X E5" 3 ,3»’n/J3-511}H-x;1~1! .. .§ 5" }- ‘ ' I ...- —‘.—- I . . ., VIE“ 3W 134 ABSTRACT COMPARATIVE STUDY OF PHENOLIC COMPOUNDS IN CHERRY ROOTSTOCKS BY Kyung Sang Yu Sweet cherry trees grafted on Mahaleb rootstocks (g, mahaleb) show incompatibility symptoms after several years of growth in the orchard, while those grafted on Mazzard (g. ayium) do not show such symptoms. The cause of this incompatibility is not known. Several groups of biochemical compounds have been suggested as causal agents: cyanogenic glucosides, alkaloids, proteins, amino acids, and phenolic compounds. In this study, the phenolic compounds occurring in Mazzard and Mahaleb seedlings were evaluated and compared to determine their role in cherry graft incompatibilities. Total phenolic compounds of leaves, stem, and root bark were measured at two-week intervals during 5 months of active growth. The levels of phenolic compounds in these tissues increased during the first two months, then stabilized. Mahaleb root tissues, however, showed marked variation during this period. The two rootstocks differed Kyung Sang Yu significantly in total phenolic compounds in all tissues tested in that Mazzard tissues contained 1.5 to 3 times more phenolics than Mahaleb. Both Mazzard and Mahaleb had higher levels of total phenolic compounds in the bark than in the leaves. Greenhouse-grown cherry seedlings of both species contained markedly lower levels of total phenolics than those grown in the field. Methanol extracts and centrifugal sap of fresh tissues were examined for phenolic composition. The following compounds were identified on the basis of (1) color reactions, (2) Rf's in paper chromatography and thin layer chromatography, (3) retention times in gas chromatography, and (4) ultraviolet spectra. Com- pounds found in only one of the two species are under- lined. Mazzard Leaf . . . o-coumaric acid, p-coumaric acid, caffeic acid, chlorogenic acid, p-coumarylguinic acid, coumarin, kaempferol, guercetin, dihydrowogonin, d-catechin, epi-catechin. Stem bark . . . o-coumaric acid, pfcoumaric acid, coumarin, chlorogenic acid, p-coumarquuinic acid, dihydrowogonin, decatechin, epi- catechin,‘leucoanthocyanidin. Root bark . . . o-coumaric acid, p-coumaric acid, coumarin, chlorogenic acid, p-coumarquui- nic acid, dihydrowogonin, d-catechin, epi- catechin, leucoanthocyanidin. Sap . . . coumarin, dihydrowggonin. Kyung Sang Yu Mahaleb Leaf . . . o-coumaric acid, coumarin, herniarin, kaempferol. Stem bark . . . o-coumaric acid, coumarin, herniarin, d-catechin, epi-catechin, leucoanthocyanidin. Root bark . . . o-coumaric acid, coumarin, herniarin, d-catechin, epi-catechin, leucoanthocyanidin. Sap . . . coumarin, herniarin. The possible roles of these compounds in incom- patibility are discussed. COMPARATIVE STUDY OF PHENOLIC COMPOUNDS IN CHERRY ROOTSTOCKS BY Kyung Sang Yu A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1971 Dedicated to my father and mother ii AC KNOWLEDGME NT S The author wishes to express his sincere thanks to Dr. Robert F- Carlson for his assistance, guidance, and understanding throughout these studies. A special expression of gratitude goes to Dr. Frank G. Dennis for his suggestions and encouragement throughout the study and the final editing of the manuscript. Appreciation is also expressed to Drs. Alvin L. Kenworthy, Alan L. Jones, and Matthew J. Zabik for serv- ing on the guidance committee and for the critical review of the manuscript. Special appreciation is due to his wife, Youngsook, for her encouragement, patience, and sacrifice during the course of graduate study. iii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . Vii LIST OF FIGURES . . . . . . . . . . . . x INTRODUCTION . . . . . . . . . . . . . 1 REVIEW OF LITERATURE O I O O O O O O O O 3 I. Graft Incompatibility. . . . . . . . 3 A. Definition and types. . . . . . . 3 B. Incompatibility in cherry . . . . . 4 1. Symptoms . . . . . . . . . 4 2. Effect of scion cultivars. . . . 5 3. Environmental effects . . . . . 6 4. Reciprocal grafts . . . . . . 8 C. Causes of incompatibility . . . . . 8 1. Anatomical. . . . . . . . . 8 2. Role of viruses . . . . . . . ll 3. Biochemical . . . . . . . . 14 a. Evidence . . . . . . . . 14 b. Specific compounds. . . . . 16 alkaloids. . . . . . . . 16 amino acids . . . . . . . 17 proteins . . . . . . . . l7 cyanogenic compounds . . . . 18 phenolic compounds. . . . . 18 II. Role of Phenolic Compounds in Graft Incom- patibility. O O I O O O I C O O 18 A. Physiological functions of phenolic compounds and seasonal variations. . 18 iv Page B. Evidence for the role of phenolic com- pounds in incompatibility . . . . . 21 C. Evidence for translocation '. . . . . 23 D. Possible mechanism of action in incom- patibility I I I I I I I I I I 25 III. Phenolic Compounds Identified in Cherry Tissues I I I I I I I I I I I I 29 MATERIALS AND METHODS. . . . . . . . . . . 34 I. Seasonal Variations of Total Phenolic Com- pounds I I I I I I I I I I I I 34 A. Plant material . . . . . . . . . 34 B. Extraction. . . . . . . . . . . 34 C. Quantitative determination . . . . . 35 II. Identification of Phenolic Compounds in Methanol Extracts of Greenhouse-grown Cherry Seedlings and Determination of Relative Amounts . . . . . . . . . 35 AI Plant material I I I I I I I I I 35 B. Extraction. . . . . . . . . . . 36 CI HYdrOIYSiSo I I I I I I I I I 38 D. Purification and identification. . . . 38 1. Paper chromatography . . . . . . 38 2. Thin layer chromatography . . . . 41 3. Gas liquid chromatography . . . . 42 4. Ultraviolet absorption spectra. . . 43 5. Sources of reference compounds. . . 43 E. Quantitative determination . . . . . 43 III. Identification and Relative Amounts of Phe- nolic Compounds in Centrifugal Sap . . . 45 A. Plant material . . . . . . . . . 45 B. Extraction. . . . . . . . . 45 C. Fractionation of. extract . . . . . . 46 Page RESULTS AND DISCUSSION . . . . . . . . . . 48 I. Seasonal Variations of Total Phenolic Com- pounds in Field-Grown Cherry Seedlings. . 48 II. Identification of Phenolic Compounds in Methanol Extracts of Greenhouse-Grown Cherry Seedlings and Determination of Relative Amounts . . . . . . . . . 52 A. Total phenolic compounds. . . . 52 B. Paper chromatography of hydrolyzed leaf extracts . . . . . . . . 54 C. Paper chromatography of hydrolyzed stem bark extracts. . . . . . . 66 D. Paper chromatography of hydrolyzed cherry root bark extracts. . . 72 E. Gas liquid chromatography of hydrolyzed phenolic compounds . . . . . . . 72 III. Identification and Relative Amounts of Phe- nolic Compounds in Centrifugal Sap . . . 87 A. Ether fraction . . . . . . . . . 87 B. Neutral and acid ethyl acetate fractions 95 IV. Unhydrolyzed Phenolic Compounds in the.Leaf, Stem Bark and Root Bark of Mazzard and Mahaleb Seedlings. . . . . . . . . 101 A. Stem bark extracts. . . . . . . . 101 B. Root bark extracts. . . . . . . . 108 C. Leaf extracts . . . . . . . . . lll DISCUSSION I I I I I I I I I I I I I I 118 SUMMARY AND CONCLUSIONS . . . . . . . . . 124 SUGGESTIONS FOR FURTHER STUDY OF GRAFT RELATION- SHIPS IN CHERRY CLONES . . . . . . . . . 126 LITERATURE CITED . . . . . . . . . . . . 128 vi 10. LIST OF TABLES Phenolic compounds occurring in cherry trees Weights of tissues used for extraction of phenolic compounds. . . . . . . . . Reagents used for phenolic compounds. . . Sources of reference compounds. . . . . Total phenolic compounds in Mazzard and Mahaleb tissues from greenhouse-grown cherry seedlings I I I I I I I I I I I I Color reactions of phenolic compounds extracted from Mazzard and Mahaleb leaves, hydrolyzed, and purified by paper chroma- tography . . . . . . . . . . . . Rf values of phenolic compounds extracted from Mazzard and Mahaleb leaves, hydrolyzed, and separated by two-dimensional paper chroma- tography. The compounds were rechromato- graphed together with reference compounds in six different solvent systems . . . . Rf values of phenolic compounds extracted from Mazzard and Mahaleb leaves, hydrolyzed, and separated by two-dimensional paper chromatography . . . . . . . . . . Spectral characteristics of partially puri- fied, hydrolyzed compounds from Mazzard and Mahaleb leaves, compared with those of ref- erence compounds . . . . . . . . . Relative amounts of phenolic compounds in Mazzard and Mahaleb leaves based on spot size and color intensity on two—dimensional paper chromatograms after application of FeCl3-K3Fe(CN)6 reagent . . . . . . . vii Page 33 36 40 44 53 57 58 59 60 62 Table Page 11. Color reactions of phenolic compounds extracted from Mazzard and Mahaleb stem bark, hydrolyzed and chromatographed on a two-dimensional paper chromatogram . . . . 69 12. Rf values and relative concentrations of phenolic compounds extracted from Mazzard and Mahaleb stem bark, hydrolyzed and chroma- tographed on a two—dimensional paper chroma- togram . . . . . . . . . . . . . . 70 13. Color characteristics of phenolic compounds extracted from root bark of Mazzard and Mahaleb, hydrolyzed and chromatographed on a two-dimensional paper chromatogram. . . . . 75 14. Rf values and relative concentrations of phe- nolic compounds extracted from Mazzard and Mahaleb root bark, hydrolyzed and chroma- tographed on two-dimensional paper chroma- tograms. . . . . . . . . . . . . . 76 15. Retention times and concentrations (mg/g fresh weight) of phenolic compounds extracted from leaves, stem bark, and root bark of Mazzard and Mahaleb seedlings as determined by gas liquid chromatography . . . . . . '. . . 86 16. Comparison of hydrolyzed phenolic compounds in extracts of leaf, stem bark, and root bark of Mazzard and Mahaleb seedlings . . . . . . 88 17. Color reactions of phenolic compounds in the ether fraction of Mazzard and Mahaleb sap extract. The compounds were chromatographed on two-dimensional paper chromatograms and sprayed with phenolic reagents . . . . . . 92 18. Rf values of phenolic compounds in the ether fraction of Mazzard and Mahaleb sap extracts on a two-dimensional paper chromatogram . . . 93 19. Retention times and concentrations of coumarins extracted from centrifugal sap of Mazzard and Mahaleb as determined by gas liquid chroma- tography . . . . . . . . . . . . . 94 viii Table Page 20. Color reactions of phenolic compounds occur- ring in neutral and acidic ethyl acetate fractions of Mazzard and Mahaleb sap. Com- pounds that occurred in the ether fraction were omitted . . . . . . . . . . . . 98 21. Comparison of phenolic compounds in Mazzard and Mahaleb sap extract . . . . . . . . 102 22. Color reactions of unhydrolyzed phenolic com- pounds occurring in Mazzard and Mahaleb root and stem bark extracts . . . . . . . . 105 23. Rf values of unhydrolyzed phenolic compounds occurring in extracts of Mazzard and Mahaleb root and stem bark. Compounds were chroma- tographed on a two-dimensional paper chroma- togram . . . . . . . . . . . . . . 106 24. Color reactions and Rf values of unhydrolyzed phenolic compounds occurring in Mazzard and Mahaleb leaf extracts. Spots occurring in stem bark were omitted . . . . . . . . 114 25. Comparison of unhydrolyzed phenolic compounds in extracts of leaf, stem bark, and root bark of Mazzard and Mahaleb seedlings . . . . . 115 26. Comparison of known phenolic compounds in extracts of leaf, stem bark, and root bark of Mazzard and Mahaleb seedlings . . . . . . 117 ix LIST OF FIGURES Figure l. 10. 11. Procedure for extraction phenolic compounds . . Procedure for extraction centrifugal sap . . . and purification of and purification of Seasonal variations of total phenolic compounds in field-grown cherry seedlings. . . . . . Paper chromatograms of hydrolyzed pounds extracted from Mazzard and leaves . . . . . . Paper chromatograms of hydrolyzed pounds extracted from Mazzard and stem bark . . . . . Paper chromatograms of hydrolyzed pounds extracted from Mazzard and barkI I I I I I I phenolic com- Mahaleb phenolic com- Mahaleb phenolic com- Mahaleb root Gas liquid chromatogram of a mixture of stan- dard phenolic compounds Gas liquid chromatograms compounds extracted from leaves . . . . . . Gas liquid chromatograms compounds extracted from stem bark . . . . . Gas liquid chromatograms compounds extracted from root bark . . . . . of hydrolyzed phenolic Mazzard and Mahaleb of hydrolyzed phenolic Mazzard and Mahaleb of hydrolyzed phenolic Mazzard and Mahaleb Paper chromatograms of phenolic compounds in the ether fraction of Mazzard and Mahaleb sap extracts . . . . . Page 37 47 50 56 68 74 79 81 83 85 90 Figure Page 12. Paper chromatograms of phenolic compounds in the neutral fraction of Mazzard and Mahaleb sap extracts . . . . . . -, . . . . . 97 13. Paper chromatograms of phenolic compounds in the acid fraction of Mazzard and Mahaleb sap extracts . . . . . . . . . . . . . 100 14. Paper chromatograms of unhydrolyzed phenolic compounds in Mazzard and Mahaleb stem bark . . 104 15. Paper chromatograms of unhydrolyzed phenolic compounds in Mazzard and Mahaleb root bark . . 110 16. Paper chromatograms of unhydrolyzed phenolic compounds in Mazzard and Mahaleb leaf tissues . 