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'4' ..I.. 1' II C'- iplh'hl' This is to certify that the thesis entitled Isolation, Identification, and Relationship to Sex ExPression of the Gibberellins of Cucumis melo and Cucumis sativus presented by DELBERT DEAN HEMPHILL, JR. has been accepted towards fulfillment of the requirements for Ph .1) 0 degree in Biochemistry M Mtge/UL. Maint- 01'an Date 5/12/71 0-7839 Masai-£15” ABSTRACT ISOLATION, IDENTIFICATION, AND RELATIONSHIP TO SEX EXPRESSION OF THE GIBBERELLINS OF CUCUMIS MELO AND CUCUMIS SATIVUS By Delbert Dean Hemphill Jr. Gibberellins and other phytohormones are known to alter sex expression in many plant species. Exogenously applied gibberellin preparations increase maleness in.Q. sativus, even inducing staminate flowers in homozygous gynoecious lines. In contrast, similar gibberellin treat- ments have no effect on sex expression in the closely related species 9. yelp. However, staminate flowers have been induced on gynoecious Q. 9219 plants by grafting melon scions onto either rootstocks or interstocks of several other species. Several lines of indirect evidence and a few experi- ments on endogenous gibberellin content have indicated that monoecious Q. sativus seedlings contain more gibberellin activity than gynoecious seedlings at the same growth stage. This investigation was undertaken to determine the nature and levels of endogenous gibberellins in a wide variety of Q. sativus and Q. 2319 sex types. Gibberellin activity was determined in.severa1 bioassays after either aqueous tmffer or organic solvent extraction of both free acidic Delbert Dean Hemphill Jr. and enzyme-releasable gibberellins. Mature dry seeds, etiolated seedlings, and green plants of each sex type at a wide range of growth stages were used. Monoecious and andromonoecious.§. sativus seeds, etiolated seedlings, and green plants were found to contain higher levels of free acidic and total (free acidic plus enzyme-released) gibberellin than did the gynoecious at all growth stages tested. In monoecious and andromonoecious (but not gynoecious) green plants, free gibberellin reached a maximum at the growth stage corresponding to flower initiation at the cotyledonary and first leaf axils. Vernalization of gynoecious seed resulted in staminate flower induction in plants produced from this seed; extracts of one week old seedlings contained increased amounts of gibberellin relative to non-vernalized controls. In contrast to Q. sativus, seeds and green plants of monoecious and andromonoecious lines of Q. gglg contained far less gibberellin activity than those of gynoecious and hermaphroditic lines. This finding is consistent with the failure of exogenous gibberellins to induce staminate flowers on gynoecious g. gglg plants. Seeds and leaves of two pumpkin varieties which, as rootstocks or interstocks, induce staminate flowers in gynoecious Q, mgl2.scions were found to contain less gibberellin than any 9. gglg sex type. Gibberellin.is hypothesized to be the I'rnale hormone" of Q. sativus but to play no role in _q. melo sex expression. Delbert Dean Hemphill Jr. The predominant gibberellin of Q. sativus was found to be A1 by thin-layer and gas-liquid chromatography and mass spectrometry. Small quantities of gibberellins A3, A“, and A7 are also present. The types and proportions of gibberellins did not vary with sex type. Gibberellins A1, A3, and A5 were identified in Q. mglg. A gibberellin Al-B-D-glucoside was isolated from the n-butanol fraction of an extract of monoecious g. sativus seed. This compound may account for a portion of the enzyme-releasable gibberellin activity in both seeds and seedlings. The n-propyl esters of gibberellins A1 and A3 were isolated from a neutral fraction of an extract of monoecious Q. sativus seed. The compounds were characterized by thin- layer chromatography and mass spectrometry. The identity of the A3 ester was confirmed through comparison of the isolated compound with an authentic sample: mass spectra, infrared spectra, melting points, and migrations in three solvent systems were identical. Growth promoting activity of the A3 ester was very low in several bioassays. ISOLATION, IDENTIFICATION, AND RELATIONSHIP To SEX EXPRESSION OF THE GIBBERELLINS OF OUQUNIS MELO AND CUCUMIS SATIVUS By Delbert Dean Hemphill Jr. A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1971 ACKNOWLEDGMENTS The author expresses appreciation to Professor Harold M. Sell for the suggestion of the problem and his advice and continued encouragement during the course of this work. To Dr. L. R. Baker, my thanks for his advice, aid in preparation of this thesis, and provision of plant material used in this work. Thanks are also due to my parents and my wife for their financial and moral support. The financial support of the Herman Frasch Founda- tion and the Department of Biochemistry is gratefully acknowledged. V 11 TABLE OF CONTENTS LIST OF TABLES . . . LIST OF FIGURES . . . LIST OF ABBREVIATIONS . INTRODUCTION . . . LITERATURE REVIEW . . Cucumber Genetics and Morphology Muskmelon Genetics and Morphology . Environmental Effects on Plant Sex Expression . Effects of Gibberellins and Anti-Gibberellins on Sex Expression . Effects of Auxins on Sex Expression Effects of Ethylene on Sex Expression . Z I Cytokinins and Sex Expression . . . Effects of Various Other Chemicals on Sex Expression . Effects of Grafting on Sex Expression . . . Occurrence of 'Bound' or 'Conjugated' Hormones in Plants . . . MATERIALS AND METHODS . Background of Plant Material 0 Growth, Care, and Harvesting of Plant Materials Gibberellin Extraction Procedures Purification of Gibberellins from Crude Extracts Bioassays . . Instrumental Techniques RBULTS O O O O O Gibberellin Activity in C. sativus: Etiolated Seedlings . Gibberellin Activity in C. sativus: grown Plants Gibberellin Activity and Sex Expression of Chilled C. sativus Seeds . Gifim MII nActivity in C. melo Seeds and Greenhouse- Gibberellin Activity in'Pum pEIn Seeds and Leaves 111 Page vii 10 10 12 14 16 22 27 33 35 40 40 41 an 48 53 59 66 Page Identification of Free Acidic Gibberellins in C. sativus . . . 83 Identification of Free Acidic Gibberellins in C. melo . . . 96 Identification of Neutral Fraction Gibberellins in C. sativus. BiologicaI Acti ivity of a Neutral Gibberellin from C. sativus . . . 121 Characterization of a Highly Polar Gibberellin from g. sativus . . . . . . . . . 127 . . . . 135 . . . . 1GB . . 106 DISCUSSION . . . . . . . BIBLIOGRAPHY . . . . . . . iv LIST OF TABLES Table Page 1. Gibberellin activity in phosphate buffer extracts of andromonoecious monoecious, and gynoecious C. sativus seed (dry). . . . 67 2. Gibberellin activity in phosphate buffer extracts of etiolated andromonoecious g. sativus seedlings. . . . . . . 69 3. Gibberellin activity in phosphate buffer extracts of 22 day old etiolated andromonoe- cious, monoecious, and gynoecious g. sativus seedlings . . . . . . . . . 69 4. Free acidic gibberellin activity in phosphate buffer extracts of greenhouse-grown monoecious, andromonoecious, and gynoecious Q. sativus plants . . . . . . . . . . 78 5. Flower sex types in chilled and control 9. sativus plants, monoecious and gynoecious lines 0 O O I O O O O O O 79 6. Effects of chilling of seeds on free acidic gibberellin activity in phosphate buffer extracts of monoecious and gynoecious lines or _C_. sativus e e o e e e e e 80 7. Free acidic gibberellin activity in methanol extracts of dry seed of four 9. melo sex types . . . . . . . . . . 82 8. Free acidic gibberellin activity in phosphate buffer extracts of four week old greenhouse- grown Q. melo plants of four sex types . . 82 9. Free acidic gibberellin activity in methanol extracts of seeds of two varieties of pumpkin and leaves of one variety . . . . . 82 10. Analysis of mass spectrum in Figure 13 . . 89 11. Analysis of mass spectrum in Figure 1“ . . 96 1'." .EJ Table 12. 13. 14. 15. 16. 17. 18. 19. Analysis of mass spectrum in Figure 15 . . Analysis of mass spectrum in Figure 20 . . Analysis of mass spectrum in Figure 21 (N1). TLC Rf values of CA3 n—prOpyl ester, N1, and N2 in three solvent systems. . . . . Analysis of mass spectrum in Figure 26 . . Analysis of mass spectrum in Figure 27 . . Biological activity of the GAB n-propyl ester (N1) from g. sativus . . . . . Genetics of Cueumis sex expression . . . vi Page 96 106 110 110 126 126 127 140 LIST OF FIGURES Figure 1. 2. 3. 5. 6. 7. 9. 10. 11. 12. 13. Structures of the thirty-two known free acidic gibberellins . . . . . . . . . Structures of known conjugated gibberellins and representative examples of other phytohormones and growth regulators. . . . . . . Flow sheet for organic solvent extraction of gibberellins from C. sativus and C. melo seeds and vegetative tissue. . . . . . . Flow sheet for phosphate buffer extraction of gibberellins from C. sativus and C. melo seeds and vegetative tissue. . . . . . . Standard curve for the dwarf pea bioassay. . Standard curves for the dwarf corn bioassay . Response to gibberellin standards in cucumber hypocotyl bioassay . . . . . . . Response to GA3 standards in the barley aleurone amylase bioassay . . . . . . . . Free acidic and total (free acidic plus enzyme- released) gibberellin activity in etiolated monoecious, andromonoecious, and gynoecious 9. sativus seedlings at several growth stages. Free acidic gibberellin content of greenhouse- grown monoecious, andromonoecious, and gynoecious Q. fiatiznfi plants at several growth stages. . Thin—layer chromatogram of gibberellin extract from 100 g of andromonoecious Q. sativus seed. Gas-liquid chromatogram of a methylated gibberel- lin extract of andromonoecious 9. sativus seed and a mixture of methylated gibberellin standards. Mass spectrum of Cl, a gibberellin—like compound isolated from andromonoecious Q. sativus seed. vii Page #6 50 56 58 61 63 72 76 88 91 Figure la. 15. 16. 17. 18. 19. 20. 21. 22. 23. 2h. 25. 26. 27. 28. 29. Mass spectrum of C2, a gibberellin-like compound isolated from andromonoecious Q. sativus seed. . Mass spectrum of C3, a gibberellin—like compound isolated from andromonoecious Q. sativus seed. . Thin-layer chromatogram of gibberellin extract of 100 g of gynoecious Q. melo seed . . . . Infrared spectrum of M1, a gibberellin—like com- pound isolated from gynoecious 9. melo seed . . Infrared spectrum of M2, a gibberellin—like com- pound isolated from gynoecious Q. melo seed . . Mass spectra of M1 and M2, gibberellin-like com- pounds isolated from gynoecious Q. melo seed . . Mass spectrum of M3, a gibberellin-like compound isolated from gynoecious Q. melo seed. . . . Thin-layer chromatogram of N1 and N2, neutral gibberellin-like compounds isolated from Q. sativus seed . . . . . . . . . . Mass spectrum of N1, a neutral gibberellin—like compound isolated from g. sativus seed . . . Mass spectrum of authentic gibberellin A3 n-propyl ester. 0 O O O 6 O O O O O 0 Infrared spectrum of N , a neutral gibberellin— like compound isolated from 9. sativus seed . . Infrared spectrum of authentic gibberellin A3 n-propyl ester. . . . . . . . . . Mass spectrum of TMS-N (first component), a neutral gibberellin-Ii e fraction isolated from g. sativus seed. 0 e o o o e e o 0 Mass spectrum of TMS-N (second component), a neutral gibberellin-Ii e fraction isolated from g. sativus seed . . . . . . . . . Thin-layer chromatogram of a highly polar acidic gibberellin (B1) isolated from g. sativus seed . Thin-layer chromatograms of the hydrolysis pro- ducts of a highly polar acidic gibberellin isolated from Q. sativus seed and several sugar 8 tandards o o o o o o e o o O 0 viii Page 93 95 98 101 103 105 108 112 114 116 118 120 123 125 129 131 Figure Page 30. Thin-layer chromatograms of the hydrolysis pro- ducts of a highly polar gibberellin isolated from g. sativus seed, D-glucose, and GAl. . . 133 ix LIST OF ABBREVIATIONS CCC (2-chloroethyl)-trimethylammonium chloride IAA B—indole-B—acetic acid NAA a-naphthaleneacetie acid 2,4-D 2,u-dichlorophenoxyacetic acid TIBA 2, 3, 5-triiodobenzoic acid Ethrel 2-chloroethy1phosphonic acid, also Ethephon ABA abscisic acid MH maleic hydrazide CAI, 0A2, :39. gibberellin A1, gibberellin A2 239. GA3 gibberellin A3 or gibberellic acid Am andromonoecious Gyn gynoecious TLC thin-layer chromatography GLO gas-liquid chromatography IR infrared IL-9 N,N-dimethylaminosuccinamic acid INTRODUCTION INTRODUCTION Sex expression in the cucurbits, including cucumber and muskmelon, is a complex characteristic, subject to many genetic and environmental influences. Since there are three types of flowers found in cucurbits (perfect, male, and female) and any one plant may bear any combination of these three, theoretically there are a total of seven possible sex types. Cultivated varieties of muskmelon are most often andromonoecious, bearing male and perfect flowers on the same plant. There are several genes conditioning sex expres- sion or flower sex type in both cucumber and muskmelon. In contrast to mammalian sex genes, they are not closely linked; each of the several genes segregates independently. Although the muskmelon and cucumber are closely related species of the same genus with a similar array of sex types, the genetics Of sex expression is different for each species. This is exhibited in divergent inheritance of sex-linked character- istics and in different responses to plant growth regulators. Several plant growth regulators, including some phyto- hormones, are known to affect plant sex expression when exogenously applied. Gibberellins, auxins, cytokinins, and ethylene are phytohormones which can alter plant sex expres- 81on, including that of the cucurbits. 2 3 The gibberellins exert myriad effects on plant metabolism and their occurrence in plants is probably ubiquitous. They appear to be involved in mitosis, cell enlargement, and cell differentiation. The gibberellins were first discovered as metabolites of the fungus Gibberella fujikuroi, the pathogen responsible for a foot-rot disease of rice characterized by excessive shoot elongation. The active substance isolated from the fungus, and later found to be a mixture of several structurally similar compounds, affected growth in many plants. Various gibberellins were subsequently identified as natural constituents of plants. To date, a total of thirty-two gibberellins, all sharing a common gibbane skeleton and exhibiting some biological activity, have been isolated from a wide variety of sources. The structures and occurrence of each of the first twenty-nine gibberellins have recently been reviewed by Lang (1970) and will not be dealt with here. Gibberellins A30 and A31 (Murofushi g§_§1., 1970) and A32 (Yamaguchi at al., 1970) were recently isolated from seeds of Calonyction aculeatum and peach, respectively. Many plants contain several of the gibberellins as well as gibberellin metabolites. Recently, naturally occurring gibberellin derivatives known as ”bound" gibberellins have been isolated, including several gibberellin-glucose com— Plexes. The structures of the thirty-two known gibberellins and the known “bound” gibberellins are presented in Figures 1 and 2. Gibberellins often exert a masculinizing effect on plant Se): expression. This is true for cucumber, but not for muskmelon. Heteroauxin (indoleacetic acid) was discovered as a promoter of cell elongation, but was soon found to affect cell division and cell differentiation as well. Auxins often feminize many plants including cucumber. The cytokinins were discovered as a factor occurring naturally in the vascular tissue of tobacco stems which promoted pith tissue cell divi- sion. Cytokinins have since been found in many species and are known to alter sex expression in several plants. Ethylene gas, considered by many physiologists to be a phytohormone, increases the numbers of female flowers in many species including cucumber and muskmelon. The structures of naturally occurring heteroauxin and cytokinin (zeatin) are seen in Figure 2. The study of the biochemical control of plant sex ex- pression is of commercial as well as theoretical interest. To control sex expression through genetic or chemical means would enable plant breeders to establish inbred lines for development of commercial Fl hybrids. These inbred lines would also be invaluable for research into the genetics and biochemistry of sex expression and the advantages of hybrids in agriculture are well known. Cucumber and muskmelon pre- sent a unique opportunity for study of the biochemistry of sex expression due to the establishment of genetically pure 111188 of different sex types. In spite of the close relation- ship between these species, important differences have been found which imply differences in the mechanism of sex expression in the two species. In cucumber, but not muskmelon, Figure 1 Structures of the thirty-two known free acidic gibberellins. [Gibbane skeleton numbering system ] 10 0'4 Inc £8. o»... x o: :6 ”xv .