llHllllllllllllllllllllll’Illllllllllmi‘llllllllllillllllllll L mmnr 3 1293 00084 3668 Michigan State University This is to certify that the dissertation entitled Effects of Heat Shock on Disease Resistance and Related Metabolism in Cucumber presented by Bruce Allen Stermer has been accepted towards fulfillment of the requirements for Ph.D. Botany & Plant Pathology degree in ngui Major professor Date M4 M5 U is an Affirmative Action/Equal Opportunity Institution 0.12771 MSU LIBRARIES “ RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. EFFECTS OF HEAT SHOCK ON DISEASE RESISTANCE AND RELATED METABOLISM IN CUCUMBER BY Bruce Allen Stermer A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1984 ABSTRACT EFFECTS OF HEAT SHOCK 0N DISEASE RESISTANCE AND RELATED METABOLISM IN CUCUMBER By Bruce Allen Stermer A brief heat shock induced resistance to the scab pathogen, glagg; sporium cucumerinum, in cucumber plants normally susceptible to the fungus. Immersion of seedlings in a 50 C water bath for 40 or 50 seconds was found to be the optimal treatment for the induction of resistance. Plants inoculated with g; cucumerinum as soon as 3 h after the heat shock exhibited increased resistance to the fungus; a 12 h interval from heat shock to inoculation allowed for development of maximum resistance. 'The resistance was still fully effective when plants were inoculated 48 h after heat shock. All scab susceptible cultivars that were tested became more resistant to g; cucumerinum after heat shock. There was a direct correlation between the activity of soluble peroxidase induced by heat shock and the resistance induced by the same treatment. Heat shocked cucumbers had an increase in activity of the same isoperoxidases seen to increase in cucumbers with systemic resistance induced by prior Colletotrichum lagenarium inoculation. 'The relationship of heat shock induced resistance to other stress responses and the role of peroxidases in induced resistance is discussed. Within 6 h after the heat shock there were increases in the produc- tion ethylene and in its precursor l-aminocyclopropane-l-carboxylic acid. Heat shock also enhanced the accumulation of extensin, a hydroxyproline-rich glycoprotein found in the cell walls of the seedlings. Inoculations of heat shocked seedlings with §;_cucumerinum 24 h after the shock resulted in further enhancement of extensin. Cell walls from heat shocked seedlings were more resistant to degradation by enzymes from Q; cucumerinum than were cell walls from unshocked seedlings, but increased lignin deposition did not appear responsible. The accumu- lation of extensin after heat shock and its crosslinking by peroxidase is discussed as a possible mechanisms of resistance to §._cucumerinum. ACKNOWLEDGMENTS I am indebted to my major professorlhn Ray Hammerschmidt for his insightful guidance, enthusiastic encouragement, and understanding friendship. Drs. Derek Lamport and Hans Kende are gratefully acknowledged for their technical help with many of the experiments in this dissertation. Also, I thank Drs. Robert Scheffer, Derek Lamport, and Dennis Fulbright for serving on my graduate committee. Special thanks go to Carla Thomas for her unending support and encouragement throughout my studies. ii TABLE OF CONTENTS Page LIST OF TABLES ........................... V LIST OF FIGURES ............... . ........ . . . Vl GENERAL INTRODUCTION .................... . . . . 1 SECTION I EFFECTS OF HEAT SHOCK ON CULTIVAR AND NONHOST RESISTANCE IN CUCUMBER SEEDLINGS Abstract .............................. 8 Introduction ............................ 9 Materials and Methods ........................ 10 Results ............................... 11 Discussion ............................. 14 References ............................ . 18 SECTION II HEAT SHOCK INDUCES RESISTANCE TO CLADOSPORIUM CUCUMERINUM AND ENHANCES PEROXIDASE ACTIVITY IN CUCUMBERS Abstract .............................. 21 Introduction ............................ 22 Materials and Methods ........................ 23 Results ............................... 25 Discussion ............................. 33 References ............................. 39 SECTION III Page HEAT SHOCK INCREASES THE SYNTHESIS OF ETHYLENE AND ENHANCES THE ACCUMULATION 0F HYDROXYPROLINE-RICH GLYCOPROTEIN IN CUCUMBER SEEDLINGS Abstract . . . ........................ . . . 44 Introduction ........................... . 45 Materials and Methods. . . . . . . . . . ..... . . . ...... 46 Results. . . . . . . ...................... . . 49 Discussion ............................ . 57 References ...................... . . . . . . . 60 RECOMMENDATIONS ........................... 63 iv Table LIST OF TABLES SECTION I Page The effect of a heat shock prior to inoculation on lignin deposition and disease development in cucumber seedlings . . . . . . . . . . . . . . . . . . . . 15 SECTION II Resistance to Cladosporium cucumerinum induced in different cultivars of cucumbers by preinoculation heat shock . . . . . . . . . . . . . . . . . . . . . . . . 31 SECTION III Effect of heat shock on ACC levels in cucumber seedlings. . . . . . . . . . . . . . . . . . ...... . 51 Effect of a heat shock 24 h prior to inoculation on lignin deposition in cucumber epidermal cell walls . . . . 55 Figure LIST OF FIGURES SECTION I Lignin deposition in epidermal peels from heat shocked and unshocked halves of a cucumber hypocotyl inoculated with Helminthosporium carbonum . SECTION II The effectiveness of different heat treatments in protecting cucumber seedlings against 9; cucumerinum Protection of cucumber plants against C; cucumerinum by heat ShOCk. I I O O O I O I O O O I O O O O O O O The effect of challenge inoculum concentration on resistance observed after heat shock . . . . . . . . The effect of the time of challenge on resistance observed after heat shock.. . .. . .. . .. .. . The enhancement of peroxidase activity after heat treatments at various temperatures and durations . . Time course of peroxidase activity enhancement after heat ShOCk O O O O I O O O O O O O I O O O O O O O O Electrophoretic separation of anodic peroxidase isozymes from cucumber tissue with or without induced resistance to Cladosporium cucumerinum . . . vi Page . 13 26 27 28 3O 32 34 35 Figure SECTION III Page Effect of heat shock on ethylene production by cucumber seedlings . . . . . . . . . . . . . . . . . . . . 50 Time course for the accumulation of cell wall hydroxyproline after heat shock. . . . . . . . . . . . . . 53 Release of reducing sugars from cell walls of heat shocked and unshocked seedlings by wall degrading enzymes 0 O I O I O I O O O O O O 0 O O O O O O O O O O O O 56 vii GENERAL INTRODUCTION Heat shock has been widely used by plant pathologists. Kunkel reported in 1936 that dormant trees could be cured of yellows diseases by immersing tissues in a 50 C water bath for three to four minutes (9). In the following years heat treatments proved to be one of the most successful methods to eliminate viruses and yellows agents from infected plants (5). A summary of the therapeutic use of heat treatments on plant viruses has been published in a review by Hollings (5). Heat shock also has been used in studies of disease resistance mechanisms, e4», the heat treatment of uninfected plants to increase their suscep- tibility to viruses and fungi. Yarwood found that immersion of bean leaves in hot water for a few seconds before inoculation increases their susceptibility to various viruses and fungi (18). Later work demon- strated that a brief heat shock could reduce disease resistance in many types of plants (2,17). Researchers attributed the decrease in resis- tance of heat shocked plants to a blocking of defense mechanisms, such as phytoalexin production (6). Section I of this thesis describes the use of heat shock to suppress pathogen-induced lignin deposition in cucumber cell walls and simultaneously increase susceptibility to fungi. Hider interest in heat shock began with Ritossa's paper in 1962 (12L. This paper showed that transient chromosome modifications, indi— cative of active gene loci, were dramatically induced in Drosophila by a brief heat shock. However, very little progress was made towards understanding the phenomenon until 1974 when it was discovered that heat shock induced the synthesis of a small number of proteins and reduced normal protein synthesis (15). Since this time, heat shock has received considerable attention in model studies of gene expression in Drosophila (1). Analogous responses to heat shock were reported for cultured avian cells, bacteria, protozoans, yeast, and plants in 1978 (see ref. 13 for an excellent review). Thus, the heat shock response appears to be ubiquitous. In addition to heat shock other stress agents also induce heat shock proteins in various organisms, including amino acid analogs, metal ions, anoxia, viral infection, certain ionophores, and various antibiotics (13). The common occurrence of the heat shock response has been strengthened by the demonstration that antibodies to chicken heat shock protein cross-react with similar proteins of Drosophila, yeast, man,mouse, and frog (7% Previous studies have shown that stress of cucurbits caused by a prior infection can render susceptible plants resistant to subsequent attack from many different pathogens. This induced resistance has many similarities with the resistance described in Section II where cucumbers develop the ability to resist §;_cucumerinum infection approximately 24 h after a heat shock. Disease resistance induced in plants by prior infection has received considerable attention and is the subject of many reviews (8,11,14). Changes in epidermal cell walls appear to be involved in the mechanism of induced resistance in cucurbits against fungi. Correlated with the induction of disease resistance are an enhancement of cell-wall-associated peroxidase activity and lignin depo- sition (8). Increases in extensin, a hydroxyproline-rich glycoprotein of plant cell walls, is also associated with disease resistance in cucurbits (3,4). The involvement of cell wall modifications in the resistance induced in cucumber by heat shock is examined in Section III. Recent reviews discuss extensin and its role in plants and also sum- marize knowledge about the role of lignification disease resistance (10,16). REFERENCES Ashburner, M. and Bonner, J. J. (1979). The induction of gene activity in Drosophila by heat shock. Cell 17, 241-254. Chamberlain, D. N. (1972). Heat-induced susceptibility to non- pathogens and cross-protection against Phytophthora megasperma var. splag in soybean. Phytopathology 62, 645-646. Esquerre-Tugaye, M. T., Lafitte, C., Mazau, D., Toppan, A. and 5. Touze, A. (1979). Cell surfaces in plant-microorganism inter- actions. 