113 xi INTRODUCTION Mahaleb (Prunus mahaleb) and Mazzard (P. avium) are the two rootstocks generally used for sweet cherry (a. azigm) cultivars. In the nursery Mahaleb seedlings are vigorous, uniform, and easy to bud. Sweet cherry cultivars budded on Mahaleb grow well in the nursery and for the first 4 to 6 years in the orchard. When the trees come into production, however, many show graft-incompati- bility. Cultivars budded on Mazzard do not show this abnormality (Argles, 1937). Several groups of compounds have been suggested as the causal agents of graft-incompatibilities including cyanogenic glucosides (Gur, 1968b), alkaloids (Mothes, 1955), proteins (Crane, 1945), amino acids (Tschiersch, 1963), and phenolic compounds (Thiel, 1954; Gur, 1968b). Phenolic compounds are ubiquitous and yet specific in higher plants. Many phenolic compounds are phytotoxic (Pridham, 1960), and their inhibitory roles have been shown in germination (Hemberg, 1961; Evenari, 1961), shoot and root growth (Hemberg, 1961; Goodwin and Taves, 1950). The compounds also have been implicated in growth regulation via the IAA-oxidase system (Galston, 1969). Preliminary study showed that Mahaleb and Mazzard root- stocks differed in their phenolic composition. The pos- sible role of phenolics as causal agents of graft- incompatibility prompted us to make a detailed comparison of the compounds in Mazzard and Mahaleb seedlings. REVIEW OF LITERATURE I. Graft Incompatibility A. Definition and types Graft-incompatibility may be defined as an inherent antagonism or discordant association between certain stocks and scions (Amos, 1936). It can be due to either failure of graft unions or factors other than graft unions (Luckwill, 1962). Two types are generally recognized, which Mosse (1962) has termed localized vs. translocated incompati- bility. The first type requires direct contaCt between the incompatible tissues. Reciprocal grafts of these are also incompatible. Use of compatible interstems eliminates the incompatibility. The second type does not require direct tissue contact, but occurs despite the presence of an intermediate stem piece. Reciprocal grafts may or may not exhibit incompatibility. Examples of "localized" incompatibility are the pear variety C8 on quince A (Mosse and Herrero, 1951) and Oullin's Gage plum on Myrobalan B (Herrero, 1951). Examples of "trans- located" incompatibility include Hale's Early peach on Myrobalan B (Mosse, 1955) and lemon on sour orange (Cala- van gt_31., 1951). The incompatibility reaction may be immediate, in which case the graft union either does not form or is short lived as in Cox's Orange Pippin on Malgs theifera (Luckwill, 1962), and ornamental cherries (P, serrulata) on Mahaleb (Amos et al., 1936). Alter- natively, it may be delayed until the plant is several years old, as is the case in certain sweet cherry clones budded on Mahaleb (Grubb, 1938). In fruit trees, decline often occurs when the trees begin to fruit (Garavel, 1954; Jimenez, 1957). B. Incompatibility in cherry 1. Symptoms. The two principal rootstocks for sweet cherry cultivars are wild seedling types of Prunus azigm_(Mazzard) and P. mahaleb (Mahaleb). Mahaleb seed- lings, which are easily budded and which produce vigorous trees in the nursery, have shown symptoms of incompati- bility with sweet cherry cultivars (Grubb, 1938; Simons and Carlson, 1968). The symptoms generally occur only after 8 to 10 years. This fact, together with differences in response in different climates, has raised questions as to the true nature of this incompatibility. However, observations on growth rates and tree losses in many countries confirm that sweet cherry cultivars grafted on Mahaleb are generally shorter lived than those grafted on Mazzard (Argles, 1937; Garner, 1967; Raptopoulus gt_gl., 1959; Hilkenbaumer, 1952; Howe, 1927; Quinn, 1935). Symptoms include constriction of the stock immediately below the graft union, premature defoliation, and a decline in vigor. Starch accumulation at the union is a characteristic symptom of incompatibility. However, vascular connections between stock and scion appear to be normal (Herrero, 1951). 2. Effect of scion cultivars. Cultivar effects on graft-incompatibility have not been carefully studied in sweet cherry/Mahaleb combinations. Symptoms do occur in many sweet cherry cultivars budded on Mahaleb. Trees often grow vigorously for a few years, then decline or die in a 5- to 24-year period (Garner, 1967; Howe, 1927; Fowler, 1933; Amos §t_§1,, 1936). In Michigan, however, Carlson (unpublished data) observed the different responses among sweet cherry cultivars on Mahaleb. Cer- tain cultivars such as "Emperor Francis," "Hedelfingen," and "Stark's Black Hardy Giant" frequently show incompati- bility symptoms, whereas cultivars such as "Vista," "Schmidt," and "Windsor" appear to be more compatible with Mahaleb. Philp (1930) reported a case of complete incompatibility among cherry cultivars. In California the sweet cherry 'Chapman' showed lack of affinity with the sour cherry 'Stockton Morello' but this could be overcome by double working. Ornamental cherry trees also showed incompatibility on clonal selections of Mahaleb (Amos gt_al., 1936). Trees on the Mazzard selections grew vigorously but varieties budded on Mahaleb died by the end of the second season. At East Malling, Herrero (1951) noted that trees of the sweet cherry cultivar 'Frogmore' budded on rootstock F250 (sour cherry selection) were dying in four years. No cause could be found, so death was considered due to incompati- bility. Grubb (1938) reported three cultivars 'Early Rivers,‘ 'Governor W00d,‘ and 'Waterloo,‘ incompatible with Mahaleb selections, F6/1/2, F8/1/10, and F8/1/12. 3. Environmental effects. Cherry trees are rather exacting with regard to climatic and soil requirements, growing best on light, moist, well-drained loams. Most of the commercial production in the United States is con- fined to the Great Lakes area and the Pacific Coast where moderate temperatures and humidity occur (Westwood, 1966). The behavior of sweet cherries on Mahaleb seems to be closely related to environment. Failure of trees budded on Mahaleb has been reported in England (Garner, 1967), New York (Howe, 1927), Tennessee (McClintock, 1930), Virginia (Anthony et_al., 1937), Michigan (Simons and Carlson, 1968), Canada (Anonymous, 1945), Germany (Hilken- baumer, 1952), Greece (Raptopoulos, 1959), and South Australia (Quinn, 1935). Such trees are short lived and unproductive, whereas trees budded on Mazzard are vigorous, productive, and long lived. However, trees have been successfully grown on Mahaleb in Utah (Coe, 1945) and California (Philp, 1930). In Utah, Coe (1945) found that Mahaleb was definitely superior to Mazzard. In California some growers preferred Mazzard, others Mahaleb (Philp, 1930). Also in California, Brooks (1950) recognized that the best rootstocks for sweet cherries varied according to soil and climatic conditions. There, the percentages of rootstocks used commercially for sweet cherries were Mazzard, 65%; Mahaleb, 30%; and Stockton Morello, 5%. These striking differences might be related to environmental factors. Soil drainage in particular seemed to be related to tree losses. In Utah, where the orchard soil was a coarse gravelly loam with good drainage and aeration, trees on Mahaleb outgrew and out- produced those on Mazzard (Coe, 1945). Upshall e£_31. (1950) believed that poor soil drainage was the main cause of tree loss. In the Niagara district of Ontario, the authors found that most tree losses (sweet cherry/ Mahaleb) occurred in orchards with poorly drained soils, whereas in well-drained soils, trees on Mahaleb out- yielded and grew more than those on Mazzard. Although the basic causes of graft-incompatibility are genetic, symptom expression may be governed by environmental factors (Chang, 1937). Soil drainage conditions and other environmental factors may thus accelerate or delay expression of incompatibility in sweet cherry trees on Mahaleb. Possibly there is no true incompatibility between these two species; trees may fail simply because Mahaleb cannot tolerate poor soil drainage. In the same area, however, sour cherry (Prunus cerasus) trees tolerate poor drainage conditions better than sweet cherries. Therefore an inherent factor in sweet cherry accelerates tree decline. 4. Reciprocal grafts. There are a few reports on reciprocal grafting of sweet cherry on Mahaleb. Hedrick (1914) reported that reciprocal grafts of Mahaleb on any cultivated sweet cherry failed at Geneva, New York. How- ever, Cummings g£_21. (1933) found that Mazzard actually grew better on Mahaleb stock than on its own roots, whereas Mahaleb grew well on both its own roots and on Mazzard during ten years' trial in Vermont. C. Causes of Incompatibility 1. Anatomical. The underlying causes of graft incompatibility are unknown (Mosse, 1962). Special attention has been paid to anatomical studies on the assumption that the graft union is a mechanical barrier to translocation. Chang (1937), from his studies of compatible and incompatible combinations of pears, plums, peaches, and cherries, noted that incompatible unions reduced the flow of dyes and water. Starch accumulation occurred at the graft union in incompatible combinations (e.g., 'Durondeau' pear/Quince F). Starch depletion was also noted in the roots of the incompatible combinations Hale's Early peach/Myrobalan B and 'Frogmore' cherry/F250 sour cherry (Herrero, 1951). In simulated graft union studies using grafting, banding, and scoring techniques, sweet cherry "Napoleon" on Mahaleb was found to contain higher levels of starch than those on Mazzard (Carlson and Yu, 1969). McClintock (1948), from his study of incompatibility between peaches and Mariana plum found that the roots died first and that the phloem tissues of the plum stock and the peach scion failed to unite. He concluded that this mechanical obstruction at the union was caused by a lack of translocation to the roots lead- ing to their death. Nitrogenous materials were reported to also accumulate at the graft union in incompatible combinations. This interference with translocation appears to be due to structural defects at the graft union, including one or more of the following: 1. A layer of parenchymatous tissue at the line of union as in 'President' on common plum and some pears on quince rootstocks (Chang, 1937; Proeb- sting, 1926) rather than normal differentiated tissue. 10 2. Distortion of vascular tissue at the line of union in pear/quince (Herrero, 1951; Proebsting, 1926). 3. Gummy masses at the union in certain stone fruits (Proebsting, 1928). 4. A cork layer at the union as in 'Reeve's' peach on Myrobalan B (Herrero, 1951). The initial reaction that leads to vascular dis- continuity appears to be related to cambial activity at the point of union. Herrero (1951) noted that primary causes of discontinuity were necrosis of some cells in the cambial region in pear/quince and a slowing down of cambial activity at the line of union in plums. Mosse and Scaramuzzi (1956), working with pear/quince grafts, found that the first necrotic symptoms appeared, not in the cambium, but in l- to 2-year-old phloem tissues. The necro- sis then spread to ray and to cambium cells, breaking vas- cular continuity in the union. The cause of the degener- ation of phloem tissues was not clear. However, the fact is noteworthy that these abnormalities occurred most often at the end of the growing season, when the maximum trans- location of metabolites was downward. Herrero (1951) and Mosse and Scaramuzzi (1956), from their anatomical studies, considered that the causes leading to necrosis and breaks in cambial continuity were biochemical rather than anatomical, and were related to seasonal metabolic changes in the tree. 11 Histological differences between two incompatible tissues were suggested as a causal factor in tree decline, but Herrero (1951) could not correlate such differences, such as relative amounts of vessels, fibers, parenchyma cells and rays, or the size of these elements with the degree of compatibility. Chang (1937) suggested that different growth charac- teristics of the stock and scion were a cause of incompati- bility. His work suggested that differences in periods of cambial activity, seasonal growth patterns, and growth rates between stock and scion might be responsible. Herrero (1951), however, believed that these charac- teristics were not directly associated with the primary causes of incompatibility. 2. Role of viruses and mycgplasma. The presence of latent viruses or virus complexes in stone fruits has been demonstrated by Milbrath et_al. (1945). Several cases have been reported in which viruses were responsible for incompatibility. Toxopeus (1936) noted that sweet orange (Citrus sinensis) failed on sour orange (Citrus aurantium). Interestingly, failure was limited to Java and South Africa; the same combination was successfully grown elsewhere. Later, the diseases called 'tristeza,‘ or 'quick decline' was noted in other localities in con- junction with sour orange rootstock. Subsequent study 12 showed that viruses were responsible for these diseases and for graft failure (Fawcett gt_al., 1946; Bitters et_al,, 1953). A similar case was reported in apples. The clonal stock Spy 227 proved to be incompatible with certain varieties of apples (Shaw and Southwick, 1944). Gardner §E_§1. (1946) pointed out that there was a similarity between this phenomenon and tristeza disease of citrus. Weeks (1948) demonstrated that the toxic principle was viral in nature. Symptoms of x-disease (buckskin) in sweet cherry on Mahaleb are similar to incompatibility symptoms. Trees infected with this disease, which is caused by a mycoplasm, develop necrotic tissues at the union and distal portions of roots. The trees usually die quickly in late summer after infection. Sweet cherry trees on Mazzard infected with x-disease decline 4 or 5 years before they die. Tissues at the graft union usually remain normal until the tree dies (Granett and Gilmer, 1971; Parker et_§1., 1963). Mosse (1962) has pointed out the similarities between the symptoms of virus diseases and symptoms of incompati- bility. The "translocated" type of graft incompatibility, in particular, has many features in common with virus- induced graft-incompatibility including: 1. Absence of mechanical weakness of the union; 2. Phloem degeneration; 13 3. Accumulation of starch above, and its absence below, the union; 4. Reduced bud-take; 5. Variability of one-year-old trees; 6. Blistering stem tissues. However, all "translocated" incompatibilities do not appear to be due to virus. In the case of the incompatible combination, 'Hale's Early' peach on Myrobalan B, where the evidence is strong that trans- locatable factors are responsible, the virus did not seem to be a causal factor. Herrero (1951) tested the possibility of virus infection by grafting scions from incompatible trees to trees of the compatible combination Hale's Early/Brompton. The test was negative.