< n.( £8 ruse. on: U N m We: 3 Figure 2 Structures of known conjugated gibberellins and representative examples of other phytohormones and growth regulators. bNfiv owom owmwumnm nausea Iaamocmummoooamlnun Inlo 0 N6 m6 nahmocmummoosamlnu u Into ..Hmmocmummougmuan a Into sass owumomnmnmaoocw n40 Iahmocmuamoogminl a imlo 9 lgibberellins have been used to induce male flowers in female plants. Grafting of muskmelon scions onto pumpkin rootstocks can induce male flowers in female muskmelon, but this does not hold for female cucumber scions. Thus, it is of interest to determine the relationship, if any, between endogenous gib- ‘berellin activity and sex expression in the two species. The objectives of the research presented here were to quantitatively assay and identify the gibberellins occurring in a wide range of muskmelon and cucumber sex types atyvarious growth stages and to correlate the quantity or types of gib- berellins with the sex expression and genetic makeup of the plants. The principles guiding this research and the reasons for postulating that endogenous gibberellins are involved in male sex expression in cucumber are further elucidated in the following literature review. LITERATURE REVIEW A. Cucumber Genetics and Morphology Sex expression in the cucumber (Cueumis sativus L.) is marked by a wide variety of sex types. Monoeciousl, andro- monoecious, gynomonoecious, trimonoecious, androecious, gynoe- cious, and hermaphroditic lines either occur in the wild or have been established in breeding programs. However, Atsmon and Galun (1960) have established that pistillate, staminate, and perfect flowers all pass through a bisexual stage. Divergence is then accomplished by inhibition of development of pistil or stigma. The genetic control of these sex types is complex and the environment may also affect the sex type. Atsmon and Galun (1962) noted that when the female tendency of the cucumber is strong, floral buds develop adjacent to yOung leaves, the reverse being true for more male plants. They postulated that environmental and genetic factors which affect sex tendency change the leaf age-flower ‘bud.relationship and that this may be related to the hormonal 'balance in the vicinity of the developing floral primordium. lMOnoecious: staminate and pistillate flowers occurring on the' same plant; andromonoecious: staminate and perfect ¥ flohmers on the same plant; gynomonoecious: pistillate and Perfect on the same plant; trimonoecious: pistillate, per- fect: , and staminate on the same plant; androecious: staminate fltnvears only; gynoecious: pistillate flowers only; hermaphro- ditic: perfect (hermaphroditic) flowers only. 10 11 Several genes are known.to condition cucumber sex types. The first of these, denoted quy Rosa (1928), conditions the potentiality for pistillate flowers. When M is substituted by its homozygous recessive allele mm, bisexual flowers replace pistillate flowers. Galun (1961) concluded that allele 9 also increases the number of staminate flowers. Kubicki (1969d) concluded that M must act by inhibiting sta- men production while 9 conditions simultaneous development of pistils and stamens. Another set of genes governs the intensity of femaleness in monoecious and gynoecious cucumber plants. The alleles of this locus have been designated 3 and F (Tkaczenko, 1935), or g; and 33+ (Galun, 1961), or 393 and Ac}: (Shifriss, 1961a). Kubicki (1969a) discovered other alleles of this locus. The allele conditioning monoecism without a continuous pistillate stage was designated.gg§+, monoecism with continuous pis- 1 tillate stage acr , while gynoecious lines contain allele ggfiF. Kubicki concluded that the physiological action of the several alleles of locus 32; is connected with the quantity or‘activity of an.ovary-producing hormone. Another gene conditioning femaleness which segregates independently of 393 is designated 3 (Kubicki, 1969b). The dominant allele 2 conditions formation of a high number of Lpistillate nodes, while 2 governs formation of relatively fewer pistillate nodes in monoecious and gynoecious sex types. Kubicki (1969c) assumed androecism to be governed by a Single recessive gene _a_ which is independent of 2.25:. Andros- cious plants would then represent the genotype acr+acr+aa. IKZC sun Ira CIDU ‘Uidu ‘ iii “0)“ i.‘v“ Z°'c: in.“ aka up" 12 Kubicki (1969e) has also established that gene 2;, which conditions trimonoecism, acts through the control of superior ovary development. Bisexual flowers in trimonoe- cious plants differ from those occurring in hermaphroditic plants in both origin and structure. Trimonoecious perfect flowers are derived from modification of staminate flowers rather than replacing female flowers. These results permit the assumption that sex determina- tion in cucumber is influenced by two different groups of genes. One group, including loci 323, F, and A, controls the ratio of ovary-bearing flowers to staminate flowers. The second group, loci M and 23, controls floral structure, giving rise to epigynous (inferior ovary) and hypogynous (superior ovary) bisexual flowers, respectively. B. Muskmelon Genetics and Morphology The muskmelon (Cucumis melo L.) appears to have the potential for the same array of sex types as cucumber. How- ever, androecious and trimonoecious lines have not been reported. Perfect flowers are usually born singly on small side branches arising from the main stem or major branches. Staminate flowers usually occur in groups on main stem or lmador branch nodes (McGlasson and Pratt, 1963). Rosa (1928) found that monoecism in muskmelon is domin- ant over andromonoecism and this difference is conditioned by £1 single allelic pair. Poole and Grimball (1939) discovered hermaphroditic forms and proposed a two gene explanation of muskznelon sex expression: Ag is monoecious, _Ag gynomonoecious, 13 {fig andromonoecious, fig hermaphroditic. However, these authors included trimonoecious and gynoecious plants under the heading gynomonoecious. Kubicki (1969f) explored this problem and concluded that at least four sets of genes were involved. M (A) conditions formation Of pistillate flowers in monoecious plants, while recessive allele m (a) is associated with perfect flowers. Gene 9 leads to formation of staminate flowers in monoecious plants, while lines con- taining its homozygous recessive allele gg would have perfect flowers. Complementary genes 2:; and 2;; condition formation of bisexual flowers with superior ovaries. The dominant Egg greatly strengthens the effect of recessive Egg_in sti- mulating ovary development. Muskmelon is the closest relative to cucumber among the cultivated cucurbits and the similarity of sex types in these species is an example of this relationship. The gene pair Mm governs floral structure in both species as well as in watermelon. However, similarities in sex types need not indicate similar biological mechanisms. Gynoecism in cucumber is a partially dominant property, but gynoecism in muskmelon is totally recessive. This is presumably due to a difference in the dominance of sex genes in the two 8Pecies. This individuality in genetics of sex expression 18 also supported by differences in the effects of environ- ‘menatal factors and exogenously applied phytohormones on sex d1 fferentiation in cucumber and muskmelon. 14 C. Environmental Effects on Plant Sex Expression Environmental factors, including day-length, tempera- ture, moisture, and amounts of certain elements, are known to enhance or modify sex expression in a wide range Of plants. The HeslopuHarrisons (1957) found that carbon monoxide induced pistillate and perfect flowers in androecious plants. Czao (1957) reported that carbon monoxide treatment of cucumber seedlings increased the pistillate/staminate flower ratio while phytohormones, fertilizer, and high soil moisture also increased female flower development. Janick and Stevenson (1955) reported that high tempera- tures caused increased maleness in monoecious and some gynoecious spinach plants, whereas Heslop-Harrison (1957) concluded that, in general, low temperature favors pistil- late flower formation. Ito and Saito (1957a) reported that high night temperature favors staminate flower formation in Japanese cucumber, while Kooistra (1967) induced staminate flowers on predominately female cucumbers with high night temperatures. Bukovac and Wittwer (1961) found that low temperatures accelerated formation of pistillate flowers in pickling cucumber. Nitrogen levels also affect sex expression. Tibeau (1936) found that high nitrogen levels favor pistillate flowers in hemp and Thompson (1955) reported similar results ‘witli spinach. Tiedjens (1928) and Hall (1949), working ‘WitPl cucumber and gherkin, respectively, found that high nitrogen levels promoted pistillate flowers and low .L as ..l 15 nitrogen levels promoted staminate flower production. Brantley and warren (1960) concluded that high levels of nitrogen resulted in an increased proportion of perfect flowers in andromonoecious muskmelon. In contrast, Kooistra (1967) reported that high nitrogen favOred formation of sta- minate flowers in predominately female cucumbers. Another important environmental factor in sex expres- sion is photoperiod. Many plants specifically require long- or short-day conditions to induce flowering and pis- tillate/staminate ratios are often affected. Schaffner (1921, 1923, 1925) found that hemp plants underwent sex reversion under short-day (winter) conditions and that several other species including Humulus, Myrica and Plantggo also experienced sex reversions under the influence of an altered photoperiod. Thompson (1955) promoted pistillate flower formation in spinach under a long-day regime and andromonoecious muskmelons produced an increased proportion Of perfect flowers under long-days (Brantley and Warren, 1960). In contrast, Vergeley 22 al. (1967) promoted femaleness in.hemp with short days and Nitsch £2.21- (1952) reported that long days promoted male sex expression in gherkin and squash, while short days favored femaleness. Working with cucumber, Tiedjens (1928) and Ito and Saito (1957a) found that long days favor staminate flower Production. Atsmon gt _a_1_. (1967) found that continuous light inhibited pistillate flower formation in gynoecious 16 cucumber while staminate flowers in monoecious cucumber were inhibited by a twelve-hour light regime. Comparing these results with those produced by exogenous application of gibberellins, the authors suggest that continuous light inhibits femaleness through an effect On endogenous gib- berellins. At least in cucumber, it is clear that short days, low temperatures, and high soil moisture favor femaleness while conditions involving greater stress such as long days, high temperatures, and drought reduce female tendency. D. Effects of Gibberellins and Anti-Gibberellins on Sex Expression The effects of gibberellins on flowering often resemble effects of photoperiod, and there may be a relationship between long- and short-day regimes and levels of endogenous gibberellins. 1. Gibberellin-induced sex modifications in plants. Reports of sex modification in many plants under the influence of gibberellins have been widespread during the last decade. Interest particularly focused on the application of gibberellins to hemp and cucumber, and in the latter case, gibberellin-induced staminate flowers have been used to self-pollinate gynoecious plants. Atal (1959) first reported 8 sex-modifying effect of gibberellins on hemp, noting that seedlings of female plants treated with 100 ppm of gibberellin Produced staminate flowers. This result was confirmed by Verseley gt 9;. (1967). Davidyan (1967) found that l7 gibberellin treatment of 25 to 35 day old gynoecious hemp plants caused formation of monoecious plants. In the case of one variety, this change was transmitted to the progeny. Kutuzova (1968) also confirmed sex conversion in hemp, but a concentration of 0.03% gibberellic acid was needed for optimal results in inducing male flowers. 0n the other hand, Khryanin (1969) found that two applications of only 0.0025% gibberellin converted gynoecious hemp to monoecious. He concluded that gibberellin derepressed certain genes parti- cipating in flower formation. A scattering of results have also been reported for other species. Splittstoesser (1970) found that in monoecious Cucurbita moschata L., gibberellin increased the proportion of staminate flowers and delayed fruit-set. The masculinizing effect of gibberellin may be exerted in processes subsequent to flower initiation. Phatak gt al. (1966) reached this con- clusion after finding that addition of 25 ppm of the potassium salt of gibberellic acid to a root culture of a stamenless tomato mutant caused development of anthers and viable pollen. Glushchenko (1970) found that gibberellins applied to five hyacinth varieties resulted in increased growth and branching of pollen tubes. In contrast to these reports, Herich (1960) noted that application of gibberellin to hemp caused a slight increase 11! female tendency. In another case (Shifriss, 1961b) the Distillate/staminate flower ratio of monoecious caster beans was increased by a gibberellic acid spray. In addition, 18 Bose and Nitsch (1970) reported that GA inhibited male flower formation and weakly promoted feiale flower formation in £2223 acutangula. The effects of various gibberellins on sex expression of cucumber have been widely investigated. Most of this research has focused on commercial interest in the applica- tion of gibberellins to development of inbred gynoecious lines as hybrid parents. The earliest report on the effect of gibberellins was by Galun (1959a) who found that GA3 applied to young cucumber leaves altered sex tendency toward maleness in gynoecious and monoecious plants. For monoecious plants this effect was attributed to an increase in number of nodes preceding the first flower, thus shifting flowering to a normally staminate stage. Peterson and Anhder (1960) induced staminate flowers on the gynoecious line MSU 713-5 with GA3' Pykhtina (1968) found that spraying cucumber seedlings with 0.2% gibberellin increased both the total number and the staminate/pistillate ratio of flowers. How- ever, the high level of gibberellin used in this experiment appears to rule out an interpretation of the results as in- dicative of an endogenous role for gibberellin in staminate flower formation. Hayase and Tanaka (1966) reported that for the gynoecious line MSU 713-5, the younger the seedling tand.higher the gibberellin dosage, the larger the number of 8taminate flowers induced. Stambera and Zeman (1969) reported Silnilar results on staminate flower production and also in- hibition of pistillate flower initiation. 