1320705.. SIS OF 331$wa CELL WALLS IN VITRO Thesis for the Begree 03‘ 2‘5. 8. EiééiiHEGM STATE Ui‘é‘éE “:53th 3mm LEE 1.957 Wm'fl'wc W”)! - 5r ,, M4 was.» .3. 1 .3 3.: R 3:” fwiid‘figan State I. ‘f v _ r a ‘_ #2 L114 1 malty M — 'fl '7 WI» _' wm‘ug’o ‘1 j i" “r r a 1m. .3 ml.“ THES‘” i. 1.3- I A K Y Michigan State A University -5 M -___- ABSTRACT AUTOLYSIS OF PLANT CELL WALLS IN VITRO by Su-hwa Lee A prerequisite for elongation of plant cells is softening of the rigid plant cell wall. This study was initiated to see if autolysis could be attained in vitro in the belief that autolysis may be related to softening. Using a method developed earlier for the isolation of pure primary cell walls of corn (Zea mays)coleoptile tissue, it was found that the walls isolated by a modification of this method contain bydrolytic enzymes. This finding of autolysis of plant cell wall in vitro has not been pre- viously reported. During an eight hours incubation in water at 37° and pH 6.5, about ten per cent of the cell wall is rendered soluble, appearing in solution as reducing sugar and an as yet unidentified partially dialyzable glucose polymer. Glucose appears to be the sole monomeric product of autol~ ysis, but amounts only to one per cent of the initial cell wall weight, or approximately ten per cent of the weight of the solublized portion. It is of interest that twenty per cent of the initially soluble polymer becomes an insoluble white flocculent precipitate on freezing and thawing--that is, it retrogrades. The polymer(s) in many Su-hwa Lee respects resemble degradation products of callose. Further studies of their physical and chemical properties are needed. Since invertase is known to be associated with the cell wall, this enzyme was used as a criteria for the general enzymatic activity of the cell wall preparations under study. Most preparations were capable of hydrolyzing 0.025 umoles of sucrose per hour per mg of cell wall. The amount of reducing sugar released by autolysis, increased linearly with incubation time and with the amount of cell wall material used. The release of polymer follows the same pattern. Boiling the cell wall prepara- tion before incubation reduces the autolysis by a factor of three to six. In studies of the pH Optimum for autolysis phosphate, acetate and tris buffers were used. Acetate or phosphate ion apparently have some interaction during the incuba- tion period. For eacmple, phosphate buffer increased by almost twofold.the amount of reducing sugar released, as compared to a distilled water control at about the same pH (Table 3). The pH optimum curve, which has maxima at 5.5 to 6.5 might be interpreted as the result of effects of acetate and phOSphate buffer. It is also possible that the autolysis of cell walls involves more than one enzyme as suggested by the double optimum with respect to pH. Su-hwa Lee Cell walls prepared from whole coleoptiles showed higher autolytic activity than those from decapitated coleoptiles. This preliminary finding suggests a cor- relation between autolytic capacity and capacity for elongation growth. AUTOLYSIS OF PLANT CELL WALLS IN VITRO By Su—hwa Lee A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Botany and Plant Pathology 1967 ACKNOWLEDGMENTS I wish to express my sincere appreciation for the suggestion of this problem, and for the-guidance of Drs. Robert S. Bandurski and Aleksander Kivilaan, without whom I could not have accomplished the work presented. Also I am very much indebted to Drs. Norman E. Good and Anton Lang, for their critical reading of the thesis. Finally, I wish to express my gratitude to the United States Atomic Energy Commission (Contract-No. AT(ll-1) 1338) and to the National Science Foundation (622069) for financial~ support of this research. 11 TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . .3 . . . . . . . . . . ii LIST OF TABLES . . . .- . . . . . . . . . v LIST OF FIGURES . . .- . . . . . . . .. . . vi INTRODUCTION . . . . . . . . . . . . . . 1 LITERATURE REVIEW . o o o o' o o o o o o o ’4 Breakdown of the plant cell wall . . . .. .~ . A Elongation and softening of primary-cell walls . A Dissolution of secondary cell walls . . . . 6 Autolysis of bacterial cell walls . . . . . . 7 Autolysis of fungal cell walls . . . . . . . 8 AUTOLYSIS OF PLANT CELLS WALLS IN VITRO . . . . . 10 Material and methods .» . . . . . . . . . lO Preparations of Acetone powder of cell wall . . 