‘ A similar case was found in incompatible trees of Victoria/President/ Myrobalan B. Normally compatible President/Myrobalan B trees can be made incompatible by topworking with Victoria. However, buds taken from these incompatible trees were fully compatible when budded directly on the Myrobalan B rootstock (Mosse, 1961). These transmission studies do not rule out entirely the possibility that some undetected viruses are responsible. If a virus is involved, the specific requirements for certain grafting combinations are difficult to explain. l4 3. Biochemical. Anatomical investigation of the graft union of incompatible stock-scion combinations has revealed several facts (Herrero, 1951; Mosse, 1955, 1962; Mosse and Scaramuzzi, 1956). First, cambial breaks occur suddenly at the end of season. The reaction appears to be correlated with the time of maximum translocation of metabolites from the top. Histological characters such as the size and amount of vessels, fibers, parenchyma cells, and the width of ray initials and distance between rays are not critical factors. The abrupt change in starch content was often noted in the adjacent tissues at the union of incompatible combinations, even if there were no apparent structural defects at the union (Mosse, 1962). These observations suggest that the causes of graft-incompatibility are biochemical rather than anatomical. Thiel (1954) agreed that the underlying causes of pear/quince incompatibility were probably biochemical in nature. Robitaille and Carlson (1970) came to the same conclusion in their study of graft union behavior of certain Malus and Prunus species. a. Evidence. De Stigter (1956) studied graft- incompatibility using the graft combination, muskmelon (Cucumis melo), and malabar gourd (Cucurbita ficifolia). The combination of muskmelon as scion and malabar gourd as stock failed completely when the stock was defoliated. However, when stock leaves were left intact, the graft 15 union was strong and phloem degeneration did not occur. In a further study, he double-worked the compatible com- bination cucumber (Cucumis sativus L.) on gourd (Cucurbita ficifolia) using different lengths of melon stem pieces as interstocks. A long intermediate stem caused incompati- bility, but when the intermediate was extremely short, the combination remained healthy. The author concluded that either the muskmelon lacks specific substances for malabar gourd or the composition of metabolites was changed during transport through melon tissue. These changes were assumed to be minimized when the intermediate was very short. Calavan g£_31. (1951) showed that the graft com- bination of lemon/sour orange required the presence of sour orange leaves to overcome incompatibility. Mosse's work (1960a, 1955) with plum and peach suggests that graft transmissible factors or toxins interfere with normal growth. She induced incompatibility between two normally compatible clones by top working with another variety. For example, the compatible com— bination 'President'/Myrobalan B became incompatible when top worked with the variety 'Victoria' (Mosse, 1961). The peach variety 'Hale's Early' is compatible with the plum rootstock Brompton but incompatible with Myrobalan B. When a ring of bark of Myrobalan B is grafted on the stem of the stock of 'Hale's Early'/Brampton, symptoms of 16 incompatibility appear on "Hale's Early' scion (Mosse, 1960b). The author suggested that some translocatable factor was responsible for incompatibility. Relative position affects compatibility of certain graft combinations. 'Conference' pear was compatible with either Quince A or pear C8 when single grafted. When double grafted using all combinations of three clones as scion/interstock/stock, the combination Con- ference/Quince A/C8 and all other combinations in which any tissue of Quince A occurred above C8 grew very badly, whereas the combination Conference/C8/Quince A grew well (Mosse and Herrero, 1951). This work also suggests that some substances which cause incompatibility move in a polar manner. Incompatibility may also be triggered by repro- ductive process. Jimenez (1957) showed that the graft combination Carica goudotiana (paw paw)/Carica cauliflora was normal until male inflorescences were produced, when both stock and scion declined. However, male trees grew vigorously if the inflorescences were removed. This again suggests that certain biochemical factors are involved in incompatibility. b. Specific compounds. Alkaloids: In studies with herbaceous plants, specific substances such as alkaloids are postulated as 17 causal factors in graft failure. Mockaitis (1969) attributed the graft failure between Ipomoea violacea/ Ipomoea nil to a specific indole alkaloid compound of the scion. Mothes et_al. (1955) considered nicotine to be the causal factor in incompatibility in grafts of Atropa belladona/Nicotiana rustica. The former is free of nicotine while the latter contains much nicotine. How- ever, if Atropa belladona scions were grafted on the low nicotine containing stock, such as Nicotiana glauca, the combinations grew vigorously. Amino acids: Tschiersch (1963) found a relationship between graft failure and the presence of a particular amino acid in some plants of the Leguminosae. Canavalia ensiformis contains the amino acid, canavanine. The plant cannot be grafted to Phaseolus vulgaris L., which is closely related but free of canavanine. The author suggested that canavanine caused the death of P. vulgaris. Proteins: In explaining the origin of virus in grafting, Darlington (1944) suggested that a stable and useful protein with one plant genotype can act as a destructive agent with another. Crane (1945) proposed from the observation of apple 'Lord Lambourne' grafted on certain stocks that abnormalities in the scion may be due to the invasion of cells of one variety by the proteins of another, which behave as virus in the scion 18 tissues. However, there has been no confirmation of these proposals. Sheldrake and Northcote (1968) noted several enzymes in xylem sap and suggested that phloem sap may contain enzymes. When these enzymes are translocated to foreign tissues (stock or scion), they may influence metabolic activity. Cyanogenic compounds: The cyanogenic glucoside prunasin was suggested as a causal factor in incompati- bility of pear/quince grafts (Gur, 1968b). According to Gur's theory, vascular discontinuity at the graft union is due to the accumulation of hydrogen cyanide, a break- down product of prunasin, which is a natural component in quince, but absent from pear tissues. Phenolic compounds: Phenolic compounds have been suggested as causal agents of incompatibility (Gur, 1968b; Thiel, 1954; Williams, 1953; Buchloh, 1958). A separate section is devoted to studies dealing with these compounds. II. Role of Phenolic Compounds in Graft Incompatibility A. Physiological functions of phenolic compounds and their seasonal variations The physiological significance of phenolic compounds is uncertain. Some consider them to be end products of metabolic activity, serving only as cell wall materials (Bate-Smith, 1958). One definite role of phenolics is 19 to furnish plants with diverse color. The complex color- ation in flower petals and in fruits is known to aid in fertilization and seed distribution (Harborne, 1967b). In all vascular plants, the phenolic compounds are essential for structural features such as tracheids, vessels, and fibres. In higher plants, the immediate precursors of phenolic compounds are the aromatic amino acids, phenylalanine, and tyrosine, which are in turn synthesized via the shikimic acid pathway from carbo- hydrates. The first products of deamination are cin- namic acid and p-coumaric acid, which then serve as precursors of lignin, flavonoids, coumarin, and other types of phenolic compounds (Koukol and Conn, 1961; Neish, 1961). The deposition of lignin in xylem tissues maybe one form of cellular excretion of metabolic by- products. Reznik (1960) pointed out that higher plants, unlike microorganisms or animals, do not have an efficient system for excretion of metabolic by-products. Instead, they practice a type of 'local excretion' into the vacuoles and cell walls. Phenolic compounds might have a protective function in plant tissues. For example, Perrin and Bottomley (1962) identified a phenolic compound 'pisatin' from pea pods, which inhibits the multiplication of an invading fungus. When cactus tissue was mechanically injured, it responded by formation of lignin, which prevents rotting 20 of the tissue (Steelink et al., 1967). Feldman and Hanks (1965) reported a quantitative difference in bound phenolic compounds between citrus cultivars, one of which is sus- ceptible to the burrowing nematode (Radopholus similis) and the other tolerant. After infection, the bound phe- nolics increased 27-300% in the tolerant cultivars while decreasing 16-34% in the susceptible. Koeppe gE_al. (1969) thought that phenolic compounds may be important in mediating the effects of environmental conditions. Under stress conditions, such as high ultra violet radiation (Lott, 1960; Koeppe gp_al., 1969), 2, 4, dichlorOphenoxy- acetic acid application (Dieterman gt_al., 1964), lack of boron (Watanabe gp_al., 1964), or low nitrogen (Harborne, 1964), the concentration of certain phenolic compounds increases. In more recent years, evidence has been presented that the phenolic constituents play a part in dormancy of buds (Hemberg, 1961; Hendershott and Walker, 1959). The phenolic compounds appear to affect plant growth via the IAA-oxidase system (Hare, 1964; Tomaszewski, 1964; Galston, 1969). The dynamic status of these compounds has been documented. Hillis and Swain (1959) analyzed leaves of Prunus domestica for total phenols, leucoanthocyanins and flavonols at intervals during the growing season and found that the amounts increased rapidly until the leaves 21 reached maximum size and then decreased. In a study of seasonal levels of phenolic acids in two ferns, Glass and Bohm (1969) found a rapid increase during the early stages of growth. In apple shoots, Harvey (1925) found that phloridzin was most abundant in the apex of the shoot, where metabolic activity is high. However, the maximum concentration lagged about 2 weeks behind the maximum rate of growth. B. Evidence for the role of phenolic compounds In incompatibility Gur (1968b) investigated incompatibility of pear cultivars on quince rootstocks and concluded that certain substances moved from the quince rootstocks and were changed into toxic components in the pear bark. Pruna- sin, a cyanogenic glucoside, occurs in the quince but not in the pear. According to Gur's theory prunasin diffuses upward across the union and is hydrolyzed in the pear bark. The liberated hydrocyanic acid inhibits cambial activity and prevents vascular continuity. The liberation of hydrocyanic acid, however, is dependent on the presence of arbutin, a cofactor for B-glucosidase. The compatibility was quantitative and was inversely correlated with arbutin content of pear cultivars. Gur (1968b) also postulated that in the bark of pear varieties the ratio of free to bound phenolic compounds was related to the degree of incompatibility with Quince A rootstock. 22 Buchloh (1960) believed that some factors inhibit lignification at the graft union and that these factors are responsible for vascular discontinuity. In the early stage of graft-union formation, cell walls of stock and scion are joined by a common middle lamella consisting of pectic material. The next step is the formation of the secondary cell wall consisting of cellulose and hemi- cellulose. In further development, the pectic substances of the middle lamella disappear and are gradually replaced by lignin. However, in the incompatible graft-combination, after the pectic material decomposes, the middle lamella is not replaced with lignin. Buchloh also noted that adjoining cells, and sometimes cell walls, were dis- colored in both compatible and incompatible unions. The pigment disappears later in compatible unions, but in incompatible unions, the pigment remains, forming a dark brown precipitate. The pigment appeared to be hydrolysed phenolic compounds which subsequently were polymerized to brown precipitates. In the course of development of the graft union, lignification was markedly inhibited when the brown precipitate formed. Buchloh, therefore, concluded that lignification was inhibited either by reactions which give rise to the formation of the brown colored compounds or by the compounds themselves. Thiel (1954) suggested also that the discoloration of 23 tissues and death of cells of incompatible pear/quince graft unions were toxic effects of oxidized phenolic com- pounds liberated from arbutin. C. Evidence for translocation In order for phenolic compounds to be effective in inducing incompatibility, they must reach the tissues of the graft partner. Demonstration of graft transmission is therefore a prerequisite for their implication as causal agents, unless one assumes that cell-to-cell contact is sufficient. The evidence for graft trans- mission varies, depending upon the species under study. Friedrich (1958) found no evidence for translocation of polyphenols in reciprocal grafts of apple and pear. Hillis and Swain (1959) could find no direct relationship between phenolics in leaves and those in woody tissues of Prunus domestica. The authors suggested that translocation of phenolic compounds between the tissues was unlikely. In Eucalyptus wood, polyphenolic compounds were not trans- located from one tissue to another but were formed ifl.