‘;v .g'nfli"u *‘f;§:’l.‘ ‘.. Eldzau‘ml. \5 r! -w.m~'.' 9'.“ ‘ amt-F. 5'. at . w ‘4‘» w L R LIB.1’3;‘1RY Michigan State University This is to certify that the thesis entitled Mechanism of action of the primary determinant of pathogenicity from Helminthosporium victoriae Date 0-169 presented by Kanak R. Samaddar has been accepted towards fulfillment of the requirements for Ph.D. Plant Pathology degree in QZZuW/ia Major proéssor Dr. Robert P. Scheffer December 13, 1967 ABSTRACT MECHANISM OF ACTION OF THE PRIMARY DETERMINANT 0F PATHOGENICITY FROM HELMINTHOSPORIUM VICTORIAE by Kanak R. Samaddar The objectives of this study were to explain the differential effect of Helminthosporium victoriae toxin on susceptible and resistant oat plants, and to determine the primary site of action. The possibility that the initial toxin lesion is in the plasma membrane was examined by the use of intact tissues and cell—free systems. Toxin caused an increase in 02 uptake by-leaf (both green and etiolated) and root tissues of susceptible but not of resistant plants. Susceptible coleoptiles and aleurone cells did not show a significant respiratory increase after toxin treatment, Phosphorylative and oxidative capacities of mitochondria isolated from susceptible seedlings were not affected by toxin. Apparently, the Krebs cycle is not the primary site of action and increase in tissue respiration is a secondary event in the action of toxin. Toxin caused loss of electrolytes from all types of susceptible tissue tested. Electrolyte loss presumably re- flects a permeability change in the cell membrane. All soluble electrolytes in susceptible leaf tissue were lost 8 Kanak R. Samaddar to 12 hours after toxin treatment, indicating damage in both plasma membrane and tonoplast. Toxin stopped uptake of exogenous amino acids and Pi by susceptible but not resistant tissue. Incorporation of P32 into organic P and Cl4 amino acids into protein was blocked in susceptible but not in resistant tissue. Active ribosomes from rabbit reticulocyte cells, which are affected by all known inhibitors of protein synthesis, were not affected by toxin. Apparently, the effect of toxin on protein synthesis in tissues is another secondary effect. A breakdown in transport of materials to synthetic sites could explain the effect of toxin on incorporation of amino acids, Similarly, the lack of Pi incorporation into organic com- pounds could result from disruption of transport to the active site of synthesis, Root hair cells exposed briefly to traces of toxin could not be plasmolysed in hypertonic solutions. This is a known effect of membrane damage, Apparent free space increased in susceptible but not in resistant roots after toxin treat— ment. The increase was evident within 30 minutes, and reached 80%.free space after 2 hours exposure to toxin. Apparent free space increase after toxin treatment suggests membrane damage. Kanak R . SaMaddan: When cell wall-free protoplasts were exposed to 0.16 ug toxin/ml, protoplasmic streaming stopped and all plasma membranes of susceptible protoplasts broke within 1 hour. Resistant protoplasts were not affected. Filipin, ribonuclease, and 2,4-dinitrophenol affected both HV—toxin susceptible and resistant protoplasts, while toxin affected only the susceptible Avena protoplasts. HV-toxin seems to have a strong affinity for membranes of susceptible cells and the effects are more drastic than those of any other substances tested. Experiments with dry seeds and excised aleurone cells indicated that toxin susceptibility and resistance are ex— pressed by resting, fully differentiated, triploid aleurone cells long before metabolism leading to germination is irre— versibely activated. These data plus the data on membrane effects indicate that in this model case resistance is based on constitutive factors. It now appears that toxin combines with an unknown component in the susceptible cells, resulting in disorgani— sation of surface. It is possible that all other observed effects of toxin are secondary to membrane damage. The resistant cell membrane appears to lack the receptor or sensitive sites. MECHANISM OF ACTION OF THE PRIMARY DETERMINANT OF PATHOGENICITY FROM HELMINTHOSPORIUM VICTORIAE by Kanak R. Samaddar A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1967 GLMQ TO MY PARENTS ACKNOWLEDGMENTS I wish to thank Dr, R. P. Scheffer, my major professor, for his guidance and constant encouragement during this study and in the preparation of this manuscript. Other members of my guidance committee, Drs. J. E. Varner, N. E° Good, and Jo L° Lockwood, furnished critical evaluation of the manuscript; Mr. 0° C. Yoder gave much help and encouragement, Mr, P. G. Coleman photographed the figures, and Mrs. Jacqueline K, Yoder typed the original manuscript. I wish to thank each of these people, I am grateful to Dr. A. J. Morris for his guidance in experiments with reticulocyte ribosomes, and to Dr. J. E. Varner for valuable suggestions and use of equipment in several phases of this study, The National Science Foundation (U.S,) furnished financial support (Grants GB-l448 and GB-6560). U.S. Educa— tional Information (India) awarded a Fulbright Travel Grant. iii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . . . . . . . . viii LIST OF ABBREVIATIONS , . , . , , . . . . . . , . . . ix INTRODUCTION . . . . . . . . . . . . . . . . . . . . 1 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . 4 MATERIALS AND METHODS . . . . . . . . . . . . . . . . 12 Host plants and fungus cultures , , . , , . . . . 12 Bioassay . . . . . . . . . . . . . . . . . . . . l3 Isolation and purification of toxin . . . . . . . l3 Cl4-labeled toxin preparation . . . . . . . . . . l4 Isolation of cellulase . . . . . . . . . . . . . 16 Preparation of mitochondria . . , , . . , . . . . l6 Respiratory measurements . . . . . . . . . . . . 17 Phosphorylation by mitochondria , . . . . . . . . l9 Phosphate uptake and incorporation in tissue , . 20 Cl4-amino acid uptake and incorporation in tissue 21 Preparation of reticulocyte ribosomes . , , , , , 22 Amino acid incorporation by cell free ribosomes . 23 Preparation of protoplasts . . , . . . . . . . , 24 Ion leakage . . . . . . . . . . . . . . . . . . . 25 Apparent free space . . . . . . , , . . , . , . , 25 m-amylase production by aleurone cells . . . . , 27 RESULTS . . . . . . . . . . . . . . . . . . . . . . . 29 Effect of toxin on respiration of tissue . . . . 29 Effect of toxin on ion leakage from tissue . . . 31 iv TABLE OF CONTENTS (Continued) Effect of toxin on mitochondrial oxidation and phosphorylation , on P Effect Of toxin in tissue . . Effect of toxin incorporation Effect Effect Effect Effect DISCUSSION LITERATURE of toxin of toxin of toxin of toxin CITED , on amino acid uptake and on on on on 32. . . . . . . plasmolysis . . apparent free space of tissue free protoplasts seed germination uptake and incorporation Page 33 35 41 46 50 53 6O 7O 82 LIST OF TABLES Table Page 1. Comparative responses of different susceptible oat tissues to toxin . . . . . . . . . . . . . 30 2, Effect of toxin on loss of electrolytes from susceptible oat leaf tissue . . . . . . . . . 34 3. Inorganic phosphate and oxygen uptake by mitochondria from susceptible oat seedlings . 37 2 4. Effect of toxin on P3 uptake and incorporation by oat leaf tissue . . . . . . . . . . . . . . 39 5. Effect of toxin on uptake of C14 amino acids by susceptible and resistant oat roots . . . . . 42 6. Effect of toxin on incorporation of Cl4—leucine into TCA precipitable fractions by susceptible and resistant leaf tissues . . . . . . . . . . 45 . . . . l4 7. Requirements for optimal incorporation of C - valine into protein by reticulocyte ribosomes 47 8. Effects of toxin and 2,4—dinitrophenol on plasmolysis of root hair cells in hypertonic solutions . . . . . . . . . . . . . . . . . . 49 9. Effects of toxin and various metabolic inhibitors on apparent free space of oat roots . . . . . 54 10. Effect of toxin (0.16 ug/ml) on protoplasts from host and non-host plants . , , , , . . . . . . 56 ll. Comparative effects of toxin and 2,4—dinitrophenol on oat protOplast survival . . . . . . . . . . 59 vi LIST OF TABLES (Continued) Page 12, Effect of sodium bisulfite (8 x 10—4 M) on the toxicity of toxin (0.16 ug/ml) to susceptible oat protoplasts . . . . . . . . . . . . . . . 61 13. Effect of toxin on germination of oat seeds . . 63 14. Effect of immersion in water and drying on subsequent germination of oat seeds . . . . . 64 15. Effect of toxin and gibberellic acid on production of m-amylase by g, victoriae susceptible and resistant oat aleurone cells 67 vii LIST OF FIGURES Figure Page 1. Elution profile for HV-Toxin from Bio Gel P—2 column . . . . . . . . . . . . . . . . . . . 15 2, Effect of toxin on loss of electrolytes from Helminthosporium victoriae resistant and susceptible coleoptiles . . , . , . . . . . 32 3. Effect of toxin on 02 uptake by mitochondria from g, victoriae susceptible oat seedlings 36 4. Effect of toxin on P32 uptake and incorporation in resistant and susceptible tissues . . . . 4O 5. Effect of toxin on amino acid incorporation into protein in a cell free system . . . . . . . 48 6. Effect of toxin (0.16 ug/ml) on apparent free space (AFS) of susceptible and resistant oat r00tS . . . . . . . . . . . . . . . . . . . 52 7. Effect of toxin concentration on survival of resistant and susceptible protoplasts . . . 58 8. Effect of toxin on a-amylase synthesis by oat aleurone cells . . . . . . . . . . . . . . . 68 9. Effect of toxin on GA-induced synthesis of a—amylase, plotted by the Linweaver-Burk method . . . . . . . . . . . . . . . . . . . 69 viii LIST OF ABBREVIATIONS ADP: Adenosine diphosphate AFS: Apparent free space ATP: Adenosine triphOSphate COA: Coenzyme A CV: Cultivar DNA: Deoxyribosenucleic acid DNP: 2,4-Dinitrophenol EDTA: Ethylenediaminetetraacetic acid GA: Gibberellic acid GSH: Glutathione (reduced) GTP: Guanosine triphosphate HEPES: N-2—hydroxyethylpiperazine—N'—2-ethanesulfonic acid NAD: Nicotinamide adenine dinucleotide OD: Optical density PDA: Potato—dextrose agar Pi: Inorganic phosphate RNA: Ribosenucleic acid TCA: Trichloroacetic acid TPP: Thiamine pyrophosphate Tricine: N—tris(hydroxymethyl)methylglycine Tris: Tris(hydroxymethyl)aminomethane ix INTRODUCTION There is increasing evidence that many of the harmful effects associated with parasitism have a Chemical basis, In recent years, the discovery of highly potent, host—specific, pathogen—produced substances provides firm support to the concept of Chemical determinants of plant diseases (42), At least five plant infecting fungi are now known to produce compounds that are primary determinants of pathogenicity (51). These pathogen-produced factors, commonly called toxins, can induce symptoms of infection in the absence of the pathogen itself. In each case loss of ability of a fungus isolate to produce its toxin was associated with loss of pathogenicity, In each case, the substance is toxic only to the host of the toxin—producing fungus (51). The importance of such host— specific toxins as models for study of disease is evident, Many complexities of host-parasite interaction are bypassed, and a specific compound can be used to study disease develop- ment and disease resistance. This is a direct approach to the study of parasitism, disease development, and disease resistance in plants, One such substance, produced by Helminthosporium victoriae Meehan and Murphy, the causal organism of Victoria blight of oats, is toxic to susceptible oat cultivars, but is harmless to resistant oat cultivars and to all other non- host plants tested (42). The toxin was isolated and partially