10 Incubation and Fractionation of Wall Components after Autolysis . . . . . .3 . 13 Determination of Total Reducing Sugars and Glucose o o o o o o o o o 0‘ o ‘ o o o 1’4 Results . . . . . . . . . . . . . . l9 Autolytic Weight Loss of the Cell Wall During Incubation . . . . . . 19 Products of Cell Wall Autolysis . . . . . . 19 Time Course StUdieS o o o o 0' o o o o o 26 Effect of pH on Autolysis . . . 26 Invertase Activity Associated with the Autolytic System . . . . 33 Autolysis of Cell Wall as a Function to its Concentration . . . 33 Effect of Indoly-3- -Acetic Acid on Autolysis . . 38 Effect of Decapitation on Autolysis of Cell walls 0 ' o o o o o o o o o o o o o 38 111 DISCUSSION . . SUMMARY . . . LITERATURE CITED iv‘ Page Al ‘47 148 Table 1. LIST OF TABLES Autolytic weight loss of cell walls during incubation at 37°C . . . . . . . . . Weight loss of cell walls and alphacel during different treatments . . . . ., . . . . Effect of potassium, phosphate, and magnesium on cell wall autolysis . . . . .. . . .- . A preliminary single experiment on the effect of Indolyl-B-acetic acid on cell wall autolysiS* .- A preliminary single experiment comparing activity of cell walls prepared from decapitated and intact corn coleoptiles . . . .. . .. . Page 15 27 32 39 A0 Figure 1. LIST OF FIGURES Schematic outline of the procedure for isola- tion of cell wall fragments . . . . . . . Standard curve for glucose with the Somogyi 'method . . .. . . . . . . . . . . . Diagram of paper~chromatograms of cell wall substances solubilized during incubation . . Diagram of paper chromatograms of the hydrolysis products of the dialyzable polymer . . . . Diagram of paper chromatograms of the non- dialyzable polymer after three per cent nitric aC1d hydroly818 o o o ’ o o o o o o o o ‘ Time course of autolysis of the cell wall preparation .‘ . . .3 . .- . .3 . . .‘ .. Effect of pH on autolysis of the cell wall preparation . .- . . . .. . . . . . .- Time course for invertase activity associated With the C611 walls 0 o o o ' o o . o o ' o ‘ Autolysis as a function of cell wall concentra- tion 0 o o 0'. o o o 0,9 0'0 .30 vi Page ll 17 2O 22 2A 28- 30 3A 36 INTRODUCTION As early as 1931, Heyn proposed a possible mechanism for extension growth which involved an increase in cell wall plasticity. Tagawa and Bonner (1957) proposed that rigidity of the cell wall is a function of the number of calcium bridges present in pectic acid. The role of auxin in loosening the cell wall was thought to be due to the reduction of calcium bridges in pectic acid. However, Cleland (1960) concluded that "the removal of calcium cross linkages is not mediated by auxin and that the calcium bridge hypothesis is incorrect." Actually, a large body of evidence indicates that auxin-induced growth in various plant tissues is accompanied by changes in both the cell wall and the cytOplasm, and in nucleic acid metabolism. For example, Liao and Hamilton (1966), employing an auto- radiographic technique, have demonstrated-in Allium cernuum that the IAA-Clu is localized in the nucleus. Thus the mechansim of action of IAA is still unknown, except that the primary site of action may be the nucleus and following that a vast chain of events including cell wall softening is initiated. Results of several intensive investigations demonstrate that the plasticity of the cell wall is an important factor in cell enlargement. Of major interest is a mechanism which will account for the alterations in wall plasticity asso— ciated with growth. Speculating on the way in which plasticity of the wall could be increased, Frey-Wyssling (1950) proposed that the cutting or dissolving away of the fiber reticulum could bring parts of the wall into a semi- liquid state, allowing the remainder of the reticulum to extend under the turgor pressure of the protoplast. Thus, although we are uncertain as to the mechanism, one of the prerequisites of growth is elongation or softening of the primary wall. The cell wall develops as plants are growing. In bacteria, fungi or higher plants, disappearance or softening or degradation of the cell walls is involved at different stages in different tissues. The possibility of an auto— lytic breakdown has been considered in bacterial or fungal cell walls (Mitchell and Moyle, 1957; Arima ggJil., 1965). In vivo, isotOpe feeding experiments have indicated that similar processes