§i§2 from carbohydrates (Hillis and Carle, 1960, 1963). Bate- Smith (1962) believed that polyphenolic constituents were formed from simple precursors in the particular cells in which they occurred. In the examination of sieve tube exudate, he could find no trace of polyphenolic compounds (leucoanthocyanins and flavonols), although these were abundant in leaf tissues. 24 There are, however, several reports suggesting the possibility of translocation. For example, Hergert and Goldschmid (1958) suggested that quercetine and taxifolin were synthesized and glucosylated in the leaves, then transported down the phloem and laterally to the bark and heartwood. Many of polyphenolics found in oak bark origi- nate in the leaves, according to Hathaway (1959). The author suggested that compounds such as gallocatechin and leucodelphinidin were translocated by the sieve tube sys- tem to the cambium and then condensed to tannins which were stored in the bark. In the study of phenolics in the solanaceae, coumarins appeared to be formed in the root from tyrosine and translocated into the shoot (Kala, 1956). Gorz and Haskins (1962) noted movement of coumarins across the graft union in clover, while MacLeod and Prid- ham (1965) demonstrated the translocation of phenolic compounds which were introduced into the apical leaves of Vicia faba. The general translocation rates for phenolics approximated those for C14 assimilates. The glycosidic derivatives migrated more rapidly than the phenolic aglycones. The authors also showed the presence of phenolic compounds in aphid stylet exudates from the sieve tubes of Sali§_and 21213. Favre-Bonvin g£_gl. (1966) studied the effect of grafting on the distribution of coumarin in cherry. Chemical analysis of the inter- specific graft combination Prunus mahaleb/P. avium showed 25 that the normally coumarin-free P. avium roots accumulated coumarin and herniarin. Further study with the phenolic precursors, DL-phenylalanine-C14 by these authors (1968) showed that coumarin compounds moved readily between the graft partners. D. Possible mechanism of action in incompatibility Phenolic compounds may cause incompatibilities either directly as phytotoxins, or indirectly through their effects upon IAA oxidase. Phenolic compounds as phytotoxins: Many phenolic compounds are phytotoxic (Pridham, 1960). The inhibitory activity of the compounds has been recognized in germi- nation (Evenari, 1949; Van Sumere, 1960; Varga and Koves, 1959; Borner, 1960; Hemberg, 1961), in bud dormancy (Hen- dershott and Walker, 1959; Lane and Bailey, 1964), in potato tuber dormancy (Hemberg, 1958; Housley and Taylor, 1958), in shoot growth (Thimann and Bonner, 1959; Hancock g£_§1,, 1961; Tomaszewski and Thimann, 1966; Galston, 1969), and in root growth (Goodwin and Taves, 1950). Many compo- nents of the. B-inhibitor complex have been identified as phenolic compounds and hypotheses have been advanced as to their regulatory roles (Hemberg, 1961; Lane and Bailey, 1964; Moreland gp_al., 1966). Plants are known to excrete chemical inhibitors which control the growth of other plants (allelopathy). Some examples are: Artemisia 26 absinthium (Bode, 1940), Eucalyptus rostrata (Evenari, 1961), black walnut (Bode, 1958), and Johnson grass (Abdul-Wahab g£_gl., 1967). Some of these inhibitors have been identified as phenolic compounds (Rice, 1965; Abdul-Wahab g£_21., 1967; K5ves and Varga, 1958; Borner, 1957; Bautz, 1953; Grfimmer and Beyer, 1960). These compounds, in the free state, have both lipophilic and hydrophilic properties, and Frey-Wyssling (1942) suggested that they could interfere with the function of cell vacuoles and tonoplast membranes. A few of these compounds are reported to interfere with oxidative phos- phorylation (Millerd et_313, 1953; Stenlid, 1963) and with mitosis (Steinegger and Leupi, 1955; Cornman, 1957). Others may inhibit enzyme activity. In the study of the inhibitory action of coumarins on plant growth, Thimann and Bonner (1959) concluded that inhibition of growth involved their reacting with enzyme sulfhydryl groups. Mayer and Poljakoff-Mayber (1961) listed several enzymes whose activities were inhibited by coumarin. In germi- nating lettuce seeds and seedlings, this compound inhibited the activity of proteinase, lipase, and phytase. Plant tissues, then, need an efficient system to detoxify the phenolic compounds. Pridham (1960) reported that 21213 £222 seeds germinated and plants developed to maturity in the presence of relatively large quantities of arbutin. However, the corresponding aglycone, quinol, 27 inhibited germination. Van Sumere (1960) showed that ferulic acid-B-glucoside had no effect on the germination of wheat rust uredospores whereas free ferulic acid was strongly inhibitory. The glycosylation of applied phe- nolics has been reported in barley and wheat leaves (Nystrom §E_31., 1959), in tobacco leaves (Runeckles et al., 1963), leaves of Solanum species, Cestrum newellii, Raphanue sativus, Clematic lawsoniana, Lycopersicum escu- 1entum, and in Datura knightii (Harborne and Corner, 1961). Toweres 3E_31. (1958) have shown that maleic hydrazide, a compound with phenolic properties, is converted to a glucoside by plant tissues, and they suggest that this is the reason why relatively high concentrations of the reagent are needed for effective growth inhibition. Seedlings of the interspecific hybrid Lilium aruatum X Lilium speciosum are tumorous and die at an early stage of development. Asen and Emsweller (1962) found that 60% of total ferulic acid in the seed was present as the aglycone. However, in seedlings from intraspecific crosses, which develop normally, only 6% of the ferulic acid was present as the free acid, the remainder being present as the glucose ester. Thus, if a stock or a scion of a certain graft combination does not have an efficient system of detoxification, any condition which allows the accumulation of the phenolic compounds in the tissue may restrict the normal growth and development of the graft. 28 Phenolic compounds as cofactors or inhibitors of IAA oxidase: Phenolic compounds may affect the growth of stock or scion via the indole-3-acetic acid (IAA) oxidase system. IAA oxidase occurs widely in nature, and is believed to regulate the level of IAA by oxidizing it to inactive products (Hare, 1964). Phenolic compounds either inhibit or synergize with the enzyme depending on their structures. Monohydric phenols act as cofactors for the IAA-oxidase system, while dihydric phenols inhibit oxi- dation (Stenlid, 1963; Hare, 1964; Tomaszewski and Thimann, 1966). All 4' hydroxyflavonoids were cofactors for the oxidation of IAA, whereas 3', 4' dihydroxyflavonoids inhibited the destruction of IAA (Mumford eE_al., 1961; Stenlid, 1963). WOrk by Russell ep_al, (1969) suggested that phytochrome may control growth of pea seedlings by regulating the synthesis of phenolic compounds which act as cofactors in an IAA oxidase system. In pea seedlings Furuya and Galston (1965) identified kaempferol-3-glucoside and its p-coumaric ester as promoters, and quercetin-3- glucoside and its p-coumaric ester as inhibitors of IAA oxidase. In addition to flavonoids, other types of phe- nolic compounds have been shown to interact with IAA oxi- dase. Compounds which promote the enzyme are p-coumaric acid and p-hydroxy benzoic acid (Engelsma, 1964; Tomaszew- ski and Thimann, 1966; Lee and Skoog, 1965). The inhibi- tors of IAA oxidase are chlorogenic acid, caffeic acid, 29 ferulic acid, sinapic acid, scopoletin, scopolin, and 3, 4 dihydroxy benzoic acid (Gortner and Kent, 1958; Henderson and Nitsch, 1962; Tomaszewski and Thimann, 1966; Sacher, 1963; Schaeffer, 1967; Varga and Koves, 1962; Gelinas and Postlethwait, 1969; Sequeira, 1964; and Lee and Skoog, 1965). There is strong evidence that cambial activity and the differentiation of secondary xylem and phloem are controlled by metabolites and growth hormones, especially endogenous auxin, from the developing buds and leaves (Wareing et al., 1964; Larson, 1964; Evert and Kozlowski, 1967; Torrey and Loomis, 1967; Wilson, 1968; Reinders- Gouwentak, 1965). Thus, the radial growth of the stocks of certain graft combinations may be dependent upon the supply of auxins and metabolites from the scions. Phe- nolic compounds may play a role in incompatibility by regulating the auxin supply to the rootstock via the activation or inhibition of IAA oxidase (Gur and Samish, 1968a). III. Phenolic Compounds Identified in Cherry Tissues The fact that plant species contain specific phenolic constituents allows taxonomists and biochemists to detect relationships between plants and groups of plants (Bate- Smith, 1961a). Much research has been done on phenolic compounds in woody tissues of cherry. 30 Hergert (1962) has reviewed the economic importance of flavonoid compounds occurring in the genus Prunus. Flavonoid compounds in cherry wood have been studied in some detail by chemists in France and Japan. In the woody tissues of P. yedonensis, Hasagawa et a1. (1952) identified 4 flavonoid compounds: prunin, genkwanin, naringenin, and d-catechin. They (1954, 1957) also studied tissues of several other Prunus species. In the heartwood of E: 22122.11 flavonoid compounds were identi- fied by elementary analysis, boiling point, and chromato- graphic technique. These were d-catechin, naringenin, prunin, aromadendrin, eriodictyol, taxifolin, chrysin, aequinocitin, genistein, prunetin, and genistin. Mentzer e£_al. (1954) found 12 flavonoid compounds while examining ether extracts of wild cherry heartwood. These compounds were identified as chrysin, dihydrochrysin, sakuranetin, tectochrysin, and dihydrotectochrysin. Chopin gE_21. (1957) identified a flavanone from heart- wood extract as dihydrowogonin. The glucoside of dihydrowogonin was also detected in pedicels of P. cerasus (Wagner gE_gl,, 1969). Two water soluble compounds from the heartwood of cherry trees were identified by Pacheco ep_a1. (1957) as d-catechin and aromadendrin. These compounds were found to accumulate in the heartwood as the tree increased in 31 age. Chopin and Pacheco (1958) isolated the flavanonol 7-methy1aromadendrin from an ether extract of the heart- wood of P, ayigm, In the woody tissue of P, mahaleb, Pacheco (1959) identified four flavonoid compounds: naringenin, aromaden- drin, genistein, and prunetin. A year later the same author reported that he found a glucoside of aromadendrin in Mahaleb heartwood. Pacheco et_§l, (1960) reported on more flavonoid compounds present in the woody tissue of P: mahaleb. These were: a flavanone glucoside prunin, glucosides of aromadendrin, and taxifolin. Geissman (1956) studied the phenolic composition of normal and virus-infected cherry leaves. Using paper chromatographic methods, he found that the leaf tissue contained glucosides of kaempferol and quercetin, and esters of p-coumaric acid and caffeic acid. In discussing the taxononomic significance of phe- nolic compounds in the Rosaceae, Bate-Smith (1961b) noted certain common features in the distribution of phenolic compounds among genera and subgenera. For example, in the genus Prunus leucoanthocyanidins, flavonols, and hydroxycinnamic acids were found. The sub-genus 'cerasus' contained a 'cherry factor' which was identified as o-coumaric acid. Mahaleb leaves were shown to have the specific 'mahaleb factor,‘ herniarin (7 methoxy coumarin). 32 Favre-Bonvin et a1. (1966, 1968) studied the site of synthesis of coumarins by interspecific grafting between Mazzard and Mahaleb. Using the radioactive precursor 14, they demonstrated that leaves, fruits, phenylalanin-B-C and root-wood synthesized both coumarin and herniarin. The stem wood could also synthesize herniarin. For con- venience certain of these findings are summarized in Table l. 313 Aommav adfluuwou .N Aawwmwawualmanwuw ”M «.«+ vans uwudqaqo auouuanuo v.n uuua uwouuao .aooa. sudsmuou-n .a « ~.H+ vane ueliscwu known»: v neon oaudldOUIn “Howav nuasmlounn + can. odsdssuu Alanna: n can: uaNuIaOOIo o and ads: .Homav nuasmnounn .~ 6. ow «u .6WMMMHWfimmmnmmeum HM H+ N.“ uauu-sou suonuoa s cauuacuom Ammo”. na>son ou>om .a ~+ ~.H caudasoo unaudaso :3: 525-33 4 + 9319.3 Engages v. .255." 56,826 o lemma. cqauuaoo .n a . . . . .wamwduuwuwmuuuw ”M H ~.H+ Hocosasu axouuanauuou s.m..