11. Evidence for the accumulation of hydroxyproline-rich glycoproteins as a defense mechanism. Plant Physiology 64, 320- 326. Hammerschmidt, R., Lamport, D. T. A. and Muldoon, E. P. (1984). Cell wall hydroxyproline enhancement and lignin deposition as a early event in the resistance of cucumber to Cladosporium cucu- merinum. Physiological Plant Pathology 24, 43-47. Hollings, M. (1965). Disease control through virus-free stock. Annual Review of Phytopathology 3, 367-396. Jerome, S. M. R. and Muller, K. O. (1958). Studies on phyto- alexins. II. Influence of temperature on resistance of Phaseolus vulgaris towards Sclerotinia fructicola with reference to phyto- alexin output. Australian Journal of Biological Sciences 11, 301- 314. 10. 11. 12. 13. 14. Kelly, P. M. and Schlesinger, M. J. (1982). Antibodies to two major chicken heat shock proteins in widely divergent species. Molecular and Cellular Biology 2, 267-274. Kuc: J. (1983). Induced systemic resistance in plants to diseases caused by fungi and bacteria. Ig_The Dynamics of Host Defense, J. A. Bailey and B. J. Deverall, eds., Academic Press, New York. pp. 191-221. Kunkel, L. O. (1936). Heat treatments for the cure of yellows and other virus diseases of peach. Phytopathology 26, 809-830. Lamport, O. T. A. and Catt, J. N. (1981). Glycoproteins and enzymes of cell walls. Ig_Plant Carbohydrates, Vol. II, N. Tanner and F. A. Loewus, eds., Encyclopedia of Plant Physiology, New Series, Vol. 138, Springer-Verlag, New York. pp. 133-165. Matta, A. (1980). Defenses triggered by previous diverse invaders. _I_n Plant Disease, Vol. V, J. D. Horsfall and E. G. Cowling, Edsm, Academic Press, New York. pp. 345-361. Ritossa, F. (1962). A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 18, 571-573. Schlesinger, M. J., Ashburner, M. and Tissieres, A. (1982). Heat Shock: From Bacteria to Man. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. Sequeira, L. (1979). The acquisition of systemic resistance by prior inoculation. Ig_Recognition and Specificity in Plant Host- Parasite Interactions, J. M. Daly and I. Uritani, eds., Japan Science Society Press, Tokyo. pp. 231-251. 15. 16. 17. 18. Tissieres, A., Mitchell, H. K. and Tracy, U. (1974). Protein synthesis in salivary glands of Drosophila melanogaster. Relation to chromosome puffs. Journal of Molecular Biology 84, 389-398. Vance, C. P. Kirk, T. K. and Sherwood, R. T. (1980). Lignifi- cation as a mechanism of disease resistance. Annual Review of Phytopathology 18, 259-288. Yarwood, C. E. (1958). Heat activation of virus infections. Phytopathology 48, 39-46. Yarwood, C. E. (1956). Heat-induced susceptibility of beans to some viruses and fungi. Phytopathology 46, 523-525. SECTION I EFFECTS OF HEAT SHOCK ON CULTIVAR AND NONHOST RESISTANCE IN CUCUMBER SEEDLINGS SECTION I EFFECTS OF HEAT SHOCK ON CULTIVAR AND NONHOST RESISTANCE IN CUCUMBER SEEDLINGS ABSTRACT A brief heat treatment of cucumber seedlings immediately prior to inoculation with either Helminthosporium carbonum or Cladosporium cucu- merinum temporarily induced susceptibility to these fungi; inoculations 24 h after heat shock demonstrated that resistance had returned. The heat shock did not appear to produce any permanent damage to seedlings. The ability of seedlings to deposit lignin at points of infection was associated with both cultivar and nonhost resistance. Heat treatments which induced susceptibility prevented the epidermal cell walls of seedlings from lignifying after inoculation. The data suggest that resistance, but not susceptibility, requires active host metabolism. INTRODUCTION Heat shock has been used by many researchers to manipulate the expression of resistance to fungi. Generally, heat shock applied prior to inoculation has prevented or delayed disease resistance in plants. Such heat shock can inhibit cell wall alterations, such as papilla formation (1), and also reduce phytoalexin production (8) and hyper- sensitive cell death (6,12). However, heat shock can also prevent or delay the susceptible response to the fungi that produce host-selective toxins; the shock reduces plant sensitivity to the toxin (2,3,11). The common denominator in all these effects of heat shock on disease resistance appears to be the temporary halt of many active processes. Thus, depending on which process requires active metabolism, heat shock may block resistance or susceptibility. Heat shoCk also has effects at the molecular level including the gg_ggyg synthesis of ”heat shock proteins" and the reduction of normal protein synthesis (10). Rapid lignification of epidermal cell walls is linked to disease resistance against some fungi (14). In cucumbers, lignin deposition is associated with resistance to the fungal pathogen Cladosporium cucu- merinum (5,7). This study uses heat shock as a tool to I) examine the association of lignin deposition with cultivar and nonhost resistance in cucumber seedlings, and 2) to investigate whether the seedlings require active metabolism for resistance or susceptibility. 10 MATERIALS AND METHODS Plant and fungal material Cucumber seedlings (Cucumis sativus Ls) resistant (cv SMR-SB) or susceptible (cv Marketer) to the fungus Cladosporium cucumerinum Ell. and Arth. were used. Seeds were germinated and grown for 5 days in darkness at 22 C in rolled-up germination paper (Anchor Paper Co., St. Paul, MN) before treatment (4). Helminthosporium carbonum Ull. race 1 and §;_cucumerinum were grown on V-8 agar and potato dextrose agar, respectively, at 18 C (13). Heat shock and inoculation of seedlings The seedlings were given a heat shock by immersing their apexes and hypocotyls in a 50 C water bath for 40 seconds while holding on to the roots. The shocked seedlings were then placed in‘a covered 10 cm glass petri dish that contained one piece of moistened filter paper (9 cm diameterL, Fungal cultures were gently rubbed with a bent glass rod in the presence of some water to dislodge spores. The spore suspension was filtered through 2 layers of cheesecloth, and the concentration of spores was adjusted to 106 spores per ml for g; cucumerinum and 105 spores per ml for H; carbonum. Seedlings were inoculated immediately or 24 h after heat shock by placing a line of 3 to 5 ul drops of spore suspension along the entire length of the hypocotyl. Histochemical staining The epidermis peeled from seedling hypocotyls was stained for lignin with phloroglucinol-HCl (9%. A red chromogen is formed when phloroglucinol in HCl comes in contact with the cinnamyl aldehyde sub- units of lignin. Separate epidermal tissues were stained with cotton ll blue in lactophenol to visualize fungal structures (13). The stained epidermal peels were examined with a light microscope for evaluation of cucumber cell wall lignification and fungal development. RESULTS Effects of_prior heat shock on resistance to Helminthosporium carbonum The corn pathogen fiL_carbonum germinated a few hours after inocu- lation and formed appressoria within 6 to 10 h on untreated cucumber seedlings. By 18 h after inoculation, phloroglucinol-HCl staining produced a strong red color reaction in the cucumber cell walls around appressoria. Growth of the fungus into the epidermis stopped at about 24 h, and hyphal development in the tissues was restricted to the stained (lignified) areas. Both cultivars gave the same result. In contrast, the production of lignin was totally suppressed in plants that were inoculated with i carbonum immediately following a heat shock. The fungus readily penetrated the cucumber epidermis, and intracellular hyphae grew well and often entered adjacent cells by 24 h after inoculation. Nithin forty-eight hours after inoculation, the fungus had ramified through tissues; aerial mycelium was produced at inoculation sites by 72 h. Later, 5; carbonum produced conidia on the cucumber hypocotyls. Although the cucumber tissues regained their ability to lignify by 48 h after the heat shock, and much lignin was present in the infected tissues after this time, the response by the seedlings apparently was too late to stop the fungus. Seedlings that were inoculated 24 h after heat shock gave a typical lignification response and were resistant to the fungus. The 12 lignification of cell walls and the growth of H; carbonum in seedlings inoculated 24 h after heat shock were identical to the response of unshocked control seedlings. These delayed inoculations showed that normal resistance returned within 24 h after heat shock. The heat