Characterized (41). Several lines of evidence show that the toxin is the key factor in the development of disease (42, 51). Among the many cellular responses that occur quickly after g, victoriae toxin treatment are: increased 0 uptake 2 (49); decreased incorporation of Cl4 amino acids and uridine into trichloroacetic acid precipitable cellular fractions (51); and rapid loss of electrolytes (61). There are several reasons for believing that these effects are secondary to the initial lesion. Oxygen uptake by isolated mitochondria was not affected (49). Toxin uptake appears to be a simple process, not affected by wide ranges of temperature or by metabolic inhibitors. The peptide resulting from toxin break- down inhibited toxin uptake, suggesting a competition for receptor sites present in susceptible cells (50). Our present data suggest that the toxin causes a primary lesion in the plasma membranes of susceptible cells. The basis of resistance may be a lack of receptors or sensitive sites in the membrane (51). The purpose of this study was to investigate the biochemical and physiological effects of g, victoriae toxin on susceptible and resistant oat plants. An attempt was made to determine a primary site of action of toxin. Background information for this thesis came from previous work done in several laboratories, and is reviewed in detail by Scheffer and Pringle (42, 51). The possibility that the initial toxin lesion is in the plasma membrane was examined by the use of intact tissues and cell free systems. The effects of toxin on respiration, ion leakage from tissues, cell plasmolysis, uptake of solutes, apparent free space, behavior of cellawall— free protoplasts, mitochondria and ribosomes were determined. The data indicate that toxin affects membrane physiology and transport systems of susceptible but not of resistant cells. Finally, an effort was made to study seed germination and cer— tain early processes of seed germination as affected by toxin. LITERATURE REVIEW Pathogen produced substances that are determinants of pathogenicity are now well established in at least five model cases. These compounds, commonly known as "host—specific toxins," reproduce all visible and all known biochemical symptoms of infection. Several lines of evidence show that these toxins are the key factors in development of the respec- tive diseases. Several detailed reviews relating to pathogen-produced determinants of disease and their effects on host plants have been published (42, 51, 64). Discussion herein will be restricted to the literature dealing with biochemical responses of plants to infection, and to the toxin produced by Helminth05porium victoriae. Meehan and Murphy (35) were first to report a toxic metabolite produced by g, victoriae that is involved in Victoria blight of oats. The selective toxicity of filtrates from g, victoriae cultures was soon confirmed (31). The toxin was isolated from culture filtrates and purified as a water soluble white powder that was biologically highly active, but very unstable (41). Purity of the preparation was shown 4 by ionophoresis, countercurrent distribution analysis, and Chromatography in various systems (41, 42). The toxin molecule contains a cyclic secondary amine (C NO) known as 17H29 "victoxinine,' combined with a peptide consisting of aspartic acid, glutamic acid, glycine, valine, and leucine. The com- plete toxin has a molecular weight between 800 and 2000 but the exact structure is still unknown, largely because of problems with lability (41, 42). The toxin after purification retained its host-specificity. Concentration of the toxin required for complete inhibition of susceptible root growth was 0.0002 ug/ml, which is a very high biological activity (42, 51). Since the toxin produces all known biochemical responses of host plants to infection, many studies were made correlating these metabolic derangements with disease develop- ment. Much attention was given to toxin induced respiratory increase in susceptible leaf and root tissues (26, 45, 49) as a possible early event in action of toxin. Such respiratory responses depend on age of the plant and on experimental conditions. The response of older plants to toxin was less marked than that of younger ones (26). When treated tissues were washed in a large volume of suspending fluid, there was no increase in respiration (3, 51). Furthermore, leaf and root tissues showed different degrees of response to toxin as measured by 0 uptake (49, 50). 2 Following an earlier suggestion by Allen (2), it was suggested that toxin acts primarily as an uncoupler of respiratory oxidation from phosphorylation (9, 45). The rela— tive insensitivity of toxin-treated tissue to 2,4-dinitrophenol (DNP) was the only evidence in favor of this hypothesis (26, 45). Several lines of evidence, however, indicate that the action of the toxin is entirely different from that of DNP. Unlike toxin, DNP is nonspecific, and is less effective than toxin as a stimulator of respiration (45). Moreover, decreased sensitivity to DNP can result from changes other than uncoup— ling of respiration from phosphorylation (28). Evidence that the oxidative responses of tissue to toxin are secondary came from studies with isolated mitochon- dria from oat seedlings. Purified toxin did not influence succinic acid oxidase activity of mitochondria, even when very high toxin concentrations were used (49). There are conflicting and uncertain reports on the effect of toxin on phosphate uptake. Mitochondria from toxin-treated susceptible plants showed a loss of respiratory control and a low ADP to oxygen ratio. However, toxin did not affect Pi uptake when toxin was added to mitochondria previously isolated from healthy plants (9, 62). Insensitivity of mitochondria to toxin was confirmed by other experiments. Solutions containing high levels of toxin had no effect on permeability of mitochondria to elec— trolytes (9). The data available indicate that uncoupling of oxidation from phosphorylation is not the primary metabolic site of toxin action. As a possible explanation for augmented respiration, Krupka (26) suggested that ascorbic acid oxidase is activated by toxin. Homogenates from toxin—treated tissue oxidized ascorbic acid much faster than did control homogenates, and this was accompanied by decreased levels of ascorbic acid in treated tissue. However, much of the oxidation of ascorbic acid by the homogenate was nonenzymatic and the rise in ascorbate oxidation occurred after the respiratory rate had passed its maximum. Therefore, the role of ascorbic acid oxidase in toxin induced respiratory increase is open to ques- tion. Activation of ascorbate and other possible terminal oxidases could be a secondary effect of disturbance (49, 51). The failure to implicate respiratory increase as a primary cause of disease development led to other hypotheses. One Characteristic effect of toxin treatment is the rapid leakage of cellular materials from susceptible tissue (61). The leachings include amino acids and other nitrogen compounds, carbohydrates, sodium, phosphorus, and potassium (9). The rate of leakage of these materials shows a low temperature coefficient and is not dependent on oxygen. Moreover, a very dilute culture filtrate can cause such permeability Change, which is expressed earlier than the respiratory increase (61). Mitochondrial membranes, however, do not appear to be affected (9). On the basis of these findings Wheeler and Black (61) suggested that permeability Change is a primary effect of the toxin that could lead to respiratory increase by upsetting the balance of salts and other cellular metabolites within the cell. The possible role of permeability Changes in respira- tory increase, however, is not Clear. Loss of electrolytes might be a secondary effect of alteration in physico-chemical structure or function of plasma membranes of susceptible cells. Another early effect of toxin is the inhibition of incorparation of Cl4 amino acids and uridine into TCA precip- itable macromolecules (51). The decrease in incorporation of these compounds following toxin treatment appears to be more sensitive than any other metabolic reSponses tested. However, it is not possible to conclude that the toxin acts primarily as an inhibitor of protein synthesis. Since amino acid and uridine uptake by cells is carrier mediated, the results might reflect a disturbance in uptake and transport rather than in synthesis. Data on the effect of toxin on cell free protein synthesizing systems are not available. Toxin uptake studies first indicated that the primary Site of action of toxin might be at the cell surface (50). Toxin uptake as measured by the final toxicity was not affected by temperature over a wide range (5-370), by various metabolic inhibitors, or by osmotic concentration of assay solutions. Toxin uptake therefore does not depend on cellular metabolism and appears to be a simple physical process. This is compatible with the fact that toxin-induced ion leakage is not tempera— ture and oxygen dependent (61). As a working hypothesis it was postulated that toxin uptake by susceptible tissue involves adsorption to a receptor site that is lacking in resistant tissue. Several Circumstantial evidences support this hypothe— sis. Toxicity was greatly decreased when sodium bisulfite was present in the toxin assay medium. Bisulfite does not have a direct effect on the toxin, but it may alter a receptor, resulting in decreased adsorption of toxin. Toxicity was also decreased when breakdown products of the toxin were added to assay solutions containing intact toxin (50). Since victoxinine alone did not have an inhibitory effect, this might be interpreted as competition between the free peptide moiety and the complete toxin molecule for receptor Sites. 10 Studies on the nature of disease resistance in this model system provide further evidence in support of the receptor theory. Since toxin is the primary determinant of disease (42, 51), resistance to disease in this model case is resistance to toxin. In explaining the nature of resis— tance, Romanko (45) postulated that resistant tissue inactivates toxin more efficiently than does susceptible tissue. Scheffer and Pringle (50) could not confirm this result. Toxin could not be recovered from either susceptible or resistant tissues and the residual toxin solution lost very little activity. This led them to propose that toxin resistance is due to lack of the receptor that is present in susceptible tissue. Observations on toxin action so far are compatible with the receptor hypothesis but there has been no effort to determine the exact location of the receptors in susceptible cells. Interaction of the toxin with the cell surface indi- cates the plasma membrane as a possible site. Electron microscopic evidence of toxin induced disruption of plasma membrane of susceptible cells supports this idea (30). These results, however, were obtained with crude culture filtrates after long exposures (24 hours) of tissues to toxin, and therefore should be accepted with caution. Moreover, membrane ll disorganization caused by interaction with specific ligands such as toxin may not result in visible disruption even when normal functioning may be lost. MATERIALS AND METHODS Host plants and fungus cultures.