¢..n cauouuoso .Howmwauwauwwmwum ”M H ~.H+ soco>~au axouuanauu s.m..v sououmauqx Ahmed. Ituoanox .A + + oco>aauouu axonuoa h uxouvxnuv m..v swuocsum Aoowflmowwomwuwauw” + ova-ounam h cfiouoasou :«uoanou Anmmav «rounds: .H + + oco>~auonw auouvsnauu h.m..v caouuacoo oco>aau ixonuos s hxouv>SAv m..v swcatxcvo no Avmmav uomuco: + oco>aau >uonuol h Sacha»: m cau>u500uuua .vmasc nucuuomflwowwomwuaqaw” . oco>aau auouusnao ~.m cauaunu lemma. assuanox H~ lcauouuusvouossaav a n+ ~+ H+ Hococn>oau axouuagduuou n.m..v..n caucuaxda lawmac oucnoam .n + soaoaa>aflu sxouusnso k.m :Axacanocaa Remedy ciaouna: Asmmav ooocuom .N .H n+ ~+ s. Hococ¢>aau axouusnauu h.m..v neuucooq-ou4 .hmmav diamond: + ococn>aau >xouo>nduuou n.m..v..n ao>uou60auu Amomav wanna: Aramav admonu + unend>aau axonuoa m xxouvanwu h.m sas0m010uu>nwo Ammohwmwwwnwumucwu + o:0:¢>nau >xozuua h >w0uvsneo m..v canes-usxvm awmmwwvanuwnwuu HM ~+ ~+ ova-ounao h cwsoosaudz caughm Runway atnmcnaz .a ~+ H+ unocd>uau hxouvhnauu n.m..v cacoOCwuoz Avmmss nonuco: + anon-page axonuo- a Rhone»: m lasaaunuouucuouusnsa. nanouuuocwm lemma. uoauco: + ococa>aau auouuanau k.m .cauauzoouusnso. :«uAlooocam Ahmad. ooonuum .Anmmas «savanna + canuouuuxu :enoouqo ovuoco>auh coo: xudn used can! gain uuflu nausea-mom ousuushum aduallau all: calico nodanua .m laden .m .uoouu uuulno aw usauusuoo qusonaou ouflocosm .H sands MATERIALS AND METHODS I. Seasonal Variations of Total Phenolic Compounds A. Plant material One-year-old Mahaleb and Mazzard seedlings were planted in the field in May, 1969. Uniform nursery-grown seedlings were obtained from Hilltop Nursery, Hartford, Michigan. At two-week intervals three trees of each species were harvested and fresh weights of leaves, roots, and stems recorded. Leaves, stem bark, and root bark were put immediately in the oven and dried overnight at 85°C. The dried tissue was ground to pass through a number 4 mesh screen and stored in closed bottles until extraction. B. Extraction One gram of ground tissue was shaken for 3 hours with 50 ml 80% aqueous methanol. The solvent was then decanted and the same procedure repeated twice for a total of 150 m1 solvent. The extract was filtered through Whatman No. 1 filter paper and made up to final volume of 200 ml. 34 35 C. Quantitative determination The method used for measurement of total phenolic compounds (Swain and Hillis, 1959) involves the reduction of compounds containing sexivalent tungsten and molybdenum to colored products. A 0.1 m1 aliquot ( = 500 ug tissue) of the sample solution was diluted with 6.9 ml distilled water. After mixing, 0.5 m1 of 0.25 N Folin-Ciocalteau reagent (Bray and Thorpe, 1954) was added. The samples were shaken for 3 minutes and then 1.0 m1 of 1N Na2C03 solution was added. The solution was again thoroughly mixed, made up to 10 ml with distilled water, and stored one hour in the dark before measurement of absorbance in a Klett-Summerson photoelectric colorimeter, using a No. 66 red filter (725 mu). For a standard curve, ferulic acid was used. II. Identification of Phenolic Compounds in Methanol Extracts, and DeterminatiEn of Relative Amounts A. Plant material Uniform Mazzard and Mahaleb seedlings were planted in the greenhouse May 30, 1969, and grown until October 30, when they were placed at 35°F to satisfy the chilling requirement. At the end of January, 1970, the seedlings were again planted in the greenhouse and grown until May. They were then removed from the pots, washed thoroughly with tap water, and separated into leaves, stems, and 36 roots. Leaves were harvested with petioles attached. Bark tissues were obtained by cutting the stems and roots into small segments and peeling the bark from the wood. The wood was discarded. Both leaves and bark were weighed and frozen in liquid nitrogen. Special care was taken to discard any dead tissues such as the epidermal layer. The samples were kept frozen until used. B. Extraction The weighed samples (Table 2) were macerated in 400 ml absolute methanol, and the macerate was filtered through Whatman No. 1 paper. Each sample residue was extracted by shaking for 16 hours successively with five 200 m1 portions of methanol on an oscillating shaker. The combined methanol extracts (1400 ml) were filtered through celite and stored in brown bottles (34°F). The detailed extraction procedure is shown in Figure 1. Table 2. Weights of tissues used for extraction of phenolic compounds. Species Tissue Fresh weight (g) Mazzard leaf 100 stem bark 77 root bark 35 Mahaleb leaf 100 stem bark 100 root bark 77 37 Fresh sample Homogenize for 5 minutes with abso- lute methanol (400 m1) and filter through Whatman No. 1 paper. 1 I Methanol Residue Shake for 16 hours with 5 x 200 m1 methanol and filter through Whatman No. 1 paper. | j Methanol Residue Discard Combine methanol extracts and filter through celite. f 1 Methanol Residue r—— ——-—.— Evaporate, suspend Discard residue in 100 m1 phosphate buffer (pH 8) and extract with petroleum ether (5 x 50 ml). r l §m££g£_ Petroleum ether Adjust pH to 3 with conc. Discard H2504 and extract with 10 x 50 m1 ethyl acetate. 1 Eth 1 acetate Agpeous solution, Evaporate and dissolve Discard residue in 20 m1 methanol. Methanol (I) —Paper chromatography of unhydrolyzed Evaporate 15 g (f.w.) phenolic compounds. equivalent methanol extract and dissolve residue in 60 ml hot water and centrifuge for 10 min. 4 I I Supernatant Residue Add 60 ml 1 N HCl and hydrolyze Discard 1 hour on a boiling water bath. Extract hydrolysate with ether (5 x 50 m1). Ether Aquebus solution Evaporate and dissolve Discard in 5 ml methanol. Methanol (II) Paper chromatography and gas liquid chromatography of hydrolyzed phenolic compounds. Figure 1. Procedure for extraction and purification of phenolic compounds. 38 C. Hydrolysis Aliquots of the crude methanol extract representing 15 g (f.w.) of tissue were evaporated under vacuum, and the residues were dissolved in 60 m1 hot water. The water was centrifuged to remove the chlorophyll precipi- tate, and the supernatant was extracted with chloroform (5 x 20 ml) to eliminate carotenoids, chlorophyll, and lipids. The water phase was transferred to a 300 ml volumetric flask, and 60 m1 of l N HCl was added. The solution was then heated for one hour on a boiling water bath. After cooling to room temperature the aqueous solution was extracted with ether (5 x 50 ml). The ether extract was evaporated and the residue dissolved in 5 m1 methanol (Fig. 1). This methanolic solution was used directly for either paper chromatography or for gas liquid chromatography. D. Purification and identification 1. Paper chromatography. Twenty to fifty pl (60 to 150 mg fresh weight of tissue) of extract was spotted on the upper left-hand corner of Whatman No. 1 papers (46 x 57 cm) for chromatography. The diameter of the spots (less than 1 cm) was minimized by using a 5 ul pipette and a stream of air. The first solvent was n-butanol: acetic acid: water, 6:1:2 (BAW) which irrigated the long direction of the paper for 17 to 18 hours at room 39 temperature. The air-dried paper was then run for 3.5 to 4 hours in 2% acetic acid (HOAc) in the short direction. The air-dried chromatograms were examined under ultraviolet light before and after exposure to ammonia vapor. Replicate chromatograms were then treated with 4 different reagents (Table 3). One chromatogram was dipped in a freshly prepared solution of equal volumes of 0.5% FeCl and 0.5% K Fe(CN)6, then rinsed with 2% 3 3 HCl solution followed by distilled water. The phenolic compounds appeared as blue spots on the chromatogram. A second chromatogram was sprayed with diazotized p-nitroaniline in 2 N HCl, 5% NaNO and 20% sodium 2: acetate (w/v) in a ratio of 1:10:30. A third chromato- gram was sprayed with freshly prepared Hoepfner's reagent (5% NaNO2 plus 5% acetic acid). With this reagent phe- nolic acids give characteristic colors. A fourth chromato- gram was Sprayed with 2 N NaOH, and after 3 to 5 minutes it was viewed under ultraviolet light. Coumarins gave a characteristic greenish yellow fluorescence. The chromatogram was then sprayed with DPNA, which reacts with coumarins to form distinctively colored products. In order to prepare enough material for further characterization, the crude methanol extract was applied as a line on Whatman No. 3 paper. Chromatograms were developed with 2% acetic acid for 3.5 hours, then air- dried and examined under ultraviolet radiation. The 40 Table 3. Reagents used for phenolic compounds. Reagents Composition Specificity Reference FeClB-K3Fe(CN)6 0.5% Fe Cl and All phenolic Keppler 0.5% K3Fe(aN)6(1:l,v/v) compounds (1957) Diazotized p- 0.5% p-nitroaniline Most phenolic Swain nitroaniline 5% NaNOz compounds; (1953) (DPNA) 20% sodium acetate characteristic (1:10:30, v/v) colors with phenolic acids Vanillin-HCl 1% vanillin in Leucoantho- Swain ethanol, and conc. cyanidins et al. HCl (1:1, v/v) Catechins (1959) Tetrazotized 0.5% benzidine in dil. Phloroglucinol- Smith benzidine HCl, and 10% NaNo2 resorcinol (1960) (1:1, v/v) type compounds NaBH4-HC1 2% NaBH4 in methanol Flavanones Horowitz Fume chromatogram (1957) with HCl vapor Hoepfner 5% NaNOz and 5% acetic Phenolic acids Walker acid (1:1, v/v) (1962) Folin-Ciocalteau 10 g sodium tungstate All phenolic Swain and 2.5 g sodium molyb- compounds et a1. date in 70 m1. Add (1959) 5 ml phosphoric acid Waldi (85%) and 10 ml conc. (1965) HCl. Boil and add 15 g lithium sulphate and 1 drop of bromine. Made up to 100 ml with water (stock solution). 0.25 N Folin-Ciocalteau, then 1 N Na2C03 2 N NaOH-DPNA 2 N NaOH, then DPNA Coumarins Swain (1953) 41 distinctive color bands were marked with a pencil. A few more bands were detected when the paper was sprayed with FeC13-K3Fe(CN)6 and DPNA reagents. Zones from identical unsprayed chromatograms were cut out and eluted by shaking with 3 successive 100 ml portions of 95% ethanol. The eluates were filtered and evaporated in a rotary film evaporator under vacuum. Each fraction was rechromatographed on Whatman No. 1 paper in BAW (first direction) followed by 2% acetic acid (second direction). Bands which fluoresced under ultra- violet light, or which reacted with DPNA and FeC13-K3Fe(CN)6 on parallel chromatograms, were cut from the chromatograms, and eluted with ethanol (3 x 10 ml). The eluates were filtered through Whatman No. 1 paper and evaporated under vacuum. The residues were dissolved in 0.5 m1 ethanol, spotted on Whatman No. 1 paper, and chromatographed one dimensionally in the following solvent systems: butanol: acetic acid: water, 6:1:2 (BAW) 2% acetic acid (HOAc) butanol: pyridine: water, 10:3:3 (BPW). 2. Thin layer chromatography. Aliquots (5 ul) of the partially purified compounds were spotted on Eastman thin layer plates (silica gel G) and run in one direction in the following solvent systems: 42 benzene: methanol: acetic acid, 45:8:4 (BMA), 11% methanol in CHC13, toluene: ethyl acetate: formic acid, 5:4:1 (TEF). The chromatographic chamber was lined with filter paper. When the solvent reached 10 cm above the origin, the plates were removed and air-dried. They were then viewed under ultraviolet and visible light before and after the application of phenolic reagents. 3. Gas liquid chromatography. Samples were gas chromatographed on a 6 foot by 2 mm column of 2% QF-l on Chromosorb W using a Packard gas chromatograph Series 7300 equipped with a hydrogen flame ionization detector. Column temperature was 160°C and those of detector and injection block were 250°C. Nitrogen was used as a carrier at a flow rate of 40 ml per minute. Methylation was based on the method of Schlenk and Gellerman (1960). The methylation apparatus consisted of three test tubes with side arms. A stream of nitrogen gas was saturated with ether in the first tube and this carried the CHZN2 generated in the second tube into the sample tube where esterification took place. The components in the three tubes were as follows: Tube 1: ether (20 ml), Tube 2: 0.7 m1 carbitol (diethylene glycol), 0.7 m1 ether, 1.0 ml 60% KOH, Tube 3: sample (equivalent of 3 g fresh weight) in 1 m1 ethanol, 3.0 ml ether, 0.3 ml methanol. 43 The reaction was begun by adding a small quantity of N methyl-N-nitroso p-toluene sulfonamide (Diazald) in 0.5 ml ether to tube number 2. The tubes were stoppered and the reaction was allowed to proceed for 3 to 10 minutes until a yellow tinge appeared in tube number 3. After evaporating the solvent, the methylated sample was dissolved in 0.5 ml acetone and 0.6 pl aliquots were injected into the gas chromatograph. Flavonoid compounds were silylated by adding 50 U1 Tri-Sil (Pierce Chemical Co., a premixed trimethylsilyl- ation reagent containing hexamethyldisilazane and tri- methylchlorosilane in pyridine) to a 15 ml conical centri- fuge tube which contained 1 mg of the standard compound. After 15 minutes at room temperature, this solution was injected directly into the column. 4. Ultraviolet absorption gpectra. Absorption spectra of the purified compounds in 95% ethanol were obtained with a Beckman DB spectrophotometer. 5. Sources of reference compounds. The sources of the reference compounds used are given in Table 4. E. Quantitative determination In most cases, the compounds were so close to each other on paper chromatograms that it was difficult to elute and quantify them by absorption spectroscopy. The coumarin derivatives did not give responses similar to 44 Table 4. Sources of reference compounds. Source Compounds S. A. Brown Umbelliferone Department of Chemistry Aesculetin Trent University Herniarin Peterborough, Ontario A. Grouiller Taxifolin Institut National des Sciences Aromadendrin Appliques de Lyon 7 Methyl aromadendrin J. Favre-Bonvin Dihydrowogonin Museum National d'Histoire Prunetin Naturelle, Paris Pinocembrin Genistein H. Erdtman Chrysin Kungl. Tekniska Hogskolan Tectochrysin Stockholm, Sweden Sigma Chemical Company Naringenin St. Louis, Missouri Naringin Salicin Chlorogenic acid Rutin Scopoletin Kaempferol Hesperetin Eastman Organic Chemicals o-Coumaric acid Rochester, N.Y. Ferulic acid Quercetin Aldrich Chemical Company Apigenin Caffeic acid p-Coumaric acid R. F. Carlson Coumarin Department of Horticulture 45 other phenolic compounds when the eluates were treated with common Folin-Ciocalteau reagent or with DPNA, which also complicated the measurement. Therefore, visual estimation of the relative amounts of phenolic compounds was made. The scale was based on the area and intensity of the individual spots on a two- dimensional paper chromatogram after it was sprayed with FeCl -K3Fe(CN)6 or with 2 N NaOH plus DPNA. A spot of 3 o-coumaric acid of Mazzard leaf extract was arbitrarily assigned a value of 5, and other spots were rated 1 to 12 in relation to the size of the o-coumaric acid spots. III. Identification and Relative Amounts of Phenolic Compounds in Centrifugal Sap A. Plant material Uniform one-year-old seedlings grown in the green- house for 5 months were used. In the case of Mazzard, 128 gram of fresh weight was used and for Mahaleb, 110 grams. B. Extraction The sap was extracted as follows (Goldschmidt and Monselise, 1968). Stems were cut into 5 cm segments and placed in centrifuge tubes with bases down. Twenty per cent methanol was added to cover the basal ends of the stems, and the tubes were centrifuged at 1,500 x g for 46 20 minutes. The methanol was evaporated and the remaining aqueous solution diluted to 20 ml with water (Fig. 2). C. Fractionation of extract The aqueous solution from (B) above was extracted first with ether (3 x 10 ml) and then with ethyl acetate (4 x 10 ml). The remaining aqueous solution was acidified to pH 2 and extracted again with ethyl acetate (4 x 10 ml). Each fraction was evaporated and the residue dissolved in 2 m1 methanol. These methanol fractions were used for paper and for gas liquid chromatography as described in Section II above. 