shock treatment did not appear to produce any permanent damage to seedlings. The effects of heat shock were not only temporary, but the effects were also localized. This was demonstrated by heat shocking only half of the hypocotyl. Irregardless of whether the apical or basal half was used, only the portion given a heat shock lost the ability to lignify and lost resistance; the unshocked portion gave a normal resistant response (Fig.1). Effect of_prior heat shock on resistance to Cladosporium cucumerinum The fungus germinated several hours following inoculation on either cultivar; the response of SMR-58 (resistant) was lignification around sites of attempted penetration by 18 h, but the cell walls of Marketer (susceptible) did not contain phloroglucinol positive material at this time. The response of SMR-58 to _Q._ cucumerinum was very similar to the response of either cultivar to H; carbonum. Development of Cngugg; merinum was stopped in the resistant cultivar by 24 h after inocu- lations. However, §_._ cucumerinum continued growth and later ramified through the tissues of the susceptible cultivar. When the normally resistant cultivar SMR-58 was inoculated with g; cucumerinum immediately after heat shock, no lignin deposition was observed at 24 h after inoculation. In addition, growth of the fungus into the tissues was similar to that seen for the susceptible cultivar. Delayed inoculations showed that the effects of heat shock on cucumber l3 Fig. 1. Lignin deposition in epidermal peels from the heat shocked and unshocked halves of a cucumber hypocotyl inoculated with Helmintho- sporium carbonum. The apical half of a cucumber seedling (SMR-58) was heat shocked (40 seconds at 50 C), and entire length of the hypocotyl was then immediately inoculated. Twenty-four hours after inoculation epidermal peels were stained with phloroglucinol-HCl and photographed at 400X magnification. A, peel from a heat shocked portion of hypocotyl; B, peel from an unshocked portion of hypocotyl. l4 resistance to g, cucumerinum was also temporary; however, full resis- tance to g, cucumerinum returned slower than did resistance to H; carbonum (Table 1). Additional observations of effects of heat shock on disease resistance Two further observations are noteworthy. First, although normal resistance returned approximately'24 h after heat shock, both H; carbonum and Q; cucumerinum continued to grow through tissues with recovered resistance when inoculated immediately after the shock. Apparently the fungi can overcome the hostfs resistance once an initial barrier is breached or a certain stage of fungal development is reached. Secondly, the cultivar susceptible to g; cucumerinum demonstrated an unexpected increase in resistance when inoculated 24 h after heat shock. The resistance induced by heat shock had many similarities to systemic induced resistance in cucumbers. Studies on the disease resistance induced by heat shock are presented in the following sections. DISCUSSION Heat shock delayed the expression of both cultivar and non-host resistance in cucumber seedlings. This effect was temporary, lasting less than 24 h, and was localized to the tissues actually shocked. A heat shock immediately before inoculation, however, did not provide any protection for the _C_. cucumerinum-susceptible cultivar against the pathogen. The major effect of heat shock on plant-pathogen interactions can be explained by the temporary halt of most active plant metabolism (10). In interactions where host susceptibility appears to be an active process, such as diseases involving host-selective toxins, a heat shock 15 mumtmuoe A++V .mcwcwcum usapp A+V .mcvcpmum opumtoam Au\+v .mcmcweum c: Any .cowumpauocp toamm ; mm umumapc>mu .mcrcvmum x>uos A+++V .mcwcwmum "opmom we've; statuanmmcwuxm m>wmcwuxw mco: +++ u u ++ mcoc w>wmcmuxm o>mmcmpxo m>wmcouxm +++ u u i one: unusaopm>mu mmwmmwu +++ :owuvmoaoc cmcmWP mmlmzm mcoc aucmsaopm>wo ommmmwu u+++ acowuvmonmu cwcmwp emanate: Ezcwcw52u=u .u Eacontmo .: Ezcwtoe=o=o .u Esconamo .: Eacwtm53uao .u Escontwo .u Espaoocw Eapzoocn EspaoocH tm>pupau mxoogm new; tween mxoosm new; Levee xoosm use; Lapin ; em nope—:uocH xpmpmrumeew swampauocfi uaozuw: noun-aoocm mmcwpuomm Lassauso cw acmEQopm>wc mmummvc use coauwmoaou cwcmvp co comumpzuocw o» Learn goosm new; c ya acumen use H asses 16 can prevent or delay the normal susceptibility of plants (2,3,11). Alternatively, if host defense is an active process, then heat shock will prevent or delay the normal resistance of plants (6,8,12). Because heat shock could block cultivar and nonhost resistance but not suscep- tibility to §_._ cucumerinum, it suggests that resistance to i carbonum and _C_._ cucumerinum in cucumber seedlings requires active host meta- bolism. Heat shock also prevented the deposition of fungal-induced lignin in cucumber cell walls. There was a strong association between lignifi- cation and disease resistance. The prevention and later recovery of host resistance after heat shock was always correlated with the preven- tion and later recovery of the hostfs ability to deposit lignin. This is consistent with earlier work that indicated cell wall lignification by cucumbers is an important active defense against fungi (5,7L. Heat shock has several uses as a tool to study plant-pathogen interactions. ‘Treatment of plants with a heat shock prior to inocu- lation can affect the success of the pathogen; this technique has been used with increasing frequency in studies of host plant responses to pathogen development in tissues (1,6,8). Chemical inhibitors of plant metabolism may have the same effect, but confusion can arise in inocu- lated tissues as to whether the effects seen are due to the action of the inhibitor on the host or on the pathogen. The study presented here shows unequivocally that one or more heat-sensitive structures or processes within cucumber seedlings are necessary for disease resistance. Heat shock does not simply kill treated tissues either because seedlings recover resistance and continue to grow. 17 Another use of heat shock that requires further study is the possible determination of active susceptibility in the plant. In the limited work reported, heat treatments have reduced the susceptibility of plants to fungi producing host-selective toxins (2,3,11), but have increased susceptibility to fungi not known to produce a host-selective toxin (6,8,12). Heat shock could provide a simple test of whether a toxin or a nontoxic compatibility factor (suppressor) is involved in a disease. More plant-pathogen systems need to be examined to see if this observation holds up. REFERENCES Aist, J. R. and Israel, H. N. (1977). Effects of heat-shock inhibition of papilla formation on compatible host penetration by two obligate parasites. Physiological Plant Pathology 10, 13-20. Bronson, C. R. and Scheffer, R. P. (1977).. Heat- and aging- induced tolerance of sorghum and oat tissues to host-selective toxins. Phytopathology 67, 1232-1238. Byther, R. S. and Steiner, G. H. (1975). Heat-induced resistance of sugarcane to Helminthosporium sacchari and helminthosporoside. Plant Physiology 56, 415-419. Hammerschmidt, R., Acres, S. and Kuc J. (1976). Protection of cucumber against Colletotrichum lagenarium and Cladosporium cucu- merinum. Phytopathology 66, 790-793. Hammerschmidt, R. Lamport, D. T. A. and Muldoon, E. P. (1984). Cell wall hydroxyproline enhancement and lignin deposition as an early event in the resistance of cucumber to Cladosporium cucu- merinum. Physiological Plant Pathology 24, 43-47. Hazen, B. E. and Bushnell, H. R. (1983). Inhibition of the hyper- sensitive reaction in barley to powdery mildew by heat shock and cytochalasin 8. Physiological Plant Pathology 23, 421-438. Hijivegen, T. (1963). Lignification, a possible mechanism of active resistance against pathogens. Netherlands Journal of Plant Pathology 69, 314-317. Jerome, S. M. R. and Muller, K. O. (1958). Studies on phyto- alexins. II. Influence of temperature on resistance of Phaseolus vulgaris towards Sclerotinia fruticola with reference to phyto- 18 10. 11. 12. 13. 14. I9 alexin output. Australian Journal of Biological Sciences 11, 301- 314. Johanson, D. A. (1940). Plant Microtechnique. McGraw-Hill, New York. Key, J. L., Lin, C. Y. and Chen, Y. M. (1981). Heat shock proteins of higher plants. Proceedings of the National Academy of Science. USA 78, 3526-3530. Otani, H., Nishimura, S. and Kohmoto, K. (1974). Nature of specific susceptibility to Alternaria kikuchiana in Nijisseski cultivar among Japanese pears. 111. Chemical and thermal protec- tion against effects of host-specific toxin. Annals of the Phyto- pathological Society of Japan 40, 59-66. Tomiyama, K. (1967). Further observations on the time requirement for hypersensitive cell death of potatoes infected by Phytophthora infestans and its relation to metabolic activity. Phytopatho- logische Zeitschrift 58, 367-378. Tuite, J. (1969). Plant Pathological Methods. Burgess Publi- shing, Minneapolis. Vance, C. P., Kirk, T. K. and Sherwood, R. T. (1980). Lignifi- cation as a mechanism of disease resistance. Annual Review of Phytopathology 18, 259-288. SECTION II HEAT SHOCK INDUCES RESISTANCE TO CLADOSPORIUM CUCUMERINUM AND ENHANCES PEROXIDASE ACTIVITY IN CUCUMBERS 20 ABSTRACT A brief heat shock induced resistance to the scab pathogen, 91399; sporium cucumerinum, in cucumber plants normally susceptible to the fungus. Ihnmersion of seedlings in a 50 C water bath for 40 or 50 seconds was found to be the optimal treatment for the induction of resistance. Plants inoculated with §;_cucumerinum as soon as 3 h after the heat shock exhibited increased resistance to the fungus; a 12 h interval from heat shock to inoculation allowed for development of maximunlresistance. The resistance was still fully effective when plants were inoculated 48 h after heat shock. All scab susceptible cultivars that were tested became more resistant to g, cucumerinum after heat shock. There was a direct correlation between the activity of soluble peroxidase induced by heat shock and the resistance induced by the same treatment. Heat shocked cucumbers had an increase in activity of the same isoperoxidases seen to increase in cucumbers with systemic resistance induced by prior Colletotrichum lagenarium inoculation. ‘The relationship of heat shock induced resistance to other stress responses and the role of peroxidases in induced resistance is discussed. 