——Oat cvs. Park and Clinton were used in most experiments. Park is susceptible and Clinton is resistant to H, victoriae and to its toxin. In certain experiments corn hybrid K61 X Pr and sorghum CV. Colby were used. Seeds were germinated on moist filter paper and seedlings were grown in the laboratory at 220 in White's nutrient solution (41, 50), or in vermiculite plus White's nutrients. In some cases larger plants were used. These were grown in the greenhouse in peat—soil—sand (1:1:1) mixture watered daily with a soluble commercial fertilizer. Cuttings were taken from 7 to 10 day old plants with 2 or 3 fully expanded leaves. Unless otherwise stated, the first fully expanded leaf above the primary leaf was used as a source of leaf tissue. A highly virulent strain of H, victoriae was used for toxin production. The fungus was grown in 1 liter Roux bottles, each containing 200 ml modified Fries no. 3 basal medium (31, 41). Cultures were seeded with small pieces of mycelial mat grown on PDA Slants and incubated without 12 13 shaking at-22O for 3 weeks. Myrothecium verrucaria, isolate 460, obtained from Dr. Mary Mandels of the Quartermaster laboratory at Natick, Mass., was used for production of cellulase. The stock culture was maintained on PDA slants. The fungus was grown in shake culture (190 strokes per minute) in a modified Whitaker's salt medium (65), supplemented with 0.1% glucose and 1%yWhatman, Chemically pure grade cellulose. Cultures were harvested after 14 days at 220, when cellulase in the medium was high. Bioassay.--Inhibition of seedling root—growth was used as the standard bioassay (41, 50). Hulled oat seeds were ger— minated for 24 hours on moist filter paper. The assay made use of serial dilution of toxin in White's solution, buffered at pH 5.0 with MCIlvaine's buffer. Five ml of each dilution and 5 germinated seeds were placed in each 60 X 15 mm petri— dish. The assay was incubated 48-72 hours at 22°. The highest dilution which limited susceptible root growth to 1.0 cm or less was considered the dilution end point. Control roots grew to a length of 5-6 cm under these conditions. Isolation and_purification of toxin.—-Toxin was isolated from culture filtrates of H, victoriae by the method of Pringle and Braun (41). Culture filtrate was concentrated in vacuo, equal parts of methanol was added, and the precipitate 14 was discarded. After removal of methanol in a flash evaporator, the filtrate was extracted 3 times with nfbutyl alcohol. The combined butanol extract was concentrated in_ vacuo, an equal volume of methanol was added, and the solution was passed through an alumina column. The toxin adsorbed on alumina was eluted with 1% acetic acid. At this stage of isolation, toxin was relatively stable at pH 3.5 and the con— centrated solution was stored at 40. This preparation gave complete inhibition of root growth of susceptible oat seedlings at 0.0016 ug/ml. Further purification was achieved by pasing the alumina eluate through a Bio—Gel P—2 column. A ten ml solution was placed on a 25 X 1.5 cm column. The column was prepared, washed, and developed with distilled water. The effluent was collected in 10 ml portions by a volumetric fraction collector. Aliquots of each fraction were serially diluted with water and assayed to develop an activity elution profile (Fig. l). The maximum activity as measured by bioassay was in fractions 3 and 4. Fraction 3 gave complete inhibition of susceptible seedlings at 0.0004 ug/ml. This preparation, therefore, was 4 times more active than the eluate from the alumina column. Cl4-labeled toxin_preparation.--The fungus was grown on modified Fries no. 3 basal medium (31) supplemented with 15 O :A 2 E 6 - . Acnvm! - 3 6 - CPM n. __ -l 5 E i «z. 4 - — 2 " E E S ‘ ‘\\\ u — I -_' O o \k g\\ .— 2 \ \ .— U < E h- I’ "“"-ue.....,,.,m \ m/ I I I I "0......1 " I I I Ed I 3 5 7 9 FRACTIONS 10 ml LI I I l I I I I I I 0.4 0.3 4 6 3 0.8 0.5 0.5 AAG/WAL Fig. l.--Elution profile for HV—toxin from Bio Gel P-2 column. Column (25 x 1.5 cm) was prepared and washed with distilled water. The column was loaded with 5 m1 of eluate from an alumina column, and developed with distilled water. 16 sucrose-U—C14 (specific activity, 5 mC/mM). The final radio— activity of the culture medium was adjusted to 200 uc/l. Toxin was isolated and purified as described above. Maximum toxicity and radioactivity was in fraction 3 (Fig. 1) from the Bio-Gel P—2 column. Isolation of cellulase.—-Culture filtrates of M, verrucaria were concentrated to 0.1 volume in a flash evaporator at 370, and filtered. The filtrate was fraction- ated with solid (NH SO by the method of Green and Hughes 4)2 4 (l8). Precipitate that formed at 35% saturation was discarded after centrifugation at 30. The precipitate that formed from 35—70% saturation was dissolved in a small volume of water and desalted in a 10 x 1.5 cm Sephadex G-25 column. The column was prepared, washed, and developed with 0.1% NaCl: 1 ml fractions were collected. Maximum cellulast activity was in the second brown fraction eluted from the column. Aliqnots (200 ul) of this fraction were stored in separate vials at —200. There was no appreciable loss of activity of the prepa— ration after 2 months in storage. Preparation of mitochondria.-—Mitochondria were isolated from leaves and coleoptiles of etiolated oat seedlings grown for about 1 week on paper towels moistened with White's nutri— ent solution. Pierpoint's method as modified by Wu and Scheffer 17 (68) was further modified. Tricine (N—tris (hydroxymethyl) methylglycine) or HEPES (N-2—hydroxyethy1piperazine-N'-2- ethanesulfonic acid) was substituted (1?) for tris(hydroxymethyl) aminomethane in Pierpoints extraction and washing media. Shoots were cut into small pieces and were chilled at 20 for one hour. The pieces were then ground for 2 to 3 minutes in a mortar with half their weight of washed sea sand and with a volume of extraction fluid twice the weight of the tissue~ (m1=g). The homogenate was filtered through 4 layers of Cheese- Cloth. The filtrate was centrifuged for 10 minutes at 1,000 X g, and the pellet was discarded. The supernatant was recen- trifuged for 20 minutes at 15,000 X g and the pellets containing the mitochondria were suspended in washing medium equal in volume to the original weight of the shoots (m1=g). After a second centrifugation at 15,000 X g for 20 minutes, the supernatant was discarded and the final washed pellet was suspended in 0.25 M sucrose solution for the experiments. All preparations were done at 30-40, using previously Chilled glassware and solutions. Respiratory measurements.--The Warburg apparatus was used to determine oxygen uptake, following standard procedures (57). For experiments on oxygen uptake by whole tissue, 0.1 to 0.5 9 tissue samples were placed in each Warburg flask 18 containing 2 ml of various reaction mixtures with or without toxin. Reaction mixtures for various experiments included White's solution with or without sucrose, McIlvaine's phosphate-citric acid buffer diluted 1:20 (pH 5.8), and 0.05 M phosphate buffer (pH 5.8). In certain experiments tissues were pretreated with toxin, washed, and placed either in a flask on moist filter paper or in 2 ml phosphate buffer (pH 5.8). All tests were at 250, with duplicate flasks for all treatments, and with air as the gas phase. The first manometric readings were taken after 10 to 15 minutes equili- bration, and subsequent readings were taken at intervals of 15, 30 or 60 minutes. Oxygen uptake results were based on dry weight of tissues or on oxygen uptake/flask. In experiments with mitochondria, each flask contained 2.0 ml total volume reaction mixture (pH 7.3) with or without toxin, including 0.4 ml mitochondrial suspension. Flasks containing the reaction medium were Chilled before the mito- chondrial suspension was added. Reaction vessels contained the following materials in final concentrations as indicated: P0 . 2 4 0.0075 M; ATP, 0.001 M; cytochrome C, 0.1 mM; Substrates (potassium salts), 0.025 M; sucrose, 0.2 M; KH 0.07 M; MgSO4, NAD,0.13IMM TPP, 0.13 mM; COA, 0.013 mM; malonate, 0.025 M; and toxin, 0.16 to 0.016 ug/ml. For P:O ratio determinations, 19 0.2 m1 hexokinase solution (containing 2 mg/ml in 0.5 M glucose) was added to the flask side arm and tipped into the reaction mixture after equilibration. Equilibration time was 10 minutes, except when P:O ratios were determined, when the time was shortened to 5 minutes. All experiments were run at 300. Readings were taken at 10 minutes intervals. Activity was expressed as oxygen uptake/flask. Phosphorylation by mitochondria.—-Phosphory1ation by mitochondria was measured by disappearance of orthophosphate from the reaction medium (16). After 40 minutes of oxygen uptake, 2 ml Chilled, 10% TCA was added to the reaction fluid in each Warburg flask, and a 2 m1 sample of the resulting mix- ture was removed for determination of phosphate. Samples were centrifuged at 15,000 X g for 15 minutes at2'2O to remove precipitates, and 1 m1 supernant was diluted with 9 ml chilled water. From this dilution 0.2 m1 samples were removed and added to 8.4 ml water, to which was further added 1 ml 2.5% ammonium molybdate and 0.4 ml 0.25% aminonaptholsulfonic acid. After 15 minutes incubation, color development was measured at 660 mu with a Klett Summerson colorimeter. Two phosphate samples were run for each Warburg flask. Separate flasks were used to determine Pi at the beginning of each experiment; after equilibration in the Warburg water bath, TCA was added 20 to the mixture before hexokinase was tipped in. Phosphory- lation was expressed as Pi uptake in u moles per flask. Phosphate_uptake and incorporation in tissue.--Tissue samples were placed in flasks containing 2 m1 1 mM acetate buffer (pH 5.5), 15 ug chloramphenicol and 200 no p32. Flasks were incubated at 250 on a Dubnoff metabolic shaker (40 oscillations/minute) for 2 hours. Samples were then rinsed thonmnflmy with cold 1 mM KH2P04 solution, and ground in boil- ing 80% ethanol. The homogenate was centrifuged at 10,000 X g for 10 minutes and the supernatant concentrated to 2 m1. A 50 ul aliquot was Spotted on Whatman no. 1 paper. Marker P32 was spotted in each paper for tracing the movement of phosphate. Descending chromatographs using nebutyl alcohol: propionic acid:water (1246:620:874 v/v) were developed for 40 hours (6). Chromatograms were air dried and the radioactive zones were detected with a Nuclear Chicago monitor. Radio— autographs of the dried Chromatograms were made on Eastman Kodak X—ray films. Individual radioactive zones were removed, glued to planchets and counted in a Nuclear Chicago low back— ground gas flow counter. Total uptake was calculated by adding the counts of all spots developed from a single origin. The amount of radioactivity in the inorganic and organic phosphate Spots was expressed as percent of total uptake. 21 l . . . . . . C 4 —amino aCid uptake and incorporation in tissue.-— Uptake was measured as the amount of labeled compound taken from the medium and retained by tissues after repeated washings (48). Roots from 10 seedlings were incubated in 1.0 ml DL—leucine—l-Cl4 or DL-valine—U-Cl4 (pH 7.0). Concentrations of the amino acid solutions varied from 0.001 to 0.05 M, with specific activities from 0.5 to 1.0 uc per m1. Chloramphenicol (15 ug) was added to each flask and the mixture was incubated for l to 3 hours at 220 with gentle shaking. The roots were then washed for 1 hour in running tap water, blotted gently, placed in tightly stoppered tubes with 0.5 ml 95% ethanol, and extracted with shaking for 12 hours. Aliquots of the ethanol extracts were counted on planchets in a Nuclear Chicago gas flow counter for determination of uptake. For incorporation studies 0.5 9 tissue samples were infiltrated with DL—leucine-l-Cl4 or DL-valine-l-Cl4 (approx. 