47 Stems Cut to 5 cm lengths Immerse in 20% methanol solution Centrifuge 20 minutes, 1500 x g I I Aqueous methanol Stems Evaporate methanol, Discard add water to 20 ml and extract with wet ether (3 x 10 ml) l Ether Aqueous solution Evaporate ether Extract with ethyl and recover residue acetate (4 x 10 ml) in 2 m1 methanol Methanol (1) I l Ethyl acetate Aqueous solution Evaporate and Acidify to pH 2 dissolve residue with conc. H2804, in 2 m1 methanol extract with ethyl acetate (5 x 10 m1) Methanol (2) [ I Ethyl acetate Residue Evaporate and Discard dissolve residue in 2 m1 methanol Methanol (3) Figure 2. Procedure for extraction and purification of centrifugal sap. RESULTS AND D ISCUSS ION I. Seasonal Variations of Total Phenolic Compounds in Field—Grown Seedlings Mazzard tissues were found to contain higher levels of phenolic compounds than Mahaleb tissues (Fig. 3). Average amounts for all sampling dates for leaf, stem bark, and root bark of Mazzard were 55, 141, 129 mg equivalents of ferulic acid per gram dry weight respec- tively, compared with 17, 82, and 86 mg for Mahaleb. Leaves. Among the tree tissues sampled, the leaf tissue contained the least amount of methanol extractable phenolic compounds. One week after bud break,-the small leaves contained low amounts of phenolic compounds. As the leaves grew, the phenolic compounds increased. In Mahaleb the phenolic level increased slowly until the latter part of June, then remained constant to the end of September. In Mazzard the level of phenolic compounds increased at a more rapid rate until early July and then stabilized (Fig. 3). Stem bark. Mazzard contained 1.5 to 2 times more phenolics in the bark than Mahaleb. As in the leaf tissue, Phenolics increased slightly to the first part of June, then remained relatively stable after July 1. 48 49 Figure 3. Seasonal variations of total phenolic com- pounds in field-grown cherry seedlings. Average values of phenolics in leaf, bark, and root were significantly different at 1% level between two rootstocks. per g d.w. compd d I O 5 DJ 0 O O b O 50 o—oMazza rd LEAF °---°Mahaleb /.V ” U"°"-o-__-o.o—’ ’°‘- -O--_o—-—-O I Q’ l l l _J BARK _ May June July Aug Sept 51 Root bark. Phenolic compounds increased in bark of Mazzard root until late June and then leveled off. Bark of Mahaleb root showed the greatest variation in phenolics of the tissues tested. Two peaks in concentration were found, one in the latter part of June and the other at the end of August. During the first two weeks after planting, the level of phenolics dropped from 86 to 60 mg, then increased rapidly until June 24. The concentration declined in mid-July, reaching a low of 46 mg per gram of tissue in mid-August. A smaller rise was noted in late August through early September. Seasonal variations of phenolic compounds have been studied in plum (Hillis and Swain, 1959), oak (Feeney, 1968), and fern (Gloss and Bohm, 1969). Phenolics increased rapidly during the early part of the growing season, then declined after the leaf attained its maximum size. The bark and woody tissues in plum showed less variation than the leaf during the growing season. Transport of some phenolic compounds has been reported (Gorz and Haskins, 1962; Favre-Bonvin, 1968; Macleod and Pridham, 1965). However, it is unlikely that all the phenolic compounds in root or bark were translocated from the leaf tissues, because these tissues contained a high level of phenolic compounds before bud break. In higher plants, the immediate precursors of phenolic compounds are the aromatic amino acids 52 phenylalanine and tyrosine, which are in turn synthesized via the shikimic acid pathway from carbohydrates (Brown, 1964). In Mahaleb the supply of precursors in the top may affect the concentrations of phenolic compounds in the root tissues. However, in Mazzard, the root tissue may be able to mobilize local storage metabolites for synthesis of phenolics. The sudden drop of phenolics in Mahaleb root in August is difficult to explain. The phenolic compounds may have been transported to other tissues or changed into methanol insoluble forms. The patterns of lignification could differ in the two species. In Mahaleb active lignification may lag behind growth but be coincident with it in Mazzard. II. Identification of Phenolic Compounds in Methanol Extracts of Greenhouse- Grown Cherry Seedlimgs, and Determination of Relative Amounts A. Total phenolic compounds The total methanol extractable phenolic compounds in fresh leaf, stem bark, and root bark tissues of Mazzard and Mahaleb varied from 200 pg equivalents of ferulic acid per gram fresh weight in Mahaleb leaf to 5667 pg in Mazzard root bark (Table 5). This was based on one sampling in May following 4 months of growth in the greenhouse. Mazzard tissues consistently contained more phenolics than Mahaleb tissues, in agreement with the previous experiment. 53 Table 5. Total phenolic compounds in Mazzard and Mahaleb tissues from greenhouse-grown seedlings.a Total phenolic compounds Species Tissues (pg ferulic acid equivalents/g fresh weight) Mazzard Leaf 583 Stem bark 5175 Root bark 5667 Mahaleb Leaf 200 Stem bark 2517 Root bark 2833 aAverage of 3 determinations based on ferulic acid standard curve. However, the absolute values were approximately 10% of those obtained in the field study on a dry weight basis. The difference may be due to differences in sample preparation or in growing conditions. The drying of samples in the first study may have released more 'bound' phenolic compounds. Phenolic compounds in the living tissues usually occur as glucosides, esters, or other bound forms (Harborne, 1964; El-Basyouni eE_31., 1966). The free phenolic compounds can be released by hydrolysis. Beck (1964), studying isoflavones in clover leaves, found extremely low levels of isoflavones when the tissue was extracted immediately with boiling ethanol. If the crushed leaves were allowed to stand for less than one minute before extraction, high levels of isoflavones were obtained. 54 Environmental factors might also account for the discrepancy. Light intensity, light quality, and nutri- tional status are known to affect the synthesis of phenolic compounds in intact plants and tissue slices (Siegelman, 1964). Lott (1960) reported that field-grown tobacco plants contained more phenolic compounds than greenhouse- grown plants. He attributed the differences to the high intensity of ultraviolet light in the field. Ultraviolet light increases the levels of phenolic compounds in tabacco and sunflower (Koeppe eh_al., 1969). Thus, the high levels of phenolic compounds in field-grown cherry seedlings may be due to higher ultraviolet irradiation in the field. B. Paper chromatogrhphy of hydrolysed leaf extracts Paper chromatograms of hydrolysed extracts of Mazzard and Mahaleb leaves are illustrated in Figure 4. Most of the compounds moved rapidly in BAW (6:1:2), suggesting that they were aglycones. Fourteen spots were distin- guishable on chromatograms of Mazzard leaf extracts after spraying with FeCl3-K Fe(CN)6 reagent. In Mahaleb 3 extracts 7 spots were noted. All compounds were color- less under visible light; however, some fluoresced under ultraviolet light. The color reactions of phenolic com- pounds from both species are shown in Table 6. Rf values are given in Tables 7 and 8, and relative quantities in 55 .sflmwuo may um pouuomm mmB Aunmwm3 nmmwm me om ou used Im>flswmv muomuuxw mo an muuflne .Anmamnmzv m no ApHmNNmzv N sues pmxume mum mowommm mco on pmuofiuummu macaw .mmwommm anon ou coeeoo mumz muoam poomnm .mmcwa smxoun zuHB pmxume muomm swap mwsmucfi whoa mum3 mmcea HHHOm shes emxume macaw use .omom mm sues matcoomm tam Amuauev 3am an“; “muem pmmon>m© muw3 mamnm0pmfion£o one .mm>mma amamnmz paw pumuumz Eoum pmuomuuxm messomeoo oaaoswsm pomaaonpmz mo memumoumEouno Hmmmm .v musmflm 56 AIuwsqmv uomuuxw no a: om .Anmamnmzv 3 no AUHMNNmSV N nuw3 pmxnmfi mum muomm OHMHoQO wwwomdm .mmsmmflu neon on :oEEoo mumz muOdm pmomnm .mmsfla smxoun nuw3 venues mmonu cmnu nuanced Hempma CH ucmm Imum muoz mmsfla pflHOm nuw3 pmxumfi mu0dm mne .omom em nuns snocoumm tam 34m apes umpnm pedoam>mo mums mamnmoumeonno one .xpmn Emum nmamnmz new peanut: Eonm pmuomuuxw monsOQEoo oaaoswnm pmnmaoupmn mo mfimnmoumEonno Hemmm .m madman 422.22 5.: L35 me u .. .. ... 3 9 I. .00" I.“ . a. a. .. a '6» ‘LINNIE . 9) MVB 69 v .soflumuoc Mom 0 manna owmn .ucmmmmu HUmI mmmz nuw3 sofiuommu uoHoo m m>mm mpcsomEoo mnu mo ecozm U ma U u m U U U s3ocxss mam U ya U U m U U U s3osxc5 Ham U »m U m m U U U ascends 0am on U U B m U U U s3ocnss mm U U U u m U nov ADV ssosxss mm U SH U U m an U U s3osncs em U m U U m U U U :3ocxss om ma NH Sm » m U U U canomumo v o o > segues o nmmum > > cauuacume am an U >mn smn U UM U U swumesoo m U U U 5m m kuwum w 3 once onumesoolo n .mmmmmmm U U U U m U U U szosxcs omN U u U U m U U U s3ocncs mam U U OH ya m U ADV ADV c3ocxcs mHN U U U Va m N U U c3ocxdd FAN U y ya nmn m um U U ascends can U NM Ma 0 m U m> m> c30cxcs mam U ya ya w m U um um saocxss mN U S U >muU¢w m >um >mum U neon onumfidooum mm U U a yum m SH 0H 0H sesomosoupmnflp «N a o we 0 m o o o ennumumo e U Ma U sen» U Hwowum U nomv sHHMEdoo m U U U 5m m Hmowum w 3 neon ofiumsdoouo H cumuumz no: can ume azmo + onzovmmmx momz m «zen mz+ I coaumofimflusmpfl .os unecm> nummom momz z~ unnomm z~+ m>numucme uomm unwed unnamw> unwed >9 n.m.smumoumeouno Hemmm Hmsofimsmeaplo3u n so pmnmmumoumeowno one pouaa Iouohn .xumn Seam nwamnmz use unease: eoum pmuomuuxo mossomsoo owaoconm mo meowuommu Moaoo .HH wands 70 Table 12. Rf values and relative concentrations of phe- nolic compounds extracted from Mazzard and Mahaleb stem bark, hydrolyzed and chromato- a graphed on a two-dimensional paper chromatogram. Spot Tentative Rf value Relative No. identification BAW (6:1:2) 2% HOAc concentration Mazzard l o-coumaric acid .85 .42 +++ 3 coumarin .89 .65 + 4 catechin .62 .40 ++ 24 dihydrowogonin .89 .00-.39 ++++++++++++ ZS p-coumaric acid .85 .37 +++ Z9 unknown .86 .00 ++ 215 unknown .88 .05 ++++ Zl6 unknown .76 .31 +++ 21? unknown .75 .65 + 218 unknown .87 .13 +++ 219 unknown .78 .42 . + 220 unknown .77 .55 + Mahaleb 1 o-coumaric acid .91 .41 + 3 coumarin .91 .75 +++ H4 herniarin .90 .62 +++++++++ 4 catechin .62 .40 ++ H6 unknown .79 .48 + H7 unknown .81 .55 + H8 unknown .89 .86 + H9 unknown .70 .60 + H10 unknown .87 .62 ++ H11 unknown .64 .08 + H12 unknown .77 .28 + _1_ aSee Table 7 for notations and chromatographic con- ditions. 71 of the phenolic compounds present in the Mahaleb leaf tissues, namely o-coumaric acid, coumarin, herniarin, and two unknown spots H6 and H7. Compound 4 was found in both Mazzard and Mahaleb bark. It was colorless under ultraviolet light with or without ammonia, but gave a pink color with vanillin-HCl reagent, which is specific for flavonoids with a phloro- glucinol nucleus such as catechin and leucoanthocyanidins (Swain and Hillis, 1959). The absorption spectrum of the compound had the characteristics of d-catechin with maximum absorption at 280 mu and minimum at 250 mu (Jurd, 1962). Rf values were comparable with those of catechin reported by Luh gt_al. (1967). Therefore compound 4 was identified as d-catechin. The living bark tissues of the rootstocks differed as to type of phenolic compounds. Mazzard bark appeared to be high in cinnamic acids and flavanones (p-coumaric acid, o-coumaric acid, and dihyrowogonin) while Mahaleb bark was exceptionally high in coumarins. The spots marked v, g, and b in Figure 5 did not appear to be phenolic compounds, for they were negative to FeCl3-K3Fe(CN)6 and DPNA reagents. However, v and g were useful markers for Mazzard bark extracts, since they gave distinctive colors (v, violet; 9, green) under ultra- violet light. The two spots were absent from Mazzard leaf extracts. 72 D. Paper chromatography of hydrolyzed cherry root bark extracts Phenolic compounds found in root bark were similar to those found in bark of stem tissues (Fig. 6). Color reactions, Rf values and relative concentrations of phe- nolic compounds from both seedlings are shown in Tables 13 and 14. Eleven phenolic spots appeared on the paper chromato- gram of Mazzard extracts, and ten spots on that from Maha- leb. Root bark tissues of both rootstocks contained the three common phenolic compounds which were found in stem bark of both Mazzard and Mahaleb, namely o-coumaric acid, coumarin, and catechin. In Mazzard, dihydrowogonin was the dominant compound followed by 215, o-coumaric acid, and p-coumaric acid. In Mahaleb, herniarin was the dominant compound but cou- marin and catechin were also prominent. E. Gas liguid chromatography of hydrolyzed_phenolic compounds Gas liquid chromatography was used to confirm and extend the results obtained with paper chromatography. The standard phenolic compounds and the unknown compounds were methylated with CH (Schlenk et al., 1960). 