2] 22 INTRODUCTION Certain environmental and biological stresses of plants applied prior to challenge by a microorganism can often alter the outcome of subsequent host-parasite interactions. For example, preinoculation heat shock stress of plant tissue has been demonstrated to induce a state of susceptibility to fungi normally non-pathogenic on the shocked plant (6,14). 0n the other hand, stress caused by prior, limited infection of one leaf of cucumber plants with bacteria, fungi or viruses has been shown to induce systemic resistance against bacteria, fungi and viruses (17). In cucumber, the systemic induced resistance was associated with a systemic enhancement of peroxidase activity, enzymes which often exhibit increased activity after certain types of stress. Hhen heat shocked cucumber plants were inoculated immediately after the shock with a nonpathogen the plants were found to be temporarily susceptible to the nonpathogen (26, section 1 this thesis). Inoculation of other cucumber plants at 24 h after heat treatment demonstrated that resistance to the nonpathogen had returned (26, section 1 this thesis). Further studies found that when cucumbers susceptible to scab, incited by Q; cucumerinum, were inoculated with this fungus 24 h after heat shock the plants were also much more resistant to the pathogen. This paper reports the induction of resistance to _C_._ cucumerinum by heat shock and the association of enhanced peroxidase activity with the induced resistance. A preliminary report has been published (27). 23 MATERIALS AND METHODS Culture of plants andgpathogens Cultures of Cladosporium cucumerinum Ell. and Arth. and Colleto- trichum lagenarium race 1 (PassJ Ell. and Halst. were maintained on potato dextrose agar and V-8 agar, respectively, at 18 C in the dark. Conidial spore suspensions were prepared from 7 to IO-day-old cultures. Suspensions were filtered through 2 layers of cheesecloth and the spore concentration determined with a hemocytometer; For most experiments, cucumber (Cucumis sativus LJ plants were grown in the dark in 2 layers of rolled germination paper (10). Five days after sowing, the seedcoats were removed, and the seedlings were heat treated and replaced in the germination paper. In other experiments, cucumber plants were grown in vermiculite in a growth chamber (18 hr photoperiod, 20 C). The scab- susceptible cultivar "Marketer" was used unless stated otherwise. Heat shock treatments Seedlings were treated by dipping their cotyledons and hypocotyls in a water bath. Inoculations and disease ratings Seedlings were inoculated by spraying with a spore suspension of g; cucumerinum (3x105 spores ml'l) 24 h after the heat shock treatment unless stated otherwise. ‘The etiolated seedlings were rolled up again in the germination paper and incubated at 22 C. Light grown seedlings vvere inoculated and incubated as described (11). Individual plants were rated for disease by a method modified from Hammerschmidt et al. (10) 4 days after inoculation; O to 10, >10 to 30, >30 to 6D and >60% of the h)flaocotyl area covered by lesions was rated a O, l, 2 and 3, 24 respectively. Averages were based on 18 to 22 plants per treatment for each experiment. All experiments were replicated at least twice. Resistance-inducing inoculations were performed by infiltrating the first true leaf of green plants with a suspension of c_.1agenarium spores (1x105 spores ml’l) as previously described (13). Extraction and assay of solublegperoxidases Tissue extracts for peroxidase assays were prepared from the apical 2 cm of 20 hypocotyls (minus the cotyledons). The hypocotyl segments (which were frozen at -20 C until used) were homogenized in 2.0 ml of ice-cold 0.5M sucrose-0.01 M phosphate buffer (pH 6.0) and then centri- fuged (10,000 xg) for 20 minutes at 4 C (13). The clear supernatant was decanted and used for peroxidase determinations” Peroxidases were extracted from green plants as previously described (13). Protein content was estimated by the method of Bradford (3). Peroxidase activity was assayed using guaiacol as the hydrogen donor. The reaction mixture, consisting of 1.5 ml of guaiacol solution (0.56% in 0.1M phosphate buffer, pH 6.0) and 1.5 ml of peroxide solution (0.6% in distilled water), was added to a cuvette immediately prior to the addition of the enzyme extract (0.1 ml) (21). The reaction was followed colorimetrically at 470 nm. Enzyme preparations were diluted to give changes in absorbance of 0.1 to 0.2 absorbance units ml’l. Activity was expressed as the increase in absorbance at 470 nm min'lmg'1 protein. Electrophoretic separation ofJeroxidase isozymes Non-denaturing vertical slab gel electrophoresis was carried out using 10% polyacrylamide resolving gel (pH 8-8) and a 4% polyacrylamide 25 stacking gel of 1 mm thickness (16L. Samples to be analyzed were in sucrose-phosphate buffer and contained a trace of bromophenol blue dye. Electrophoresis was performed at 10 mA per slab gel. Peroxidase isozymes were detected by soaking the gels in o-dianisidine (1 mM in 0:1 M acetate buffer, pH 4.5) for 30 to 60 minutes. The gels then were rinsed in distilled water and placed in 0.60% peroxide to visualize the peroxidases. RESULTS Effect of the temperature and duration of heat shock on inducing resistance In general, the higher the temperature of the shock and the longer its duration at a given temperature the greater was the resistance induced against 9; cucumerinunt However, when the heat treatments were increased to the point where they irreparably damaged the plant, as determined by watersoaking of tissues (60 seconds at 50 C and 30 seconds or longer at 52.5 C) resistance was reduced or not induced (Fig. 1). A 50 C heat shock for 40 or 50 seconds was the optimal treatment of those tested for the induction of scab resistance (Fig. 2). ' Effect of inoculum level on heat shock induced resistance Protection against.§g cucumerinum induced by a 50 C shock for 40 seconds was evident at all spore concentrations used. Although inocu- lation at 3X106 spores ml'1 produced the most consistent observations of reduction in disease symptoms, the induced resistance was greater at lower inoculum levels (Fig. 3). 26 3b '21’500 :9 47.50 '22- m I H 50.00 4: LL] :2 I- . D 52.50 I I 1 l 20 30 40 50 60 DURATION OF HEAT SHOCK (secowos) Fig. 1. The effectiveness of different heat treatments in protecting cucumber seedlings against.§g_cucumerinum. Five day old etiolated seedlings were heat shocked in a water bath at different temperatures for various durations. The plants were challenged 24 h after heat shock with g, cucumerinum (3X105 spores ml'l) and incubated in germination paper in darkness. The seedlings were rated for disease 4 days after inoculation; 0 to 10, >10 to 30, >30 to 60 and >60% of the hypocotyl area covered by lesions was rated as 0, 1, 2 and 3, respectively. 27 Fig. 2. Protection of cucumber plants against g; cucumerinum by heat shock. The plants were shocked for 40 seconds at 50 C and challenged with g; cucumerinum (3X105 spores ml'l) 24 h later. A, dark grown seedlings 4 days after inoculation; B, plants grown in a lighted growth chamber at high relative humidity at 4 days after inoculation. 28 co <0 0 o l I I a I O T | l mu 0 I °/. DECREASE IN DISEASE POI (H .n. L” C) IC) (3 IC) 1* l T""‘T 3 I SXIOS 3x|05 3x|04 INOCULUM CONCENTRATION (sroaes PER wu Fig. 3. The effect of challenge inoculum concentration on resistance observed after heat shock. Resistance was induced by a 40 second shock at 50 C, and the _C_._cucumerinum challenge inoculation was 24 h after heat treatment. Disease ratings were made 4 days after the challenge as described'hiFig.1. 29 Time course for heat shock induced resistance By inoculation at various times after heat shock, resistance against g, cucumerinum was found to develop rapidly (Fig. 4). Plants inoculated as soon as 3 h after heat shock treatment demonstrated increased resistance to the fungus. Because there was a 12 to 18 h delay after inoculation before §_._ cucumerinum begins penetration of the plant, the actual onset of protection was probably 15 to 21 h after heat shock. A 12 h interval from heat shock treatment to inoculation allowed for development of maximum resistance. ‘This resistance was still fully effective when plants were inoculated 48 h after heat shock. Hhen inoculated after 48 h the unshocked control plants showed a gradual increase in resistance to the fungus. This resulted in a reduction in the disease rating when calculated as percent deerease compared to control. Heat shock induced resistance in different cucumber cultivars All of the scab susceptible cultivars that were tested became more resistant to g, cucumerinum after heat shock (Table 1). Effect of temperature and duration of heat shock on enhancement of peroxidase activity There was a direct correlation between peroxidase activity induced by heat shock and scab resistance induced by the same treatment (Fig. 5). The higher the temperature of the shock and the longer its duration the greater was peroxidase activity enhancement. As seen with induced scab resistance, temperature treatments which damaged the plants reduced the magnitude of the response. 