0.5 mM, 105 counts/ml) for 10 minutes and then incubated for l to 4 hours at 220. After incubation, tissues were extracted 3 times with hot 80%.ethanol and ground in a mortar with 80% ethanol. The homogenate was centrifuged at 10,000 X g for 15 minutes. The pellet was washed 3 times with 80% ethanol, followed by 3 washings with 5% TCA at 00. The TCA insoluble precipitate was washed once with 80% ethanol, once with 22 absolute ethanol, twice with hot ethanol:ether (3:1 v/v), and once with anhydrous ether. The pellet was then suspended in l N NaOH at 900 for 1 hour and centrifuged. Aliquots of the supernatant were counted on glass planchets to determine incorporated radioactivity (40). Preparation of reticulocyte ribosomes.—4Male New Zealand rabbits (ca. 7 lb each) were made reticulocytic by four daily subcutaneous injections of 0.175 ml 2.5% neutralized phenylhydrazine per lb body weight. On the sixth day after injections were started, the animals received an intravenous injfection containing 2000 International Unit heparin and 75 mg Nembutal. Blood was collected by heart puncture, washed, lysed and fractionated by the procedure of Allen and Schweet (l). Reticulocyte ribosomes were collected by centrifugation at 78,000 X g for 90 minutes. The supernatant was saved for enzyme preparation. The ribosomal pellet was suspended in a washing solution containing 0.25 M sucrose, 17.5 mM KHCO and 3 2 mM MgCl using a Potter homogenizer. After a second 2! centrifugation at 78,000 X g for 90 minutes, the washed ribo- somal pellet was suspended in a small volume of cold 0.25 M sucrose and used immediately or stored under liquid nitrogen. Ribosomal concentrations were determined spectrophotometri— cally (56). 23 The enzyme was prepared by the addition of powdered (NH to the 78,000 X g supernantant. The protein fraction 4'2504 that precipitated between 40 and 70% saturation at 00 (37) was dissolved in 0.1 M tris—HCl buffer (pH 7.5) containing 1 mM GSH. Soluble protein was reprecipitated with (NH4)ZSO4 (70% saturation). The final precipitate was dissolved in a small volume of solution containing 0.02M triS-HCl buffer (pH 7.5), 1 mM EDTA, 1 mM MgCl and 1 mM GSH. It was dialysed 2. overnight against 100 volumes of the same solution. The dia— lysed enzyme preparation was stable for 2 to 3 weeks when stored at —180 in 0.02 M GSH. A Spinco model L-2 ultracentri- fuge was used at 50 for high speed centrifugation. All other manipulations were done at O to 40. Amino acid incorporation by cell free ribosomes.-—The complete cell free system used to determine amino acid incor- poration contained, in 1.0 ml: 0.25 mM GTP; 1 mM ATP; 5 mM phosphoenolpyruvic acid; 40 ug pyruvate kinase; 0.05 mM L-valine-Cl4 (10 uC/Umole); 0.05 mM each of 19 other amino acids; 20 mM GSH; 50 mM KCl; 4 mM MgCl 50 mM tris-HCl buffer 2; (pH 7.5); 2 mg ribonucleoprotein; and 4 mg supernatant enzyme fraction (37). Duplicate tubes with and without toxin were incubated at 370. Radioactive protein was prepared for analysis by 24 adding 15 mg carrier bovine serum albumin to each tube, followed by precipitation with 5% TCA. The precipitate was washed by resuspension and resedimentation in 5% TCA. The pellet was dissolved in 0.5 ml 1 N NaOH, reprecipitated with 5% TCA, washed once, and suspended in acetone containing 0.01 N HCl. The precipitate from the acetone suspension was washed with a mixture of acid—acetone and ether (2:3 V/V) and finally with diethyl ether. The powder obtained was transferred to a glass counting vial with 0.5 m1 1 N NaOH. When the protein powder had completely dissolved, 15 ml counting mixture was added with vigorous Shaking. The counting mixture contained 7 g 2,5-diphenyloxazole, 150 mg l,4-bis-2—(5-phenyloxazoly1) —benzene, 50 g napthalene and 26 g thixotropic gel powder dissolved in 200 ml toluene, 30 ml ethanol and 800 ml p-dioxane. Radioactivity was measured with a Packard model 3003 liquid scintillation spectrometer. Preparation of protoplasts.——Protoplasts were isolated by cellulase treatment (46). Coleoptiles from seedlings grown in dim red light were harvested when they were approximately 25 mm long. The epidermal cells were removed from each coleop- tile in 4 to 6 clean strips, peeling from the base to the tip with fine jeweler's forceps under dim red light. Sections 1 cm long were then cut from the remaining tissue and the primary 25 leaf was removed. Tissue was then cut into 1 mm sections. About 80 such sections were placed in a mixture containing 200 ul enzyme solution (prepared as described above) and 200 ul of 1.0 M mannitol buffered with 0.025 M NaH2P04 (pH 6.5). After 2 hours incubation in the dark at 220, 2 ml 0.5 M mannitol buffered at pH 6.5 was added. Protoplasts settled to the tube bottom in 10 minutes, and the supernatant was removed with a pipette. Protoplasts were washed a second time in this way to remove cellulase. Ion leakage.-—Toxin treated or control tissue samples were rinsed in glass distilled water, enclosed in washed cheesecloth bags, and incubated on a shaker (90 strokes/ minute) in 100 ml glass distilled water at 22°. Conductivity of the ambient solution was measured at intervals with a model RC l6B1 Industrial Instruments conductivity bridge (14), using a dip type cell (k=l.0). Specific conductivity was expressed as reciprocal ohms (mhos). Apparent free space.-—Apparent free space of excised roots was determined by the India ink tagging method of Bernstein and Nieman (7). Roots were rinsed in distilled water, then equilibrated in 0.035 M mannitol solution (50 ml/g root tissue) for 0.5 to 2 hours, with 4 or 5 Changes of solution. During the last minute of equilibration, 1 m1 26 India ink suspension (1 part/10 parts 35 mM mannitol solu— tion) was added to each 20 m1 equilibrating solution. The preparation was stirred for a few seconds before roots were removed, drained, and transferred to 100 ml glass distilled water for exodiffusion. Exodiffusion solutions were stirred for 5 minutes, and samples were taken at intervals of 5, 10, 15, 30, and 60 minutes. Optical densities of 1:100 dilution of equili- brating solutions and exodiffusion media were determined with a colorimeter at 500 mu. A correction for the volume of equilibrating solution adhering to root surfaces was calculated from the Optical density and volume of equilibrating and exodiffusion media (7). Mannitol concentrations in equilibration and exodiffusion media were determined by iodometric titration as described by Butler (11). Mannitol adhering to the root surface was subtracted from total mannitol quantity in the exodiffusion medium to obtain the amount of solute in free space. Toxin causes leakage of anthrone positive carbohydrates (9), which might give an error in mannitol determinations. Therefore a correction for toxin treated samples kept in water was applied. Apparent free space was calculated from the diffusible solute (mg) in the exodiffusion medium and the concentration (mg/ml) of the 27 equilibrating solution, expressed as percentage fresh weight of root tissue. a-amylase productionpby aleurone cells.——Gibberellic acid induced s—amylase production in oat aleurone cells was determined by the method of Chrispeels and Varner (12). Oat seeds were cut transversely and the embryo halves were dis- carded. The endosperm halves containing the aleurone cells were sterilized with 1% sodium hypochlorite for 20 minutes, rinsed several times with sterile distilled water, and preincubated on sterile moist sand in petri dishes at 220 in the dark. After 2 or 3 days preincubation, 10 half seeds were transferred aseptically to 25 m1 Erlenmeyer flasks con— taining 2 m1 incubation medium with or without toxin. The incubation medium contained 2 uM acetate buffer (pH 4.8), 200 uM CaCl 15 ug Chloramphenicol, various concentrations 2. of gibberellic acid, and toxin. Toxin and chloramphenicol were filter sterilized and added to the autoclaved solution containing the other components. Sterile techniques were followed throughout. The half seeds were incubated at 250 on a Dubnoff metabolic shaker (40'oscillations/minute). After incubation, 1 ml water was added and the flasks were decanted. The half seeds were rinsed with 2.5 ml water and the rinsings were combined with the decanted medium. The half seeds were 28 ground to thick paste in a porcelain mortar with a little sand and 0.8 ml 0.2 M NaCl. The homogenate was diluted with 4.0 ml of the same solution and centrifuged at 2000 X g for 10 minutes. The decanted supernatant was considered as extract. a—amylase assays for the media and the extract followed the methods of Shuster and Gifford (53). Water was added to suitable volumes of medium or extract (0.02 to 0.2 ml) to make a total volume of 1.0 ml. The reaction was started by adding 1.0 ml starch solution to the reaction mixture. The reaction was allowed to continue for 1 to 5 minutes; it was stOpped by adding 1.0 m1 iodine reagent (12). Five ml dis- tilled water was then added, stirred, and the Optical density (O.D.) read at 620 mu. The decrease in O.D. at 620 mu is directly proportional to a—amylase activity; the response is linear with time in the 35 to 75%.range in O.D. reduction. The assay was calibrated with purified malt a-amylase and was found to have a conversion factor of 2.7 ug m—amylase/unit O.D. change. Starch and iodine reagents were prepared by the method of Jones and Varner (23). RESULTS Effect of toxin on respiration of tissue.--Experiments were designed to determine the comparative effects of toxin on respiration of leaves, roots, coleoptiles, and aleurone cells. Both green and etiolated leaves and coleoptiles were used. Seedlings for these experiments were grown in the dark, in diffuse light, or in dim red light. Samples (0.5 g) were vacuum infiltrated with toxin solution (0.16 ug/ml) or water at pH 6.5 for 20 minutes. Etiolated leaves were infiltrated in the dark. After infiltration, samples were placed in War- burg flasks on moist filter paper and equilibrated for 15 minutes at 250. Results are expressed as percent change in 02 uptake/g of fresh tissue. When dry weight was used as the basis of comparison, the results were comparable. Results of 2 experiments showed that the several tissues differed in sensitivity to toxin (Table l). Toxin caused greatest increase in respiration in leaf tissues; the response of roots was somewhat less. Coleoptile and aleurone cells had little or no respiratory response to toxin. Green and etiolated leaves had comparable responses to toxin. 29 30 Table l.——Comparative Responses of Different Susceptible Oat Tissues to Toxin Increase in oxygen uptake ‘% Tissue type Green leaf 125 :.20 Etiolated leaf 100 :'15 Green coleoptile 10 :_10 Etiolated coleoptile 15 :_10 Root 50 :'15 Aleurone cells 15 + 10 Tissue samples (0.5 g) were vacuum infiltrated with toxin (0.16 ug/ml) or water for 20 minutes and placed on moist filter paper in Warburg flasks. Oxygen uptake was determined manometrically for 1 hour at 25°. Oxygen uptake is compared with non-treated controls in each case. 31 Effect of toxin on ion leakage from tissue.-—A Characteristic feature of toxin treated tissue is increased loss of electrolytes to the ambient solution (61). Since coleoptile and aleurone cells did not show significant increase in 02 uptake after toxin treatment, loss of ions was chosen as a possible indicator of action of toxin on these tissues. Tissue samples were vacuum infiltrated with toxin (0.16 ug/ml), enclosed in cheesecloth bags, and placed in flasks containing 100 ml glass distilled water on a shaker. Coleoptile tissue of susceptible toxin treated oats released electrolytes at a much faster rate than did the controls and the treated resistant tissue (Fig. 2). Eight hours after toxin treatment, the specific conductance of the ambient solution of treated susceptible tissue was 5 times greater than that of any other treatment solution. Similar results were obtained with aleurone cells, although the response was somewhat slower. Twelve hours after toxin treatment, the ambient solution containing susceptible aleurone cells had 3 times higher specific conductance than that of solutions with resistant cells or non—treated cells. In another experiment, electrolyte loss after toxin treatment was compared with the total soluble electrolytes 32 5 "E? 4 _ x W IO 2 E 3 I- III t) Z L‘- 2 ‘J _ D D g 50: c"__,. __o u 0—7 X—f H I — ”MW 04’:::::H?mflfiflfim ' . /Q_..