 25 m M citrate-phosphate buffer, p H 5.5. One ml of substrate was incubated with 0.5 ml of enzyme, usually for 2 h at 37~ in a shaking waterbath a n d the reaction was terminated by the addition of 4 ml of absolute ethanol. The cloudy solution was centrifuged in the cold for 1 5 m i n at 1 0 0 0 x g and an aliquot (0.1 0.5 ml) of the resulting clear supernatant assayed for pentose residues by the orcinol-ferric chloride-HC1 method of Dische (1962). Absorbance was determined at both 560 n m and 670 n m and a correction was made for interfering hexoses whenever the absorbance at 560 n m was more than half the absorbance at 670 nm. One enzyme unit is defined as the a m o u n t of enzyme which causes the release of 10 gg of pentose residues during the standard 2-h incubation. Viscometricxylanase assays were performed with barley aleurone cell wall arabinoxylan as a substrate in a Cannon-Fenske viscometer. Reactions were run at pH 5.5 at 40 ~ Five m1 of substrate were mixed with 5 - 2 5 ~tl of enzyme and the viscosity of the resulting mixture determined at 2 rain intervals. Typical results are shown in the inset in Fig. 6. One enzyme unit is defined as the a m o u n t of enzyme which causes a change of 1.0 in the relative viscosity of the arabinoxylan solution.
Determination of Sugars Cell walls were resuspended, mixed with an equal volume of 4 N trifluoroacetic acid, and hydrolyzed for 1 h at 121 ~ in sealed hydrolysis ampules. Media were cleared by centrifugation at 12,000 x g for 10 min and an aliquot was used for the determination of pentose (Dische, 1962). An aliquot of the medium was hydrolyzed in 2 N trifluoroacetic acid for 1 h at 120~ and the trifluoroacetic acid removed by evaporation. The sugars were derivatized and separated by gas liquid chromatography (Albersheim et al., 1967). Inositol was included as a standard.
The effect of GA3 on the degradation of the aleurone cell wall was determined by measuring the total pentose residues in the wall and in the incubation medium. Accumulation of pentose residues in the med i u m - s h o w n in Figure 1 - b e g a n 1 0 - 1 2 h after the start of the incubation of the layers with GA 3. The orcinol method employed to determine pentose levels estimated not only the free sugars but also the pentose residues in larger molecules. After 64 h of incubation the medium contained 5 0 0 - 6 0 0 gg of pentose per aleurone layer. In the absence of GA3, release of pentose residues did not start until 48 h after the beginning of incubation. The molecular size of the pentose-containing molecules in the medium was estimated by subjecting the medium to gel filtration chromatography on Sephadex G-50. The results (Fig. 2) indicate that 18 h and 25 h after the start of the incubation a large proportion of the pentose residues was present in molecules excluded from the gel. The presence of free arabinose and xylose in the incubation medium was confirmed by paper chromatography (see Fig. 2 in Chrispeels et al., 1973). Analysis by gas liquid chromatography of the sugar residues in these released molecules indicated that xylose accounted for 60% of the total pentose residues and arabinose for 40% (data not shown). The medium also contained large amounts of free sucrose, glucose and fructose but these were not quantitated. The presence of these sugars in the medium is the result of the GA3-enhanced release of sucrose from the aleurone cells (Chrispeels et al., 1973). Since the cell walls of wheat endosperm are known to be rich in arabinoxylan (Mares and Stone, 1973) we examined the possibility that the small endosperm fragments, which invariably cling to isolated aleurone layers, were the source of the pentose residues in the medium. Using aleurone layers which contained much less and much more than the usual amount of starchy endosperm we observed no differences in the amount of pentose residues released into the medium. Cell-wall breakdown was demonstrated directly
W.V. Dashek and M.J. Chrispeels : GA~ and Cell-wall-degrading Enzymes in Barley Aleurone
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Fig. 4. Metabolic stability of [l*C]arabinose labeled macromolecules in the cytoplasm and the cell wall of aleurone layers. The layers were labeled with [14C]arabinose for 6 h, rinsed, and further incubated with 50 mM arabinose either with or without GA3. Samples were harvested at the times indicated and the radioactivity in the cytoplasmic macromolecules and the cell wall determined. Open symbols: control closed symbols: GA3
by measuring the total pentose residues in the isolated cell walls after hydrolysis of the walls in 2 N trifluoroacetic acid. Each aleurone layer lost about 400 gg of cell wall pentose during a 64-h incubation (Fig. 3). This number agrees reasonably well with the amount of pentose which accumulated in the medium. In the absence of GA3 cell-wall pentose remained unchanged for up to 48 h of incubation. It was not possible to determine exactly at which time GA 3induced cell-wall dissolution began because of the variability between the samples. The loss of hexoses from the cell wall was not determined.