3O IN DISEASE 45 C.’ 30- 20 ’ ID I “Yo DECREASE l I l I SIIZ 24 48 72 96 HOURS AFTER HEAT SHOCK Fig. 4. The effect of the time of challenge on resistance observed after heat shock. Resistance was induced by a 40 second shock at 50 C. Disease ratings were made 4 days after the g, cucumerinum challenge inoculation (3X105 spores ml'l). The disease ratings (explained in Fig. 1) were expressed as the percent decrease in disease rating of heat shocked plants when compared to unshocked control plants calculated by (1 - fig.) x 100. 3T Table 1 Resistance to Cladosporium cucumerinum induced in different cultivars of cucumber by preinoculation heat shock Treatmentof Disease rating of cultivarsa seedlings Marketer Shamrock Gemini Straight-8 Heat shockb 0.80:.23 0.51:.29 0.61i.34 0.84:.35 NO shock 2.32:.36 2.121.42 2.29i.42 2.17:.28 Disease reduction by heat shock 65.5% 75.9% 73.4% 61.3% aDisease ratings were made 4 days after challenge inoculation (3x105 spores ml'l) as described in Fig. 1. Values are means and standard errors. bEtiolated seedlings were heat shocked in a 50 C water bath for 40 seconds. 32 .2. - ' E 20- 50.00 _ E :x ' 47.50 T T? '5' ' 4/.45/.oc. I! 3 l/ , a 2 '0' 52.50 ‘3 1 l l I 20 30 40 50 50 DURATION OF HEAT SHOCK (SECONDS) Fig. 5. The enhancement of peroxidase activity after heat treatments at various temperatures and durations. Five day old dark grown cucumber seedlings were heat shocked and hypocotyl samples were taken 24 h after the shock. Peroxidase activity is expressed as change in absorbance min ‘lmg’1 protein using guaiacol as the hydrogen donor. 33 Time course for heat shock inducedgperoxidase activity Peroxidase activity increased rapidly by 24 h after heat shock (Fig. 6). The rise in enzyme activity peaked at 2 days after the shock and then decreased. The peroxidase activity in the unshocked control seedlings remained relatively constant over this same period. Separation of_peroxidase isozymes by electrophoresis Heat shocked cucumbers had an increase in activity of the fastest moving anodic isozymes when compared to control plants. The same iso- peroxidases exhibited enhanced activity in cucumber plants with systemic resistance induced by prior 9; lagenarium inoculation (Fig. 7) (13). DISCUSSION Preinoculation heat treatments of plants are known to alter the outcome of some host-parasite interactions. For example, susceptibility to fungi has been modified in many plants by a sudden rise in incubation temperature (heat shock) prior to inoculation. Heat shock pretreatment has increased the susceptibility of plants to fungal pathogens (32) and to nonpathogens (6,14). Heat shock inhibits plant defense responses to fungi (1,15,28). However, plants incubated at elevated temperatures can also become resistant to fungi that produce host-selective toxins. Loss of susceptibility to the toxin producing fungus has been associated with a loss of sensitivity to the toxin after heat treatment (4,5,19). The specific action of heat shock on disease resistance is unknown, although recent work has shown that heat shock causes the temporary inhibition of normal protein synthesis while increasing synthesis of a few ”heat shock proteins"(2,7,23). 34 I PROTEIN [\D O +4 AA470 MIN" M9' '5 I l l 1 l - 0 I2 24 48 72 96 HOURS AFTER HEAT SHOCK Fig. 6. Time course of peroxidase activity enhancement after heat shock. Dark grown cucumber seedlings were heat shocked 5 days after sowing and hypocotyl samples were taken 1 to 96 h after the shock. Peroxidase activity is expressed as change in absorbance min'lmg'1 protein using guaiacol as the hydrogen donor. Open circles represent values for heat shocked seedlings; solid circles represent values for unshocked seedlings. 35 ABCDEFG Fig. 7. Electrophoretic separation of anodic peroxidase isozymes from cucumber tissue with or without induced resistance to g; cucumerinum. For each sample 100 pg of protein was applied and electrophoresis was carried out with a 10% polyacrylamide slab gel (pH 8-8). Peroxidase isozymes were visualized with o-dianisidine as the hydrogen donor. Lane A, unshocked etiolated seedlings 6 days after sowing; B, etiolated seedlings 24 h after heat shock (40 sec at 50 C) and 6 days after sowing; C, unshocked etiolated seedlings 7 days after sowing; D, etio- lated seedlings 48 h after heat shock (40 sec at 50 C) and 7 days after sowing; E, leaf 2 of green plants; F, leaf 2 of green plants that had leaf 1 infiltrated with H20 7 days earlier; G leaf 2 of green plants that had resistance induced by infiltration of leaf 1 with Colleto- trichum lagenarium 7 days earlier. 36 A brief heat shock 24 h prior to inoculation induced a high level of resistance to _C_._ cucumerinum in normally susceptible cucumber seed- lings. .Associated with the heat shock induced resistance was enhanced peroxidase activity in the seedlings. ‘The optimal heat treatment for induction of peroxidase activity was identical to the treatment that resulted in the greatest protection against 9; cucumerinum. Moreover, for all of the different heat shock treatments tested there was close correlation between the soluble peroxidase activity extracted and the amount of resistance observed against 0. cucumerinum. The level of heat shock induced resistance seen depended on the inoculum concentration used in the challenge» Resistance resulting from heat shock was more effective at the lower inoculum levels. Earlier work showed a similar effect of inoculum concentration in cucumbers with resistance induced by g, lagenarium (21). Electrophoretic analysis of the peroxidase isozymes demonstrated that the same anodic isozymes are associated with resistance whether induced by heat shock or by prior 9; lagenarium inoculations. Previous evidence indicates that these fast moving acidic isozymes are associated with the plant's cell wall (13,25). Thus, similar mechanisms may be implicated in resistance induced by either method, and the mechanisms may involve changes in the cell wall. From the experiments described here it is not clear if the enhanced peroxidase activity is a cause or a consequence of heat shock induced resistance. Enhanced peroxidase activity has been associated with induced resistance in tobacco to tobacco mosaic virus (24,30). However, one study suggested that increases in peroxidase are not directly 37 involved in induced resistance against Pseudomonas solanacearum in tobacco (18%. Recent work with cucumbers has shown that induced resis- tance and increases in peroxidase activity are at least causally related (13L Associated with the acquired resistance of cucumber was increased levels of lignification after challenge with g, cucumerinum (11). In addition, increased levels of extensin, a plant cell wall hydroxyproline- rich glycoprotein, has been implicated in resistance to §g_cucumerinum (12). Peroxidase may be important in cucumber resistance because it is necessary for the crosslinking of extensin molecules (9) and also for the oxidative polymerization of hydroxycinnamyl alcohols to form lignin (29). Furthermore, peroxidase may contribute to induced resistance by increasing the concentration of fungitoxic free radicals formed from hydroxycinnamyl alcohols and other phenols. The mechanism by which heat shock enhances peroxidase activity and induces scab resistance is unknown. ‘The reduction of normal protein synthesis and the gg_ggyg_synthesis of heat shock proteins caused by nonlethal heat stress appears to protect the plant against the dele- terious effects of high temperatures (2). Recent research indicates that other types of stress also induce this response (8,22,31,32), suggesting that the heat shock response may be part of a more general response to stress that protects against cell damage. Perhaps disease resistance and peroxidase activity induced by heat shock are involved with this response. Disease resistance induced in cucumbers by heat shock has many similarities with resistance induced by C; lagenarium and other bio- logical agents. Heat shock may provide a good model to study the 38 mechanism of induced resistance as this system does not have the confounding effects of an inducing pathogen and can use very uniform plant tissue. Also, the heat shock induction of peroxidase activity may provide a good system to study enzyme regulation in plants. REFERENCES Aist, J. R. 8: Israel, H. H. (1977). Effects of heat-shock inhi- bition of papilla formation on compatible host penetration by two obligate parasites. Physiological Plant Pathology 10, 13-20. Altschuler, M. and Mascarenhas, J. P. (1982). Heat shock proteins and effects of heat shock in plants. Plant Molecular Biology 1, 103-115. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72, 248- 254. Bronson, C. R. & Scheffer, R. P. (1977). Heat- and aging-induced tolerance of sorghum and oat tissues to host-selective toxins. Phytopathology 67, 1232-1238. Byther, R. S. 8: Steiner, G. H. (1975). Heat-induced resistance of sugarcane to Helminthosporium sacchari and helminthosporoside. Plant Physiology 56, 415-419. Chamberlain, D. H. (1972). Heat-induced susceptibility to non- pathogens and cross-protection against Phytophthora megasperma var. sgjgg in soybean. Phytopathology 62, 645-646. Cooper, P. 8: Ho, T. D. (1983). Heat shock proteins in maize. Plant Physiology 71, 215-222. Dhinder, R. S. & Cleland, R. E. (1975). Water stress and protein synthesis. I. Differential inhibition of protein synthesis. Plant Physiology 55, 778-781. 