w: 25¢ per do per item RETUMING LIBRARY MATERIALS: \— Place in book return to ream charge from circulation recoa 6’" 2:“..‘s‘ -;".- '1- fl“\\\\ '.' 1.: “43:5,!” 3.1 waving) @93’ GIBBERELLINS AND THE PHOTOPERIODIC CONTROL OF STEM GROWTH IN THE LONG-DAY ROSETTE PLANT SPINACH BY James David Metzger A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1980 «J K\ two ABSTRACT GIBBERELLINS AND THE PHOTOPERIODIC CONTROL OF STEM GROWTH IN THE LONG-DAY ROSETTE PLANT SPINACH BY James David Metzger The long-day rosette plant spinach responds to a trans- fer from short to long days with increased petiole growth, stem elongation, and flower formation. At least the first two responses appear to be mediated through modulation of the gibberellin status. Analysis of highly purified extracts from spinach shoots by combined gas chromatography-mass spectrometry has demon- strated the presence of six gibberellins (GAS): GA53, GA44, GA19' GAl7, GAZO’ and GA29. The two major GAs were GA19 and GA These two GAs were probably responsible for most of 20' -the GA-like activity detected in spinach shoot extracts with the d-S corn bioassay. The other four GAs found in extracts from spinach shoots occurred in much smaller amounts. Struc- tural considerations suggest that the six GAs identified in spinach shoots are related in the following metabolic path- way: GA53 + GA44 + GA19 + GAl7 + GA20 + GA29. The changes in the levels of five of these GAs in rela- tion to photoperiodic treatment were examined by combined gas James David Metzger chromatography-selected ion current monitoring. Long-day treatment caused a 5-fold decline in the level of GA19' while GA20 and GA29 increased almost 7-fold during the same period. In absolute terms, the level of GA20 increased from 0.8 ug per 100 g dry weight in short days to 5.5 pg per 100 g weight after 14 long days. The levels of GAl7 and GA44 did not change significantly with long-day treatment. These results are consistent with the idea that GA19 is converted to GAZO' and that this conversion is under photoperiodic con- trol. Since stem growth in spinach is correlated with an increase in the level of GA one major aspect of photo- 20' periodic control of stem growth might be the availability of GA20 through regulation of the conversion of GA19 to GAZO' Analysis of spinach root extracts by either combined gas chromatography-mass spectrometry or combined gas chroma- tography-selected ion current monitoring showed that roots contained only four of the six GAS found in spinach shoots: and GA . Neither GA nor GA 44’ 19’ 29 17 20 detected in root extracts. Both phloem and xylem exudate GA were GA53, GA had patterns of GA-like activity similar to those found in shoots and roots, respectively. Since foliar application of [3HJ-GA resulted in transport of unmetabolized [3Hj-GA20 20 to the roots, part of the endogenous GA20 present in the phloem must have been transported to this organ. Thus, if GA is made in, or transported to the roots, it is rapidly 20 metabolized in that organ. This is a clear indication that the regulation of GA metabolism is greatly different in James David Metzger roots and shoots. The subcellular distribution of GA19 and GA20 in rela- tion to photoperiod was also investigated. Analysis by the d-5 corn bioassay showed that chloroplast fractions from spinach leaves contained about 15% of the total GA20 found in the leaf and 1% of the total leaf GA regardless of 19’ prior photoperiodic treatment. It was concluded that photo- period does not act through a redistribution of GAs between the chloroplasts and the cytoplasm. Comparison of two methods to extract GA-like substances from chloroplasts of spinach or wheat showed that extraction by non-ionic deter- gents was not more efficient than the conventional methanolic extraction technique. ACKNOWLEDGMENTS I would like to take this opportunity to thank all those people, who, in the past five years, contributed to this effort in a concrete way or with moral support. In particular, I would like to thank my committee and especial- ly my major professor, Jan Zeevaart, for their advice and guidance. I would also like to thank Belinda Dorer for her excellent typing in preparing the manuscript. Finally, I would like to extend my deepest gratitude to my parents, Peg and Paul Metzger,and my good wife, Sue, for their constant support and love which contributed in no small way to the success of this work. ii To two fine teachers, Larry B. Adams and Guy L. Steucek. iii The research reported in this thesis was supported by the U.S. Energy Research and Deve10pment Administration and the U.S. Department of Energy under Contract EY-76- C-02-l338. iv LIST OF TABLES LIST OF FIGURES TABLE OF CONTENTS LIST OF ABBREVIATIONS CHAPTER 1. 1.1. 1.2. 1.3. ‘GENERAL INTRODUCTION AND LITERATURE SURVEY Introduction . . . . . . . . . . . . GAS: Chemistry and Analytical Techniques . . . . . . . . . . . . 