19 Differences in the activity of various gibberellins in inducing staminate flowers in cucumber have been noted by several authors. Bukovac and Wittwer (1961) found that GAu was about ten-fold more active than GA3 in induction of staminate flowers on monoecious and gynoecious plants. Hittwer and Bukovac (1962) then investigated the effects of all the then-known gibberellins on staminate flower formation in the gynoecious line MSU 713-5. They found that GA7 was the most active followed by A“, A2, and A9. GA8 was least active. These most active gibberellins all lack a 6-72 hydroxyl group and all gibberellins possessing a 0-? hydroxyl group are significantly less active. Pike and Peterson (1969) also found that a gibberellin Au/A7 mixture at 50 ppm was more effective in staminate flower induction than 0A3 at 1000 ppm and did not cause the excessive stem elonga- tion associated with CA3. Clark and Kenney (1969) found that GA13, which also lacks a C-7 hydroxyl, produced staminate flowers on a gynoecious cucumber but was less effective than gibberellins A“ and A7. In contrast to the masculinizing effect of gibberellins on cucumber, Peterson (1963) was unable to induce staminate flowers on the closely related species, gynoecious muskmelon, ‘with GAB. Kubicki (1966a) also failed to obtain male flower induction with GA3 in muskmelon and hypothesized (Kubicki, 1969g) that this failure is understandable since the * 2G1 bbane skeleton numbering system 20 differentiation of staminate flowers in muskmelon is governed by a gene other than that in cucumber. In summary, research with cucumber, hemp, and other species has demonstrated that exogenous application of gib— berellins Often, but not always, has a masculinizing effect on the treated plants. However, this is only the most in- direct evidence for a role, directly or indirectly, of gibberellins as the "male hormone" of these species. Other indirect evidence is found in studies which appear to cor- relate high gibberellin levels, long internodes, and increasing male tendency (Atsmon gt al., 1967). 2. Anti—gibberellins and sex expression in plants. Other evidence for a role of gibberellins in sex expres- sion comes from experiments with growth retardants which are either known or presumed inhibitors of gibberellin biosynthesis or action. Ota (1962) found that (2-bromoethyl)-trimethy1- ammonium bromide increased femaleness in cucumber while Mitchell and Wittwer (1962) noted that allyltrimethylammonium tromide induced increased formation of pistillate flowers in .monoecious cucumber plants. Hayase and Tanaka (1967) found 'that (Z-chloroethyl)-trimethylammonium chloride (CCC), a 81313131in inhibitor of gibberellin biosynthesis, did not affect gibberellin-induced staminate flower induction in cucumber. Tanaka _e_t §_J_._. (1970) reported that 000 and N,N-dimethy1- aIllinosuccinamic acid (B-9) shifted cucumber sex expression towuamd.femaleness, while Mishra and Pradhan (1970) found that CCC decreased staminate flower but increased pistillate 21 flower formation in cucumber. Other species have exhibited similar responses. CCC inhibits staminate flower production in squash (Abdel-Gawad and Ketellapper, 1968), while Phatak gt 31. (1966) determined that CCC induces abnormal pollen growth in tomato. In con- trast to the failure of exogenous gibberellins to induce staminate flowers on gynoecious or other sex types of musk— melon, Halevy and Rudich (1967) found that B-9 induced a female shift in andromonoecious muskmelon. Subsequently, Rudich 23 31. (1970) reported that B-9 applied to young muskmelon seedlings inhibited staminate flower production for the first two to three weeks of the flowering period. These results, contrasted with the results of gibberellin application, indicate that the role, if any, of gibberellin in muskmelon sex expression is indirect. 3. Endogenous gibberellins and sex expression. 'A more direct approach to the role of gibberellins in sex expression is to relate the endogenous content to sex type and study any differences in gibberellin metabolism in various sex types. Kamienska (1966) determined that the endogenous gibberellin-like activity of male poplar plants is greater than that of female poplars. Of direct interest here are the two studies made on levels of gibberellins in gynoecious and monoecious cucumber. 41:8mon gt 9;. (1968) used shoot diffusates from four day Old seedlings and root exudates from six week old plants. Al though different parts of the plant were assayed at the 22 two growth stages and there was no attempt to identify the endogenous gibberellins, the gibberellin levels in the monoe- cious line were significantly higher at both growth stages than in the gynoecious line. Recovery of applied 3H-GA1 from monoecious plants was also much greater than the recovery from gynoecious ones, indicating a relative gib- berellin destruction or inactivation in the gynoecious line. Hayashi 23.21- (1967, 1971) harvested etiolated cucumber seedlings of closely related monoecious and gynoecious lines at several growth stages over a period of zero to eighteen days. The gibberellin content of whole seedlings was deter- mined in three bioassays. The monoecious line contained significantly higher gibberellin levels at all growth stages than the gynoecious. GA1 was identified as the primary active component, although trace amounts of gibberellins A3, A4, and A7 were also present. Thus, the gibberellins most active in inducing male tendency when exogenously applied do not make up the bulk of the endogenous gibberellin activity. E. Effects of Auxins on Sex Expression 1. Sex modification by exogenous auxins. The phytohormone 3-indole-3-acetic acid (IAA) or hetero- auxin, like gibberellins, affects many phases of plant life. There have been numerous reports of effects of heteroauxin on :flowering. Those dealing particularly with sex expression in cucumber and other plants are reviewed here. Several 8VII-“IL".hetic auxins which have not been shown to occur in plants, but which induce auxin-like responses in bioassays, have 23 also been shown to affect plant sex expression. These include d-naphthaleneacetic acid (NAA) and 2,4-dichloro- phenoxyacetic acid (2,4-D). Wittwer and Hillyer (1954) found that spraying young seedlings of the curcurbit 'Table Queen' squash with NAA increased the pistillate/staminate flower ratio. Heslop- Harrison (1956) noted that application of NAA to the third node of young male hemp plants caused them to bear female flowers up to the thirteenth node. He postulated that flower sex type may be regulated by the auxin level at the time of flower primordia differentiation. The same author (1957) stated that, in general, exogenous auxins accelerate appearance Of female flowers and increase the numbers of female flowers in plants. Saito (1957) found that NAA at 10 ppm induced a female sex transition in normally male strobili of Japanese red pine and knack pine. Mallik gt a1. (1959) determined that 100 ppm of NAA applied to mango just previous to onset of flower budding caused a doubling in the number Of female flowers. NAA, 2,4-D, and p-chlorophenoxyacetic acid have been used to increase the pistillate/staminate ratio and total fruit set in the ribbed gourd (Satyanarayana and Rangaswami, 1959). Vergeley st 31. (1967) found that both IAA and NAA caused increased femaleness in hemp, reproducing the effect Of‘ short days. Bose and Nitsch (1970) reported that NAA Inrcamoted femaleness in Luffa acutangula, while Saeed and Akbar (1970) reported that IAA induced female flowers in watzermelon. 24 In contrast to these reports of a feminizing effect of auxins, Champault (1969) used IAA and NAA to produce staminate flowers on newly formed female inflorescences of Mercurialis aunnua. These staminate flowers were mor- phologically identical to genetically male flowers. As with gibberellins, much of the early work with auxins focused on application of the hormone to development of commercial breeding lines of cucumber. Laibach and Kribben (l950a,b) found that application of a 0.1% paste of either NAA, IAA, or 2,4-D to the leaf or petiole stump of young cucumbers caused increased numbers of pistillate flowers. Hittwer and Hillyer (1954) also found that spraying young cucumber seedlings with 100 ppm of NAA greatly in- creased the pistillate/staminate ratio of two cucumber varieties. Galun (1956) treated monoecious cucumber seeds with 10"3 to 10'”1 ppm of NAA, causing a reduction in the number of nodes preceding the first pistillate flower. vernalization of the seed had a similar effect. Ito and Saito (1956a,b) found that application of 2,4-D, NAA, or IAA to cucumber seedlings following cotyledon «expansion caused retardation of staminate flower formation and acceleration of pistillate flower formation. The same tauthors (1957b) also reported that 10 ppm of NAA increased Pietillate flowers even under environmental conditions favoring staminate flowers. Galun (1959b) also reported increased femaleness of cucumber due to NAA and found that 25 monoecious and gynoecious plants responded differently to removal of young leaves, whereas mature leaves of monoecious plants contain a larger content of auxin inhibitor than mature leaves of gynoecious plants. Choudhury and Phatak (1960) also found that IAA and NAA at 100 ppm increased the number of female flowers in cucumber, but that at lower con- centrations, IAA increased production of staminate flowers. Galun.g£ 21- (1962,1963) cultured young monoecious floral buds at a morphologically bisexual stage and supplied either IAA or GA3 in the medium. Ovaries developed only in IAA-supplemented media; GA3 reduced the IAA effect. In experiments with field-grown cucumbers, Kubicki (1966b) sprayed monoecious plants with NAA or gibberellin. NAA checked development of male flowers and stimulated that of female flowers. Gibberellin was found to inhibit pistillate flower development, and counteracted the NAA effect. Alvim and Quinones (1967) confirmed that NAA caused increased pistillate flowers in cucumber, but found no influence of NAA on flower morphology and development. The effects of auxins on cucumber discussed thus far have been on monoecious or gynoecious plants. However, .Kubicki (1969c) induced a few pistillate flowers in an androecious line with IAA and induced perfect flowers at the expense of staminate flowers in a trimonoecious line using NAA (Kubicki, l969e). In muskmelon, the results of auxin application on sex expression are not as clear. Brantley and Warren (1960) 26 found that under masculinizing long-day conditions, NAA decreased the number of both staminate and perfect flowers in andromonoecious muskmelon without inducing pistillate flowers and that NAA increased both staminate and perfect flowers under a short-day regime. Kubicki (l969f) noted that NAA induced more pistillate flowers in a monoecious muskmelon and more perfect flowers in an andromonoecious line, but these effects were attributed to NAA promotion of side shoots where these flowers occur. The author con- cluded that NAA had no specific influence peg as on sex organ differentiation in muskmelon. 2. Anti-auxins and sex expression in plants. Presumed inhibitors of auxin biosynthesis or action have also been found to affect flower sex expression, es- pecially 2,3,5-triiodobenzoic acid (TIBA). Laibach and Kribben (l950a) reported that TIBA had no effect on cucumber sex expression, while Hittwer and Hillyer (1954) found that 25 ppm of TIBA sprayed on cucumber seedlings greatly increased the pistillate/staminate ratio. Ito and Saito (1956a), on the other hand, found that TIBA slightly retarded female flower formation in cucumber. Heslop-Harrison and Heslop-Harrison (1957) reported no effect of TIBA on hemp sex expression while Choudhury and Babel (1969) found that TIBA greatly increased the number of pistillate flowers in 8 monoecious line of the cucurbit bottle gourd. Thus, TIBA effects are not consistent and shed little light on the ro 1e of auxins in sex expression. 27 3. Endogenous auxin and sex expression. As with gibberellins, there have been few studies of endogenous auxin content of female versus male lines of the same species. Galun (1959b) found no significant difference in auxin content between monoecious and gynoecious cucumber lines, although an auxin inhibitor appeared to occur in higher concentrations in the monoecious line. Hashizume (1960) reported that the content of auxin-like substances of Egyptomeris japonica decreased in flower-bearing portions of the plant during flower initiation, but increased in flowers during growth. He found a higher auxin content in female than in male flower-bearing portions during the sex differentiation period. Conrad and Mothes (1961) found approximately thirty-fold more auxin in female than in male hemp plants. Galun.§t 31. (1965) found about twice as much IAA in a hermaphroditic cucumber compared to a closely-related andromonoecious line. Thus, some evidence exists for a role for endogenous auxin in female sex expression in these plants, but the evidence is not as strong as for the role of gib- berellin in male sex expression. .F. Effects of Ethylene on Sex Expression '1. Ethrel and sex expression. The effects of ethylene on plant growth have been widely noted and the compound has been recognized as a phy- t0110rmone, perhaps arising in 3132 from enzymatic oxidation Of methional (Yang, 1967). Effects of ethylene on plant sex expression have been widely investigated, usually by means 28 of exogenous application of the presumed ethylene-generating compound 2-chloroethylphosphonic acid (Ethrel, CEPHA, Ethephon). Several reports of ethylene evolution from Ethrel lend support to attributing its effects on plant growth to ethylene. Cooke and Randall (1968) found that baSic aqueous media cause slow ethylene evolution from Ethrel at room temperature and rapid, quantitative release at 75°C. Ethylene is also evolved when Ethrel is added to extracts of etiolated pea seedlings (Warner and Leopold, 1969). Yang (1969) reviewed some pro- posed mechanisms for water or base catalyzed Ethrel decom- position. Shannon and De la Guardia (1969) applied Ethrel to cucumber plants and found that increasing amounts of ethylene were given off with increasing Ethrel dosage. Ethrel effects on sex expression have been observed in several species. Rudich gt 31. (l969a) found that applica- tion of Ethrel to monoecious squash (Cucurbita.pgpg L.) and andromonoecious muskmelon increased the number of pistillate flowers (squash) and perfect flowers (melon) and decreased staminate flower production in both. Pistillate flowers occurred at normally staminate lower nodes in squash and perfect flowers occurred on the main axis of the muskmelon where, normally, only staminate flowers are found. The same authors (1970) also found that treatment of squash and inuskmelon seedlings with Ethrel completely inhibited staminate flower production for the first three weeks of the growing period. Karchi (1970) determined that Ethrel, unlike auxin, was effective in promoting female sex expression in 29 muskmelon. In andromonoecious, monoecious, and hermaphroditic lines Ethrel promoted pistillate and inhibited staminate flowers. Pistillate flowers were produced in the andro- monoecious and hermaphroditic lines and perfect flowers were initiated in the monoecious line. Coyne (1970) and Splittstoesser (1970) reported that application of Ethrel to Cucurbita moschata varieties led to an increased production of pistillate flowers; Splittstoesser also found that Ethrel counteracted the masculinizing effect of gibberellin. Robinson.§£ a1. (1970) found that four cultivated Cucurbita species, 9. 