39 10. 11. 12. 13. 14. 15. 16. 40 Fry, S. C. (1982). Isodityrosine, a new cross-linking amino acid from plant cell wall glycoprotein. Biochemistry Journal 204, 449- 455. Hammerschmidt, R., Acres, 5. & Ku’c, J. (1976). Protection of cucumber against Colletotrichum lagenarium and Cladosporium cucu- merinum. Phytopathology 66:790-793. Hammerschmidt, R. & Kuc, J. (1982). Lignification as a mechanism for induced systemic resistance in cucumber. Physiological Plant Pathology 20, 61-71. Hammerschmidt, R., Lamport, D. T. A. & Muldoon, E. P. (1984). Cell wall hydroxyproline enhancement and lignin deposition as an early event in the resistance of cucumber to Cladosporium cucu- merinum. Physiological Plant Pathology 24, 43-47. Hammerschmidt, R., Nuckles, E. M. & KIIc, J. (1982). Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiological Plant Pathology 20, 73-82. Heath, M. C. (1979). Effects of heat shock, actinomycin D, cyclo- hexamide and blasticidin S on nonhost interactions with rust fungi. Physiological Plant Pathology 15, 211-218. Jerome, S. M. R. & Muller, K. O. (1958). Studies on phytoalexins. II. Influence of temperature on resistance of Phaseolus vulgaris towards Sclerotinia fructicola with reference to phytoalexin out- put. Australian Journal of Biological Science 11, 301-314. Keleti, G. & Leder, H. H. (1974). Micromethods for the Biological Sciences. Van-Nostrand Rheinhold Co., New York. 166 pp. 17. 18. 19. 20. 21. 22. 23. 41 MC, J. (1982). The immunization of cucurbits against fungal, bacterial, and viral diseases. In Plant Infection, Ed. by Y. Asada, N. R. Bushnell, S. Ouchi and C. P. Vance, pp. 137-153. Japan Scientific Societies Press, Tokyo, and Springer-Verlag, New York. Nadolny, L. & Sequeira, L. (1983). Increases in peroxidase are not directly involved in induced resistance in tobacco. Physio- logical Plant Pathology 16, 1-8. Otani, FL, Nishimura, S. & Kohmoto, K. (1974). Nature of specific susceptibility to Alternaria kikuchiana in Nijisseiki cultivar among Japanese pears. III. Chemical and thermal protection against effect of host-specific toxin. Ann. Phytopathol. Soc. Jpn. 40, 59-66. Richmond, 5., K60, J. 8: Elliston, J. E. (1979). Penetration of cucumber leaves by Colletotrichum lagenarium is reduced in plants systemically protected by previous infection with the pathogen. Physiological Plant Pathology 14, 329-338. Ridge, I. & Osborne 0. A. (1970). Hydroxyproline and peroxidases in cell walls of Pisum sativum. Regulation by ethylene. Journal of Experimental Botany 21, 843-846. Sachs, M. H., Freeling, M. 8. Okimoto, R. (1980). The anaerobic proteins of maize. Cell 20, 761-767. Schoffl, F. & Key, J. L. (1982). An analysis of mRNAs for a group of heat shock proteins of soybean using cloned cDNAs. Journal of Molecular and Applied Genetics 1, 301-314. 24. 25. 26. 27. 28. 29. 30. 31. 32. 42 Simons, T. J. & Ross, A. F. (1970). Enhanced peroxidase activity associated with induction of resistance to tobacco mosaic virus in hypersensitive tobacco. Phytopathology 60, 383-384. Smith, J. A. & Hammerschmidt, R. (1984). Association of enhanced peroxidase activity with induced resistance of muskmelon and water- melon. Phytopathology 74, 874. Stermer, B. A. & Hammerschmidt, R. (1982). Effects of heat-shock on varietal and nonhost resistance in cucumbers. Phytopathology 72, 969. Stermer, B. A. & Hammerschmidt, R. (1983). Heat shock enhances peroxidase activity in cucumbers and induces resistance to glggg; sporium cucumerinum. Phytopathology 73, 818. ‘Tomiyama, K. (1967). Further observations on the time required for hypersensitive cell death of potatoes infected by Phytophthora infestans and its relation to metabolic activity. Phytopatho- logische Zeitschrift 58, 367-378. Vance, C. P., Kirk, T. K. & Sherwood, R. T. (1980). Lignification as a mechanism of disease resistance. Annual Review of Phytopatho- logy 18, 259-288. Van Loon, L. C. (1976). Systemic acquired resistance, peroxidase activity and lesion size in tobacco reacting hypersensitively to TMV. Physiological Plant Pathology 8, 231-242. Hebster, P.ln (1980K. “Stress" protein synthesis in pea root meristem cells? Plant Science Letters 20, 141-145. Yarwood, C. E. (1956). Heat-induced susceptibility of beans to some viruses and fungi. Phytopathology 46, 523-525. SECTION III HEAT SHOCK INCREASES THE SYNTHESIS OF ETHYLENE AND ENHANCES THE ACCUMULATION OF HYDROXYPROLINE-RICH GLYCOPROTEIN IN CUCUMBER SEEDLINGS 43 ABSTRACT Cucumber seedlings heat shocked for 40 seconds at 50 C developed increased resistance against the fungal pathogen Cladosporium cucu- merinum. By 6 h after the heat shock there were increases in the production ethylene and in its precursor l-aminocyclopropane-l- carboxylic acid. Heat shock also enhanced the accumulation of extensin, a hydroxyproline-rich glycoprotein found in the cell walls of the seed- lings. Inoculations of heat shocked seedlings with §g_cucumerinum 24 h after the shock resulted in further enhancement of extensin. Cell walls from heat shocked seedlings were more resistant to degradation by enzymes from £1 cucumerinum than were cell walls from unshocked seed- lings, but increased lignin deposition did not appear responsible. The accumulation of extensin after heat shock and its crosslinking by peroxidase is discussed as a possible mechanisms of resistance to C; cucumerinum. 44 45 INTRODUCTION The response of plants to heat shock has recently received much attention. Many studies have examined the cytological, biochemical and molecular events in plants occurring within an hour after the heat shock. These events include the halt of cytoplasmic streaming (8), suppression of plant defenses against disease (9), and the rapid production of "heat shock proteins“ (1). However, little is known about the long term response of plants to heat shock. One exception is the induction of disease resistance; 15 to 24 h after heat shock cucumber seedlings will develop resistance to the fungal pathogen Cladosporium cucumerinum (20L. Concomitant with the induction of disease resistance in cucumbers is a marked enhancement by heat shock in the activities of peroxidase isozymes located in cell walls (section II this thesis). Although the mechanism by which heat shock enhances peroxidase activity and also induces disease resistance is unknown, recent research indi- cates that peroxidase may play an important role in the induced resis- tance (section II this thesis). Cell wall modifications appear to be involved in the mechanism of resistance in cucumbers against 5;; cucumerinum. Hammerschmidt and We found that the epidermal cell walls of plants with systemic induced resistance were lignified more rapidly and to a greater extent than were controls in response to attack by g, cucumerinunI(5). Also, a recent study has shown that, in addition to enhanced lignification, an enhanced accumulation of bound extensin in cucumber cell walls was associated with cultivar resistance to g; cucumerinum (6). 'Thus, increased lignin 46 deposition and extensin accumulation may be involved in the resistance induced by heat shock. Ethylene is often produced by plants undergoing various types of stress, such as mechanical or radiation injury, infection by micro- organisms, or temperature deviations (21).* Heat shock may also induce the production of ethylene, but this has not been examined. Inter- estingly, among the many plant processes reported to be stimulated by ethylene are the enhancement of extensin accumulation and peroxidase activity (2,10,18). The purpose of this study is to examine some prolonged effects of heat shock on cucumber seedlings, and to inves- tigate the possible basis of disease resistance induced by heat shock. MATERIALS AND METHODS Cucumber seedlings Cucumis sativus L. cv Marketer) were used in all experiments unless stated otherwise. Seeds were germinated and grown for 5 days in darkness at 22 C in rolled germination paper (4). Heat shock and inoculation of seedlings The seedlings were given a heat shock by immersing their apexes and hypocotyls in a 50 C water bath for 40 seconds while holding on to the roots. Inoculation with the fungus Cladosporium cucumerinum Ell. and Arth. was by spraying seedlings with a spore suspension (3X106 spores per ml) 24 h after heat shock. g; cucumerinum is a pathogen of cv Marketer; .After treatment, the etiolated seedlings were rolled up again in the germination paper and incubated until harvest. 