occur in higher plants (Maclachlan and Young, 1962 and Maclachland and Duda, 1965). The aim of this study was to investigate the softening process of the primary plant cell wall by enzymatic autolysis in vitro. First, highly purified corn coleoptile cell wall was prepared by the method of Kivilaan 213;. (1959), as dia- gramed in Figure 1. Cell wall preparations were washed free of glycerol with absolute alcohol, acetone and ether at low temperature resulting in a white amorphous powder. Thus the wall preparation utilized was made exactly as described by Kivilaan eg_al., except that glycerol was removed with solvents at temperatures of -10 to -20°. This material, designated as cell wall was suspended in buffers or distilled water. and incubated. Autolytic weight losses and the products of autolysis were determined. The amounts of reducing sugars were determined colorimetrically, sugars were identified by chromatography, and weight losses were determined by weighing the cell wall material either on an electrobalance or on an analytical balance. The heat lability of autolysis and its exhibition of a pH optimum at near neutral pH indicate the involvement of a typical enzymatic reaction. It is of interest that cell wall prepared from whole coleoptiles had higher autolytic activity than those prepared from decapitated coleoptiles. LITERATURE REVIEW Breakdown of the Plant Cell Walls Neumfiller (1958) stated that polysaccharides serving as structural elements in plants could not be broken down by enzymes of endogenous origin, but anatomical evidences have indicated that this is not true. Easu (1953), for example, has cited several instances which may be inter— preted as digestion of cell wall material in_si£u, This is especially true, for the primary cell wall at the growing and expanding stages of the cells. Elon tiongand Softening of the rimary Oell Wall That auxin, indoly1-3-acetic acid (IAA) increases the rate of cell elongation in Azen§_cole0ptiles by causing the cell walls to become more easily stretchable and more plastic was first suggested by Heyn (1931). This theory lost popularity for a time but in the late 1950's regained it again, as, for example, in the studies of Tagawa and . Bonner (1957), employing a "coleoptile bending machine." In their experiments, IAA and potassium ion were able to increase the deformability by increasing the plastic com— ponent, whereas calcium ion reversed these effects. Plastic extension of the primary cell wall in enlarging plant cells is thought to require relaxation of the interwoven structure A of the pre-existing wall as well as deposition of new wall material (Setterfield and Bayley, 1961). It is possible that relaxation of the old wall which has usually been attributed to fiber—orientation, could also be facilitated by the breakage of bonds. That bond breakage and "plastic flow" is involved has been demonstrated by Lockhart (1966 and personal communication). With sections excised from the third internode of 8—day-old etiolated pea epicotyls, Maclachlan and Young (1962) were able to show in reality that wall catabolism and wall anabolism are correlated with growth. Their data lead to the conclusion that about one-third of the original insoluble material is dissolved during the time period of their experiment. They prOposed that an extensive breakdown of pre-existing wall, rather than the synthesis of new wall, should be regarded as an essential feature of plasticization in enlarging cells. During growth, new wall material is intercalated in amounts which, depending on substrate availability, may or may not replace the material dissolved. Experiments with pollen grains support a relationship between degradation of cell wall material and plasticization of the cell wall. Matchett and Nance (1962) suggested that, when degradation does not result in lysis of the growing tip of the pollen tube, it may cause an increase in plasticity of the pollen tube wall. This can be interpreted as strengthening the argument for the existence of a relationship between degradation of cellu- lar material and plasticity of the primary cell wall. Dissolution offiSecondary Cell Wall Hartig (1853) and Flach (192A) reported that the dissolution of the end walls was a gradual process originat- ing in one spot in the middle of the wall and spreading from this place to other parts of the wall. Esau (19A0), in attempting to