2229 L., g. maxima Duch., Q. moschata Duch. ex Poir, and g. m1§Ea Pang., responded to Ethrel treatment by developing only pistillate flowers for extended periods. Ethrel treatment was not successful in inducing pistillate flowers in vegetative plants of several wild Cucurbita species. The only wild species responding to Ethrel were those which flowered at an early stage of development when untreated. This indicates that Ethrel does not influence floral initiation in cucurbits, but rather influences development after initiation. Ram g; 31. (1970) found that Ethrel sprayed on male hemp plants induced over ninety percent female or bisexual flowers. The plants reverted to staminate flowering after six weeks. Interest in alteration of cucumber sex expression with Ethrel has produced several reports in the last two years. Mcflurray and Miller (1968, 1969) found that 120 ppm of Ethrel applied to monoecious plants resulted in as many as 30 nineteen continuous pistillate nodes. Rudich g§_§1. (1969, 1970) reported that Ethrel application to monoecious plants at the second leaf stage inhibited staminate flower produc- tion for the first three weeks of the flowering period and stimulated pistillate flower formation on normally staminate nodes. Similarly, Robinson 31 a1. (1969) found that cucumbers sprayed at the first or third leaf stage produced only pis- tillate flowers. Stamen formation was inhibited and the authors postulated an anti-gibberellin role for Ethrel. Lower and Miller (1969) reported similar results for monoecious cucumber and other cucurbits at the first leaf stage. Iwahori 21 a1. (1969, 1970) also noted that 50 ppm Ethrel applied at the first leaf stage was effective in producing earlier and larger numbers of female flowers in cucumber. 2. Effects of free ethylene. Ethylene itself has occasionally been used to alter flowering. The earliest report was by Rodriguez (1932) who found that ethylene gas induced flowering in pineapple under otherwise non-inductive conditions. Iwahori gt a1. (1970) found that 50 ppm of ethylene induced female flowers in monoecious cucumber. These reports provide evidence for a feminizing effect of ethylene on plants and indicate that endogenous ethylene Inay also play a role as a plant sex hormone. Studies on endogenous ethylene levels versus sex expression are not available at this time. The question of the role of auxins and ethylene in sex 31 expression is complicated by indications that auxin appli- cation gives rise to ethylene production in plants. Thus, effects attributed to auxin may, in fact, be due to ethylene. Zimmerman and Hilcoxon (1935) felt that auxin application might stimulate plants to evolve ethylene. Abeles and Rubinstein (1964) found that rates of ethylene evolution were closely allied with auxin levels in nine species, including pea, bean, maize, and tobacco, and hypothesized that acceleration of abscission by NAA or IAA might be due to evolved ethylene. However, in apple and pear trees with ripening fruit, NAA was found to decrease ethylene evolution. Burg and Burg (1966) noted that IAA induced ethylene evolution in a variety of stem sections. Ethylene gas was found to produce effects on the sections which had previously been attributed to auxin. Fuchs and Lieberman (1968) found that IAA enhanced ethylene evolution from Alaska pea seedlings, but gibberellin had no effect. Shannon and De la Guardia (1969) demonstrated that application of either IAA or NAA to young cucumber seedlings led to increased ethylene evolution with increased auxin dosage. G. Cytokinins and Sex Expression Sex expression in some plants has been altered by exogenous application of cytokinins, but in all reports to (Late, synthetic cytokinins, rather than the naturally occurring phytohormones, have been used. The first report Of sex modification was by Catarino (1964) who demonstrated that 500 ppm of kinetin applied to inflorescences of 32 Kalanchoe crenata stimulated development of the calyx and gynoecium while inhibiting development of corolla and androecium. Durand (1966) found that daily spraying of Mercurialis annua with one ppm of kinetin transformed staminate flowers into pistillate flowers. The inflorescence structure was not modified. IAA and gibberellin abolished the kinetin effect but had no positive effect of their own. Negi and Olmo (1966) and Gargiulo (1968) found that the synthetic cytokinin 6-(benzylamino)-9-(tetrahydropyran- 2-yl)-9H-purine converted male flowers of a grape vine to perfect flowers. Negi and Olmo also found that indole- butyrate, TIBA, CCC, fl-naphthoxyacetate, IAA, NAA, and GA3 had no effect on the flowers. Bose and Nitsch (1970) found that N6-benzyladenine and l-(m-chlorophenyl)-3- phenylurea strongly stimulated formation of female flowers in 1. acutangula even under long days. , In contrast to these reports of a feminizing effect Of cytokinins, Abdel-Gawad and Ketellapper (1968) reported inhibition of pistillate flowers in squash with N6-benzyladenine. This is the only known report of cytokinin- induced sex alteration in cucurbits. However, a role for cytokinins in cucurbit sex expression appears possible since endogenous cytokinins have been identified in seeds of two species. Gupta and Maheshwari (1970) found that extracts of pumpkin (Cucurbita pepo L.) seeds contain three OYtOkinins, one of which may be zeatin. Extracts of inunature watermelon seeds also contain three cytokinins, 33 possibly including zeatin and zeatin ribotide (Prakash and Maheshwari, 1970). As with auxins, cytokinin effects on plants may be involved with ethylene production. Abeles 25,51. (1967) found that high concentrations of either kinetin or N6-benzyladenine applied to bean explants doubled ethylene production. Fuchs and Lieberman (1968) reported that 10"8 to 10'“ M kinetin also stimulated ethylene production in Alaska pea seedlings. H. Effects of Various Other Chemicals on Sex Expression Abscisic acid (ABA), the newest phytohormone, has not been reported to alter sex expression in plants. However, ABA often counteracts the growth-promoting effects of gibberellins, auxins, and cytokinins, and ABA treatment enhanced production of ethylene by Citrus leaves (Cooper 22.51., 1968) and bean explants (Abeles, 1967). In view Of these effects, ABA may also affect sex expression. Several other compounds are also known to affect sex expression in plants. Some of these effects are probably ”pharmacological" and not related to hormone levels; effects of other compounds such as maleic hydrazide (MH) may be due to a role as hormonal antagonists. Naugoljnyh (1955) reported that seeds treated with methylthionine Chloride produced far more pistillate flowers than did the controls. Mehanik (l958a,b) found that acetylene gas greatly stimulated pistillate flower formation in young cucumber plants. Weston (1960) observed induction of 34 staminate flowers in genetically gynoecious hop (Humulus lupulis L.) plants fOIIOWing treatment with a-(Z—chloro- phenylthio)propionic acid. Lange (1961) used 2,3-dichloro- isobutyrate and 2,2-dichloropropionate to induce pistillate flowers on two hermaphroditic papaya lines. Colchicine has been reported to feminize hemp plants (Wichert-Kolus, 1969). Bose and Nitsch (1970) found that morphactin com- pletely inhibited pistillate and strongly increased sta- minate flower formation in Luffa acutangula. Hillyer and Wittwer (1959) reported complete suppres- sion of staminate flowers in acorn squash with MH. Choudhury and Phatak (1959) and Prasad 21 a1. (1966) obtained increased numbers of pistillate flowers on monoecious cucumber with MH, while Prasad and Tyagi (1963) obtained the same effect of MB on bitter gourds. I. Effects of Grafting on Sex Expression Grafting of a scion of one sex type onto a stock of another sex type has been found to induce sex reversal in the scion and/or stock. Limberk (1954) induced sex reversal cfi'staminate hop stocks with pistillate scions while Mockaitis and Kivilaan (1964) induced staminate flowers in gynoecious muskmelon scions with andromonoecious musk- inelon and monoecious pumpkin rootstocks. Rowe (1969) induced small numbers of staminate flowers in gynoecious IHUSkmelon scions with pumpkin, squash, monoecious and gynoecious cucumber, and andromonoecious and hermaphroditic muskmelon rootstocks. The induced staminate flowers were 35 not normal, containing a rudimentary stigma. The effect of pumpkin rootstocks was duplicated by use of pumpkin inter- stocks, indicating that the stimulus for staminate flower formation came from the pumpkin leaves rather than the roots. The sex reversal effects of grafting are presumably due to transport of a plant growth regulator, perhaps one of the phytohormones, from stock to affected scion. J. The Occurrence of "Bound" or "Conjugated" Hormones Findings that endogenous phytohormones are often associated with carbohydrates, amino acids, or other com- pounds has led to a new class of phytohormones, the ”bound" or "conjugated" hormones. Gibberellins, in particular, have been found over the last decade to occur in 'aqueous— soluble' fractions of plant extracts, indicating complexing with polar compounds, or in neutral fractions, indicating conjugation of the gibberellin carboxyl group.‘ More recently, some of these ”bound" gibberellins have been iso- lated and identified. Hayashi 21 a1. (l962a,b) found that the neutral fraction of methanol extracts of potato tubers contained gibberellin- like compounds but these were not isolated or identified. Ogawa (1966a) and Hashimoto and Rappaport (1966a) reported neutral gibberellin-like substances in Sechium edule seeds and bean seeds. Pegg (1966) reported gibberellin-like sub- stances with basic and neutral properties in tomato seeds and seedlings . Water- or n-butanol-soluble gibberellin—like substances have been found in developing seeds of Sechium edule 36 (Ogawa, 1966a), in seeds of Japanese morning glory (Pharbitis n11), lupine, and peach (Ogawa, 1966b), in Pharbitis purpurea (Reinhard and Sacher, 1967a,b), and in developing bean seeds (Hashimoto and Rappaport, 1966a). Murakami (1962) found water-soluble gibberellins, including possibly a GA3 glucoside, in seeds of Japanese morning glory and Japanese wisteria. Sembdner g3 a1. (1964) found several free acidic gibberellins and five other gibberellin—like substances in Phaseolus coccineus. One of the latter, on acid hydrolysis, yielded gibberellic acid and carbohydrates as well as ninhydrin-positive compounds. Sembdner and Schreiber (1965) found three polar gibberellins in Shoot apices and flower buds of tobacco. On acid hydrolysis, one of these yielded gibberic acid (GA breakdown product), sugars, and various amino acids ingluding the carboxyl— reactive serine and methionine. Evidence for the presence of conjugated gibberellins has also come from enzymatic release of gibberellin from plant tissues and extracts. The earliest report was by McComb (1961) who found about six times as much gibberellin activity in ficin-hydrolyzed runner bean extracts than in non-ficin treated extracts. Jones (1964) obtained release of increased amounts of gibberellins from seeds of £33 gays and Phaseolus multiflorus with ficin while Pegg (1966) used .ficin to liberate unique gibberellins and increased total 8‘1 bberellin from tomato seed and seedling proteins. Reinhard and Sacher (1967c) used emulsin and ficin to release 37 gibberellins A8, A5, and A3 from seeds and fruit walls of Pharbitis purpurea, while Jones (1968) obtained release of gibberellins from pea tissue with ficin. Hayashi gt,§1. (1971) obtained increased amounts of gibberellin after hydrolysis of cucumber extracts with ficin and a—glucosidase. The occurrence of conjugated gibberellins has also been studied by following incorporation of exogenously applied gibberellins into other gibberellin-like substances. Murakami (1961) found that applied GA3 was incorporated and converted by young cucumber leaf disks into a butanol-soluble compound which produced 0A3 and glucose upon hydrolysis with s-glucosidase. The same phenomenon was observed with tissue sections from several other plants including peanut, sweet potato, and pear. Hashimoto and Rappaport (1966b) found that applied GA1 induced a large increase in the neutral gibberellin-like fraction of been seeds. Kende (1967) found that physiological concentrations of 3H-GA1 applied to dwarf pea induced label into an acidic gibberellin-like compound and several neutral compounds. No lasting attachment of the 0A1 to macromolecules was observed. Barendse 23 g1.(l968) injected 3H-GA1 into excised fruit of pea and morning glory and followed the label during development of the seeds. A large part of the label became associated with the aqueous fraction, with two new acidic compounds, and with neutral compounds which gave labelled gibberellin A1 on mild acidic hydrolysis. The structures of relatively few conjugated gibberellins have been elucidated. The source materials have often been 38 seeds, where concentrations of these novel gibberellins are often particularly high. Schreiber 21 a1. (1966) iso- lated 2-O-acetyl-GA33 from the mold Gibberella fujikuroi. 3-O-a-D-glucopyranosyl-GA8 was isolated from both immature and mature seeds of Phaseolus coccineus (Sembdner gfip§1., 1968; Schreiber 21'31., 1967; Sembdner, 1970) and from immature seeds of Pharbitis nil (Yokota 93,§1., 1969a,b). A 0A8 glucoside has also been isolated from shoot apices of Althaea ggsgg (Harada and Yokota, 1970). 2-O-a-D—gluco- pyranosyl-GA3 was also found in immature seeds of Pharbitis 211 (Tamura §17§1., 1968; Yokota g§_a1., l969a; Yokota 21,31., l969b), as well as the 3-O-s-D-glucopyranosides of GA26 and GA27 (Yokota 21,21., l969a,b). The role of conjugated gibberellins in plant metabolism remains unclear but some evidence exists for a storage func- tion in the case of seeds. Radley (1958) reported that free gibberellins in bean seeds fall drastically with increasing maturity. Hashimoto and Rappaport (1966a) reported an increase in butanol—soluble and neutral fraction gibberellins during bean seed development. Sembdner gglg1. (1968) found that GA8 glucoside increased during bean seed development while free gibberellins decreased. Only the glucoside was present in dry mature seeds. They also noted that maturing bean seeds incorporated labelled gibberellins .A3 and A6 into A8 and A3 glucosides. Reinhard and Sacher 01967a) reported an increased amount of bound gibberellin 3g1bbane numbering system used for conjugated gibberellins 39 in both seed and fruit wall of Pharbitis purpurea during development. There have also been reports that conjugated gib- berellins were converted into free gibberellins during seed germination in tomato (Pegg, 1966), pea and morning glory (Barendse g; g1., 1968), Althaea rosea (Harada and Yokota, 1970), and bean (Sembdner 21,§1., 1968). Aung §£_§1. (1969) found that transfer of tulip bulbs from 13° to 18°C, favorable for shoot and root growth, resulted in a rapid decrease in the level of bound gibberellins together with an increase in free gibberellins. Among the phytohormones the occurrence of conjugated forms is not unique to the gibberellins. Andreae and Good (1955) obtained incorporation of exogenous IAA into IAA- aspartate in pea seedlings and Klémbt (1960) isolated indole- 3-acetylaspartate from three species of plants. Klfimbt (1961) and Zenk (1962) obtained incorporation of labelled IAA into indoleacetylglucosides in wheat coleoptiles and Colchicum neapolitanum, respectively, and Varga and Bito (1968) found IAA-glucose conjugates in bean hypocotyl tissue. Indoleacetylinositol esters have been isolated from 123 ma 8 (Bandurski 33.21., 1969; Nicholls, 1967; Labarca 2§,§1., 1965). Winter and Thimann (1966) found that labelled IAA became bound to a protein in Azggg coleoptiles. Koshimizu .22 31. (1968) isolated a B-D-glucopyranoside of abscisic field from immature seeds of Lupinus luteus. The roles of these IAA and ABA conjugates are unclear although storage and. membrane transport functions have been postulated. MATERIALS AND METHODS Background 9: Plant Material 1. Cucumber (Cueumis sativus L.) Gynoecious, monoecious, and andromonoecious lines were used. The gynoecious line MSU 713-5, developed by Peterson (1960), is homozygous for gynoecious sex expression under normal field and greenhouse conditions. It was developed from a cross between a gynoecious segregate of the Korean variety 'Shogoin' (PI 220860) and the monoecious pickling variety 'Wisconsin SMR 18'. Two generations of inbreeding were achieved by self-pollinating predominately female seg- regates. Three additional self-pollinated generations were obtained by inducing staminate flowers in strictly gynoecious plants with gibberellin (Peterson and Anhder, 1960). The monoecious line used, MSU 736, is predominately staminate-flower bearing and was chosen for high male intensity. Only staminate flowers are produced on the first flower-bearing nodes under greenhouse conditions. The andromo- noecious variety 'Lemon' was obtained from Dessert Seed Co. 2. Muskmelon (Cueumis mg1g L.) Gynoecious, andromonoecious, monoecious, and hermaph- I'Oditic lines were used. All lines were obtained by IMP. L. R. Baker, Dept. of Horticulture, M. S. U. or from commercial sources. 