47 Measurement of ethylenegproduction Immediately after the heat shock four seedlings were placed in a 50 ml flask containing 2 ml of water; the flask was then sealed with a serum bottle cap and incubated in darkness at 22 C. Four replicate flasks were used for each treatment. At various time intervals 1.0 ml gas samples were removed from each flask with a tuberculin syringe, and the concentration of ethylene in the sample was determined by gas chromatography (12L. The accumulation of ethylene over time was measured and adjustments were made in calculations for ethylene removed during previous samplings. Estimation of 1-aminocyclopropane-I-carboxylic acid (ACC) levels The apical 2 cm of seedling hypocotyls (minus cotyledons) were excised and homogenized in 2.0 ml of buffer containing 100 mM K-Hepes (pH 8.0), 4 mM dithiothreitol and 0.4 mM pyrodoxal phosphate. The sample was then centrifuged at 12,000 RPM in a 85-34 rotor for 15 minutes at 4 C. The ACC levels in the supernatant were then estimated by determining the amount of ethylene produced from ACC after 13 yitgg_ conversion of OJIInl samples in the presence of alkaline sodium hypo- chlorite in a sealed test tube (14). The ethylene was determined by gas chromatography as described above. Estimation of bound extensin levels The levels of cell-wall-bound extensin, a hydroxyproline-rich glycoprotein, were estimated by determining the amount of hydroxyproline remaining in extracted cell walls. The apical 2 cm of hypocotyl tissues were excised and ground to a powder in liquid N2, then extracted with ca. 10 ml per gram fresh wt of the following: .015M Na-phosphate pH 64) 48 (once), 0.5M NaCl (twice), 1.0 M NaCl (twice), and distilled deionized water (five times). ‘The extracted cell walls were freeze-dried and 5 mg samples were hydrolyzed in 0.4 ml of 5.5M HCl at 110 C for 18 h. The hydrolysate was then reduced to dryness under nitrogen, resuspended in 0.5 ml of distilled deionized water, and 0.2 ml aliquots were removed for two determinations of hydroxyproline content by a spectrophotometric assay previously described (13). Histochemical staining for lignin The epidermis peeled from the apical 2 cm of a seedling's hypocotyl was stained with phloroglucinol-HCl (1% phloroglucinol in methanol/ concentrated HCl, 1:1) to visualize lignin deposition (11). Peels from at least four seedlings were examined with a light microscope for each observation. Enzymic degradation of cucumber cell walls Cell walls were prepared from the apical 2 cm of seedling hypo- cotyls (minus cotyledons) at 48 h after heat shock (40 seconds at 50 C). The excised tissues were ground with liquid NZ to a powder, extracted three times with ca. 10 ml per gram fresh wt of 50 mM phosphate (pH 6.0), and then freeze-dried. Cell wall degrading enzymes were produced by g; cucumerinum in liquid nutrient media containing 0.5% pectin and 0.5% polypectate or 150% cucumber cell walls as the carbon source (19). After six days on reciprocal shaker at 22 C the culture fluids were filtered through 4 layers of cheesecloth and centrifuged (12,000 g) for 15 minutes at 4 C. The filtrates were then dialyzed against water overnight. ‘The partially purified filtrates were stored in scintillation vials at -20 C. The 49 Macerozyme solution was prepared fresh before each experiment. A one mg per ml solution of the Macerozyme powder (Kinki Yakult Mfg. Co., Japan) was passed through a Sephadex G-50 desalting column to remove contami- nating reducing sugars. The enzymic degradation was carried out in a 12 ml conical centri- fuge tube containing 5 mg of cucumber cell walls, 0.25 ml of phosphate buffer (0.2M, pH 6.0) with .004% merthiolate, and 0.75 ml of enzyme preparation (16). The reaction mixture was incubated at 25 C for 16 h; then OLIInl aliquots were removed for estimation of the reducing sugars released (15). Values for control tubes containing only cell walls or only enzyme preparation were subtracted from the total amount of reducing sugars found. RESULTS Ethylenegproduction after heat shock A brief heat shock markedly increased the production of ethylene by cucumber seedlings. Ethylene accumulation was approximately two-fold higher by 6 h after heat shock with the shocked seedlings when compared to unshocked controls (Fig. 1%. The increased production of ethylene was evident for at least 15 h in heat shocked seedlings. ACC levels after heat shock ACC levels were also higher in heat shocked seedlings. ACC levels were approximately two-fold higher in heat shocked seedlings than in unshocked seedlings by 6 h after the shock (Table I). Assays for ACC synthase must be considered inconclusive due to the low amounts of 50 |.25— ; 3 _ LI. T c.» {30.75- _l C) IE E _— f N 00.25— l l l l l 3 6 9 l2 I5 25 HOURS AFTER HEAT SHOCK Fig. 1. Effect of heat shock on ethylene production by cucumber seed- lings. Seedlings were heat shocked (40 seconds at 50 C) and four seed- lings were placed in each 50 ml flask and sealed. One ml gas samples were removed at intervals and the ethylene concentration was determined by gas chromatography. Adjustments were made in subsequent measurements for the ethylene removed. Data represent the mean for two experiments each with four replicates per treatment. Open circles represent values for heat shocked seedlings; solid circles represent values for unshocked seedlings. 5T Table 1 Effect of heat shock on ACC levels in cucumber seedlings ACC (nmoles‘g‘1 fresh wt)a Hours after heat shock Treatment 3 6 Heat shockb 1.44 z .19 2.58 i .20 Unshocked control 1.26 i .11 1.20 i .22 aApical hypocotyl samples were excised at three or six hours after heat shock, homogenized, and centri- fuged. The supernatant was used for determination of levels of l-aminocyclopropane-l-carboxylic acid (data means::SE for two experiments). b40 seconds at 50 C. 52 activity extracted from tissues. However, preliminary results indicate heat shock increased levels of activity (data not shown). Accumulation of bound extensin after heat shock There was a marked increase in hydroxyproline content of extracted cell walls from heat shocked seedlings. Virtually all the hydroxy- proline in extracted cell walls is found in extensin. Thus, there was an increased accumulation of bound extensin in the seedling cell walls after heat shock. The amount of hydroxyproline increased in treated cell walls relative to control seedling cell walls during a 72 h period after the heat shock (Fig. 2). When heat shocked and unshocked seedlings were inoculated with the pathogen C_. cucumerinum the pattern of hydroxyproline accumulation was changed. The fungus caused a slight decrease in the rate of hydroxy- proline accumulation in both shocked and unshocked seedlings for up to 48 h after inoculation. However, between 72 and 96 h after inoculation there was a rapid rise in the hydroxyproline content of cell walls from both treatments. By 96 h after inoculation, the heat shocked seedlings, whether inoculated or not, had strikingly higher hydroxyproline levels than control seedlings (Fig. 2). The hydroxyproline content of the epidermis of seedlings also increased after heat shock. Assays of epidermal peels and of the underlying tissues indicated a comparable enhancement in both. Lignin deposition in heat shocked and unshocked seedlings Normally susceptible cucumber seedlings with heat-shock-induced resistance to g; cucumerinum showed only a slight increase in lignin deposition. In contrast, cucumber seedlings of the resistant cultivar 53 244.0 x0 3 _l _I 8 T 5’? m 3.0- z _I C) a: Q )- )< C) ‘5 2 0 >- ' _ 3: 0'3 3 IO" I I I J I 0 24 48 72 96 HOURS AFTER HEAT SHOCK Fig. 2. Time course for the accumulation of cell wall hydroxyproline after heat shock. Cucumber seedlings were heat shocked (40 seconds at 50 C) and some were inoculated (ARRON) 24 h later with Cladosporium cucumerinum (106 spores per ml). At one day intervals the apical 2 cm of hypocotyls were excised for analysis. The hydroxyproline content of cell walls was estimated colorimetrically after acid hydrolysis. The data represents the mean for three experiments. (H) unshocked, uninoculated; (On-O) unshocked, inoculated; (o-o) heat shocked, uninoculated; (Ono) heat shocked, inoculated. 54 showed considerable lignification after the challenge inoculation (Table 2). The heat shocked seedlings produced sporadic flecks of phloroglucinol-HCl positive material without a challenge inoculation, probably due to the formation of "wound lignin" after the shock. How- ever, increases in the amount of lignification seen in the normally susceptible seedlings after the inoculation were similar in heat shocked and unshocked seedlings. The presence of lignin in epidermal peels was confirmed by CuO oxidation. Analysis of the epidermal degradation products by thin layer chromatography showed a marked increase in p-hydroxybenzaldehyde from inoculated seedling of the resistant cultivar (SMR-58) compared to uninoculated seedlings at two days after inoculation. Oxidation of epidermal peels of the susceptible cultivar (Marketer) after inoculation with g; cucumerinum yielded very low levels of p-hydroxybenzaldehyde for either the heat shocked or the unshocked seedlings. This indicates there was no significant increase in fungal-induced epidermal lignification due to a heat shock 24 h prior to inoculation. Vanillin and syringalde- hyde were not detected in the oxidation products during these experi- ments. Enzymic degradation of cell walls from heat shocked and unshocked seedlings Cell walls from heat shocked seedlings were more resistant to degradation by'§;_cucumerinum culture filtrates than were cell walls from unshocked seedlings (Fig. 3). A 55% reduction in degradation of cell walls from heat shocked seedlings was seen when the cell walls were treated with culture filtrates produced by'§;_cucumerinum on the 55 .mcrcawum m>Pmcmuxm A++++V .mcpcwupm mementos f++v .ccwcmoum anus? A+V .ucpcvmpm oramcoam AI\+V .mcvcvmum o: AIV "upwom mcwumt Atatuwatmup=u asachmE:o:o .u spa: covpmpzoocw Leave mama mpmma Pastmnaam to mcpcwoum FuzI-ocwosrwdtopsa m-Pm: ppmo Postwupam tmaeaoao cw cowuwmoaou cmcmwp co comumpauoca ou copra ; em xuogm new; a mo uowwem N opnwh 56 I-' J... (I) O I I a. O l l N O l I nw0L GLUCOSE EQUIVALENTS - ML"'-HOUR" 01 o I l PECTIN-PECTATE CELL WALL MACEROZYME Fig. 3. Release of reducing sugars from cell walls of heat shocked and unshocked seedlings by wall degrading enzymes. Two days after the seedlings were heat shocked (40 seconds at 50 C) the apical 2 cm. of seedling hypocotyls (minus cotyledons) were ground to a powder with liquid N2. Five mg cell wall samples were incubated with one ml of buffered enzyme preparation for 16 h at 25 C. Aliquots (051 ml) were then assayed for reducing sugars. Bars on the left of a pair represent cell walls from unshocked controls; bars on the right represent cell walls from heat shocked seedlings (mean t SE). 