2N2 Among the different standard phenolic compounds studied, only the cinnamic acid series was successfully methylated. Since the flavonoid compounds did not respond to 73 .cfimwuo osu no oouuomm mo3 Ausmfioz smoum me om on ucoao>flsvov uomuuxo a: om .Anoaonozv m no Apnonumzv N cues ooxume ouo muomm Damaoomm mofloomm .moaoomm cuon on coEEoo ouo3 muomm pooonm .moCflH coxoun nowz ooxume omocu soap mucsoao Homnoa CH “somoum ouos mocHH oflaom nuflz ooxuoe muomm one .odom mm guns mancoomm mam Amuaumv zsm can: umuflm oomoHo>oo oHoB meoumoumaouno one .xuoa noon noamnoz tam pumNNmz Eonm wouomuuxo mocsomEoo owaosonm coumaoupma mo mEoumoumEouno Homom .o onsmflm 74 88¢. #008 <—KZ:I26)MVC tsONflOE 75 v .COAumuo: mom o oanoe oomn .Humu mmoz cuaz coauooou uoHoo m o>om monsooEoo on» no ocozm U ya U m so U U U c3osxcs mam U ya U m o U U U czocxcs 0am UH ya U m 0+9 U U U ssocxcs mm U U m m .x w 25 A3 gosxcs mm U ya U m U U U U czocxss on U ya U m m U U U c3ocxcs mm U U >um U “ma mum > > swuoacuon vm mo ya a m m U U U cflsomumo a U U. sod U sod Hows on lav coumesoo m U U U m so duo» » 3 nova oeumssooto a mg U U ya m yum U U U cacomozounmnfic VN U U U m U U U U s3ocxcs mmN U ya U m U U U U s3osxcs NNN U U U m U m > > s3ocxcs HNN U wa U m U ya x U s3osxcs haw U wa U m uma U U U szocxcs oau U mm 0H m oum U m> m> c3ocxcs mam U U U m omuo+> >mum >mum U Uflom canoesoolo mu o U U m u U U U canoouoo v U U >m mm so Hoooa U U cflumssoo m U U U m so How» >0 3 ofiom ofluoasoolo H 3 How and um: ozoo oozovmomx momz m uaflcm> nmooom momz zm mano «zoo ZN mz+ u cowuoofiwaucoofl .oc o>wuouco9 poom unmwa oanfimfi> unmfla >5 n.o.&oumouofiou£o Hoomo Hocoamcosflonozu o co ponomumoumeouso poo comma nouomn .noamnoz can oumuuoz mo xuon noon scum oouoonuxo monsooeoo oaaocono mo mcowuooou MOHOU .MH oanme Table 76 14. Rf values and relative concentrations of phenolic compounds extracted from Mazzard and Mahaleb root bark, hydrolyzed and chromatographed on two- dimensional paper chromatograms.a Rf value Spot Tentative Relative No. identification BAW (6:1:2) 2% HOAc concentration Mazzard 1 o-coumaric acid .90 .49 +++ 3 coumarin .90 .66 + 4 catechin .64 .42 + Z4 dihydrowogonin .91 .16-.38 +++++++++++ 25 p-coumaric acid .89 .40 ++ 215 unknown .89 .05 ++++ 216 unknown .77 .32 + 217 unknown .70 .67 + Z21 unknown .89 .57 + 222 unknown .75 .51 + 223 unknown .71 .40 + Mahaleb l o-coumaric acid .93 .42 + 3 coumarin .92 .72 +++ 4 catechin .61 .40 +++ H4 herniarin .91 .52 ++++++++++++ H6 unknown .80 .50 + H7 unknown .82 .62 + H8 unknown .89 .06 + H9 unknown .70 .60 + H10 unknown .89 .60 + H13 unknown .49 .30 + aSee Table 7 for notation and chromatographic con— ditions. 77 methylation, silylation was employed. Different columns and a wide range of temperatures were used, but no response could be demonstrated by this method. Figure 7 shows a trace obtained with the methyl esters of the standard phenolic compounds. Five compounds were chromatographed successfully. Coumarin was eluted first, followed by o-coumaric acid, p-coumaric acid, caffeic acid, and herniarin. Two peaks of different concentration were observed with o-coumaric acid, with p-coumaric acid, and with caffeic acid. This probably was due to impurities or to incomplete methylation. Typical gas chromatograms for leaf, stem bark, and root bark extracts are shown in Figures 8, 9, and 10. Retention times and concentrations of phenolic compounds are given in Table 15. o-Coumaric acid occurred in all tissues of both rootstocks, and similar levels were present in both. Leaves contained higher levels of the compound than did the bark tissues. p-Coumaric acid occurred in all Mazzard tissues tested, but the level of the compound was much higher in the bark than in the leaves. Coumarin was present in all the tissues tested. In stem and root bark, Mahaleb con- tained 8 to 10 times more coumarin than did Mazzard. Caffeic acid was detectable only in Mazzard leaves. The level was 0.ll mg per gram fresh weight of leaves. Her- niarin occurred only in Mahaleb tissues. As in the case Figure 7. 78 Gas liquid chromatogram of a mixture of stan- dard phenolic compounds. Compounds were methylated before chromatography on 2% QF-l on Chromasorb W. Column temperature was 160°C. Coum = coumarin; O-C o-coumaric acid; p-C = p-coumaric acid; Caf caffeic acid; Her = herniarin. 79 Mixture of Standd rd O-C A Phenolic Compds Comm 1 Z A A I A f V f V ' v I a 3 4 Minutes 80 Figure 8. Gas liquid chromatograms of hydrolyzed phenolic compounds extracted from Mazzard and Mahaleb leaves. Compounds were methylated before chrom- atography on 2% QF-l on Chromasorb W. Column temperature was 160°C. 1 = coumarin; 2 = o-coumaric acid; 3 = p-coumaric acid; 4 = caffeic acid; 5 = herniarin. ‘ 81 Mazzard Leaf I h Mahaleb leaf Figure 9. 82 Gas liquid chromatograms of hydrolyzed phenolic compounds extracted from Mazzard and Mahaleb stem bark. Compounds were methylated before chromatography on 2% QF-l on Chromasorb W. Column temperature was 160°C. 1 = coumarin; 2 = o-coumaric acid; 3 = p- coumaric acid; 4 = caffeic acid; 5 = he niarin. Mazzard Bark 83 Mahaleb Bark Figure 10. 84 Gas liquid chromatograms of hydrolyzed phe- nolic compounds extracted from Mazzard and Mahaleb root bark. Compounds were methylated before chromatography on 2% QF-l on Chroma- sorb W. Column temperature was 160°C. 1 = coumarin; 2 = o-coumaric acid; 3 = p- coumaric acid; 4 = caffeic acid; 5 = herniarin. Mazzard R009 85 d Maha leb Root 86 Table 15. Retention times and concentrations (mg/g fresh weight) of phenolic compounds extracted from leaves, bark, and root bark of Mazzard and Mahaleb seedlings as determined by gas liquid chromatography.avb Retention Leaf Stem bark Root bark time(min) Compoundc Maz Mah Maz Mah Maz Mah coumarin .86 .76 .76 .08 .64 .06 .52 o-coumaric acid 1.24 .21 .22 .06 .08 .08 .06 p-coumaric acid 1.61 .13 - .28 - .26 - caffeic acid 2.80 .11 - - -' - - herniarin 3.10 - .44 - .50 - .38 aAverage of two determinations. b2% QF-l on Chromasorb W, column temperature 160°C and flow rate 40 ml/min. cCompounds were methylated before chromatography. 87 of coumarin, all the tissues tested contained a high level of the compound. Mahaleb stem bark had the highest level with 0.5 mg per gram fresh weight followed by leaves with 0.44 mg and root bark with 0.38 mg. Table 16 sum- marizes the hydrolyzed phenolic compounds occurring in leaf, bark, and root bark tissues of Mazzard and Mahaleb seedlings. III. Identification and Relative Amounts of Phenolic Compounds in Centrifugal Sap A. Ether fraction The ether fraction of the sap showed several phenolic substances (Fig. 11). In Mazzard, ten spots appeared on two-dimensional paper chromatograms. Four of these (3, 5, 6, 7) had the same color characteristics and Rf values as compounds occurring in Mahaleb extracts. Compound 3 was readily identified as coumarin on the basis of color reactions and Rf values. Compounds 5, 6, and 7 gave strong color reactions with FeCl -K Fe(CN) however, 33 6’ they were not identified. Spot 24, which reacted strongly with both DPNA and FeCl3-K3Fe(CN)6 reagents, had color characteristics and Rf values identical with those of authentic dihydrowogonin. In Mahaleb, among 7 spots on paper chromatograms, only three spots appeared to be specific to Mahaleb extracts (H4, H14, and H15). Compound H4, one of the prominent spots, had the same color reactions and Rf 88 Table 16. Comparison of hydrolyzed phenolic compounds in extracts of leaf, stem bark, and root bark of Mazzard and Mahaleb seedlings. Leaf Stem bark Root bark Sggt Compound ° Maz Mah Maz Mah Maz Mah 1 o-coumaric acid + + + + + + 2 kaempferol + + — _ - _ 3 coumarin + + + + + + 4 catechin + - + + + + 24 dihydrowogonin + - + - + _ 25 p-coumaric acid + - + — + - 26 caffeic acid + - — _ _ _ Z7 quercetin + - — _ - _ 28 chlorogenic acid + - — — _ _ 29 unknown + - + _ _ _ 210 unknown + - — _ - - le unknown + - — _ _ _ 212 unknown + - — - _ - 213 unknown + - — - - _ 214 unknown + - — _ _ - 215. unknown - — + _ + _ 216 unknown - - + - + _ 217 unknown - - + - + _ 218 unknown - - + - _ - 219 unknown - - + _ _ _ 220 unknown - - + _ - _ 221 unknown - - — _ + - 222 unknown - - — _ + _ 223 unknown - — - _ + _ H4 unknown - + — + _ + H5 unknown - + - - _ H6 unknown - + — + — + H7 unknown - + — + - + H8 unknown - - - + _ + H9 unknown - - — + - + H10 unknown - - — + - + H11 unknown — — _ + _ _ H12 unknown - - _ + _ - H13 unknown - - - - _ + 89 .CHmHHo onn um pouuoom moz AnoHonoz CH 0 m.m UCo UHoNNmz CH uano3 nmoum .m «.0 on unoH |m>quov uomuuxo H: OOH .mxooumuoou noon on COEEoo oHoB muoom ooomnm .moCHH Coxoun nUHB ooxHoE muoom Con» munsoeo Homuoa CH uComouo oHoB moCHH UHHOm nuHB ooxume muoom one .Umon wm zuHs Homecomm 6cm AmuHuoU 3mm nqu nmnflo poooHo>oU ouo3 mEoumouoEonno one .muomuuxo oom nonnoz UCm Unonnmz mo CoHuoonm Honpo on» CH mUCsooEoo UHHOCono mo mEonoumEouno Hoomo .HH onsmHo 42.2.32 .umrpu.aH > m nmvowum 0 U czocxcs «Hm U > oo U mum >um >um cHuchnmn em U U U m U U U C30Csz e U U U m you w >H C30Csz o um » mm mum m 3m 3m Czocxcs m U CoH soH U wepm U U CHuoEsoo m a U U U U M U U CzoCsz mNN I U U U ma > > C30CxC5 BNN I U U mo U mum U a3ocxcs own I U mH mm U U U C30Csz mNN I U U m U U a Czocxcs own I »m w m e U U CHComozouoonHo vN U U U m U U U CzoCsz e U U U m U o >H CzoCsz 0 mm » em mum U 3m 3m C3OCxC5 m I now U U me U U CHHoEsoo m ououumz mcaoaucom «zoo+ «zoo onzovoomx momz mmz+ I . . mooz ZN +MHUoo ZN COHuooHUHuCoUH .OC o>HuouCoe poom uanH oHnHmH> uanH >D «.munomoou UHHOCono nuH3 oomoumm nno mamumoumsouno Moooo HoCoHUCoEHUIozu Co nonooumouoeouno ouoz monsoosoo one .uoouuxo oom noHonoz UCo vuouuoz mo COHuumum uonuo onu CH monsooeoo oHHOConm mo mCOHuoooH HOHOU .eH oHnme 93 Table 18. Rf values of phenolic compounds in the ether fraction of Mazzard and Mahaleb sap extracts on a two-dimensional paper chromatogram.a Spot Tentative Solvent No. identification BAW (6:1:2) 2% HOAc Mazzard 3 coumarin 0.86 0.70 5 unknown 0.93 0.00 6 unknown 0.10-0.30 0.00 7 unknown 0.00 0.00-0.70 24 dihydrowogonin 0.90 0.00-0.30 224 unknown 0.85 0.00 225 unknown 0.89 0.04 226 unknown 0.88 0.51 227 unknown 0.58 0.87 228 unknown 0.70 0.70 Mahaleb 3 coumarin 0.87 0.68 5 unknown 0.92 0.00 6 unknown 0.22 0.00 7 unknown 0.00 0.00-0.57 H4 herniarin 0.87 0.53 H14 unknown 0.86 0.64 H15 unknown 0.87 0.72 aSee Table 7 for notations and chromatographic con- ditions. 94 Table 19. Retention times and concentrations of coumarins extracted from centrifugal sap of Mazzard and Mahaleb as determined by gas liquid chroma- tography.a Retention time (min) Relative Compound concentration 2% ov-1 1% 53-30 (mg/g f.w.) Mazzard sap Peak 3 3.7 - - Mahaleb sap Peak 1 0.2 1.3 11 Peak 2 0.5 3.4 24 Standard compound Coumarin 0.2 1.3 Herniarin 0.5 3.4 Column temp. (°C) 220 180 Detector and injector temp. (°C) 250 250 N flow rate (ml /min) 4 o 80 aAverage of 2 determinations. 95 phloem tissues. The reported presence of hydrolytic enzymes in xylem sap could result in accumulation of free aglycones (Sheldrake and Northcote, 1968). B. Neutral and acid ethyl acetate fractions After extraction with ether, the sap was further extracted with ethyl acetate without changing the pH. The aqueous layer was then acidified and again extracted with ethyl acetate. The content of phenolic compounds was essentially the same in all 3 fractions. In Mazzard, one additional spot (229) appeared in the neutral fraction (Fig. 12, Table 20) and 4 more in the acid fraction (Fig. 13, Table 20). In Mahaleb, Spot H16 occurred in the acid fraction (Fig. 13, Table 20). Most of the dihydrowogonin, coumarin, and herniarin appeared in the ether fraction. However, spots 5, 6, 7, H14, H15 occurred in all three fractions. The occurrence of phenolic compounds in Mazzard and Mahaleb sap supports the theory that they can be trans- located. Phenolic compounds are thought to be metaboli- cally inert, non-translocatable secondary metabolites, which are synthesized in situ_from non-aromatic pre- cursors such as carbohydrates (Hillis and Cale, 1962; Bate-Smith, 1962). This may be true for certain compounds which are limited to specific tissues. For example, anthocyanin appears in petals and fruit tissues but may 96 .CHmHHo onu no oouuoom mos Anonnoz CH 0 m.m UCo UHMNNMZ CH uano3 nmoum m v.w on pCoH Io>quov uomunxo H: 00H .mxooumuoou nuon on CoEEoo ouoB muoom Uooonm .moCHH Conoun nUH3 ponHmE muoom Conn munsoeo HomHoH CH uComoHo oHo3 moCHH UHHow nuH3 ooxuofi muoom one .ooom wm nufiz oaecoomm 62m Amnoumv zam zoos umuHo ooooHo>oo ouo3 mEonouoEouno one .muoouuxo oom nonnmz UCo puouuoz mo COHuoon HouusoC onu CH monsooeoo UHHOCono mo mEonouoEOHno Hoomo .NH oHCwHo 97 2: 3: 00@ 322.22 AH<¢thZV a uanH >9 ccsooeoo o.pouuHEo ouo3 CoHuooum Honuo on» CH oohusooo uonu mUCCooEOU .omm noHonoz UCo ououuoz mo mCoHuoon ououoom Hanuo UHCHom UCo HouusoC CH mCHHHCOUO mUCCooEoo UHHOCono mo mCoHuoooH HOHOU .om oHnoe 99 .CHmHHo onu um vouuoom mo3 AnoHonoz CH 0 m.m Unouuoz CH uanoB nmoum o v.m ou uCoH Im>quov uomuuxo H: 00H .mxooumuoou nuon ou CoEEoo ono3 muoom pooonm .woCHH Coxoun nUH3 Conuoa muoom Con» mquoEo HomHoH CH uComouo oHoB moCHH UHHom nuHB ponuos muoom one .ooom mm nqu oawcoomm can Amuaumc 3mm nqu pmuHm CoooHo>o© ono3 mfionmouoeouno one .muoouuxo oom nonnoz UCo onouuoz mo COHuomum oHoo onu CH moCsooEoo UHHOCono mo mfioumouofiouno Hoooo .ma musmflo 100 u<02 and .‘. 2 : nu..." ...