determine how continuity is established f} between vessel segments found that two superposed vessel I elements were separated from each other by two cellulose layers. With the electron microscope and shadow cast material, Scott gt_§g, (1960) could not observe the dis— integration process and assumed that the end wall during disintegration of the protoplast is broken up into pieces and resorbed. That connection between sieve elements through the sieve areas involved a removal of the cell wall at the pore site was first demonstrated by Esau §t_al, (1962). The site of the future pores was delimited-by the appearance of small deposits of callose. Perforation of the pore site occurs in its center by dissolution of part of the wall and middle lamella, resulting in the union of the callose platelets. Obviously, a variety of enzymatic mechanisms must underlie the highly localized wall differ- entiation observed in the sieve plate, including the dissolution of the components of the cell wall and middle lamella and the accompanying synthesis of callose. Autolysis of Bacterial Cell Walls Autolysis and autolytic enzyme systems have been observed in a variety of Gram-positive and Gram-negative bacteria (Kronish gt_al., 196A; Mitchell and Moyle, 1957; Murray gtgal., 1959). A specific autolytic substance has been observed in the supernatant fluids of sporulating I? cultures of Bacillus terminalis (Greenberg and Halvorson, .1955). This material is relatively heat stable, non- dialyzable, and has a pH optimum in the range of 5.0-5.5. ' f'" It is precipitated at 0.A5-0.70 saturation with ammonium sulfate. The atuolytic system found in Streptococcus faecalis by Shockman g§_a1. (1961) seems to be highly specific. Well-washed walls from cells in the exponential phase will slowly autolyze in phosphate buffer or in distilled water. Lytic activity of the extracts was non— dialyzable and heat-labile. The autolytic-system does not seem to be a lysozyme, since the extracts failed to lyse Micrococcus lysodeikticus. Young and Spizizen (1963) also found autolytic enzyme activity in the cell wall of Bacillus subtilis. A heat-labile, non-dialyzable enzyme is present, degrading the cell wall, liberating nonedialyzable hetero- polymers, dialyzable mucopeptides, amino acids and glucose, and an insoluble residue. The non-dialyzable fraction, \ which constituted 67 to 75 per cent of the cell wall, is i composed of at least three heteropolymers with a molecular i weight of 15,000 to 20,000. That these autolytic enzymes play some role in wall growth and cell division has been proposed by a number of laboratories (Mitchell and Moyle, 1957; Shockman gg_§l., 1958; Weidel 22_§l-: 1960). Shockman (1965) proposed that perhaps existing bonds are broken in the more or less continuous matrix of wall polymers surrounding the cell, so that a new bit of completed precursor can be inserted, thereby lengthening the polymer chain. On the basis of evidence obtained by use of immunoflurescence, Chung g§_al,, 196A; Cole and Hahn, 1962, and Cole, 1964 and May, 1963, concluded that the bands of new wall synthesis might be initiated by such an autolytic system and correspond to the only areas of wall that are susceptible to the autolytic enzyme system. Autolysis of-Fungal Cell Walls Enzymatic lysis of fungal cell walls by other micro- organisms has been reported for a number of fungi. Horikoshi and lids (1958) associated lysis of Aspergillus by some Bacillus species with the action of a chitinase. Mitchell and Alexander (1963) demonstrated chitinase and protease activity in bacterial degradation of Fusarium cell wall. With these fungi the source of the lytic enzymes was another micro-organism. Studies on the autol- ysis of Aspergillus oryzae, Arima et al., (1965) showed that when.mycelia are suspended in water and incubated under suitable conditions, proteins, nucleic acids, and sugars are excreted into the medium as products of hydrolysis. They found that up to ”5°, autolysis occurs faster at higher temperatures. Autolysis occurred readily when the mycelia were incubated in water without glucose under anaerobic con— ditions. Mitchell and Sabar (1966) showed a direct relation- ship between fungal growth and autolytic activity. The rate of branching which occurs during development of young hyphae of Pythium, suggests that branching points may be sites of maximum lytic activity. However, both