Aleurone layers readily convert [14C]arabinose into [l*C]xylose and incorporate both pentoses into cellular macromolecules (Johnson and Chrispeels, 1973). The metabolic stability of these newly synthesized macromolecules was tested in the presence and absence of GA3. Aleurone layers were allowed to incorporate [14C]arabinose for 6 h. The layers were then rinsed and further incubated with 50 mM unlabeled arabinose and in the presence or absence of GA3. The radioactivity in cytoplasmic and cell wall polymers was determined. The results (Fig. 4) indicate that the radioactivity in cytoplasmic polymers was
254
w.v. Dashek and M.J. Chrispeels: GA3 and Cell-wall-degradingEnzymesin Barley Aleurone
secreted and/or metabolically labile, while the radioactivity in cell-wall polymers was metabolically stable. The newly synthesized cell-wall arabinoxylans were apparently not degraded in the presence of GA3 and differed in this respect from the bulk of the arabinoxylans in the wall.
GA3-induced Xylanase Activity We tested for the presence of xylanase activity by incubating aliquots of tissue extracts or incubation medium with commercially available larchwood xylan or with arabinoxylan prepared from aleurone layers, and measuring the total pentose residues in the alcoholic supernatant of the reaction mixture. Similar results were generally obtained with either substrate. Preliminary experiments indicated that xylanase activity was present in the incubation medium of aleurone layers incubated in the presence of GA3 for 48 h, and could be measured quantitatively by using sufficient enzyme to produce an absorbance of 0 . 1 - 0 . 6 units under the standard assay conditions (see Material and Methods). The release of alcohol-soluble products from the alcohol-insoluble larchwood xylan or aleurone-layer arabinoxylan could be caused by the activity of both exo- and endopeptosanases. Both types of enzymes are present in barley malt (Preece and MacDougal, 1958) and produced by aleurone layers incubated with GA3 (Taiz and Honigman, 1976). The extent to which both types of enzymes contributed to the formation of reaction products was determined by measuring the molecular size of the alcoholsoluble reaction products on a Sephadex G-50 column. The results (Fig. 5)
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Fig. 6. Time course of the appearance of endoxylanase activity in the extract and the incubation medium of aleurone layers incubated with GA 3. No detectable xylanase activity was found in the absence of GA3. The inset shows the raw data on the decrease in running time of the reaction mixture in the viscometer. The reaction mixture consisted of 4 ml of cell wall arabinoxylan and 5 ml or 10 ~tl of enzyme from a 48-h medium
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The results presented in this paper demonstrate that GA3 induces the breakdown of the walls of aleurone cells of barley. During a 64-h incubation the aleurone cells lost a/3 of their cell wall pentose (about 400 gg per layer) while free pentoses and pentose-containing macromolecules accumulated in the medium (about 500 lag per layer). Since arabinoxylan makes up 80% of the aleurone cell wall (McNeil, 1975) this finding constitutes an analytical confirmation of ultrastructural (Jones, 1969; van der Eb and Nieuwdorp, 1967) and cytochemical (Taiz and Jones, 1970) evidence concerning the GA 3-enhanced breakdown of the wall. Our results also confirm the finding of Taiz and Honigman (1976) that GA3 induces the formation and release of an enzyme or enzymes with xylanase activity. Barley malt contains several enzymes which degrade pentosans (Preece and MacDougal, 1958) and GA3 induces the formation and the release of both exo- and endopeptosanases in aleurone cells. The enzyme(s) detected by our assays degraded commercial larchwood xylan as well as cell-wall arabinoxylan; degradation was followed colorimetrically through the formation of alcohol-soluble products, or viscometrically. Since the products of the reaction were larger than individual sugars we concluded that the enzyme measured by both procedures-colorimetric and viscometric- was an endoxylanase. The possibility that exoxylanase contributed to the formation of reaction product could not be excluded. Xylanase activity appeared in the medium after 26-30 h and release from the tissue continued for 12-16 h. Tissue extracts contained small amounts of endoxylanase (after 24 h of incubation) but there was no accumulation in the tissue of a large amount of endoxylanase prior
256
W.V. Dashek and M.J. Chrispeels: GA3 and Ce11-wall-degrading Enzymes in Barley Aleurone
to its release into the medium as is the case with ribonuclease (Chrispeels and Varner, 1967) and fl(1,3)-glucanase (Jones, 1971). Taiz and Honigman (1976) found equivalent amounts of endoxylanase in the tissue and in the medium at the time of maximum enzyme release (48 h). The discrepancy between these two observations may be due to differences in the seeds. The relationship between cell-wall breakdown and enzymatic activity remains unresolved. The accumulation of free pentoses and macromolecular pentose in the medium indicates that both exo- and endopentosanases are involved in cell-wall breakdown. Ultrastructural and cytochemical experiments indicate that cell-wall breakdown has progressed considerably by 24 h (Taiz and Jones, 1970; van der Eb and Nieuwdorp, 1967). Our own analyses indicate that breakdown starts after 12-16 h and that less than 20% of the cell-wall pentosan has been hydrolyzed at 24 h. The secretion of exoxylanase and exoarabanase begins 1 2 - 16 h after the start of incubation (Taiz and Honigman, 1976), while endoxylanase is not secreted until 12 h later. It is difficult to see at the moment how the activity of these exoglycosydases can account for the early release of macromolecular pentose in the medium. The resolution of this problem may have to await the characterization of these pentose-containing macromolecules. It is remarkable that the massive degradation of the aleurone cell wall cannot be followed by determining the metabolic lability of the newly synthesized cell-wall pentosans. Secretion of labeled macromolecules is a rapid process which is completed in 3 0 - 6 0 rain (Chrispeels, 1976) and most of the decrease in cytoplasmic radioactivity can therefore be attributed to metabolic lability of macromolecules. The results indicate that the cell wall contains two arabinoxylan components: one which is readily degraded when the tissue is incubated with GA3, and one which is relatively stable. Taiz and Jones (1973) made a: similar observation when they treated aleurone layers with a mixture of hydrolytic enzymes. They found that the inner layer of the wall was quite resistant to degradation by fungal enzymes which removed all of the outer portion of the wall. We suggest that the newly synthesized pentosans are added to this resistant cell wall layer. Dr. K.D. Johnson of San Diego State University carried out the experiment described in Figure 4. We thank him for allowing us to incorporate this experiment in our paper. The work was supported by the US. Energy Research and Development Administration (ERDA) contract E(04-3)-34/159.