«¢ Res Ck 8: TO): M”- e“. “0 0 l l l 2 4 6 8 TIME AFTER TREATMENT (HR) Fig. 2.-—Effect of toxin on loss of electrolytes from Helminthosporium victoriae resistant (Res) and susceptible (Sus) coleoptiles. Tissue samples (0.5 g) were infiltrated with toxin solution (0.16 ug/ml) for 10 minutes, and suspended in glass distilled water. Electrolyte loss was determined from conductivity of the water. d=susceptible toxin treated tissue; o=susceptible control; L=resistant toxin treated; and ~"-=resistant control tissue. 33 in tissue. One 9 healthy leaf tissue was homogenized thoroughly in a mortar with washed sea sand. The homogenate was centrifuged at 15,000 X g for 15 minutes. The pellet was washed three times; all washings were combined with the~ supernatant of the first centrifugation, and brought to 100 m1. Electrical conductivity of this solution gave the total amount of soluble electrolytes present in the tissue. All soluble electrolytes were lost from comparable tissue 8 to 12 hours after toxin treatment (Table 2). The experiment was repeated with similar results, indicating a breakdown of both plasma and vacuolar membranes after toxin treatment. Effect of toxin on mitochondrial oxidation and phosphorylation.-—Toxin increased respiration in certain tissues of susceptible plants but had no effect on succinate oxidase activity of isolated mitochondria (49). However, another significant property of mitochondria is the incorpo— ration of orthophosphate into energy rich phosphate compounds (68); effect of toxin on this system was studied. Phosphorylation efficiency (P:O ratios) of mitochon— dria from etiolated Park oat seedlings were determined with a—ketoglutarate as a substrate. The cyclic reactions beyond succinate were inhibited by adding malonate to the reaction mixture with or without toxin. Hexokinase was used to couple 34 Table 2.——Effect of Toxin on Loss of Electrolytes from Susceptible Oat Leaf Tissue Electrolyte lossa Leaching time Control Treated (hours) % % l 4 l6 2 6 28 4 8 42 8 8 96 12 10 100 aPercent loss was calculated from the total soluble electrolytes in tissues. 35 the system. The cofactors NAD, TPP, COA, and cytocrome C (were added for maximum activity. Toxin from an alumina column was used in the reaction mixture at final concentrations of 0.016 and 0.16 ug/ml. This toxin preparation, when used at 0.016 ug/ml with 20 minutes exposure time, caused 100% increase in seedling respiration. Oxygen uptake was measured manometrically, and uptake of phosphate was determined by the method of Fiske and Subbarow (16), using triplicate flasks for phOSphate at the start and at the end of each experiment. Results indicate that toxin had no effect on m—ketoglutrate dehydrogenase activity of isolated mitochondria at any con— centration used (Fig. 3). In several experiments, P:O ratios for toxin—treated mitochondria did not differ from non-treated control ratios (Table 3). . 32 . . . Effect of tOXin on P uptake and incorporation in tissue.--Transpiring leaf cuttings of resistant and susceptible 10 day old plants were allowed to take up toxin solution (0.16 ug/ml) or water for 4 hours. Following toxin treatment, 0.5 9 leaf samples were incubated with buffered P32 solutions for 2 hours, washed thoroughly with cold 1 mM KH PO , and 2 4 extracted with hot 80% ethanol. Radioactivity (Cpm) of the ethanol extract was used to estimate total uptake. Counts of the several spots on paper Chromatograms gave the distribution 36 150'- 100*- . TOX' 0-016 AID/ml A TOX- 0.16 AIS/ml OCCH‘TROW pl 02 / FLASK 50 M . I. Omen-M" “Mum“ E N D O G “Wm“... l l l J 10 20 30 40 TIAAE IHIN Fig. 3.—-Effect of toxin on 02 uptake by mitochon— dria from H, victoriae susceptible oat seedlings. Reaction mixture contained the following in u moles/ flask: sucrose, 400; a-ketoglutarate, 50; KH2P04, 140; M9804, 15; ATP, 2; cytochrome C, 0.2; DPN, 0.26; TPP. 0.26; COA, 0.026; malonate, 50; and 100 u moles glucose containing 2 mg/ml hexokinase. Solution was buffered with 0.05 M tricine (pH 7.3). 37 .mmuscHE om Ou om Eoum Umwum> mmEau coauommm .Lomm mxmmam M MD mommuw>m mum wmoam> .xmmam mom mEOum 1 .Oxmums cwmmxo .Xmmam Hem mmHoE j .mxmums Hon .oom mm3 enoumuOmEmB .HE\m1 0H.o mp3 EHxOB .Am.h mmv TEHUHHD 2 mo.o QDAB Umuwmmon mmz coausaom .Ommcflxoxwfl HE\mE N mcflcflmucoo mmoosam mOHOE d ooa pom “om .mumcoame ammo.o .¢OO “om.o .mme “om.o .oaz “«.0 .o msons00uao um .eaa “ma .eommz u03 .sommms hom .memsmesHmOmeua hoos .OmOHUSm “xmmaw mom mOHOE 1 CH .mcHBOHHOw Ono owcfimucoo OHouxHE cofluomwmm m.m m.mH m.¢m cflxoe m.m m.m m.mm HOHDCOO .v m.H v.ma N.NN aflxoe m.H H.ma H.vm Houucoo .m m.N ®.HH m.om , GHxOB m.m m.m h.mH Houucoo .m N.m m.ma m.>m CHxOB H.N m.HH H.¢N HOHDCOU .a OH on . O m as cwmwx m m a: a EOE mom .0: .u o.m o x u o o x u .m me u a unmeaummxm mmcaaommm umo maflflumwomom Eoum mHHUCOSUOuHS wn OxmumD cmmhxo cam wumflmmozm UflcmmuocHII.m magma 38 of phosphate in organic and inorganic fractions. Results showed reduction in uptake and incorporation of P32 into organic phosphate compounds after toxin treatment of susceptible tissue (Table 4). Assuming the uptake of sus- ceptible control tissue as 100%, toxin treatment caused about 90% decrease in P32 uptake in susceptible tissue, 'while uptake was not inhibited in resistant treated tissues. Controls and treated resistant tissues incorporated 20 to 30% of the total P32 into organic phosphate, while incorpo— ration was completely blocked in treated susceptible tissues (Fig. 4). Similar results were obtained with root and coleoptile tissues. All experiments were done three times with essentially the same results. In another experiment, ethanol extracts of susceptible and resistant tissues were spotted on Whatman no. 1 paper until all the spots had approximately equal counts. The paper was developed Chromatographically and an autoradiograph was made. Labeled organic P—compounds occurred in extracts of control and treated resistant tissues but little or none were found in extracts of toxin treated susceptible tissue (Fig. 4). While this work was in progress Black and Wheeler (9) reported decreased P:O ratios by mitochondria from tissue 39 Table 4.—-Effect of Toxin on P32 Uptake and Incorporation by Oat Leaf Tissue Radioactivity Oat type and Total uptake Inorganic P Organic P treatmenta cpm cpm cpm Susceptible Control 12,895 9,555 3,245 Susceptible plus Toxin 1,560 1,215 120 Resistant Control 14,400 10,160 3,550 Resistant plus Toxin 15,800 11,990 3,780 aToxin concentration was 0.16 ug/ml. Each tissue sample was 0.5 g, pre-treated with toxin or water for 4 hdurs and incubated with P32 for 2 hours. Treatments were duplicated. 4O "' RES 80" 40 “ n. N 0° 0 o\ " "' sus 80- 4o- . I H Cl: Tox CI! Tox Fig. 4.--Effect of toxin on P32 uptake and incor— poration in resistant and susceptible tissues. Tissue samples (0.5 g) were pretreated with 0.16 ug/ml toxin or water for 4 hours and incubated with P for 2 hours. 41 previously treated with toxin, but no effect of toxin added to mitochondria isolated from untreated tissue (9, 62). Failure of Pi incorporation in susceptible treated tissue may result from disruption of cellular membranes and Pi transport. Effect of toxin on amino acid uptake and incorporation.—- Uptake of exogenous amino acids in tissues is a carrier mediated transport across the membrane (20). Most solutes that cross the membrane are retained in the cell after repeated washings, which removes solutes from apparent free space. Cells with damaged plasma membranes should retain less solutes after washing. Therefore amino acid uptake and retention in roots treated with toxin were compared with uptake and retention in untreated controls. Roots were treated with toxin solution (0.16 ug/ml), for varying periods, incubated for l to 3 hours in labeled amino acid solutions, washed for 1 hour and extracted with ethanol. The radioactivity (Cpm) in ethanol extracts was made as a measure of intracellular free pool amino acids. Toxin treatment caused a decrease in active uptake and retention of Cl4 amino acids in susceptible but not in resistant tissues (Table 5). Susceptible roots exposed to toxin for only 30 minutes had 90% decrease in the active trans-membrane uptake of labeled solutes. Similar results 42 Table 5.—-Effect of Toxin on Uptake of Cl4 Amino Acids by Susceptible and Resistant Oat Roots Uptake of C14 Valine (50 mM) Leucine (1 mM) EXP?:::e)tlme Treatmenta SUS RES SUS RES ’ cpm cpm cpm cpm 30 Control 9,280 10,950 1,540 ... Toxin 585 11,240 255 ... 60 Control 9,570 9,525 1,700 ... Toxin 580 10,040 270 ... 120 Control 9,985 10,320 1 960 1,815 Toxin 460 10,895 190 1,795 aReaction mixture (1.0 ml) contained amino acid as indi- cated; 1 drop of Chloramphenicol solution (0.5 mg/ml); 30 mM phosphate buffer (pH 7.0), and 250 mg fresh root tissue. After incubation for 2 hours at 220 on a shaker, tissue was washed 1 hour with water and extracted with 0.5 ml ethanol. Aliquots (0.1 ml) on planchets were counted. Toxin concen- tration was 0.16 ug/ml. The valine-U-C was 1.0 uC/ml. The leucine—l-Cl4 was 0.5 uC/ml. 43 were obtained with labeled valine and leucine at concentra- tions from 0.001 to 0.05 M. After 30 minutes exposure to toxin, most cells became totally permeable to solutes, since solutes entering these cells were readily lost during the washing period. The effect of toxin on incorporation of Cl4 amino acids by tissues was determined by measuring the amount of radioactivity retained in TCA precipitable cellular fractions. In one set of experiments roots were exposed to 2 concentra— tions of cl4-1eucine or cl4-valine. After removal of aliquots of the ethanol extracts for assay of uptake, the roots were homogenized in hot 80% ethanol and processed as described for leaf tissue samples. For incorporation in leaf tissues, cuttings from 10 day old susceptible and resistant plants were allowed to take up toxin (0.16 ug/ml) or water for 4 hours. Following toxin treatment, 1 cm leaf discs were punched and treated as follows. Randomized duplicate or triplicate batches of discs weighing 0.5 g each were placed in 2 ml labeled amino acid solutions at several concentrations, and infiltrated i3 vacuo for 10 to 15 minutes. The tissue samples were then incubated on moist filter paper in petri dishes for 4 hours at 220. After incubation the discs were processed as described in methods, and the 44 radioactivity of the NaOH hydrolyzed protein fraction was determined. Incorporation of Cl4 amino acid by resistant tissue was not affected by toxin whereas incorporation by susceptible tissue was practically stopped (Table 6). Comparable results were obtained with root and leaf tissue thus confirming some previously published data (51). The results could indicate some effect of toxin on protein synthesis, especially at the translational level. Uptake studies, however, indicated that the failure of incorporation as measured by this method might be due to non-availability of such solutes at the site of synthesis. Attempts have been made to obtain a cell—free protein synthesizing system from oat seedlings. This preparation had such low synthetic activity that no conclusions were possible (51). However, if toxin is a general inhibitor of protein synthesis, insensitivity of resistant plants could be based on inability of toxin to enter such cells. If this assumption is true, ribosomes from any organism, should respond to toxin action when freed from their cellular barrier. To test this possibility, the effect of toxin on Cl4-Valine incorporation by reticulocyte ribosomes was determined. Ribosomes and the supernatant enzyme fraction were 45 Table 6.