mycelial growing tips and cell wall synthesis may equally account for the realtionship between autolysis and growth. Evidence by Lloyd and Lockwood (1966) also suggests that an autolytic mechanism can account for soil mycolysis. When soil was separated from fungal mycelium by membrane filters having pores small enough to prevent passage of intact organisms, living hyphae of several fungi were completely or partially lysed whereas dead hyphae were not lysed. Complete autol— ysis of living hyphae of Glomerella cingglata or Hemintho- sporium victoriae was induced by exposure to antifungal antibiotics when the hyphae were kept starved. AUTOLYSIS OF PLANT CELL WALLS IN VITRO Material and Methods Preparation of Acetone Powders of the Cell Wall Glycerol pellet of cell wall preparation.--Cell wall fragments of corn coleoptiles were prepared according to the method previously described by Kivilaan g£_al., (1959). A summary of the steps involved is given in Figure 1. Michigan 300 hybrid corn (196A) was soaked for 20 hours in tap water. After germination for A to 5 days in the dark, coleOptiles were harvested and frozen until used for cell wall preparation. In some cases coleoptiles were decapi- tated by removing approximately three millimeters of the tip at least five hours prior to harvest. In other cases, whole coleoptiles together with shoot apices were harvested. In such preparations the coleoptile tissue constitutes about two thirds of the total fresh weight. All manipula- tions were done in a growth room under red light. Frozen tissue was homogenized in glycerol, repeatedly washed free of subcellular particles and cytOplasm with the same medium and cell wall fragments collected by centrifuga- tion (Figure 1). Cell wall fragments made up five per cent of the final pellet, the rest being glycerol. 10 t T"- 11 Figure 1.--Schematic outline for the isolation of cell wall fragments. Michigan 300 hybrid corn (196“) was soaked in water for 20 hours and germi— nated for A days in the dark. Coleoptiles were harvested and frozen until used for cell wall preparation. Coleoptile tissue (25 gm) was homogenized in a Servall "Omnimixer" for two 5- minute intervals, at 16,000 r.p.m. together with 180 m1 of redistilled glycerol and 37 gm of glass beads 200 u in diameter (Minnesota Mining and Manufacturing Company, Saint Paul, Minnesota). 12 CORN COLEOPTILES GLASS BEADS, GLYCERIN I GHOMOGENIZED FOR IO Io I5 MINUTES HOMOGENATE <1 ALLOWED TO STAND FOR 30 MINUTES, DECANTED SUPERNATANT FLUID RESIDUE 4 FIRST CLASS BEADS. FILTRATION PARTICULATE PLANT MATERIAL DISCARD FILTRATE RESIDUE I PLASTICS, NUCLEI, CYTOPLASM, ETC. CELL WALL FRAGMENTS, CONTAMI NANTS DISCARD <1 RESUSPENSION AND SECOND FILTRATION FILTRATE RESIDUE I DISCARD <1 RESUSPENSION AND THIRD FILTRATION FILTRATE RESIDUE I DISCARD <1 RESUSPENSION AND FOURTH FILTRATION FILTRATE RESIDUE I DISCARD CELL wALL FRAGMENTS I <1 CENTRIFUGATION CELL WALL PREPARATION 13 'Preparation of an alcohol-acetone powder from the cell wall glycerol pellet.-—Glycerol was removed from cell wall fragments by suspending the pellet in at least one hun— dred times its weight of cold absolute ethanol, and the cell wall collected by filtration (filter, E—8B Precipitation apparatus, Tracerlab, Boston) on Whatman No. 5A0 ashless filter paper. Following extensive washing with acetone and peroxide-free ether in the cold, the preparation was recovered and dried overnight under vacuum over phosphorus pentoxide. The resulting white granular powder was Stored over anhydrous calcium sulfate at -20°C and used as a cell wall preparation when needed. All procedures were conducted at -5 to -10° and did not require longer than twenty to thirty minutes. Incubation and Fractionation of WSIILComponents aTter Autolysis In most of the autolysis studies the dry cell wall powder was suspended in glass distilled water (10 mg per ml), a drop of toluene added and the suspension then incu- bated at 37°. The reaction was stopped by boiling for three to five minutes in a water bath, cooled, the residue collected by filtration under reduced pressure on Whatman No. 540 filter paper, washed with a small amount of water, dried to constant weight, and weight losses determined by a Cahn electrobalance, by an analytical balance, or both. Clear filtrate was kept overnight at -10°, thawed, and the resultant white precipitate formed, collected by centri- fugation at 1000 g for