® 0 ® 32.2.22 91 .O_U<. astov xnmn Eoum ouonuoz mo pomuuxo H: OH .nnonnozv n no Announozv N nuHB Coxume oum moHooom oCo ou pouoHHumoH muoom .moHooom nuon on COEEoo ouo3 muoom poomnm .moCHH Coxoun nuHB ConHoE muoom Conn omCouCH oHoE oHoB moCHH UHHom nqu Conume muoom one .040: mm nuH3 meCooom UCm ANHHHUV Sdm nuHs umuHm poooHo>oo ouoB mEoumoumEonno one .nuon Eoum nonnmz CCm UHoNNoZ CH monsooeoo UHHOCono UonHoupmnCd mo mEonouoEouno Hoooo .VH ousmHo 104 ® .. a. 2:0 % 2:. notes—us. _ iguana—2 ¥¢H5oov poonnxo H: mH .nnoHonmzv n no nononumzv N nqu ooxnoE ono moHooom oCo on oouoHuumoH muoom .moHooom noon on COEEoo onoz mooom nooonm .omom mm nuns sancoomm cam ANHHHGU zam nqu umHHm oooOHo>op onoB mEonouoEonno one .xnon uoou noHonoz oCo ouounoz CH moCsooEoo UHHOCono pouoHouomnCs mo meonmonmEonno Hoooo .mH onCmHo 110 Up... 2.00 £02930: uHCooU uoouuxo H; om .oaom mm nuns snocoomm was Amunuov zam nuHS umnHm ooooHo>oo ono3 mEonoumEounu one .mosmmHu mmoH nonnoz oCo possum: CH mpCsooEoo UHHOCono oonHonphnCs mo mEonmouoEOHno Hoooo .GH museHo 113 (p: 2.00 322.22 u COHuoonHuCooH .02 mm “Bonn mHnnmn> sauna >3 m>numucma noon HGUGD GOMHUflmh HOHOU m.oouuHEo ono3 nnon Boom CH mCHHHCooo muoom .muoonuxo mooH noHonoz oCo ouonnmz CH mCHHHsooo mUCCooEoo OHHOCono wouwHOHomnCs mo mosHo> mo oCo mCOHuoooH HOHOU .qm oHnoe 115 Table 25. Comparison of unhydrolyzed phenolic compounds in extracts of leaf, stem bark, and root bark of Mazzard and Mahaleb. Mazzard ’ Mahaleb 5:3? Compound Stem Root Stem Root Leaf bark bark Leaf bark bark 1 unknown - + + - + + 2 unknown - + - - + _ 3 unknown - + + - + _ 4 unknown - + + - + - 5 unknown - + - - + - 6 epi-catechin + + + — + + 7 d-catechin + + + - + + 8 leucoanthocyanidin - + + - + + 9 leucoanthocyanidin - + + — + + 10 leucoanthocyanidin - + + - + + 211 unknown - + + - _ _ 212 unknown - + + _ - _ 213 unknown - + + - _ - 214 chlorogenic acid - + - - - - ZlS chlorogenic acid + + - - - - 216 chlorogenic acid + + - - - _ 217 chlorogenic acid - + + - - - 218 unknown - + - - _ - 219 p-coumarquuinic acid + + - — — _ 220 p-coumarquuinic acid - + + _ _ _ 221 unknown - + + _ _ _ 222 unknown - + + _ - - 223 unknown - + - _ _ _ 224 unknown — _ + - _ _ 225 unknown - — + - - _ 226 unknown + - - - - _ 227 unknown + _ - - - _ 228 caffeic acid + - - _ - - 229 unknown + _ - _ _ _ 230 unknown + _ - _ _ _ 231 unknown + - - — _ - H11 unknown - - - - + + H12 leucoanthocyanidin - - - _ + + H13 unknown - - — - + + H14 unknown - - - + + + H15 herniarin - - - + + + H16 unknown - - - + + + H17 unknown - - — + + + H18 coumarin - - - ? + ? H19 unknown - - - _ _ + H20 unknown — - _ + _ _ H21 unknown - - - + _ _ H22 unknown - - - + _ _ 116 widespread in their occurrence. For example, all Mazzard tissues sampled contained chlorogenic acid, p-coumaryl quinic acid, catechin, and epi-catechin. The Mahaleb tissues shared herniarin and possibly coumarin, and 3 other compounds (H14, H16, H17). The differences noted between leaf and bark tissues indicate that all the phe- nolic compounds found in bark tissues are not translocated from the leaves. However, some of the phenolic compounds may be translocated, especially coumarin and herniarin, and possibly p-coumarquuinic acid and chlorogenic acid. Table 26 summarizes the phenolic compounds identi- fied from the studies of sections II, III, and IV. 117 .noHono: u no: mononuo: u no:o CHUHComoonuCoooCoH CHnoouooIHoo CHnoouooIo CHuoonoCo HonomoEoon CHComo3onomnHo I I +-+-+ +-+-+ +-+-+ I+-+-+ I I I I I I I + I +-++-+-+I I + I + I + I moHOCo>oHo + I + I + I + I CHnoHCnon + I + + + + + + CHnoEsoo mCHnoECOU oHoo UHComonoHno oHoo UHCHCUHmnoECOUIo oHoo UHomnoo oHoo OHnoECooIo oHoo UHnoECooIo I I I +-+ I I I II II +-+I II +-+I +~+ I +-++-+-+ moHoo UHHOCono no: No: no: No: no: no: ono: oNo: omm xnmn boom xnmn Coum noon monsoosoo .mmCHHUoom noHono: oCo ononno: mo nnon noon oCo .xnon Eoum .nooH no muoonnxo CH moCsooEoo UHHOCono C30Cx mo COmHnooEOU .mm oHnoe DISCUSSION Mahaleb rootstocks, which show graft-incompatibility symptoms with certain sweet cherry cultivars, differ in phenolic composition from Mazzard rootstocks, which are fully compatible with sweet cherries. The former con- tained lower levels of phenolic compounds in their leaves, root bark, and stem bark (Fig. 3). These differences have implications when sweet cherries are grafted on Mahaleb rootstocks. Mahaleb tissues may not have an efficient system for handling large quantities of phe— nolics, which could interfere with physiological pro- cesses (Mayer g£_al., 1961; Goodwin and Taves, 1950; Stenlid, 1963; Frey-Wyssling, 1942). Mahaleb roots may be more sensitive to sweet cherry phenolics, which might accumulate in the soil as a result of leaching or decom- position of leaf tissues. There are also qualitative differences in phenolic compounds in the two rootstocks (Tables 16, 21, 24, 25, and 26). Mazzard tissues contain a greater variety of phenolics. For example, in unhydrolyzed tissues 31 com- pounds were present in Mazzard as determined by paper 118 119 chromatography, but only 22 in Mahaleb. Qualitative dif- ferences were particularly noted in phenolic acids, coumarins, and flavonoids (Table 26). Phenolic acids. Mazzard tissues contained high levels of hydroxy cinnamic acid derivatives, including p-coumaric, o-coumaric, caffeic, p-coumaryl quinic, and chlorogenic acids, whereas Mahaleb tissues contained only o-coumaric acid. In his survey of leaf phenolics in Prunus, Bate-Smith (1961b) found p-coumaric, o-coumaric, and caffeic acids in Mazzard. In Mahaleb, o-coumaric acid was the only phenolic acids identified, although the presence of p-coumaric acid was suspected. The two rootstocks may have different biosynthetic scheme of phenolic acids by differing in their hydroxyl- ation pattern of cinnamic acid, which is known to be an important precursor of phenolic acids in higher plants (Steck, 1968; Neish, 1964). Mahaleb tissues, which con- tain large amounts of coumarin and herniarin, apparently hydroxylate only the ortho position of cinnamic acid, hence p-coumaric and caffeic acids are lacking. Mazzard tissues, however, appear to be able to hydroxylate both the ortho and the para position of the benzene ring of cinnamic acid. The esters of p-coumaric and caffeic acids may regulate levels of IAA in plant tissues by influencing its decarboxylation. Tomaszewski and Thimann (1966) 120 showed that polyphenols such as chlorogenic acid and caffeic acid reduced the inactivation of IAA, whereas monophenols such as p-hydroxy benzoic acid and p-coumaric acid increased the inactivation. In Mazzard, caffeic acid was mainly located in leaf tissues, whereas p-coumaric acid was high in stem and root bark and low in leaf tissue. Thus, translocation of p-coumaric acid from a sweet cherry scion to a Mahaleb stock could con- ceivably favor decarboxylation of IAA and thereby reduce the vigor of the rootstock. Coumarins. The two rootstocks differed markedly in coumarins. All Mahaleb tissues tested contained high levels of both coumarin and herniarin, whereas Mazzard contained no herniarin and showed high coumarin activity only in leaf tissues. Favre-Bonvin §t_al, (1966) con- sidered that P. aging was incapable of synthesizing cou— marin. However, o-coumaric acid can be easily transformed to coumarin in acid solution or on exposure to light (Bate-Smith, 1961b; Edwards and Stoker, 1968). Therefore, the coumarin observed in Mazzard hydrozates may be an artifact. The inhibitory activity of coumarins in root growth, in germination and in mitosis (Goodwin and Taves, 1950; Thimann and Bonner, 1949; Mayer and Poljakoff-Mayber, 1961) suggest a role for these compounds in the 121 graft-incompatibility of P. aziEm/P, mahaleb. This is unlikely, however, for the following reasons. The major sites of synthesis of coumarins are the aerial parts, especially the young leaves (Gorz and Haskins, 1962; Favre-Bonvin g£_al., 1966, 1968). After the fourth year of grafting of E. azium on P, mahaleb, Favre-Bonvin e£_al. (1966) noted the complete disappearance of coumarins from the stocks. During this period the trees grow vigorously with no symptoms of incompatibility (Garner, 1967). At low concentrations, coumarins stimulate plant growth (Mayer and Poljakoff-Mayber, 1961). Instead of being inhibitory, they might be essential for normal growth. Their disappearance from root tissues after several years may be related to the loss of vigor of the tree. Coumarin derivatives such as scopolin and scopoletin are known to inhibit IAA-oxidase (Andreae, 1952; Gortner and Kent, 1958; Schaeffer gt;313, 1967). Thus, the absence of coumarins in Mahaleb rootstocks may allow the destruction of IAA. Alternatively, their absence may limit lignin synthesis, leading to incompati— bility. Kosuge and Conn (1961) reported that labelled coumarin was rapidly transformed into B-glucosides of o-hydroxy cinnamic acids. The latter are known to be incorporated into lignin (Neish, 1964; El-Basyouni g£_31., 1966). 122 Flavonoids. Mazzard tissues contain three flavo- noids, dihydrowogonin, kaempferol, and quercetin. The latter two are found only in leaf tissues. Kaempferol also occurs in Mahaleb leaf tissues, but quercetin was not found. Bate-Smith (1961b) found both quercetin and kaempferol in hydrolysates of Mahaleb leaves. The con- centration of quercetin in leaves of the Mahaleb seedlings used in the present study may have been too low to be detectable, or occurrence may depend upon tree age. Dihydrowogonin occurred in all Mazzard tissues tested, as well as in sap. Mature cherry trees are known to contain various other flavonoids in the heartwood (see Table 1). None of these compounds was noted in the extracts of either bark or leaf tissues of Mazzard and Mahaleb seedlings. Flavonoids act as synergists or antagonists to IAA in_zi2£g. Kaempferol conjugates promote IAA oxidation while quercetin conjugates inhibit its oxidation (Furuya and Galston, 1965). The relatively high amount of kaemp- ferol in Mahaleb suggests that it may have a higher rate of decarboxylation of IAA than Mazzard. The activity of flavonoids in IAA decarboxylation appears to depend on the hydroxylation pattern in the B ring. The effect of dihydrowogonin on IAA oxidase is difficult to estimate because of the lack of a hydroxyl group in the B ring. 123 However, the wide occurrence of the compound in Mazzard tissue and its presence in the sap suggest that it may be important in graft-incompatibility. SUMMARY AND CONCLUSIONS Phenolic compounds in leaf, bark, and root tissues from Mazzard and Mahaleb seedlings were examined. In field-grown seedlings, Mazzard tissues contained much higher levels of total phenolic compounds than did the Mahaleb seedlings during a five-month growing period. The phenolic level increased during the first two months, then stabilized for the remaining period. However, the Mahaleb root tissue varied during the season, in that the phenolic content rose rapidly at beginning of growth, then dropped sharply during the latter part of the season. Furthermore, the bark tissues contained higher levels of phenolic compounds than did the leaf tissues. These facts apparently relate to the imperfect graft of some sweet cherry cultivars on Mahaleb rootstocks. Since sweet cherry cultivars are closely related to Maz- zard, and since Mazzard contained 3 times as much total phenolic compounds than Mahaleb, it can be proposed that (l) the abnormal amounts of phenolics in sweet cherry species are toxic to the Mahaleb, (2) that the phenolics translocate across the graft union from sweet cherry to 124 125 the Mahaleb or (3) the phenolic balance between these 2 species interferes with normal growth. In summary it also should be noted that the levels of phenolic compounds were consistently higher in Mazzard than in Mahaleb tissues when grown under greenhouse con- ditions. Furthermore, the greenhouse-grown seedlings contained only 10% as much total phenolics as the field- grown seedlings. Thus, this data strongly indicates that, the cli- matic conditions under which the trees are grown play an important role in the normal synthesis of phenolic com- pounds both in the rootstock and in the scion. The so- called incompatibility conditions have been observed to be more prevalent in some fruit-growing areas than in others. Variable amounts of phenolic compounds were found in the different tissues of Mazzard and Mahaleb. For example, dihydrowogonin was found in all the tissues tested of Mazzard, but not in Mahaleb; herniarin was in all tissues tested of Mahaleb, but none in Mazzard; and caffeic acid was found only in the Mazzard leaf tissue. As in the application of balanced nutrient require- ments of plants, so must there apparently also be an internal balance of phenolic components in the 2 plants which are combined by grafting. SUGGESTIONS FOR FURTHER STUDY OF GRAFT RELATIONSHIPS IN CHERRY CLONES Seasonal levels of phenolic compounds in the phloem sap of sweet cherry/Mahaleb; The effect of dihydrowogonin and p-coumaric acid on cambial activity of Mahaleb stem; Differences in phenolic compounds of Mahaleb clones, compatible and incompatible; The effects of soil accumulated phenolic com- pounds on the growth of Mahaleb root; Nature of metabolites in phloem above and below graft union; The effect of reproductive activity on the dis- tribution of metabolites between two compatible and incompatible rootstocks; Extensive field survey of the behavior of sweet cherry/Mahaleb in relation to factors such as soil drainage and climactic conditions. 126 127 8. The relationship between the loss of synthetic ability of coumarins and the vigor of Mahaleb roots; 9. The biological effects of dihydrowogonin on IAA oxidase, root growth, germination, and stem growth; 10. Graft-incompatibility of sweet cherry/Mahaleb may be due to either lack of metabolites or toxic effects of one of the graft partners: a. Toxic action of scion on stock, b. Toxic action of stock on scion, c. Lack of metabolites in scion for stock, c. Lack of metabolites in stock for scion. This could be studied with various graft combinations between stock and scion. LITERATURE CITED LITERATURE CITED Abdul-Wahab, A. S. and E. L. Rice. 1967. Plant inhibition by Johnson grass and its possible significance in old field succession. Bull. Torrey Bot. Club. 94:486-497. Amos, J., T. N. Hoblyn, R. J. Garner and A. W. Witt. 1936. Studies in incompatibility of stocks and scion. I. 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