57 pectin-polypectate medium. A 28% reduction in degradation of cell walls from heat shocked seedlings was seen using culture filtrates from the cell wall mediunt The Macerozyme preparation, however, degraded cell walls from heat shocked and unshocked seedlings at the same rate. DISCUSSION A brief heat shock stimulated the synthesis of ethylene in cucumber seedlings and increased the accumulation of bound extensin in their cell walls. Estimates of the amounts of ACC in cucumber tissues indicated that increases in this ethylene precursor was responsible for the increase in ethylene production. The rise in ethylene production and extensin levels was not limited to the few hours immediately'following heat shock; instead, the effect of heat shock on ethylene and extensin lasted for at least 24 h. This is the first report of the long term effects of heat shock on plant metabolism. The enhanced accumulation of extensin in the cell walls of heat shocked seedlings supports the importance of this glycoprotein in cucumber resistance to _C; cucumerinum. Accumulation of extensin in cucumber seedling cell walls is associated with resistance to C; gngg; merinum. Resistant cucumber cultivars exhibited an average 61% increase in cell-wall-bound extensin content at 48 h after inoculation with C; Cucumerinum, while susceptible cultivars showed only an average 8% increase in bound extensin content (6). In another study the enrichment of bound extensin in the cell wall was associated with resistance in melon plants against Colletotrichum lagenariunI(2). Moreover, in cucumber plants with systemic disease resistance induced by a localized 58 g; lagenarium infection, there is a systemic increase in bound extensin in the epidermis (Stermer and Hammerschmidt, unpublishedL The apparent lack of lignin deposition in seedlings with heat-shock-induced resis- tance focuses more attention on the possible role for extensin in this resistance. Cell wall alterations can contribute to disease resistance by forming a barrier that is more resistant to attack by the pathogen”s cell wall degrading enzymes (17). ‘The cell walls of heat shocked seed- lings were tested to see if resistance to enzymic degradation could be involved in heat-shock-induced resistance. Results demonstrated that the cell walls from heat shocked seedlings were markedly more resistant than cell walls from unshocked controls to degradation by crude enzyme preparations from g, cucumerinum. This data further strengthens the hypothesis that cell wall alterations are important in cucumber resis- tance to CL cucumerinum. The commercial enzyme preparation degraded cell walls from heat shocked and unshocked seedlings at the same rate. Apparently, Macerozyme contains enzymes that can cleave cell wall components which Czcucumerinum filtrates can not. A possible mechanism for the disease resistance induced by heat shock that is consistent with available data can now be proposed. This mechanism may also be important in the systemic resistance induced by localized infections. Increases in peroxidase activity is closely cor- related with the onset of induced resistance whether brought on by prior infection or by prior heat treatment (7, section II this thesis). Furthermore, the peroxidase activity induced by C; lagenarium infection or by heat shock is in the same cell-wall-associated isozymes (section 59 II this thesis). Crosslinking of extensin molecules in the cell wall could result from peroxidase activity, and might be a factor in resis- tance induced by heat shock. Recent work has shown that extensin is crosslinked in plant cell walls by the coupling of tyrosine residues, probably by peroxidase (3). Because the extensin content of cucumber cell walls increases before the onset of resistance in heat shocked seedlings, it may be the crosslinking of extensin by the later increase in peroxidase activity that is crucial for disease resistance. The greater crosslinking could be responsible for the resistance of cell walls from heat shocked seedlings to digestion by g; cucumerinum enzymes. The data suggest that perhaps a heat-shock-induced increase in ethylene production could stimulate the accumulation of extensin and peroxidase in the cell wall. The subsequent crosslinking of extensin in the cell wall by peroxidase may confer resistance to attack by pathogen. Heat shock could be a useful took in the study of induced resis- tance and other plant responses linked to stress. Further studies with heat shock should increase our knowledge of the regulation of ethylene, peroxidase and extensin, and illuminate their roles in disease resistance. REFERENCES Altschuler, M. and Mascarenhas, J. P. (1982). Heat shock proteins and effects of heat shock in plants. Plant Molecular Biology 1, 103-115. Esquerre-Tugaye, M. T., Lafitte, C., Mazau, 0., Toppan, A. and Touze, A. (1979). Cell surfaces in plant-microorganism inter- actions. 11. Evidence for the accumulation of hydroxyproline-rich glycoproteins in the cell wall of diseased plants as a defense mechanism. Plant Physiology 64, 320-326. Fry, S.(L (1982K Isodityrosine, a new cross-linking amino acid from plant cell wall glycoprotein. Biochemistry Journal 204, 449- 455. Hammerschmidt, R., Acres, S. and WC, J. (1976). Protection of cucumber against Colletotrichum lagenarium and Cladosporium cucu- merinum. Phytopathology 66, 790-793. Hammerschmidt, R. and Kfic J. (1982L. Lignification as a mechanism for induced systemic resistance in cucumber. Physiological Plant Pathology 20, 61-71. Hammerschmidt, R., Lamport, D. T. A. and Muldoon, E. P. (1984). Cell wall hydroxyproline enhancement and lignin deposition as an early event in the resistance of cucumber to Cladosporium cucu- merinum. Physiological Plant Pathology 24, 43-47. Hammerschmidt, R., Nuckles, E. M. and Ku’c, J. (1982). Association of enhanced peroxidase activity with induced systemic resistance of cucumber to Colletotrichum lagenarium. Physiological Plant Patho- logy 20, 73-82. 60 10. 11. 12. 13. 14. 15. 16. 17. 6I Hazen, B. E. and Bushnell, w. R. (1983). Inhibition of the hyper- sensitive reaction in barley to powdery mildew by heat shock and cytochalasin B. Physiological Plant Pathology 23, 421-438. Heath, M. C. (1979). Effects of heat shock, actinomycin D, cyclo- heximide and blasticidin S on nonhost interactions with rust fungi. Physiological Plant Pathology 15, 211-218. Imaseki, H. (1970). Induction of peroxidase activity by ethylene in sweet potato. Plant Physiology 46, 142-174. Johanson, 0. A. (1940). Plant Microtechnique. McGraw-Hill, New York. Kende, H. and Hanson, A. D. (1976). Relationship between ethylene evolution and senescence in morning-glory flower tissue. Plant Physiology 57, 523-527. Lamport, D. T. A. and Miller, D. A. (1971). Hydroxyproline arabi- nosides in the plant kingdom. Plant Physiology 48, 454-456. Lizada, C. C. and Yang, S. F. (1979). A simple and sensitive assay for 1-aminocyclopropane-I-carboxylic acid. Analytical Bio- chemistry 100, 140-145. Nelson, N. (1944). A photometric adaptation of the Somogyi method for the determination of glucose. Journal of Biological Chemistry 153, 375-380. Ride, J. P. (1980). The effect of induced lignification on the resistance of wheat cell walls to fungal degradation. Physio- logical Plant Pathology 16,.187-196. Ride, J. P. (1978). The role of cell wall alterations in resis- tance to fungi. Annals of Applied Biology 89, 302-306. 18. 19. 20. 21. 62 Ridge, I. and Osborne, 0. A. (1970). Hydroxyproline and peroxi- dases in cell walls of Pisum sativum. Regulation by ethylene. Journal of Experimental Botany 21, 843-846. Spalding, D. H. (1963). Production of pectinolytic and cellulo- lytic enzymes by Rhizopus stolonifer. Phytopathology 53, 929-931. Stermer, B. A. and Hammerschmidt, R. (1983). Heat shock enhances peroxidase activity in cucumbers and induces resistance to C_l_a_d_o_- sporium cucumerinum. Phytopathology 73, 818. Yang, S. F. and Pratt, H. K. (1978). The physiology of ethylene in wounded plant tissues. _I_n Biochemistry of Nounded Plant Tissues, Gunter Kahl, ed., N. de Gruyter, Berlin. p. 595-622. RECOMMENDATIONS Explore the potential use of heat shock as a simple method to deduce if a host-selective toxin is involved in a plant-pathogen interaction. Determine why seedlings inoculated immediately'after heat shock do not stop the fungus later when the seedling's resistance has appar- ently returned. Examine the levels of isodityrosine, the cross-linking amino acid of extensin, to see if levels are higher in cucumbers with heat-shock- induced or systemic induced resistance. Determine if the peroxidase isozymes associated with induced resistance are capable of cross-linking the tyrosine residues of extensin molecules in m. Pursue other possible roles for peroxidase in induced resistance, such as the cross-linking of cell-wall-esterified ferulic acids. Look for increases in chitinase activity in tissues with induced resistance. Resolve if exogenous ethylene or ACC treatments can induce resistance in cucumber seedlings. Look for changes in extensin, peroxidase, and isodityrosine levels after ethylene treatments of cucumber seedlings. Examine the systemic effects of heat shock in larger plants. 63 HICHIGQN ST TE U NIV. LIBRARIES IIIIIIIIIIIIHIIIIIIIIHI 0843668 A 9300 I IIIIIII 3