five minutes. This procedure was 1A repeated three times. The pellet so obtained is referred to as non—diahysable polymer or retrogradation product. It was taken to dryness and weight determined with a Cahn electro- balance. Residue of the final supernatant fluid obtained after 1yophilization, was taken up in a minimum amount of water, pipetted to weighing pans, redried under vacuum over P205, and the weight determined by an electrobalance. This is designated as the soluble or dialysable fraction. In cases where glycerol occurred as a contaminant of the dialyzable polymer (see Table 1) it was-separated by paper chromatography and estimated semi-quantitatively by the intensity of the spot of silver nitrate reaction material. Reducing sugars, as soluble autolysis products in the fil- trate were determined colorimatrically whereas non—reducing polymers were weighed with the Cahn electrobalance. In studies of the pH optimum, the acidity of the incu- bation medium was adjusted from pH “.0 to pH 5.5 with 0.01 M acetate and 0.01 M K2H phosphate buffers, from pH 5.5 to pH 7.5 with 0.01 M K2H phOSphate buffer, and from pH 7.5 to pH 10 with 0.01 M tris and 0.01 M K2H phosphate buffers. Determination of Total Reducing Sugars and Glucose Modified Somogyi (1952) and Nelson (1944) methods were employed.for the colorimatric determination of total re- ducing sugar. The COpper reagent consisted of solution I (2A gm anhydrous Na2CO3; 12 gm KNaCuHuO6. H20: 16 gm 15 TABLE 1.-—Autolytic weight loss of cell walls during incubation at 37°C. Boiled Boiled Difference after before incubation incubation mg mg mg Cell wall preparation VII 100 100 Residue recovered after incubation 80 91 -11 In fi 1t rate: Glycerol 7 7 Reducing sugar 1.2 0.2 1.0 Non-dialyzable polymer 2.6 0.5 2.1 Dialyzable polymer 9.2 1.3 7.9 Incubation was for 8 hours at 37° using 100 mg of cell wall in a total volume of 5 ml. Weight loss of the walls was determined gravimetrically. Glycerol was identified by chromatography and estimated by difference. Reducing sugar was determined by the method of Nelson- Somogyi. Non-dialyzable polymer was estimated gravi- metrically after 1yophilization of the contents remaining in the dialysis bag. Dialyzable polymer was estimated by 1yophilization of the water exterior to the dialysis bag, then extracting glycerol with water, redrying, and weighing the retrograded polymer. 16 anhydrous NaHCO3; 1AA gm anhydrous Na2SOu; dissolved in glass distilled water and made to 800 m1), and solution II (A gm CuSOu-S H20; 36 gm NaZSOA3 dissolved in 200 m1 H2O). The ratio of solution I to solution II was A to 1. The color reagent contained 25 gm (NHu)6Mo7O2u.A H2O in A50 m1 H O; 21 m1 concentrated H280“; 3 gm Na2HAsOu.7 H2). It 2 was "aged," in an incubator at 37°C for 2A to A8 hours and then stored in a glass—stopped brown bottle. The standard curve for glucose concentration was obtained by using a 5A0 mu filter in a Klett-Summerson colorimeter and is shown in Figure 2. Glucose was determined by the glucose oxidase method (Saifer and Gerstenfeld, 1958) using the "Glucostat" reagent from Worthington Biochemical Corporation. The solvents used for the chromatography of autolySis hydroly- sates were n-butanol : acetic acid : water = A : 1 : 5 and ethyl acetate : pyridine: water = 8 : 2 : 1. Sugar spots on chromatograms were detected by aniline-phthalic acid and silver nitrate sprays. The autolysate filtrate was either applied to the paper immediately after incubation or after storage in a deep freeze. The precipitate which formed upon freezing and thawing was collected by centrifugation and dialyzed against water in dialysis tubing (Union Carbide Corp., Chicago, Illinois) previously washed free of ninhydrin and silver nitrate positive material. The contents of the dialysis tubes and the dialysis water were recovered and taken to dryness by lyophilization. The weight of 17 Figure 2.