40 41 The inbred gynoecious line MSU 1G was derived from an F2 gynoecious segregate of a monoechaus x hermaphroditic cross and was increased by self-pollination after induction of staminate flowers by grafting onto pumpkin (Rowe, 1969). The original source for the hermaphroditic line, MSU 3897, was received from Dr. B. Kubicki, Institute of Plant Genetics, Poland. The monoecious line, MSU 3898, was derived from a cross between the variety 'Iroquois' and a monoecious strain received from the Plant Introduction Service of the U. S. D. A. The cross was made by Dr. H. M. Hunger, Cornell University. The andromonoecious line used was the commercial variety 'Rocky Ford'. 3. Pumpkin (Cucurbita pepo L.) The two pumpkin varieties tested for gibberellin content were 'Small Sugar', H. Atlee Burpee Co., and 'Small Sugar Pie', Vaughn Seed CO. Both varieties successfully induce staminate flowers on gynoecious Q. 2312 (MSU 1G) scions when used as either rootstock or interstock in grafts. Growth, Care, and Harvesting 9: Plant Materials 1. Cultural conditions, etiolated g. sativus seedlings. For many gibberellin extraction experiments, etiolated (dark-grown) seedlings at various growth stages were utilized. This prevented interference with gibberellin extraction and assay procedures by excessive amounts of chlorophyll and other pigments . Generally, 30 g of seed (22° 1100 seeds) were placed in a plastic pot, covered with cheesecloth, and soaked in running 3"- ka' t, n. U ‘ ‘. H ‘fii \- 42 tap water at room temperature. When necessary (MSU 736 seed) fungicide-treated seeds were washed for five seconds in 75$ ethanol to remove the fungicide (Captan) which inter- fered with bioassays. These seeds were then washed with water and placed in pots as above. After soaking overnight, the water was drained off; the seeds transferred to glass crystallization dishes (approximately 7.5 g/dish), and again covered with cheesecloth. Five ml distilled water was added to each dish; the dishes were placed in plastic pane containing one cm of water into which the cheesecloth extended to maintain high humidity. Water was added as needed to maintain growth. The germinated seeds were grown in abso- lute darkness at a constant temperature of 27°C and harvested at growth stages from one to twenty-two days. For gibberellin extraction of ungerminated seeds, dry seeds were used, after washing (if necessary) with 75% ethanol. Seeds soaked overnight were considered to be germinated. Q. 2212 seeds were treated in the same fashion. Dark- grown g. mg1g_seedlings were not used due to limited material. 2. Cultural conditions, greenhouse-grown plants. Germinated.§. sativus seeds were placed in six-inch diameter clay pots filled with sterilized sandy soil enriched With peat moss and black top soil. Several seeds were Planted per pot and the seedlings thinned as necessary to three plants per pot. The plants were watered daily. Banks 0f':flourescent light were used to provide at least twelve 43 hours of light per day; temperature was maintained at or above 25°C. The vines were trellised about sticks placed in each pot. Plants were harvested at various growth stages by uprooting the entire plant and washing it free of soil. Entire plants were then used for gibberellin extracts. Only one growth stage of g. 2212 plants was extracted due to limited amounts of seed of the gynoecious and her- maphroditic lines. This plant material was grown under conditions similar to the above, except that raised soil beds were used (93. 1.5 sq. ft. per plant) rather than clay pots. 3. Cultural conditions, vernalized seeds. In one set of experiments, monoecious and gynoecious Q. sativus seeds were vernalized and then planted according to the procedure for greenhouse-grown plants. ,These plants were studied for any change in sex expression and gibberellin activity was measured at three growth stages. Germinated seeds were placed between several layers of cheesecloth; the cheesecloth was placed between one inch layers of sterilized, acid-washed, white sand in plastic pans. The sand was moistened and the pans were covered, Sprayed with mercuric chloride, and placed in the cold room. Temperature was maintained at 4°C for one month, (after which seeds were recovered and planted in the green- house . 44 Gibberellin Extraction Procedures 1. Organic solvent extractions. Seeds, etiolated seedlings, or entire green plants were homogenized for three minutes at 4°C with 90% aqueous acetone or 90% aqueous methanol in a Waring Blendor. The homogenate was stirred for twenty-four hours at 4°C, then filtered under vacuum. The residue was reextracted for twenty-four hours with acetone or methanol and again fil- tered. The extracts were combined, evaporated to dryness, and 5% sodium bicarbonate was added. After repeated attempts to solubilize the material, any residue was dis- solved in ethyl acetate. The sodium bicarbonate solution was extracted with ethyl acetate to remove oils, pigments, neutral gibberellins and other neutral and basic compounds. This fraction was combined with the ethyl acetate-soluble residue left after sodium bicarbonate extraction. The aqueous layer, containing acidic gibberellins, was adjusted to pH 3.0 with dilute sulfuric acid and extracted with hexane. This hexane layer was washed with distilled water, dried over anhydrous sodium sulfate, and evaporated to dryness. The aqueous layer was further partitioned against chloroform, ethyl acetate, and n-butanol. Each organic layer was treated similarly to the hexane layer. The gummy residue of each fraction was further purified by chromatog- JRaphy. All solvents used in these extractions were re- ttistilled prior to use and the residue left after evaporation 01' each solvent was bioassayed with negative results. 'Fierure 3 is a flow sheet of the complete extraction procedure. 45 Figure 3 Flow sheet for organic solvent extraction of gibberellins from Q. sativus and g. melo seeds and vegetative tissue. 1' :31 46 Seeds (15 to 100 g) Germinate, grow in water culture or greenhouse Homogenate in acetone or methanol, stir overnight | Filter J FilUrate ' Resfdue Reextract with acetone, filter 1 [I f l Filtrate ' Residue (discard) Evaporate to water phase fAdd Nahco3 ReSidue 7 Solution: pH 7.5 to 8.0 Ethyl acetate Ethyl acetate or t‘petroleum ether r ‘ I Ethyl acetate Bicarbonate phase r Dilute H2804 r-Dilute H2804 *Ethyl acetate, pH 3.0 neutral fraction r Hexane l *Hexane Aqueous IChloroform I l *Chloroform Aqueous fEthyl acetate F’ *1 *Ethyl acetate Aqueous (most free acidic gibberellins) a rn-Butanol l l *n-Butanol Aqueous (discard) (polar acidic gibberellins) *Possible active fractions Wash, dehydrate Evaporate to dryness Paper chromatography Elute in fractions Further purify , TLC 47 2. Phosphate buffer extractions. Organic solvent extractions proved unsatisfactory for green plants due to large amounts of chlorophyll which were difficult to completely remove. In partitioning, some gibberellin activity moved with the chlorophyll, and small residues interfered with bioassays. Phosphate buf- fers were used to extract acidic gibberellins from green plant material, avoiding solubilization of chlorophyll and plant lipids. Gibberellin activity recovered from phos- phate buffer extracts of etiolated material or seeds did not vary significantly from the total amount recoverable from organic extracts. Fresh plant material was homogenized for three minutes in a Waring Blendor at 4°C with 0.1 M phosphate buffer, pH 6.0. The homogenate was stirred for twenty-four hours in the cold and then filtered through cheesecloth. The filtrate was centrifuged at 15,000 x g for thirty minutes. The pH of the buffered solution was adjusted to 3.0 with dilute sulfuric acid and the solution was extracted three times with equal volumes of ethyl acetate. The aqueous phase was discarded, unless bioassayed for polar gibberellin activity. The combined ethyl acetate-rich layers were reduced,AAlz§ggg to an aqueous residue of thirty to forty m1. This phase was readjusted to pH 3.0 and again extracted hdth three equal volumes of ethyl acetate. The combined ethyl acetate phases were reduced in volume, dried over A7>A1>A5>A4>A6=A9>A8. Most other gibberellins are relatively inactive. The cucumber hypocotyl assay permits detection of those gibberellins which lack a C-7 hydroxyl (A4' A7, A9, and A13, referred to below as less- polar gibberellins) independently of A1 or A3. At a concentration of 10"2 pg per cucumber seedling, A“, A7, A9, and A13 are active whereas Al and A3 do not induce a response (Figure 7). Relative activity of the gibberellins in the barley amylase assay is as follows: A1=A3>A2>A7>Au> A5>A6>A9>A8. Hence, activity in this assay is mainly attributable to A1 and A3. These bioassays can give a strong indication of the class of gibberellins present in an extract. Thus, the cucumber hypocotyl assay (Table 1) indicates that the less-polar gibberellins are present in cucumber seed extracts to a limited extent. However, proof of their presence requires isolation and identifica- tion by other means. 4 Table 2 contains the results of dwarf pea bioassay of phosphate buffer extracts of etiolated andromonoecious Q. sativus seedlings from zero to fourteen days old. Free and ficin- and B-glucosidase-releasable gibberellins 69 Table 2 Gibberellin activity in phosphate buffer extracts of etiolated andromonoecious 9. sativus seedlings. [dwarf pea bioassay, zero to fourteen days old; cucumber hypocotyl and barley amylase bioassays of six day old seedlings; expressed in pg GA3/kg seedling fresh weight] Days after Fresh Free acidic GA Enzyme- Total GA germination wei ht released GA1 (mean) (g l 2 mean 1 2* mean 0 30.0 5.9 5.8 5.9 4.1 3.9 4.0 9.9 1 30.2 5.9 5.6 5.8 9.0 7.3 8.1 13.9 2 38.6 8.4 9.0 8.7 14.8 12.7 13.8 22.5 4 41.5 21.2 22.0 21.6 19.0 18.7 18.9 40.5 6 60.4 40.6 41.6 41.1 19.6 14.3 17.0 58.1 10 69.7 34.1 33.2 33.7 35.7 39.1 37.4 71.1 14 73.5 33.0 30.9 32.0 36.9 39.6 38.3 70.3 6a2 60.4 2.9 - - - - - - 6b3 60.4 42.5 - - 18.2 - - 60.5 1 Released after 24 hour incubation with ficin and emulsin 2Cucumber hypocotyl assay, measure of GA4/7 activity 3Barley amylase assay Table 3 Gibberellin activity in phosphate buffer extracts of 22 day old etiolated andromonoecious, monoecious, and gynoecious C. sativus seedlings. [dwarprea bioassay; expressed in mg 0A37kg seedling fresh weight Seedling Free acidic GA Enzyme- Total sex type released GA2 GA 1 2 mean 1 2 mean Andromonoecious 29.4 29.7 29.6 42.8 41.5 42.2 71.7 Monoecious 28.1 30.2 29.2 43.8 42.3 43.1 72.3 Gynoecious 22.5 23.4 23.0 33.1 30.4 31.8 54.7 1 Fresh weights: andromonoecious, 45.1 g; monoecious, 45.0 g; gynoecious, 41.1 g 2Released after 24 hour incubation with ficin and emulsin 'Jl‘ac 70 were measured. Table 2 also contains the results of barley amylase and cucumber hypocotyl assay of extracts of six day old etiolated andromonoecious seedlings. A peak of free gibberellin activity occurs here; this growth stage also corresponds to the maximum free gibberellin level in monoecious seedlings (Hayashi 21 A1., 1971). As in ungerminated seeds most activity appears to be due to 0A1 and/or GAB, but traces of the less-polar gibberellins also appear to be present. Table 3 contains the results of dwarf pea bioassay of phosphate buffer extracts of twenty-two day old etiolated seedlings of each sex type, thus extending experiments on etiolated seedlings to the practical limit for growth under these conditions. Figure 9 compiles results for dwarf pea bioassay of extracts of seeds and etiolated seedlings of three 9. sativus sex types. This Figure combines the data of Hayashi _e_t 9.1.- (1971) with that of Tables 1, 2, and 3 to indicate the relationship among free and total gibberel- lin activity, growth stage, and sex type. Andromonoecious and monoecious seeds and etiolated seedlings contained more free gibberellin and total gib- berellin at all growth stages than gynoecious seeds and seedlings. Dry monoecious seeds contained about 8.0 ug/kg ficin- and emulsin-releasable gibberellin and 2.1 ug/kg free acidic gibberellin (GA3 equivalents). In germinated monoecious seeds the amount of bound gibberellin decreased from 8.0 to 3.8 pg/kg with a corresponding increase in .71 Figure 9 Free acidic and total (free acidic plus enzyme-released) gibberellin activity in etiolated monoecious, andromonoecious, and gynoecious g. sativus seedlings at several growth stages. Idwarf pea bioassay, results expressed as ng GA3/kg seedling fresh weight] _" monoecious free on monoecious total x-nx andromonoecious free o-no andromonoecious total 2mm): gynoecious free cum 6 gynoecious total 72 com-2.2500 .2..- «>00 I"lll|'|ll'o'll|ll'e\\ 2.2.3 ...... 9.3.. §5tc3cou n<0 l T Figure 9 73 free gibberellin. During seedling growth free gibberel- lin increased rapidly, reached a maximum after six days (63 pg/kg), then decreased and leveled off in Older seedlings (25 to 30 pg/kg). The amount of ficin- and emulsin-releasable gibberellin slowly increased during seedling growth. The total gibberellin content increased rapidly to 72.7 ug/kg after six days and then stabilized. The curves for gibberellin activity in andromonoecious seedlings are similar to those for monoecious seedlings although the levels were somewhat lower. Free acidic gib- berellin also reached a maximum after six days. In contrast, gibberellin in gynoecious seedlings was no more than half the amount in the more male sex types at corresponding growth stages. Free and enzyme-releasable gibberellin exhibited no peak of activity but continued to increase slowly during growth. At the six day stage, free acidic gibberellin activity in andromonoecious and monoecious seedlings was five and eight times, respectively, that in gynoecious seedlings. If the dwarf pea bioassay results had been expressed as mg GA per seed of starting material rather than pg GA per kg fresh weight, the dif- ferences would have been greater since the seedling fresh weight of the more male sex types increased more rapidly than did that of the gynoecious. Also, the relative order of gibberellin activity in ungerminated seeds, i.e. monoecious a! andromonoecious) gynoecious, was maintained throughout seedling growth (0 to 22 days). 74 II. Gibberellin Activity in 9. sativus: Greenhouse-grown Plants Extraction of gibberellins from dark grown seedlings of three 9. sativus sex types strongly suggested that the gynoecious line contained considerably less free and total gibberellin activity than the monoecious and andromonoecious lines. Etiolated seedlings were primarily used to facilitate extraction and quantitation, but beyond the four to six day stage they may not represent an accurate picture of the gibberellin levels at corresponding growth stages in green plants (See Discussion). In order to avoid extrapo- lating results from etiolated seedlings to green plants, gibberellin activity in greenhouse-grown plants was measured. Since free acidic gibberellins proved to be a reliable indicator of total gibberellin content (Figure 9) in seeds and etiolated seedlings, enzyme-releasable gib- berellins were not measured. 