-- Effect of Toxin on Incorporation of Cl4 Leucine into TCA Precipitable Fractions by Susceptible and Resistant Leaf Tissues Tissue Treatmenta CPMb Susceptible Control 396 Toxin 57 Resistant Control 337 Toxin 350 aToxin concentration was 0.16 ug/ml, and treatment time was 4 hours. Tissues were then incubated with Cl4-leu- Cine (0.5 mM, 100 uC/ml) for 4 hours. All treatments were triplicated. bTotal fresh weight of tissue, 0.5 g/sample. CPM/O.l ml fractions of NaOH hydrolysate. 46 isolated as described in methods. Toxin preparation was an eluate from a Bio Gel P—2 column. Results of preliminary experiments (Table 7) showed that incorporation of Cl4-Valine into total protein was dependent on the components of the system. The time course of amino acid incorporation (Fig. 5), indicated that toxin had no effect on the rate of incorpo— ration. Increasing concentrations of toxin did not affect either the rate or total efficiency of incorporation. Effect of toxin on plasmolysis.-—Cells with damaged membranes will not plasmolyze when placed in hypertonic solutions. Therefore we can test whether or not toxin destroys or damages the plasma membrane ip.§i£p, The selective effects of toxin were compared with the effect of DNP on cell membranes, using concentrations of DNP known to uncouple oxidation from phosphorylation. Roots of 4 day old plants were placed in either toxin solutions (0.16 ug/ml), DNP (10 and 100 uM) or water for varying times. After treat- ment, roots were quickly rinsed in water, placed in 0.5 M mannitol solutions, and observed under a microscope. Within 20 minutes after toxin exposure, susceptible root hair cells lost the ability to plasomlyse in hypertonic solutions (Table 8). Tox1n treated resistant cells showed plasmolytic shrinkage even after 180 minutes of exposure to toxin. 47 Table 7.--Requirements for Optimal Incorporation of Cl4 -Valine into Protein by Reticulocyte Ribosomes . . a d' ' - . Reaction mixture Ra loaCt1V1tY/mg ribosome cpm Complete system 2,360 Complete, minus ATP and energy generating system 40 Complete, minus ribosomes 0 Complete, minus supernatant enzyme 275 Complete, minus amino acid mixture 360 Complete, minus GSH 215 Complete, minus MgCl 150 2 aThe complete system contained 2 mg ribosomes, 4 mg supernatant enzyme, Cl4-va1ine (specific activity 10.0 uc/ umole), and other components as described (see methods). Incubation time at 37° was 40 minutes. 48 4 I— 2 no 3 F- X :2 O a. 2 OCOMPLETE SYSTEM ‘1 a COMP.+ TOX 0.16.09/ml 0 NO ENERGY nuns-nouns" O usuuumunnm O O 3 l l l 10 20 30 'TIAAE IHIN Fig. 5.—-Effect of toxin on amino acid incorpo- ration into protein in a cell—free system. The complete system (01 contained 2 mg ribosomes, 4 mg supernatant enzyme, C 4—valine (specific activity 10 uc/hmole), and other components as described. Incubation temperature was 37°. Toxin (A) concentration was 0.16 ug/ml. o=complete system less ATP or other energy generating system. 49 Table 8.—-Effects of Toxin and 2,4-Dinitrophenol on Plasmol- ysis of Root-Hair Cells in Hypertonic Solutions Exposure time required to destroy plasmolysis abilitya Treatment Susceptible Resistant min . min . Control >180 >180 Toxin, 0.16 ug/ml 20 >180 DNP, 100 uM 90 120 DNP, 10 uM 180 >180 aExposure times were 10, 20, 30, 40 50, 90, 120, 150, and 180 minutes prior to placing cells in hypertonic solution. b . . Control roots were placed in water or deactivated toxin. 50 Resistant and susceptible untreated controls plasmolyzed normally. DNP at 100 uM acted much more slowly than toxin and destroyed the plasmolytic ability of both susceptible and resistant cells (Table 8). Effect of toxin on apparent free space of tissue.—— Apparent free space is that portion of the tissue in which solutes move by free diffusion (10); in roots this consists of cell wall and intercellular spaces. Cytoplasm is thought to be unavailable for free movement of ions because the plasma membrane is a permeation barrier. The possibility of toxin induced changes in apparent free space in roots was examined as a further measure of membrane damage. The exper- iment was based on the hypothesis that if toxin disrupts the plasma membrane, the barrier for free permeation will disappear and apparent free space should increase. Resistant and susceptible oat plants were grown in duplex staining dishes (90 x 73 x 60 mm) with removable trays. Cheesecloth was fitted with rubber bands over the bottom of the removable tray, and 30 germinated seeds were placed on the Cheesecloth. The tray was then suspended above the surface of 100 ml White's nutrient solution in the dish. Seedling roots soon grew into the nutrient solution; after 7 to 10 days at 220 there were sufficient roots for an 51 experiment. Toxin or other treating substances were added to the solution of some dishes, while others were used as untreated controls. Apparent free space was determined by the India ink tagging method (7), using 35 mM mannitol as the equilibrating solution. Data of preliminary experiments indicated that apparent free space was the same in excised roots and roots of intact plants, with different equilibration and exodif- fusion times up to 2 hours. Therefore, in most experiments roots with several different toxin treatment times were excised and equilibrated for 1 hour in 35 mM mannitol, followed by 1 hour exodiffusion in glass distilled water. Control and treated resistant roots had apparent free Space values ranging from 13 to 20%.and are therefore in good agreement with published values for Gramineae (7, 11). Within 30 minutes, free space in toxin treated susceptible roots increased to 40%. As the time of treatment increased, values also increased until after 8 hours 90 to 100% of the total root volume became free space (Fig. 6). Thus, mem— brances of more cells appear to be destroyed as toxin treatment time increases. There was no Change in apparent free space in treated resistant roots, even after 8 hours. Apparent free space increase after toxin treatment suggests 52 ..I Susceptible Resistant c-c—c—*—c I I I I I I I I 2 4 6 8 HOURS Fig. 6.——Effect of toxin (0.16 ug/ml) on apparent free space (AFS) of susceptible and resistant oat roots. Roots were treated with toxin, excised, and equilibrated in 35 mM mannitol for 1 hour, then placed in glass distilled water for 1 hour for exodiffusion. Maximum and minimum values are indicated for hour 4. 53 membrane damage. Apparent free space appears to Change more slowly after toxin treatment than does the response measured by amino acid uptake. However, the AFS measurements are for whole tissues, while the amino acid uptake experiments may include only the outer cells. The effects of toxin (0.16 ug/ml) were compared with the effects of 0.1 mM DNP, 1 mM NaF, and 1 mM NaN3(Table 9). After 2 hours exposure to toxin about 80%.Of the root volume in susceptible tissue was freely permeable, while apparent free space of resistant roots remained unchanged. NaF and NaN3 had no effect on apparent free‘space, although the con- centrations used are known to inhibit respiratory metabolism of the cell. Since solutes move through apparent free space with no metabolic energy requirement, and the results obtained with NaF and NaN3 were consistent and indicate no drastic effect on the membrane. DNP increased apparent free space in both susceptible and resistant roots, and therefore appears to affect the plasma membrane non—specifically. Effect of DNP on plasma membranes has been demonstrated by other types of eXperiments (29). Effect of toxin on free_protoplasts.——Cell wall free protoplasts were prepared from coleoptiles of corn, sorghum, and susceptible and resistant oats (46). The Spherical 54 Table 9.-—Effects of Toxin and Various Metabolic Inhibitors on Apparent Free Space of Oat Roots b % Apparent Free Space Treatmenta Susceptible Resistant Control 16 :_4 22 :_4 Toxin, 0.16 ug/ml 80 :_5 22 :_4 DNP, 0.1 mM 45 :_5 45 :_5 NaF, 1 mM 16 :_4 22 i_4 NaN3, 1 mM 20 :_2 20 :_2 aTreatment time, 2 hours; equilibration time in 35 mM mannitol, 1 hour; exodiffusion time in glass distilled water, 1 hour. bThe data were calculated as % of total root volume. 55 protoplasts were enveloped by plasma membranes and showed active protoplasmic streaming. The protoplasts were trans— ferred to microscope slides with concave cavities and treated ‘with various concentrations of toxin or inhibitors. The basic reaction mixture as 10 ul protOplast suspension plus 10 ul treatment solution, buffered'at pH 6.5. Coverslips were placed over the cell suspensions, and slides were incu— bated at 220 in a moist chamber. A microscope was used to take zero time count of intact protoplasts, followed by counts at varying intervals. SurVival percentages were based on zero time counts. Protoplasmic streaming (cyclosis) stopped in many protoplasts from susceptible oat plants within 10 minutes after exposure to toxin. Toxin at 0.16 ug/ml caused 100% bursting of such protoplasts in 1 hour (Table 10). Most broken protoplasts and the remains of their plasma membranes soon lysed and disappeared, leaving mitochondria apparently unharmed. Protoplasts from corn, sorghum, and resistant oats were not affected by toxin (Table 10). Cyclosis in the resistant protoplasts did not stop and there was no more bursting or lysis than in controls. In several experi— ments done with Slight variations in procedure, free protoplasts from susceptible and resistant plants clearly 56 Table 10.-—Effect of Toxin (0.16 ug/ml) on Protoplasts from Host and Non-Host Plants % Survival after 1 hour Protoplasts from Control Treated Susceptible oat (CV. Park) 93 0 Resistant oat (CV. Clinton) 89 92 Corna 96 94 Sorghum 95 92 a . . . All corn and sorghum CVS. are re51stant to H, Victoriae and to its toxin. 57 retained their specific differential response to toxin. Since free protoplasts lack cell walls, we can eliminate this struc- ture as a necessary site of action of the toxin. Toxin concentrations from 1.6 to 1.6 X 10—7 ug/ml were used in another eXperiment. Again toxin had a dramatic effect on susceptible but no effect on resistant protoplasts (Fig. 7). Toxin at 1.6 X 10_4 ug/ml caused 50% bursting of susceptible protoplasts in 1 hour, while 1.6 ug/mlcaused 100% bursting. Control and treated resistant protoplasts had 5 to 10% burst- ing during this time. Cyclosis was not affected in the toxin treated resistant and untreated control protoplasts to the end of the experiment. DNP was used at concentrations (10 and 100 u M) known from preliminary experiments to damage oat cuttings. Toxin was used at a concentration of 0.16 ug/ml. Again the highly specific effect of toxin was evident (Table 11). Thirty minutes after exposure to toxin84% of susceptible protoplasts were destroyed and the remainder showed no cyclosis. Within 1 hour all susceptible protoplasts were broken, while only 5 to 10%.of control and toxin treated resistant protOplasts were broken. DNP acted more slowly, and affected susceptible and resistant protoplasts equally. Cyclosis stopped in both re— sistant and susceptible protoplasts 15 minutes after exposure to DNP, and after 2 hours 48 to 62% of the protoplasts had 5% ---.-:-’.