--Standard curve for glucose with the Somogyi method. Clucose solution, 0.5 ml, contain- ing between 0 ug to 36 ug were incubated with 0.2 m1 CDpper reagent for 10 minutes in a boiling water bath. The reaction was stopped by cooling and 0.2 m1 of Nelson's color reagent added. Absorption was measured employing a Klett-Summerson colorimater with a 5A0 mp filter. JJQ GLUCOSE 50 (N 0 IO 18 I T E O / ./ ‘ O IOO zoo 300 400 500 KLETT READINGS AT 540mg l9 non—dialyzable polymer was determined on the Cahn electro- balance, and then partially hydrolyzed in three per cent nitric acid in a sealed tube at 105° overnight and chromato- graphed. Dialyzable polymer was estimated by lyophilization of the water exterior to the dialysis bag, then extracting glycerol and soluble sugars with water, redrying and weigh- ing the insoluble polymer. Results Autolytic Weight Loss of the Cell Wall During Incubation Cell Wall preparations were suspended in glass dis- tilled water and incubated at 37° for 8 hours. The reaction was stopped by boiling in a water bath for 3 to 5 minuted. The average autolytic weight loss of cell wall preparations during an eight hour incubation was approximately ten per cent (Table 1). The loss consisted of reducing sugar, and a partially non-dialyzable polymer, later identified as a glucose polymer. Products of Cell Wall Autolysis In filtrates, glucose was the only reducing sugar detected (Figure 3), when chromatographed immediately after filtration. However, on repeated freezing and thawing of the filtrate, a water-insoluble white precipitate was formed. Partial acid hydrolysis of this with three per cent nitric acid at 105° overnight, yielded again only glucose (Figure A and Figure 5). 20 Figure 3.-—Diagram of a paper chromatogram of sugars solubilized during autolysis. The filtrate obtained after incubation of cell walls was applied to Whatman No. 1 paper. A pyridine (pyridine : ethylacetate " water = 2 " 8 A 1) was used as developing solvent by the descending technique. Sugars were detected with aniline- phthalic acid. Standard sugars are GAL, galactose; GLU, glucose; FRU, fructose; MAN, mannose; ARA, arabinose; and XYL, xylose. 21 BMED BOILED BOILED AFT TER AFTER . .BEFORE STANDARD INCUBAT INCUBAT, MEAT SUGARS I IQV’I‘th O O O . . <30 :9: :MA“ ARA 22 Figure A.——Diagram of paper chromatography of the hydrolysis products of the dialyzable polymer. Dialyzable fraction of autolysate precipitate after freezing and thawing was chromatogrammed. Hydrolysis as described in the text. Procedure was the same as for Figure 3. 23 DIALYZABLE DIALYZABLE STANDARD POLYMER POLYMER SUGARS G LU O . MAN FRU .XYL 2A Figure 5.—-Diagram of paper chromatography of the non-dialyzable polymer after three per cent nitric acid hydrolysis. Non-dialyzable fraction of autolysate precipitate obtained after freezing and thawing was chromatogrammed. Procedure was the same as Figure 3. 25 NW HY DRGXSATE ”Oi-um W-UALYZ. 51mm) POLYMER POLYMER _ SUM -5.- —. F RU 26 In separate experiments using either 50 mg of cell wall or a commercial u-cellulose preparation (Alphacel of Nutritional Biochemical), the average weight losses of the insoluble residue was determined (Table 2). Alphacel incu- bated and boiled under the same conditions as the cell wall preparation loSt cxflgr 5 per cent of its weight compared to 22 per cent weight loss of the cell wall preparation. Time Course Studies The enzymatic activity of cell wall acetone powder in vitro was studied with time as a variable (Figure 6). The reaction rate was a linear function of time suggested that the release of reducing sugar is a consequence of an enzymatic process and not attributable to microbial con- tamination. Effect ofng on Autolysis Cell wall preparations were autolyzed at pH values between pH A.0 to 10 during an eight hour incubation period. Incubation was carried out at 37° by using acetic acid buffer (A.0 - 5.5); phosphate buffer (5.5 — 7.5) and tris buffer (7.5 - 10). Equivalent amounts of phosphate were present at all pH levels. Comparing Figure 7 with that of the time course studies (Figure 6), reveals that phosphate buffer increased by almost two-fold (1.9) the amount of reducing sugar released, as compared to a glass distilled water control. It is also shown in Table 3, with a 27 TABLE 2.-—Weight losses of cell walls and alphacel during different treatments. % weight loss Treatment of cell wall IX and alphacel in residue 1. Boiled (5 min.); without incubation 5 2. Boiled before incubation (37°C, 8 hours) 9 3. Boiled after incubation 22 A. Incubation without boiling 17 5. Alphacel (Nutritional Biochemical) boiled after incubation 5 50 mg sample of cell wall, or alphacel, was sus- pended in 5 ml glass distilled water and-treated as indicated. The pH of the reaction mixture was 6.8 and did not change during incubation or boiling. Each value is an average of three closely agreeing replicates. Incubation was for 8 hours at 37°. Alphacel was from Nutritional Biochemical Corporation. 