References A1bersheim, P., Nevins, D.J., English, P.D., Karr, A.: A method for the analysis of sugars in plant cell wall polysaccharides by gas liquid chromatography. Carbohyd. Res. 5, 340-345 (1967) Ashford, A.E., Jacobsen, LV. : Cytochemical localization of phosphatase in barley aleurone cells. The pathway of gibberellic acid-induced enzyme release. Planta [Bed.] 120, 80-105 (1974) Burke, D., Kaufman, P., McNeil, M., Albersheim, P.: The structure of plant cell walls. VI. A survey of the walls of suspensioncultured monocots. Plant Physiol. 54, 109-115 (1974) Chrispeels, M.J. : Biosynthesis, intracellular transport, and secretin of extracellular macromolecules. Ann. Rev. Plant Physiol. 27, 1 9 - 3 8 (1976) Chrispeels, M.J., Tenner, A.J., Johnson, K.D.: Synthesis and release of sucrose by the aleurone layer of barley: Regulation by gibberellic acid. Planta [Berl.] 113, 3 5 - 4 6 (1973) Chrispeels, M.J., Varner, J.E. : Gibberellic acid-enhanced synthesis and release of a-amylase and ribonuclease by isolated barley aleurone layers. Plant Physiol. 42, 398-406 (1967) Dische, Z. : Color reaction of carbohydrates. In : Methods in Carbohydrate Chemistry, vol. I, pp. 477 - 512, Whistler, R.L., Wolfram, M.L., eds. New York: Acad. Press 1962 Johnson, K.D., Chrispeels, M.J.: Regulation of pentosan biosynthesis in barley aleurone tissue by gibberellic acid. Planta [Berl.] 111, 353-364 (1973) Jones, R.L. : Gibberellic acid and the fine structure of barley aleutone cells. II. Changes during the synthesis and secretion of a-amylase, Planta (Berl.) 88, 7 3 - 86 (1969) Jones, R.L.: Gibberellic acid-enhanced release of fl-(1,3)-glucanase from glucanase from barley aleurone cells. Plant Physiol. 47, 412-416 (1971) Mares, D.J.o Stone, B.A. : Studies on wheat endosperm. I. Chemical composition and ultrastructure of the ceil walls. Aust. J. biol. Sci. 26, 793-812 (1973) McNeil, M., Albersheim, P., Taiz, L., Jones, R.L. : The structure of plant cell walls. VII. Barley aleurone cells. Plant Physiol. 55, 6 4 - 6 8 (1975) Preece, I.A., MacDougal, M.: Enzymatic degradation of cereal hemicelluloses. II. Pattern of pentosan degradation. J. Inst. Brewing 64, 489-500 (1958) Taiz, L., Honigman, W.A.: Production of celI wall hydrolyzing enzymes by barley aleurone layers in response to gibberellic acid. Plant Physiol. 58, 380-386 (1976) Taiz, L., Jones, R . L : Gibberellic acid, fl-(1,3)-glucanase and the cell walls of barley aleurone layers. Planta (Bed.) 92, 73--84 (1970) Taiz, L., Jones, R.L. : Plasmodesmata and an associated cell wail component in barley aleurone tissue. Amer. J. Bot. 60, 6 7 - 7 5 (1973) Van der Eb, A.A., Nieuwdorp, P.J. : Electron microscopic structure of the aleurone cells of barley during germination. Acta bot. need. 15, 690-699 (1967) Varner, J.E., Ho, D.T.: The role of hormones in the integration of seedling growth. 34th Syrup., Soc. Devel. Biol. (in press). New York: Acad. Press (1976) Varner, J.E., Mense, R.: Characteristics of the process of enzyme release from secretory plant cells. Plant Physiol. 49, 187-189 (1972)
Received 12 October 1976; accepted 17 January 1977