------;I \. so - \ 00.000.000.000;00.00.000.0008 0 O I e Sus TOX °/o SURVIVAL I 40 — e 508 Ck _ A ROS Tax 20 b- O Res CI: h- . I L I I l ‘\j o 15 164 10'2 TOXIN-PQ/m' “-6 Fig. 7.-—Effect of toxin concentration on survival of resistant and susceptible protoplasts. Intact proto— plasts were counted at zero time and after 1 hour exposure to toxin or water-(controls). I=susceptible control; O=susceptible toxin treated; o=resistant con— trol; and s=resistant toxin treated tissues. 59 Table ll.--Comparative Effects of Toxin and 2,4—Dinitrophenol on Oat Protoplast Survival. Toxin Concentration was 0.16 ug/ml Protoplast survival afterb Protoplast type Treatmenta 30 min 60 min 120 min %» % 36 Susceptible Control 94 90 90 (CV. Park) Toxin 16 0 0 DNP 100 uM 82 60 38 DNP 10 uM 88 76 45 Resistant Control 96 92 92 (CV. Clinton) Toxin 91 90 90 DNP 100 uM 80 60 40 DNP 10 uM 87 72 . 52 aSolutions were made with 25 mM phosphate buffer (pH 6.5). bCalculated as % of intact protoplasts at zero time. 60 lysed or burst (Table 11). Filipin and ribonuclease are known to cause bursting of free protoplasts (24, 27). Therefore, these compounds were tested for possible differential effects on toxin resistant and susceptible cells. Filipin at 50 ug/ml caused 20 to 30% of protoplasts to break in 2 hours, while 0.03% ribonuclease solutions caused about 5&%'bursting in 1 hour. Resistant and susceptible protoplasts were affected equally by both filipin and ribonuclease. Sodium bisulfite is known to reduce the effect of toxin on susceptible oat seedlings (50). Therefore, the effect of NaHSO on toxic action against protoplasts was tested. When 3 toxin was diluted with freshly prepared NaHSO3 solution at pH 6.5, toxic effects were delayed (Table 12). Toxin at 0.16 ug/ml caused 100% bursting of protoplasts in 1 hour. In the presence of 8 X 10-4M NaHSOB, only half the protoplasts lysed in 1 hour, and only 60% in 2 hours. This concentration of bisulfite alone did not affect protoplasts survival. Bisulfite did not completely counteract the effects of toxin, since most of the surviving protoplasts later collapsed. Cyclosis was virgorous in both water and NaHSO3 controls. The bisulfite effect on toxicity to protoplasts parallels the effect on seedlings (50). Effect of toxin on seed germination.-—Inhibition of 61 Table 12.--Effect of Sodium Bisulfite (8 X 10-4 M) on the Toxicity of Toxin (0.16 ug/ml) to Susceptible Oat Protoplasts % Survival of protoplasts after exposure Treatmenta 60 min 120 min Control 96b . ' 92b NaHSO3 control 92b 92b Toxin 0 0 Toxin + NaHSO3 63 44 aReaction mixtures (pH 6.5) contained phOSphate buffer. bCyclosis evident. 62 susceptible seedling root growth has been the basis of the standard bioassay for toxin (31, 41). This response of sus— ceptible seedlings had been used for mass screening of disease resistant mutants by several workers (21, 22, 63). However, there are no data on the effect of toxin on dry, resting seeds. Therefore, an attempt was made to determine the effect of toxin on seed germination and certain early process of seed germination. Two hundred hulled susceptible or resistant seeds were held for varying times in 20 ml of solution containing 0.16 ug/ml toxin. Controls were kept in water. Seeds were then washed in running tap water for 1 hour and incubated on moist filter paper in the dark. All treatments were duplicated. Results (Table 13) showed that 1 hour treatment caused 90% inhibition, while 30 minutes treatment caused 50 to 60% inhi- bition of germination of susceptible seeds. Treated resistant seeds germinated normally. Toxin could be acting either on resting cells of dry seeds or on the cells that are metabol- ically activated during the experimental period. To test the latter possibility, susceptible and resistant seeds were immersed in water for varying times, and then dried under vacuum over CaCl2 and P205 for 48 to 72 hours. Seeds were then placed on moist filter paper in the dark for germination. Results (Table 14) showed that germination was not irreversibly 63 Table 13.--Effect of Toxin on Germination of Oat Seeds % Germination Treatmfigf time Treatmenta Susceptible Resistant 1. Control 86 95 Toxin 8 96 2. Control 79 82 Toxin 0 86 aSeeds were immersed in toxin (0.16 ug/ml) or water (control) for indicated periods, washed in running tap water for 1 hour and then incubated on moist filterpaper in the dark. Germination %.was based on counts of 200 seeds. 64 fTable l4.--Effect of Immersion in Water and Drying on Subse- quent Germination of Oat Seeds J _ ‘ %,Germination Immersion time (hr)a Sus Res 0 90 89 4 89 90 8 82 89 12 80 84 16 50 47 aSeeds were immersed in water and dried over CaC12 and P205 under vacuum. Seeds were then resoaked for germination. Germination %.is based on counts of 200 seeds. Oat CVS. used were Park (sus) and Clinton (res). 65 activated even when seeds were held in water for 12 hours. These seeds could be dried without losing their viability and subsequent germination capacity. There was a drastic decrease in germination ability when seeds were held in water for more than 12 hours before drying. The experiment was done 3 times with similar results. An early phySiOlOgical process in cereal seed germina— tion following imbibition is the synthesis and secretion of a—amylase and other hydrolytic enzymes by aleurone cells (58). Aleurone cells are triploid, fully differentiated, nondivid— ing resting cells with very little synthetic activity. After 24 to 48 hours of imbibition, their synthetic capacity is activated by the transport of gibberellic acid from the embryo (13). The aleurone cells can be separated from the embryo and activated 3p vitro by an exogenous supply of GA (59). The possibility that toxin interferes with GA induced synthetic capacity, which results in inhibition of seed germination, was tested with half seeds devoid of embryos. Preliminary experiments showed that toxin did not have a direct effect on either GA ores-amylase activity. Oat aleurone cells were excised, allowed to imbibe for 48 hours and then incubated with GA alone or with GA plus toxin. Control susceptible and resistant aleurone cells 66 produced little or no m—amylase when GA was not added to the mixture (Table 15). In presence of 0.1 u M GA there was a great stimulation of m-amylase production. Toxin (0.16 ug/ml) completely inhibited GA induced synthetic activity in sus- ceptible cells, while resistant cells were not affected. In several experiments toxin was added to dry aleurone cells during imbibition, or along with GA after 48 hours of imbi- bition. In each case toxin treated susceptible cells failed to Show GA induced synthetic capacity. The addition of GA to aleurone cells of oat resulted in a linear synthesis of a-amylase after a lag period of 6 to 8 hours (Fig. 8). Toxin added along with GA or after 11 hours of GA induction completely inhibited the snythesis of m-amylase. In another experiment toxin was used with various concentrations of GA. When a—amylase production was plotted against GA concentration, with or without toxin, reasonably straight lines resulted (Fig. 9). Therefore, inhibition of GA-induced a—amylase production by toxin is not competitive. The results suggest that toxin does not interfere directly with GA regulated metabolism. I would expect that any com- pound toxic to the essential life processes would inhibit germination and even kill the seed if present in sufficient amounts. 67 Table 15.--Effect of Toxin and Gibberellic Acid on Production of a-Amylase by H, victoriae Susceptible and Resistant Oat Aleurone Cells ug m—amylase from aleurone cells of 10 half seeds Treatmenta Susceptible Resistant Control 7 4 6 0.1 u M GA 95 94 0.1 u M GA + 0.016 ug/ml Toxin 1 90 0.1 u M GA + 0.16 ug/ml Toxin 0 98 aSeeds were incubated with buffer, with or without 0.1 u M GA and toxin as indicated. Results are averages of dupli- cate flasks. 68 35 - 25 P D 1 m h- v: .< ;: .eeeeeese D assumes-unset) 2 I s '- ’o Ines-l. '< ' Tex . JP 5 I— ”...Aumm A IIIImquImm A assume-sun A FO‘Au-Iu-HA‘ l l I J a '2 ‘3 24 TINHE HR Fig. 8.-—Effect of toxin on a—amylase synthesis by aleurone cells. Cells from 10 half seeds were imbibed for 48 hours and then incubated with 0.1 u M GA for the periods indicated. Toxin (0.16 ug/ml) was added at times indicated by arrows, and a—amylase synthesis was measured at intervals. o=control; d=toxin added 11 hours after addition of GA; A=toxin added along with GA. 69 3. \ of A 24 I- \& ‘C. en ¢) 3 2 9 e‘é. x 04°33 DI -' (3's? 9. 1. ,+ s V o ‘s. an «..§ In 16 - x f < -l >. E '1 I a > s I 4 6 8 10 10 I 10 I/GA M' Fig. 9.——Effect of toxin on GA-induced synthesis of a—amylase, plotted by the Linweaver—Burk method. Half seeds were imbibed for 48 hours prior to GA treatment and m-amylase production was determined 24 hours later. DISCUSSION H, victoriae susceptible oat tissue has several known physiological and biochemical responses to infection; these responses can be reproduced by HV—toxin (51). Resistant oat and other tissues have no known responses to toxin. This is an ideal model case for studying disease development and disease resistance and for determining the primary site of action of a pathogenic microorganism (51, 64). One well known response, typical of diseased plants in general, is the increase in gas exchange in toxin treated leaves and roots. There are several possible explanations for "pathological respiration" of tissue. For certain dis— eases, the increase is correlated with stimulation of growth (15), but Clearly this is not the case with HV—toxin treated tissue. The primary lesion of toxin was once thought to be an uncoupling of oxidation from phosphorylation (45), because toxin treated tissue did not respond to DNP concentrations that caused increased oxygen uptake (uncoupling) by control tissue (26). However, data of this study Clearly show that 70 71 neither oxygen nor phosphorus uptake by isolated mitochondria is affected by toxin. Since toxin has no effect on phosphorylation by iso- lated mitochondria, the high respiratory rate found in treated tissues does not appear to be the rSult of a direct effect of the toxin on respiratory centers. Apparently, some system other than the Krebs cycle is the primary site of action and this in turn could affect oxidation rates in various ways. Loss of respiratory control and slightly decreased P20 ratios by mitochondria isolated from tissue pretreated with toxin (9, 62) may be a secondary effect on mitochondria by cell breakdown products. Lack of respiratory response to toxin by certain types of susceptible tissues (coleoptiles and aluerone cells) also indicates that the respiratory effect may not be a primary lesion in the toxin's action. Another effect of HV—toxin is to stop the incorpo- ration of C14 labeled amino acids into TCA insoluble cellular components, a previously observed effect (51) which I have confirmed. If a primary lesion is in protein synthesis at the translational level, there should be an inhibition of synthesis by cell—free ribosome preparations. However, ribo— somes from oats, when prepared carefully to prevent bacterial growth, had very low activity and no conclusions were 72 possible (51). Active preparations from reticulocyte cells, which are affected by all known inhibitors of protein synthe- sis, were not affected by toxin. Apparently, the effect of toxin on protein synthesis in tissues is another secondary effect. This conclusion, however, is tentative since toxin may be a specific inhibitor of activities of ribosomes from susceptible plants. Furthermore, toxin iS known to disrupt DNA and RNA synthesis as measured by inhibited incorporation of labeled uridine or thymine in tissues (51). Data on effects of toxin at the transcription