0 Table 4 and Figure 10 contain results of phosphate buffer extraction of gibberellins from one, two, four, and eight week old plants of each sex type. Figure 10 also incorporates the results for dry and germinated seeds. The gibberellin content (in pg/kg fresh weight) in one and two week old greenhouse-grown plants was only slightly higher than in etiolated seedlings at similar growth stages (Table 4). However, total free acidic gibberellin per plant was much higher since the fresh weight of light-grown plants far exceeds that of etiolated seedlings at the 75 Figure 10 Free acidic gibberellin content of greenhouse- grown monoecious, andromonoecious, and gynoecious Q. sativus plants at several growth stages. [activity measured relative to GA3 standards in dwarf pea bioassay; expressed as pg GA3/kg fresh weight of seed or entire plant] 76 cots-£5.00 .030 3.803 a n 0 n V m N — O '0 O' O. Q00 to o o o O I o o o o o o O I I o 00 00 ...-............ ...-....I-III...-.........-.33....III.O...-....- .- O...- 223 ...... mime 0 £00 -*’0 zeta-Emucou 040 I II QIIII I, O? 4 ¢ ¢ ¢ 00 m30_uooc>m O ...-.... O u:o_ueocoE O l O «somuoocoEo-vco d I I Id F igure 10 77 same stage. Gibberellin activity in monoecious and andro- monoecious plants (Figure 10) increased rapidly to a peak at one week and then descended to a relatively stationary level from two to eight weeks. In contrast, the gynoecious plants showed no corresponding early-peak in gibberellin activity and levels from two to eight weeks were only fifty to sixty percent of those in the more male plants. At one week the gynoecious plants contained only 16% as much gibberellin activity as the monoecious and andro- monoecious plants. At this stage a rudimentary male flower bud is often observed on the cotyledonary node. The results with greenhouse-grown plants were in all respects consistent with those for etiolated seedlings. Furthermore, the relative gibberellin levels in dry seeds were maintained throughout the test period for both greenhouse plants and etiolated seedlings, i.e. the gynoecious type contained lower levels of gibberellin activity compared to other sex types for seeds, germinated seeds, etiolated seedlings, and greenhouse-grown plants at all growth stages up to eight weeks. Thus, the gibberel- lin level in mature, dry seeds of a certain sex type may be a strong indicator of relative gibberellin levels in growing plants of that sex type. 78 Table 4. Free acidic gibberellin activity in phosphate buffer extracts of greenhouse-grown monoecious, andromonoecious, and gynoecious g. sativus plants. dwarf pea and dwarf corn bioassays; expressed in pg GA3 kg fresh weight; 500 to 2000 g material extracted] Sex type Growth Dwarf pea expt. no. Dwarf corn1 stage (wk) 1 2 mean d1 d5 Andromonoecious l 64 61 63 62 63 2 40 42 41 45 43 4 38 33 36 33 39 8 39 40 40 39 43 Monoecious l 57 68 63 61 72 2 39 37 38 41 40 4 41 35 38 33 37 8 38 32 35 34 37 Gynoecious l 10 9 10 10 11 2 21 22 22 22 25 4 25 21 23 21 22 8 26 26 26 28 29 1 Same extract as for Dwarf pea expt. 2 III. Gibberellin Activitygand Sex Expression of Chilled C. sativus Seeds Greenhouse-grown gynoecious (MSU 713-5) plants produced from chilled, germinated seeds were observed to see if cold-treatment of seeds would affect sex expression. Table 5 lists the total number of flowers of each sex type, including aborted staminate flowers, for control and chilled plants at the eight week stage. Monoecious plants produced from chilled seed exhibited little change in either the total number of flowers or sex ratio compared to monoe- cious controls. In contrast, gynoecious plants produced from chilled seed produced fewer flowers, but an increased 79 proportion of normal and aborted staminate flowers relative to gynoecious controls. All staminate flowers occurred at early nodes; after six weeks no staminate flowers were produced. The gynoecious controls produced about three percent (of total flowers produced) aborted staminate flowers and no normal staminates, while the gynoecious plants from chilled seeds produced about 18% normal and aborted staminate flowers. At the end of six weeks nearly one-third of the flowers produced by the chilled gynoecious plants were staminate. Table 5 Flower sex types in chilled and control C. sativus plants, monoecious and gynoecious lines. [m = sfaminate, f = pis- tillate, m8 = aborted staminate] Plant no. Monoecious Gynoecious control chilled control chilled 1 7m 7m lma, 6f 6f 2 14m 11m, 1f 5f 1m, 2f 3 13m 7m 8f 3ma, 3f 4 9m 8m lma, 8f 8f 5 12m 6m 8f 6f 6 7m 8m 5f 3ma, 7f 7 7m 8m, 1f 4f 1mg, 9f 8 6m 9m llf 6f 9 8m 16m lma, 10f 1m, 0f 10 -6m, 2f 15m 10f 2m, 1f 11 12m 11m 12f lma, 10f 12 7m 6m 10f 2m, 5f Total: 108m, 2f 112m, 2f 3ma, 97f 8ma, 6m, 63f 80 Phosphate buffer extracts of chilled and non-chilled seeds and plants indicated that for the monoecious line, gibberellin activity in seeds and plants was unchanged (Table 6). However, chilling increased gibberellin activity slightly in gynoecious seeds. At the one week stage, a very significant three-fold increase in activity was noted. At the eight week stage, there was very little difference in gibberellin activity between chilled and non-chilled gynoecious plants. Table 6 Effects of chilling of seeds on free acidic gibberellin activity in phosphate buffer extracts of monoecious and gynoecious lines of C. sativus. [dwarf pea bioassay, expressed as ug GAB/Kg fresh weight] Sex type Gibberellin Activity germinated seeds 1 week plants 8 week plants Monoecious control 7 65 35 Monoecious chilled 7 64 37 Gynoecious control 6 9 25 Gynoecious chilled 7 27 27 IV. Gibberellin Activity in C. melo Seeds of four sex types of 9. 3219 were available in sufficient quantity to measure gibberellin activity. How- ever, only the andromonoecious type was available in suf- ficient quantity for experiments at several growth stages. Only one to two ounces of the monoecious and hermaphroditic types were available, so gibberellin activity was measured only in dry seeds and four week old greenhouse-grown plants. Dwarf pea and barley amylase assays were employed to quaint us em I) and he I~ t'l 5% tea bi 81 quantitate the gibberellins. The cucumber hypocotyl assay was employed to detect less-polar gibberellins. Dry seeds of the relatively more female gynoecious and hermaphroditic lines were found to contain 43 and 40 ug/kg free acidic gibberellin, respectively, in the dwarf pea bioassay; the barley amylase assay results support these data (Table 7). In contrast, the more male andro- monoecious and monoecious lines contained only about 7 pg/kg gibberellin. These results are the converse of those for g. sativus where the gynoecious line was gibberellin deficient. Gibberellin activity in extracts of four week old gynoecious and hermaphroditic plants was higher than in monoecious and andromonoecious plants (Table 8), but the differences were not as pronounced as for the seeds. 9. 2319 plants normally abort all flowers until five weeks old, but flower buds were developing at the four week stage. Cucumber hypocotyl bioassays indicated very little, if any, activity due to less-polar gibberellins in extracts of both seeds and green plants. The small response seen with seeds and plants of the gynoecious and hermaphroditic sex types, although calculated relative to a GA7 standard, may represent the slight activity of other gibberellins 111 this bioassay. V.. Gibberellin Activity in Pumpkin Seeds and Leaves Methanol extracts of two different varieties of pumpkin seed and leaves of one variety contained 1.0, 1.1, and 2.9 ug/kg free acidic gibberellin, respectively (Table 9). Free dry . W r.) ,. One 82 Table 7 Free acidic gibberellin activity in methanol extracts of dry seed of four 9. melo sex types. [dwarf pea, cucumber hypocotyl, and barley amylase bioassays; expressed in ug GAB/kg seed] Sex type Gibberellin Activity Dwarf pea expt. no. Barley Cucumber 2 l 2 3 u mean amylasel hypocotyl Andromonoecious 6.7 5.7 6.7 9.3 7.1 7.5 none Monoecious 6.1 7.3 - .- 6.7 6.9 none Gynoecious 47 33 46 45 43 40 0.1 Hermaphroditic 40 39 - - 40 42 0.1 13ame extract as dwarf pea expt. 1 2Measure of GA4/7 activity (GA7 equivalents) Table 8 Free acidic gibberellin activity in phosphate buffer extracts of four week old greenhouse-grown.g. melo plants of four sex types. [dwarf pea, barley amylase, and cucumber hypocotyl bioassays; expressed in mg GA3/kg fresh weight] Sex type Gibberellin Activity Dwarf ea expt. no. Barley 1 Cucumber 2 I 2 mean amylase hypocotyl Andromonoecious 9.0 13.2 11.1 12.3 none Monoecious 14.1 12.5 13.3 14.0 none Gynoecious 31.0 28.0 29.5 34.6 0.1 Hermaphroditic 20.2 21.7 21.0 22.2 0.1 18ame extract as dwarf pea expt. 1 2Measure of GAu/7 activity (GA7 equivalents) Table 9 Free acidic gibberellin activity in methanol extracts of seeds of two monoecious varieties of pumpkin and leaves of one variety. [dwarf pea and dwarf corn bioassays; expressed in mg GAB/kg seed] Variety Gibberellin Activity Dwarf pea expt. no. Dwarf corn 1 2 mean 0.1 'Small Sugar' seed 0.9 1.0 1.0 1.2 'Small Sugar Pie' seed 1.2 0.9 1.1 1.1 'Small Sugar' leavesl 3.2 2. 5 2.9 - lFiuve hundred and four g leaves from four week old plants These the g Spot, 83 These levels of gibberellin activity are far lower than the gibberellin activity in any muskmelon sex type. VI. Identification of Free Acidic Gibberellins in E. sativus Free acidic gibberellins in andromonoecious, monoecious, and gynoecious 9. sativus seeds were determined by thin- layer chromatography, gas-liquid chromatography, and mass spectrometry. Figures are presented for extracts of andro- monoecious seed, but the same types and relative proportions of gibberellins were found in the other two sex types. A. Thin-layer chromatography. A TLC plate comparing a purified free acidic gibberel- lin extract of andromonoecious Q. sativus seeds with several gibberellin standards is represented in Figure 11. Gib- berellins having a C-7 hydroxyl group (Al, A3, and A5 in this Figure) do not develop dark spots when sprayed with alcoholic H2304; therefore, the dim yellow spots for these gibberellins were outlined. The andromonoecious extract produced spots at Rf values 0.69, 0.49, and 0.20. The Rf 0.69 spot corresponds closely with CA“; the Rf 0.49 spot corresponds with GA1 and/or GAB. The very faint Rf 0.20 spot, evidently due to a highly polar compound, did not correspond with any of the standards. B. Gas-liquid chromatography. Figure 12 represents a gas-liquid chromatogram of a mixture of equal parts of 0A1, GA3’ GA“, and 0A7 methyl esters and a methylated acidic gibberellin extract from andromonoecious seeds. Peaks with retention times of 84 Figure 11 Thin-layer chromatogram of gibberellin extract from 100 g of andromonoecious Q. sativus seed. [plate: Brinkmann Pre-coated Silica Gel F25“, 0.25 mm thickness, sprayed with alcoholic H2804; authentic samples of gibberellins A1, A2, A3, A4, A5, A7, A9 spotted for comparison; solvent system: benzenezn-butanolzacetic acid (70:25:5, V/V)] 85 3.31-5009: #042.“ ‘70 125:5. q 7 f). 513:? ”N -3 5' I :} in) fl ‘cl \0' ' ‘J {I :0 \0 Figure 11 86 22. 6, 7, 15, and 17.5 minutes correspond well with gib- berellin A“, A7, A1, and A3 standards, respectively. The peak corresponding with GA1 is the predominant compound present in the extract. Small peaks at 4 and 9.5 minutes do not correspond to any of the standards used. C. Mass spectrometry. Three highly purified eluates (labelled C1' C2’ and 03) from thin-layer chromatography of an andromonoecious extract, suspected to contain gibberellins A1, A3, A4 and/or A7, were crystallized and subjected to direct probe mass spectrometry. Figures l3, l4, and 15 contain the mass spectra of these samples. Table 10 contains an analysis of the major peaks in Figure 13 (Cl). The molecular ion mass (m/e 348) is identical to the molecular weight of GAl. Published spectra of the free acidic gibberellin series are not available but the mass spectra of the methyl esters of gibberellins A1 through A21 have been reported (Binks g£_§1., 1969). The spectrum of Figure 13 corresponds very closely to that for authentic methyl A1 if the necessary correction of 14 mass units is made: the spectra of Figure 13 and methyl A1 both have prominent peaks at m/e 284, 261, 243, 237, 213, 201, 199, 185, 163, 135, 121, 109, 108, 107, 105. 95, 69, 67, 55, 43, and 41. Thus, the presence of GA1 in the extract is confirmed. Table 11 contains an analysis of the spectrum of Figure 14 (02)° The molecular ion mass (m/e 346) is iden- tical to the molecular weight of GA3° This spectrum 87 Figure 12 Gas-liquid chromatogram of a methylated gib- berellin extract of andromonoecious Q. sativus seed (lower graph) and a mixture of methylated gibberellin standards (upper graph). [columnz QF-l, 200°C; two ,11 of a 1% alcoholic solution of the extract and standards were injected] 88 Detector Response O 5 10 Retention Time Figure 12 15 2O 87 Figure 12 Gas-liquid chromatogram of a methylated gib- berellin extract of andromonoecious Q. sativus seed (lower graph) and a mixture of methylated gibberellin standards (upper graph). [column: QF-l, 200°C; two “1 of a 1% alcoholic solution of the extract and standards were injected] 88 Detector Response O 5 10 Retention Time Figure 12 15 20 89 corresponds closely to that of methyl A3: prominent com- mon peaks are m/e 255, 237, 236, 227, 225, 223, 209, 195, 181, 179, 155, 136, 121, 105, 91, 77, 69, 65, 55, 44, 43, and 41. The prominent peak at m/e 136 has been assigned to cleavage of rings C and D from B (refer to Figure l) by Wulfson.g§_§1. (1965) for methyl A3 and the same ion would be expected for CA The peak at M+-63 (m/e 283) has been attributed to loss30f H20, C02, and H from ring A to give a tropyllium ion. Peaks at M+-63 are characteristic of gibberellins having a double bond at the 3,4 position of ring A, e.g. 0A3 and 0A7 (Binks 93,31., 1969). Table 12 contains an analysis of the spectrum of Figure 15 (03). The molecular ion (m/e 332) has a mass identical to that of GA“. The spectrum corresponds closely to that of methyl A“: prominent common peaks include m/e 258, 255, 242, 241, 224, 199, 197, 183, 181, 169, 157, 155, 143, 129, 128, 119, 117, 115, 105, 92, 69, 67, 65, 55, and 41. The peak at m/e 136 is attributed to loss of rings A and B (as above). The M+-63 peak associated with A and 3 A7 is not very prominent. Thus, GA“ is present in andro- monoecious seed extracts. Table 10 Analysis of mass spectrum in Figure 13. m/e M+-m/e assignment (loss of fregment) 348 0 molecular ion 330 18 H20 312 36 2 x H20 302 46 CO , H (HCOOH) 284 64 H20, H OOH 90 Figure 13 Mass spectrum of 01' a gibberellin-like com- pound isolated from andromonoecious Q. sativus seed. Figure 13 91 100— W L l 1 l L 4 8 J. ' CL cocogw N 340 300 260 220 180 140 100 60 92 Figure 14 Mass spectrum of 02’ a gibberellin-like com- pound isolated from andromonoecious Q. sativus seed. Figure 14 7‘3 95 100- 80-‘ 60-‘ 404 20- 100 140 ‘180 220 260 300 340 60 94 Figure 15 Mass spectrum of 03, a gibberellin-like com- pound isolated from andromonoecious 9. sativus seed. 95 In W l l l J l ____=.§_ 1 -—4 8 8 a, 1‘ N Figure 15 260 220 180 140 100 60 96 -Table 11 Analysis of mass spectrum in Figure 14. m/e M+-m/e assignment (loss of fragment) 346 0 molecular ion 328 1.8 H20 310 ‘ ' 26 - 2 x H20 300 6 002, Hz (HCOOH) 284 62 H20, 002 283 63 H20, 002, H 282 64 H20, C02, H2 238 108 H20, 002, HCOOH 136 210 rings A and B Table 12 Analysis of mass spectrum in Figure 15. m/e M+-m/e assignment (loss of fragment) 332 0 molecular ion 330 2 H2 (from 2-hydroxy1) 314 18 H O 286 46 082, H (HCOOH) 270 62 H20, 062 268 64 H20, 002, Hz 136 196 rings A and B VII. Identification of Free Acidic Gibberellins in C. melo The free acidic gibberellins in gynoecious, monoecious, andromonoecious, and hermaphroditic 9. mglg seed were deter- mined by thin-layer chromatography, infrared analysis, and mass spectrometry. The figures presented are for extracts of gynoecious seed, but the same types and relative propor- tions of gibberellins were found in all sex types. A. Thin-layer chromatography. A purified extract of gynoecious seed was compared with several gibberellin standards on a TLC plate (Figure 16). The only visible spot corresponds well with 6A1 and/or GAB' 97 Figure 16 Thin-layer chromatogram of gibberellin extract of 100 g of gynoecious Q. mglg seed. [platez Brinkmann Precoated Silica Gel F254, 0.25 mm thickness, sprayed with alcoholic H2804; authentic samples of gibberellins A1, A2, A3, A“, A5, A7, A9 spotted for comparison; solvent system: benzene:n-butanol:acetic acid (70:25:5, V/V)] 98 d3; l" 86’9“ i '43”( ,, "‘ '73Q51f (? ?: 1* ‘flL Aigflm / j? ‘57 Sorrel “I. ..