28 Figure 6.--Time course of autolysis of cell wall preparation. A set of tubes containing 50 mg of cell wall preparation were incubated in distilled water (2.5 ml) at 37°C for different time periods, and the filtrates were collected. Reducing sugars were determined by the Nelson-Somogyi method. 50 no REDUCING SUGAR no mg CELL WALL 29 BOILED AFTER INCUBATION vv’T"‘I vo‘TT" 0” o a,» BOILED BEFORE / ‘,.—‘ INCUBATION ‘ d 5‘.’ P ’ I s 30 Figure 7.-—Effect of pH on autolysis of cell wall preparations. The assay consisted of 50 mg of cell wall preparation in acetate, dipotassium hydrogen phosphate and tris, all 0.01 molar, total volume of 2.5 ml. Incubation was carried out at 37°C for eight hours. 80 60 ug REDUCING SUGAR/mg CELL wALL x Io jJ. I] O - PHOSPHATE (0.0l we ACETATE (0.0l M) II - PHOSPHATE (0.0! M) I — PHOSPHATE (am we TRIS (0.0| M) O - BOILED ENZYME 32 TABLE 3.-—A preliminary single experiment on the effect of potassium, phosphate, and magnesium on cell wall autolysis. K2HPOu, MgCL Assay H2O K2HPOu MgCl2 2 (0.01 m) (0.001 M) (0.01 M) (0.001 M) Klett units 137 257 85 185 Reducing sugar 9.6 18.3 6.0 13.0 20 mg cell wall preparation suspended in 1.5 m1 of K HPO (0. 01 M), MgCl (0.001 M), K HPO (0.01.ND +MgCl2 (6. 00 M); andH 2O fo§8 hours incugatign at 37°C. 33 different incubation medium, that the rate of glucose liberation in phosphate compared to water media is 1.9. Invertase Activity Associated Wiffi the Autolytic System Sucrose solution (200 umole sucrose; 1500 umole Tris pH 8.0; 500 umole MgCl in 100 m1 H 0; final pH 7.6) 2 2 was used as substrate and incubated with 50 mg of cell wall preparation fOr 8 hours at 37°. Sucrose hydrolysis was nearly a linear function of time for up to two hours at which time 80 per cent sucrose had been hydrolyzed (Figure 8). AutOlysis of the Cell Wall Preparation as a—FUnction STLits Concentration Different weights of cell wall preparation were incu— bated with the same amount of glass distilled water at 37° for 8 hours. Autolysis was a linear function of cell wall concentration (Figure 9). It was also found that the precipitate obtained from the filtrate after freezing and thawing was increased approximately linearly as the cell wall concentration increased. In tubes containing only 5 mg of cell wall, there was no precipitate at all; in 10 mg tubes, a barely noticeable precipitate; in 20 mg tubes, noticeable precipitate; in A0 mg tubes, good precipitate; in 70 mg tubes, heavy precipitate, and in 100 mg tubes, a very heavy precipitate. Tubes containing A0 and 70 mg cell wall but having been boiled before incubation showed only 3A Figure 8.—-Cell wall associated invertase activity as a function of time. Incubation of 2.5 me (5 UM) sucrose solution together with 50 mg cell wall for eight hours at 37°C. Reaction was stopped by boiling 3 to 5 minutes and reducing sugar in hydrolysate of sucrose determined by the method of Nelson-Somogyi on Klett-Summerson photometer at 5A0 mu. 35 DMN>JOmQ>I mmomUDm o\o INCUBATION h 36 Figure 9.--Autolysis as a function of cell wall con- centration. 0.5, 10, 20, A0, 70, and 100 mg of cell wall preparations were incubated with A ml glass distilled water at 37°C for eight hours. Filtrate was collected, and reducing sugar deter— mined by the Nelson-Somogyi colorimeteric method. yg REDUCING SUGAR 60‘ 50 4O 30 37 O " IO BOILED AFTER INCUBATION ’0 ’ d v" BOILED BEFORE ,a’ INCUBATION l l l 20 4o 70 mg CELL WALL IOO 38 a barely noticeable amount of precipitate in filtrates after incubation. Effect of Indoly-B-Acetic Acid On Autolysis These experiments are quite incomplete and preliminary but are included owing to their possible interest and help to others. Cell wall (50 mg/tube) was suspended and incu- “6 or 10'3 molar IAA solutions. No bated in 10'9, 10 significant effect of IAA on release of reducing sugar from the cell wall was observed during the incubation. However, on freezing and thawing of filtrates, the precipi— 9 6 tation was noticeably higher in tubes of 10- and 10' molar IAA (Table A). Effect of Decapitation on Autolysis ofTCell Wall The total amount of reducing sugar released during autolysis was found to be twice as high in cell walls pre- pared from intact coleoptiles compared to those decapitated prior to harvest (Table 5). 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