level using isolated nuclei are not available. One of the first responses to toxin in susceptible tissue is loss of cellular materials, presumably reflecting a permeability Change in the cell membrane (61). Unlike the respiratory response, all susceptible tissues respond to toxin in this way. Black and Wheeler (9) found that the leaf leachings after toxin treatment include several forms of nitrogen, amino acids, carbohydrates, and various inorganic salts. My data show that all water extractable electrolytes leaked from toxin treated tissue, while control tissue lost only 10% of the total extractable electrolytes. This suggests a disruption of both the plasma membrane and the tonoplast. It would be interesting to know whether all soluble 73 carbohydrates and amino acids leak from toxin treated tissue. If so, this effect could be an important factor in initial establishment of the pathogen in host tissue. Resistant cells do not leak cellular materials after toxin treatment; there— fore available soluble nutrients may be a limiting factor in early development of the fungus in resistant cells. Permeability Changes together with intracellular imbalance of ions in toxin treated tisSue have been considered a cause of respiratory increase. Amador and Wheeler (3) showed that toxin treated tissue when leached in distilled water had a much lower rate of respiration than similar tissue not leached. They postulated that leaching would quickly re— move vacuolar materials from the cells, and that this could account for the rapid loss of respiratory activity. However, no increase in respiration resulted when concentrated leach- ates were added back to tissue. A simple explanation for the increase in respiration after toxin treatment seems unlikely, because indirect effects on metabolism may be involved. Many substances toxic to cells affect either wall and membrane function, respiration, photosynthesis, protein synthesis, nucleic acid metabolism or control mechanisms in tissue. Since cell wall free protoplasts from susceptible tissue are highly sensitive to toxin, the wall is not a 74 necessary site of toxin action. Although toxin has significant effects on many metabolic processes in tissue, the isolated cell organelles so far tested are insensitive to very high levels of toxin. All data suggest that toxin is effective only when applied to membrane bound intact susceptible cells. Several findings strongly indicate that the site of primary lesion of toxin is in the plasma membrane. Previous data were suggestive in nature but not conclusive. Data pre- sented in this report are more conclusive, and may be summarized as follows: (i) Toxin causes rapid loss of all electrolytes from susceptible tissue. (ii) Root hair cells exposed briefly to traces of toxin cannot be plasmolyzed in hypertonic solutions. This is a known effect of membrane damage. (iii) Toxin inhibits or stops membrane regulated, active uptake of exogenous solutes such as amino acids and Pi after brief exposure. (iv) Apparent free space in tissue increases after toxin treatment. The plasma membrane is considered the per— meability barrier in tissues; disruptions are expected to lead to increased apparent free space. (v) Plasma membranes of isolated susceptible protoplasts break after brief exposure to toxin. Furthermore, there is evidence of membrane damage from the electron microscope work of Luke pp 31 (30). These workers, however, used tissues that had been exposed to culture 75 filtrates containing toxin for 24 hours; therefore the inter— pretation must be made with caution. Many plant infections result in loss of intracellular materials (8, 40, 48, 52), but relatively few studies have been made on the effects of disease on influx of eXOgenous solutes. Wood and Braun (67) showed that crown-gall tissue collects exogenous solutes more efficiently than does normal callus tissue, and that this may account for the ability of crown gall tissue to grow on a simple medium. Furthermore, the membrane of tumor cells is altered both in quality and quantity. In bacteria free tumor tissues total phospholipids decreased and an unknown phospholipid appeared (66). My results suggest that drastic damage to the plasma membrane can result in leakage of intracellular ions as well as inhibition of active uptake of exogenous solutes. A breakdown in transport of materials to synthetic sites could explain the effect of toxin on incorporation of amino acids. Similarly, the lack of phosphate incorporation into organic compounds could result from disruption of trans— port to the active site of synthesis. Influx studies therefore appear to be important for better understanding of permeability Characteristics of diseased tissues. Apparent free space increases at a Slower rate after toxin treatment 76 than is expected from the uptake data. Uptake measurements could by more sensitive than apparent free space determina— tions. However, apparent free space measurements are for whole tissues, while the amino acid uptake experiments may include only the outer cells. Since the toxin acts selectively on susceptible proto— plasts without cell walls, we can eliminate the wall as a necessary lesion site. The differential sensitivity of protoplasts obtained from host and non—host plants indicates that we are not dealing with an artifact. The possibility that the cell wall had not been completely removed is con— sidered unlikely for the following reasons: (1) some cellulase remained throughout the experiment, yet the behavior of the protoplasts did not Change with time; (ii) the protoplasts were spherical with no angular material and were quite flexible when exposed to small currents produced in the reaction mixture; (iii) no rigid shell was left after bursting; and (iv) protoplasts disintegrated rapidly in detergents, after Change of pH, and after Change in osmotic concentration of the suspending solution. The stability of isolated protoplasts depends on intact membranes, and agents affecting this structure can Cause bursting. Filipin, a polyene antibiotic, breaks 77 Neurospora protoplasts, presumably by binding with membrane sterols (24). Proteases and lipases break Bacillus megatherium protoplasts (27). Several proteases and lipases had no effect on protoplasts of oats, but basic proteins such as ribonuclease, cytochrome C and protamine caused bursting (46). These find— ings indicate basic structural differences between bacterial and higher plant plasma membranes, although all membranes are considered to be bimolecular layers with protein and lipid components. My results show that filipin, ribonuclease, and DNP affect both HV—toxin susceptible and resistant protoplasts, while toxin affects only the susceptible Avena protoplasts. HV-toxin seems to have a strong affinity for membranes of susceptible cells and the effects are more drastic than those of any other substance tested. The results suggest innate differences in plasma membranes of susceptible and resistant plants. Since cationic detergents and basic proteins caused rapid bursting of Avena protoplasts, Ruesink and Thimann (46) postulated that these substances bind with membrane anionic groups. Toxin could have a cationic functional group that binds with the membrane. It is of interest to note that toxin contains a secondary amine and is stable at low pH where all the carboxyl groups of the peptide Chain are in a protonated 78 form. Reduction of toxicity of toxin to protoplasts by NaHSO3 further supports this idea. Bisulfite could somehow protect the binding until all bisulfite is dissociated to 50;. It would be interesting to know whether or not other SH containing compounds (such as glutathione, mercaptoethanol, or thioglycolic acid) have similar protective properties against toxin. Isolation and Characterization of the receptor sites, however, are needed for conclusive evidence. Toxin action has a possible parallel in the physio- ology of senescence. Permeability Changes in cellular membranes immediately preceding or during ripening in fruits and during senescence in other plant tissues are indicated by leakage of solutes, increase in free space, and movement of liquid to intercellular spaces (19). Early work with DNP indicated that uncoupling occurred during the climacteric rise in respiration accompanying maturity (36). The uncoupling concept was dropped after data on mitochondrial reactions (44), and tracer studies (34) became available. Possible cause- effect relations between permeability Changes and other climacteric phenomena are not Clear-at present. However, recent researches indicate Changes in permeability could alter protoplasmic compartmentalization and affect relations between enzyme and substrates which could lead to respiratory increase 79 (19, 47). In the broad sense, toxin action can be viewed as a rapid and drastic onset of senescence. However, the simi- larities between the two phenomena may be more apparent than real since cytokinins delay senescence but have no effect on toxin action (32). Toxin receptors in susceptible tissue have been suggested (50), but there are no data to prove or disprove I the hypothesis. Results reported herein provide some support for a receptor hypothesis. It now appears that toxin combines with or affects an unknown component in the susceptible cell, resulting in disorganization of the surface. Such disruptions could account for all the effects of toxin described to date. The resistant cell membrane appears to lack the receptor or sensitive site, since such cells do not respond in any observ- able way. Membrane damage by HV-toxin could lead to the "bio— chemical symptoms" observed in susceptible cells. This postulation is based in part on published data for several biological systems. Studies on the action of colicines indicate that membrane damage can lead to temporarily increased respiration and collapse of synthetic systems (39). Membrane damage may affect many other cellular components because of physical and metabolic interconnections (4, 29). However, there 80 is no direct evidence ruling out interference with energy metabolism ip.ylyp_as part of the explanation of inhibited synthesis. Moreover, the possibility that the toxin is com- bining with an intracellular component resulting in quick disorganization of the surface is not absolutely negated by the data presented. HV—toxin and two other host-specific determinants of pathogenicity, one from Helminthosporium carbonum and one from Periconia circinata, are being used as models for the study of disease development and disease resistance in plants (51). Susceptibility or resistance to all these diseases is based on reaction with or lack of effect by specific toxins. Thus resistance and susceptibility to these diseases appears to be based on constitutive factors. Experiments with seed germination described herein provide further support for this concept. Toxin susceptibility and resistance are expressed by resting, fully differentiated, triploid aleurone cells long before metabolism leading to germination is irreversibly activated. This is not consistent with the popular idea that resistance requires metabolically active cells (55). Further- more, there are several reasons to question the role of phytoalexins in resistance to H, victoriae (38). The extreme specificity of HV-toxin is of interest 81 for other reasons as well. The validity of the concept of the unit membrane (43) has been questioned by several workers (25, 33). There is increasing evidence that membranes of different plants or animals differ widely in their chemical and structural organization (5, 25, 60). 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