“\ ‘ O \o' _ I z" : ‘- I 1 '0 : t,“ ' 0 | I I 0 « ..1 Figure 16 99 B. Infrared analysis. Infrared (IR) spectra (Figures 17 and 18) were obtained for two fractions (M1 and M2) crystallized from the gynoe- cious extract suspected to contain GAl and/or GAB' Results with gibberellin standards have shown that IR cannot be used for definitive structure determination with impure samples, but certain differences in the spectra of 0A1 and GA3 are useful for identifying an unknown compound ‘suspected to be one of these gibberellins. The spectra of Figures 17 (M1) and 18 (M2) each have major peaks at 3450 (2° or 3° O-H stretching), 2920 and 2950 (COOH vibration), 1750 (C=CH2 vibration), 1740 (COOH vibration), 1170 (3° OH vibration),1100 (2° OH vibration)and.g§. 900 cm‘1(0=ca2 vibration). However, the latter spectrum contains major peaks at 1020 and 1030 car1 which are lacking in Figure 17 and also peaks at 1260 (cis disubstituted C=C), 970 (cis C=C), and 770 cm‘l(cis C=C). Each of these peaks is characteristic of GAB' Thus, the TLC spot (Figure 16) corresponding to GA1 and/or GA3 appears to be due to both of these gibberellins. Another fraction (M3) was also purified, but in insufficient quantity for IR analysis. C. Mass spectrometry. Three highly purified fractions (M1, M2, M3) of an extract of gynoecious seed were subjected to direct probe mass spectrometry. Two fractions (M1 and M2) produced spectra (Figure 19) which are almost identical to those of Figures 13 and 14 which were attributed (above) to GA1 100 Figure 17 Infrared spectrum of M1, a gibberellin-like compound isolated from gynoecious Q. melo seed. 101 8.0 6.0 A l L 1 L 1 A A J 4.0 MICRONS 5.0 A J_ 3.0 L 3000 b . 1L 0 a A 3500 ._-+_-_..._._._._ __.—._+_._ _._ 1 '7 I 1500 2000 20.0 15.0 2500 25.0 MICRONS 10.0 9.0 8.0 7.5 1200 "00 1000 900 800 700 600 500 400 moueucv (CM 'l 1300 F igure 17 102 Figure 18 Infrared spectrum of M2, a gibberellin-like compound isolated from gynoecious Q. melo seed. 103 8.0 6.0 4.0 M'CRONS 5.0 3.5 3.0 2500 2000 1500 20.0 3000 3500 25.0 15.0 MICRONS 10.0 9.0 8.0 7.5 1300 1200 1100 1000 900 800 700 600 500 FREQUENCY (CM'I Figure 18 104 Figure 19 Mass spectra of M1 (upper) and M2 (lower), gibberellin-like compounds isolated from gynoecious 9. melo seed. O¢m 000 0mm ONN omH 011 OOH om 105 — 11. 11 111 11111 111 1111 1_ 1 1 Tom 10m [OOH . N [ON 1 10m 10m [OOH Figure 19 106 and GAB' The slight differences in certain peak heights are probably due to small differences in either ion source temperature or amounts of impurities. Table 13 contains an analysis of the spectrum of M3 (Figure 20). The molecular ion (m/e 330) is identical in and GA there are many peaks molecular weight to both GA 5 7‘ . in common with the spectra of both methyl A5 and methyl ? A7. However, the peak at m/e 267 (M+-63) which is 1 Allvun‘... characteristic of methyl A7 is weak in Figure 20, and the peak at m/e 286 (M+-44) is very strong, a characteristic Fawn; .. _‘ of the spectra of methyl esters of the gibberellins which lack a C—2 hydroxyl. Thus, M3 is GAS. Table 13 Analysis of mass spectrum in Figure 20 + m/e M -m/e assignment 330 0 molecular ion 312 18 loss of H 0 286 44 n 032 284 46 ' C02, H2 268 62 n 002, H20 136 194 " rings A and B VIII. Igentification of Neutral Fraction Gibbere11ins in E. sativus Methanol extraction of five kg monoecious Q. sativus seed (Figure 3) yielded gg. 20 mg of biologically active material in the neutral fraction. Repeated thin-layer chromatography and bioassay of the neutral fraction material revealed one band at Rf 0.7 (benzene: n-butanol: acetic acid, 70:25:5, v/v) which had gibberellin—like activity. 107 Figure 20 Mass spectrum of M3, a gibberellin-like com- pound isolated from gynoecious Q. melo seed. 108 300 260 220 180 100 60 100- 80* 60— 4o 20- F igure 2O ‘4’ ‘ 109 Eluates of this band were further purified on TLC and two compounds were found to be present. An apparently pure compound (m.p. 140-1410), designated N1, was resolved from the mixture by crystallization and the remainder was designated N2 (m.p. 140-1480). N1 produced a pale brown spot on TLC plates when developed with alcoholic H2504; N2 produced overlapping pale brown and pale green spots. N1 and N2 (Figure 21) are slightly less polar than most of the free acidic gibberellin standards. A. Identification of N1. After recrystallization from ethyl acetate-petroleum ether, the apparently pure Nl was subjected to direct probe mass spectrometry. The spectrum is seen in Figure 22; Table 14 contains an analysis of the spectrum. The fragmentation pattern and molecular ion (m/e 388) indicated that N1 might be a propyl ester of gibberellin A3, but spectra of gibberellin propyl esters have not been reported. Therefore, the n-propyl ester of A3 was synthesized according to the method of Sell 22 §_1_. (1959). The product had a melting point of 1400 and its mass spectrum (Figure 23) is essentially identical to that of N1. Table 15 contains a comparison of the authentic GA3 zm-propyl ester, N1, and N2 in three TLC systems. The Rf values of N1 and authentic n—propyl A3 correspond closely in each system. The infrared spectra of N1 and authentic GA3 n-propyl ,5 ter (Figures 24 and 25, respectively), are also essentially id antical . 110 Table 14 Analysis of mass spectrum in Figure 21 (N1) m/e M+-m/e assignment 388 0 molecular ion 370 18 loss of H20 342 46 " 002, Hz 329 59 ” C3H70- 328 60 " C3H7OH 325 63 " H20, 002, 301 87 " 03H7OC=O. 300 88 ' 03H70C=0H 238 150 " 03H7OC=0H, H20, 002 237 151 ' 03H7OC=OH, H20, 002B 136 252 " rings A and B 43 (base peak) 345 CHBCHZCHZ- Table 15 TLC Rf values of authentic GA3 n-propyl ester, N1, and N2 in three solvent systems. Solvent system PrA3 N1 N21 benzene:n-butanol:CHBCOOH (70:25:5) .68 .68 .72 chloroform:ethy1 acetate:CH3COOH (60:40:5) .41 .41 .41 di-isopropyl ether:CHBCOOH (95:5) .48 .49 .50 indicator. QRILC2 plates were Silica Gel F with fluorescent spots developed with 5% alcofigliio H280“. lFaater of two overlapping spots 111 Figure 21 Thin-layer chromatogram of N1 and N2, neutral gibberellin-like compounds isolated from Q. sativus seed. [authentic samples of gibberel- lins A A2, A3, A4' A5, A7, A spotted for 1' 9 comparison; plate: Brinkman Pre-coated Silica Gel F25“, 0.25 mm thickness, sprayed with alcoholic H250“; solvent system: benzene: n-butano1:acetic acid (70:25:5, v/v)] 112 ... _.-....—.- 'wmr _- . '. b H ‘4.-- ' .1 ‘ J." ”““—- dn..|~,.fi;fl:/:-HC/.’¢+r " <9 I J 7022575 (I O. "x? ' r“ 1"! 1... ' ' i \ 3 .. ‘ ~../ ' a I .. \_I ‘ k, ’M“-’- < Figure 2 1 .-. . -. '.-. “. - .-‘ 113 Figure 22 Mass spectrum of N1, a neutral gibberellin— 1ike compound isolated from g. sativus seed. 114 omN omN SN 0: omfl om on TON [OOH 22 figure 115 Figure 23 Mass spectrum of authentic gibberellin A3 n-propyl ester. 116 0mm omN OWN OHN Ora 00H om Om 10m 10m [OOH 23 /;i— gnre 117 Figure 24 Infrared spectrum of N1, a neutral gibberellin- 1ike compound isolated from Q. sativus seed. 118 20.0 25.0 15.0 MICRONS 10.0 9.0 8.0 7.5 1100 1000 900 800 700 600 500 400 4.0 WCRONS 5.0 8.0 3.0 1200 6.0 3.5 3.1]: 6 1 1 1 1 a _ f 1 1 1 2 500 2000 1 500 moumcv (CM ., 3000 3500 4000 Figure 24 119 Figure 25 Infrared spectrum of authentic gibberellin A3 n—propyl ester. 1.0 8.0 20.0 -1 -.1. _4 .. e 1 ...__+. ...-.. . 1 1-- f 6.0 150 1"“ 1500 I 1 MICRONS 800 2000 —( A 700 4_0 MICRONS 5.0 -..—4} _._¢- 120 2500 FREQUENCY (CM '1 10.0 :1. :31 1 L; z ..... 1 1 1 12,2, ..1. 1 m 1 1 1 1 . ... -.. 11.1- . 11 10 ”1 11;. 1 .1.-..4... : .....1;. ..1.....-_ A 411.1 3 l+.....+..-x4_.1%| a (J _. . 1... w _ 1 1 1 1 _ H 1. 1 1 u m 1 1 1 n 0111 A 11 1 1 .1 .211»: .I. 1... A I o 01710 v 0 . M01 9 _ . . (1111 1111 v1 11.1.1.1 r1.-. 4.1 r1117 1.111111 . 9.0 /0 non 3.0 3000 . i ; t 1 1 : ‘ i f ‘ 1 I f 5 f T . : : 1-13 81) 1200 1300 3500 7.5 011 10 .20 30 .40: " so 60 70 Lo, 4000 F igure 2 5 E! to: I'll 121 B. Identification of N2. N2, which appeared to contain N1 and another compound, proved difficult to resolve completely by either TLC or crystallization. After trimethylsilylation, N2 was sub- Jected to combined GLC-mass spectrometry. Figures 26 and 27 contain mass spectra of the two compounds resolved from trimethylsilyl (TMS) -N2; Tables 16 and 17 contain analyses of these spectra. The fragmentation pattern in Figure 26, with molecular ion of 532 and fragments due to loss of propyl and propylcarboxyl groups as well as several peaks typical of TMS derivatives, indicates the presence of the TMS diether of GA3 propyl ester. The spectrum in Figure 27 has a molecular ion of 53# and other major peaks are two mass units greater than those of Figure 26, indicating the presence of the TMS diether of GAl propyl ester. IX. Biological Activity of a Neutral Gibberellin (N11 from C. sativus The n-propyl ester of GA3 (N1) was tested for activity in four bioassays (Table 18). Activity in each assay was 10% or less of the activity of GA These figures correspond 3. well with those for synthetic propyl A3 in the dwarf pea, cucumber hypocotyl, and d1 dwarf corn bioassays (Brian £3 11,, 1967). In addition, Sell 932 9;. (1959) reported that synthetic n-propyl A3 was considerably less effective in Promoting lettuce seed germination than GA3° 122 Figure 26 Mass spectrum of TMS-N2 (first component), a neutral gibberellin-like fraction isolated from Q. sativus seed. [N2 was trimethylsilylated and subjected to GLC-mass spectrometry] 0mm 00m owN CNN 02 00.” om TON 10.». Tom .Iom rIOOH N F' igure 26 12“ Figure 27 Mass spectrum of TMS-N2 (second component), a neutral gibberellin-like fraction isolated from Q. sativus seed. Figure 27 100— 80— 60—4 I 20— 500 460 420 380 340 300 260 220 180 140 100 60 126 Table 16 Analysis of mass spectrum in Figure 26 m/e M+-m/e assignment 532 0 molecular ion 51? 15 loss of CH . 502 30 ' 2 2 CH3- #88 (weak) an " CO 473 59 ' C3a 0° £32 30 ” C3H 0H 7 " C H OC=0° 003 88 " CBH;OC=0H 442 90 ” (8H3) SiOH 028 104 ' C02, 338708 129 #03 C6H1 031 75 #57 (CH3;ZSiOH 73 “59 (CH3)331° Table 17 Analysis of mass spectrum in Figure 27 m/e M+-m/e assignment 534 0 molecular ion 519 15 loss of CH ‘ 504 30 I 2 % CH3° 090 (weak) 44 ' C0 #74 60 “ C3 70H “#7 87 u C3H700=00 006 88 " c H70C=OH 444 90 " (8H3) SiOH 005 129 ' C6H1 351 402 132 " C3H7 C=0H, 002 129 005 0881 031 75 459 ( H33231OH 73 461 (CH3)3310 127 Table 18 Biological activity of the GA n-propyl ester (N1) from C. sativus. [relative to an Equal concentration of 0A3 (0.1 mg7mI) in four bioassays] Bioassay N1 activity/GAB activity dwarf pea <.005 cucumber hypocotyl .1 dwarf corn (d1) .05 barley amylase .Ol X. Characterization of a Highlyfiyolar Gibberellin from 2. sativus Methanol extraction of five kg of monoecious Q. sativus seed (Figure 3) and subsequent purification yielded several mg of active material in the n-butanol fraction which was not due to traces of free acidic gibberellins. Following repeated TLC purification and crystallization, this material was found to be more polar than several gibberellin standards on TLC (Figure 28). The crystalline material (m.p. 170-1800) was suspected to be a gibberellin-polyol conjugate since it gave a positive Molisch test and strong bands at ca. 3#00 and 1000 to 1100 cm"1 in the infrared. Titration of the material as a monobasic acid yielded a molecular weight of 500. The remaining material was hydrolyzed with emulsin, purified by TLC, and compared with several D-sugar standards in two solvent systems (Figure 29). The hydrolyzed material was also compared with 0A1 and a-D-glucose standards in two solvent systems (Figure 30). The Rf values of the two spots 128 Figure 28 Thin-layer chromatogram of a highly polar acidic gibberellin (B1) isolated from Q. sativus seed. [authentic gibberellins A1, A2, A3, A4, A5, A7, A9 spotted for comparison; plate: Brinkmann Pre-coated Silica Gel F25“, 0.25 mm thickness, sprayed with alcoholic H250“; solvent system: benzene:n-butanol:acetic acid (70:25:5, v/v)] Figure 28 129 -m--— ...“- - 3 BR .-9 a. '4 5" 7 C1 ”“""“ " 0.5.1,: ego/1mm ‘ {74;/ I“ \~’ 130 Figure 29 Thin-layer chromatograms of the hydrolysis pro- ducts of a highly polar acidic gibberellin iso- lated from Q. sativus seed, and several sugar standards. [D-sugar standards: glucose, ribose, fructose, mannose, galactose, glucosamine; plates: Brinkmann Pre-coated Silica Gel F25“, 0.25 mm thickness, sprayed with periodate- benzidine; solvent systems: A, iso-propanol: acetic acid:water (3:1:1, v/v), B, n—butanol: acetic acid:water (4:1:2, v/v)] 151 F igure 2 9 132 Figure 30 Thin-layer chromatograms of the hydrolysis products of a highly polar acidic gibberellin isolated from Q. sativus seed, D-glucose, and GAl. [platesz Brinkmann Pre-coated Silica Gel G, 0.25 mm thickness, sprayed with Folin- Ciocalteau reagent; solvent systems: A, iso- propanolzammonium hydroxidezwater (10:1:1, v/v), B, iso-propanol:acetic acid:water (3:1:1, v/v)] u '0}- Diflwwfie GBixMt (‘3 .\ s-’ 'o Figure 30 135 ,6!)le {Muo’h'ip ,OHtl a, [launcfic 9:» 134 produced by the hydrolyzed material agree closely with those for B-D-glucose and 0A1. The gibberellin spot did not correspond to other gibberellin standards. These results indicate that the highly polar gibberellin-like material consists of a GAl-B-D-glucose conjugate. The molecular weight obtained by titration indicates the conjugate contains equimolar amounts of CA1 and glucose since the molecular weight of Gal-B-D-glucose would be 510. DISCUSSION Extraction of gibberellins from seeds and etiolated seedlings of three 9. sativus sex types has demonstrated that the gynoecious type is gibberellin deficient relative to the more male types. This difference in both free acidic and total (free acidic plus enzyme-released) gibberellin activity was observed at all growth stages. Extracts of monoecious and andromonoecious types contained 50 to 800 percent more free acidic gibberellin activity, depending on growth stage, than did the gynoecious (Figure 9). The more difficult task of measuring gibberellin activ- ity in green plants was undertaken to avoid extrapolating results with etiolated seedlings to green plants. Long days increase maleness in.§. sativus (Ito and Saito, 1957a) and gibberellin production is often associated with young leaves as well as root tips. Thus, green plants may contain higher levels of gibberellin than etiolated seedlings at the same growth stage. Since 9. sativus floral parts are initiated bisexually and then develop into either pistillate, staminate, or perfect flowers (Atsmon and Galun, 1960), hormone levels at the time of flower differentiation may be particularly important in determining sex type. Flower buds appear in 135 136 the axil of the first true leaf and often even in the cotyledonary axil. In addition, formation of staminate flowers on MSU 713-5 in response to gibberellin application is greatest if the treatment occurs at the cotyledon or first leaf stage (Kubicki, l969h; Pike and Peterson, 1969). Sex type can generally be determined this early since the ovary is usually inferior. Axils of the first leaves of monoecious plants usually bear recognizable staminate flowers which abort while quite small; normal staminate flowers appear at the second to fourth nodes. Although environmental factors cause some variation, the first true leaf (fully expanded) usually occurs at fourteen to twenty days, the second at twenty-one to twenty-six days. Thus, fully developed flowers often occur by the fourth week. The possible relationship of time of flower differentiation and gibberellin activity at that growth stage could not be satisfactorily investigated with etiolated seedlings which remain at the hypocotyl stage until three weeks old. Relative gibberellin levels in green plants were found to parallel those in etiolated seedlings (Figure 10). The high levels of gibberellin activity in the more male sex types at the one-week stage are particularly significant since this is the stage where flower initiation at the cotyledonary and first leaf axils is likely to occur. Gynoecious Q. sativus (MSU 713-5) seedlings planted in early summer and subjected to a severe cold spell of a few days duration were observed to produce unexpected 137 staminate flowers (0. E. Peterson, unpublished results). This is in contrast to normal field conditions where lower temperatures favor female sex expression in monoecious Q. sativus. It was postulated that the effect of this environmental shock might be due to.a change in either synthesis or metabolism of gibberellin or some other endogenous growth regulator. In addition, vernalization (chilling) of hazel seeds followed by incubation at 20°C increased the quantity of organic solvent-extractable gib- berellin (Ross and Bradbeer, 1968). In view of these observations it appeared that chilling of Q. sativus seed might affect gibberellin levels as well as sex expression. Greenhouse-grown monoecious and gynoecious plants were produced from chilled, germinated seeds. The staminate to pistillate ratio in monoecious plants was unaffected relative to non-chilled plants, but there was a definite induction of staminate flowers in gynoecious plants pro- duced from chilled seed (Table 5). This induction corresponded closely to increased gibberellin activity (relative to control) in one-week old gynoecious seedlings (Table 6). Thus, staminate flower induction in chilled gynoecious g. sativus plants may be due to increased gibberel- lin activity. The mechanism by which chilling increases the synthesis or release of gibberellin remains unknown. Several lines of evidence, summarized below, now exist which support the theory that endogenous gibberellins are an integral part of Q. sativus sex expression regulation and 138 deserve the title "male hormoneI of this species. Exogenously applied gibberellins have successfully induced staminate flowers in many gynoecious lines. Longer hypocotyl and internode lengths, indicators of endogenous gibberellin levels, have been correlated with more male sex types. Known inhibitors of gibberellin biosynthesis, such as CCC, increase femaleness. Shoot diffusates of four day old monoecious seedlings and root exudates of six week old monoecious plants contained higher levels of gibberellin than did those of gynoecious. Greater amounts of applied gibberellin have been recovered from monoecious than from gynoecious plants. Organic and buffer extraction of seeds, etiolated seedlings, and green plants in a wide range of growth stages has demonstrated that a monoecious and an andromonoecious line consistently contained higher levels of both free and “bound” gibberellin activity than a gynoe- cious line. Vernalization of gynoecious Q. sativus seed increased both male tendency and gibberellin activity. Kubicki (l969a) hypothesized that g. sativus sex type is determined by the balance of hormone substances 'M' and "F” in the region of the incipient flower bud. High levels of the ovary-forming substance "F" lead to pistillate or perfect flower formation while staminate flowers are formed in the presence of low levels of "F'. In view of the antagonistic effects of auxin and ethylene versus gibberellin on g. sativus sex expression, this hypothesis seems reasonable. 139 Table 19 contains a listing of the genes which are known to affect g. sativus and 9. £219 sex expression. It represents a compilation of data from several investigators (Kubicki, l969a-h; Galun, 1961; Howe, 1969). Kubicki (1969a) associates "F' with locus egg: allele 325+ condi- tions reduced accumulation of "F", ang conditions a high rate of accumulation of "F", and $251 is intermediate. According to Kubicki, the activity of this gene is increased by auxin and reduced by gibberellins, auxins being inductors, and gibberellins co-repressors in the operon system governing synthesis of ”F”. Exogenous gibberellin would then reduce the activity of "F“ in gynoecious plants to a level at which staminate flowers can be temporarily formed. Pre- sumably, high levels of endogenous gibberellin would function similarly. On the other hand, it appears equally probable that endogenous gibberellin activity is directly associated with locus egg. Of the five loci known to affect sex expression, only the occurrence of 223 can be correlated with gibberellin activity; agrfagr+ occurs in homozygous monoecious and andromonoecious lines while aggfigng occurs in homozygous gynoecious lines. Ihimozygous gynoecious and monoecious lines contain the same alleles of loci a, m, and 25. However, locus 32; may also be associated with the level of a gibberellin inhibitor, auxin, an auxin inhibitor, or some other sex determinant. Genetics of Cueumis Sex Expression 140 Table 19 A. Q. sativus Phenotype Homozygous Genotype potential for perfect or female superior female flowers female flowers intensity ovary + + androecious aa -- acr acr -- -- andromonoecious AA mm acr+acr+-- trtr monoecious AA MM acr+acr+-- trtr trimonoecious AA MM acr+acr+-- TrTr hermaphroditic AA mm achachFF trtr gynoecious AA MM achachff trtr 1 + gt according to Galun (1961) B. Q. melo Phenotype Homozygous Genotype potential for perfect or superior male flowers female flowers ovary andromonoecious GG mm TrlTrltrztr2 monoecious GG MM TrlTrltrztr2 trimonoecious -- -- trltrlTrzTr2 hermaphroditic gg mm TrlTrltrztr2 gynoecious gg MM TrlTrltrztr2 2 9 according to Poole and Grimball (1939) 141 Assuming that locus a2; is associated with either gibberellin, auxin, or 'F' levels, some predictions may be made. If gibberellin is associated with egg, homozygous androecious and trimonoecious lines, which contain allele agg+, should contain gibberellin levels similar to those in monoecious and andromonoecious lines. Homozygous hermaphroditic lines (gggfigggr) should then be relatively gibberellin deficient. Auxin can induce female flowers in androecious Q. sativus plants (agrf) (Kubicki, l969e). A hermaphroditic line has been found to contain more auxin than a closely related andromonoecious line (Galun gt $1., 1965); only alleles of locus a3; should be different in these lines. These findings indicate an association of auxin with the 9g; locus. Androecious, trimonoecious, and monoecious lines should then be auxin deficient relative to gynoecious lines. However, these predictions of relative auxin and gibberellin levels based on which hormone is associated with 9g; are not mutually exclusive since the same sex types predicted to contain relatively high gibberellin levels are also predicted to be auxin deficient. In contrast to Q. sativus, seeds and green plants of monoecious and andromonoecious Q. mglg lines were found to be gibberellin deficient relative to gynoecious and hermaphroditic lines (Tables 7 and 8). This finding is con- sistent with the failure of exogenous gibberellin to induce staminate flowers on gynoecious Q. mglg plants. Thus, gibberellin is not the I'male hormone" of Q. melo. 142 Staminate flowers have been induced on gynoecious g. melo (MSU 1G) plants by grafting melon scions onto either pumpkin rootstocks or interstocks. Thus, the active factor in staminate flower production appeared to be synthesized in, or associated with, the pumpkin foliage. Since gynoe- cious g. mglg is not gibberellin deficient relative to more male types, and exogenous application of gibberellins has not successfully induced staminate flowers in gynoecious 9. £219, a hormone or regulator other than gibberellin was suspected to be responsible for the grafting effect. Methanol extraction of pumpkin seeds and leaves of two varieties which successfully induce staminate flowers yielded levels of gibberellin activity far lower than that in any 9, mglg sex type (Table 9). Thus, the relatively small amount of gibberellin produced by pumpkin stocks would not be expected to affect Q. mglg sex expression. This is consistent with the hypothesis that gibberellin is not the “male hormone“ of g. 2219. Gene g (Table 19) plays a role in Q. melo female intensity analagous to that of gene 39; in cucumber, but the dominant allele G occurs in the more male sex types. Kubicki associates allele 9 with high levels of ”M", but it appears that gibberellin levels are not associated with 5 since gibberellin does not increase maleness in 9. £319. Since gibberellin levels are relatively high in hermaphroditic and gynoecious lines (recessive alleles gg) and low in lines containing 99, gibberellin might be 103 considered a female hormone in.§, 3212. However, this is unlikely in view of the absence of any report that exogenous gibberellin increases femaleness in g. 2219. Apparently, gibberellin has little influence on sex expression in this species. Hayashi gt 3;. (1971) demonstrated that the primary free acidic gibberellin in six day old etiolated monoecious seedlings of g. sativus is Al. Gas-liquid chromatography also indicated that A3, A4, and A7 might be present. The free acidic gibberellins of Q. sativus seed have been shown here to be A1, A3, A4, and probably A7; A1 accounts for most of the total gibberellin activity. Since all three sex types contain the same relative proportions of these gibberellins, the quantity rather than type of gibberellin appears to be important in sex expression. Gibberellins Au and A7, more active than A1 or A3 in promoting staminate flowers in Q. sativus, contribute only a small fraction of the total extractable activity. This situation may be due to easier absorption of the less-polar A4 and A7 by the plant, or A1 may represent a slightly deactivated storage or transport form while A“ and A7 are the actual active species. 9. £219 free acidic gibberellins include Al, A3, and probably A5. A1 and A3 are present in far greater amounts than A5. As in.g. sativus, the relative proportions of the gibberellins are the same for the four sex types investigated. 144 Three ”bound" or conjugated gibberellins, a glucoside and two esters, were isolated from mature seeds of monoe- cious Q. sativus (MSU 736); evidence for the presence of 'bound' gibberellin in andromonoecious and gynoecious seed was also obtained. Monoecious seed was used for isolation and identification of these compounds due to overall higher levels of gibberellins in these seeds and the availability of large seed quantities necessary to isolate several milligrams of the compounds. The original evidence for the presence of “bound” gibberellins in.g. sativus seed was extraction of increased amounts of free acidic gibberellins following incubation of tissue homogenates with ficin or a-glucosidase (Table 1). An initial decrease in this ”bound" gibberellin upon germination corresponded with increased amounts of free gibberellin. Following germination, developing seedlings synthesized increasing amounts of both free and "bound" gibberellins. The release of gibberellin by enzymes was at first attributed to hydrolysis of protein-gibberellin or carbohydrate-gibberellin bonds. Such protein- or carbohydrate- gibberellin conjugates would not be extracted with organic solvents. However, precipitation of low-molecular weight compounds with protein is a non-specific phenomenon which can be observed with gelatine solutions. Thus, much of the ficin release of gibberellin may be due to hydrolysis of protein and solubilization of gibberellin non-specifically complexed with protein by other than covalent bonds. 145 a-Glucosidase release of gibberellin may be attributed, in part, to hydrolysis of specific carbohydrate-gibberellin covalent bonds such as occur in gibberellin-B-glucosides; GAl-a-D-glucoside may contribute to the total enzyme- releasable activity. The two isolated gibberellin esters are probably not part of the enzyme-releasable activity. They were isolated from the neutral fraction and make up only a small part of the extractable activity. GAl-a-D-glucose is similar to glucosides of gibberellins A3, A8, A26, and A27 isolated from several sources. Its occurrence in dry seeds and decline during germination suggest a storage function; however, the high water solubil- ity of the glucosides suggests a transport function. The occurrence of glucosides in bleeding sap of several tree species (Sembdner 22 al., 1968) also suggests a transport function. These two functions may not be antagonistic; glucosides found in bleeding sap may be stored gibberellins in transport from storage areas to other tissues. The occurrence of neutral gibberellin propyl esters as natural constituents of plants is a unique finding. The occurrence of such compounds is surprising in view of the relative absence of esters of other plant acids. The pos- sibility that these compounds represent an artifact of the isolation procedure is small since no 03 alcohols, acids, ethers or other C3 compounds were utilized in the extraction process. Elimination of isopropanol in TLC and paper chromatography did not result in elimination of spots due 146 to the esters. The identification of the A3 n-propyl ester is beyond doubt since the synthetic compound had the same IR and mass spectra, Rf in three solvent systems, and melting point. The function of gibberellin esters in plant metabolism is unknown but a storage function seems likely since esteri- fication represents both a deactivation and depolarization. A decrease in polarity makes transport in sap less likely as a function, but transport across a hydrophobic membrane may be facilitated. Possibilities for Future Research: The results presented in this thesis point to several promising avenues for further investigations of the rela- tionship of plant hormones to sex expression. As sufficient quantities of seed become available, gibberellin activity should be assayed in other 9. 2212 and g. sativus sex types. Assays of mature seed and one growth stage of green plants, as reported above for Q. melo, do not require enormous amounts of material. Assay of gibberellin activity might also be extended to other Cucurbita species such as pumpkin, squash, watermelon, etc. as different sex types of these species become available. Assay of the activity of other hormones, e.g., auxin, ethylene, and cytokinin, in a wide variety of sex types of these species would provide additional information on the role of phytohormones in sex expression. Comparison of these results with the genetic background of the plants 147 would be of theoretical interest in regard to the biochemical genetics of sex expression. Of more immediate importance to human welfare is the probability that a complete picture of the relationship of hormones to sex expression would allow control of sex expression through hormone application. Such control might greatly facilitate development of new hybrids as well as increase productivity. From the biochemist's point of view the presence of gibberellin glucosides and esters in.§. sativus raises interesting questions about the biosynthesis of these com- pounds. Labelling studies might determine the precursor of the n-propanol fragment of the GA1 and GA3 esters. Perhaps most interesting is the question of the mechanism of gibberellin action in Q. sativus. How does gibberellin induce male floral parts, or is this a secondary effect? Are there specific receptors for gib- berellin on cell or nuclear membranes? DOes gibberellin induce DNA synthesis, RNA synthesis, or bind to an enzyme to increase its activity? 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