1.2.1. Structure. . . . . . . . . . 1.2.2. Biological Activity. . . . . 1.2.3. Purification and Quantifica- tion 0 Q - A. O O I O O O O O GAS as Regulators of Stem Growth . . 1.3.1. Correlations Between Stem Growth and Endogenous GA Content. . . . . . . . . . 1.3.2. Growth Retardants. . . . . . 1.3.3. Stem Growth in LB Rosette Plants . . . . . . . . . . 1.3.4. Cytology of the GA-response. Biosynthesis and Metabolism of GAS . 1.4.1. Biosynthesis of GAS. . . . . 1.4.2. Interconversion of GAS . . . 1.4.3. Mechanism of Action of Growth Retardants. . V Page xi xiii ll 12 13 13 14 16, 16 22 25 CHAPTER 2. CHAPTER 3. 1.4.4. Dwarfism . . . . . . . 1.5. Control of GA Biosynthesis and Metabolism . . . . . . . . . 1.5.1. Control of Enzymes . . 1.5.2. Compartmentation . . . 1.5.3. Photoperiodic Control of Metabolism . . . . . GA 1.6. GAS: Root-Shoot Interrelationships . 1.7. Statement of Purpose . . . . . THE IDENTIFICATION OF SIX ENDOGENOUS GIBBERELLINS IN SPINACH SHOOTS . . 2.1. Introduction . . . . . . . . . 2.2. Materials and Methods. . . . . 2.2.1. Plant Material . . . . 2.2.2. Bioassay . . . . . . . 2.2.3. Extraction and Purification Procedures . . . . . 2.2.4. TLC. . . . . . . . . . 2.2.5. Derivatization . . . 2.2.6. GLC. . . . . . . . . . 2.2.7. GLC-MS . . . . . . . . 2.3. Results. . . . . . . . . . . . 2.3.1. Characterization of GA-like Substances in Spinach Shoots . . . . . . . 2.3.2. Identification of GAS found in Spinach Shoots. . 2.4. Discussion . . . . . . . . . . THE EFFECT OF PHOTOPERIOD ON THE LEVELS OF ENDOGENOUS GIBBERELLINS IN SPINACH SHOOTS O O O O O C O O O D O O O 0 Vi Page 27 28 28 3O 31 36 41 42 43 44 44 44 45 ' 47 48 48 48 49 49 49 66 69 3.1. Introduction . . . . . . . . . . . . 70 3.2. Materials and Methods. . . . . . . . 70 3.2.1. Plant Material and Photo- periodic Treatments. . . . 70 3.2.2. Extraction and Purification Procedures . . . . . . . . 71 3.2.3. GLC-SICM . . . . . . . . . . 72 3.2.4. GA Treatment . . . . . . . 73 20 3.3. Results. . . . . . . . . . . . . . . 73 3.4. Discussion . . . . . . . . . . . . . 77 CHAPTER 4. COMPARISON OF THE LEVELS OF ENDOGENOUS GIBBERELLINS IN ROOTS AND SHOOTS OF SPINACH IN RELATION TO PHOTOPERIOD . . . 81 4.1. Introduction . . . . . . . . . . . . 82 4.2." Materials and Methods. . . . . . . . 83 4.2.1. Plant Cultures and Photo- periodic Treatments. . . . 83 4.2.2. Effect of Photoperiod on Levels of Extractable GA— 1ike Substances from Roots and Shoots . . . . . . . . 83 4.2.3. Identification of GAS in Root Extracts. . . . . . . 84 4.2.4. GA Content of Phloem Exudate 84 4.2.5. GA Content of Xylem Exudate. 85 4.2.6. GA Content of Spinach Roots Cultured in Vitro. . . . . 85 [14CJ-Labeling of Assimi— lates. . . . . . . . . . . 87 4.2.8. Transport of [3Hj-GA20 and C14CJ-Labeled Assimilates. 87 vii CHAPTER 5. Page 4.2.9. Carbohydrate and Protein Analysis of Phloem Exudate 89 4.2.10. Autoradiography. . . . . . . 90 4.3. Results. . . . . . . . . . . . . . . 90 4.3.1. Effect of Photoperiod on Levels of GAS in Shoots and Roots. . . . . . . . . 90 4.3.2. Identification of Root GAS . 93 4.3.3. GA Content of Phloem Exudate 94 4.3.4. Does EDTA Treatment Enhance Phloem Exudation or In- crease General Cell Leakage? . . . . . . . . . 101 4.3.5. GA Content of Xylem Exudate. 107 4.3.6. Transport of [BHJ-GA 0 and [14CJ-Assimilates rom the Shoot to the Roots . . . . 107 4.3.7. GA Content of Roots Cultured in Vitro . . . . . . . . . 117 4.4. Discussion . . . . . . . . . . . . . 117 4.4.1. Distribution of GAS Between Roots and Shoots . . . . . 117 4.4.2. Possible Sites of GA Synthesis. . . . . . . . . 120 4.4.3. Physiological Significance of GA Transport in the Phloem . . . . . . . . . . 122 4.4.4. The Physiological Signifi- cance of GA Transport in the Xylem. . . . . . . . . 122 THE SUBCELLULAR DISTRIBUTION OF GAS IN SPINACH LEAVES . . . . . . . . . . . . . 124 5.1. Introduction . . . . . . . . . . . . 125 5.2. Materials and Methods. . . . . . . . 126 viii CHAPTER 6. APPENDIX, REFERENCES 5.3. 5.4. Page 5.2.1. Plant Material . . . . . . . 126 5.2.2. Isolation of Chloroplasts. . 127 5.2.3. Extraction and Purification Procedures . . . . . . . . 128 5.2.4. Bioassays. . . . . . . . . . 129 Results. . . . . . . . . . . . . . . 130 5.3.1. Subcellular Distribution of Spinach GAS. . . . . . . . 130 5.3.2. Comparison Between Effi- ciency of Extraction of GAS from Spinach Chloro- plasts by Methanol and Solutions of Non-Ionic Detergents . . . . . . . . 130 5.3.3. An Attempt to Repeat the Findings of Browning and Saunders with Wheat Chloroplasts . . . . . . . 133 Discussion . . . . . . . . . . . . . 134 5.4.1. The Physiological Signifi- cance of the Distribution of GAS Between Chloro- plasts and the Rest of the Leaf Cell. . . . . . . . . 134 5.4.2. Comparison of Efficiency of Extraction of GAS from Chloroplasts by Methanol and Solutions of Triton X-100. . . . . . . . . . . 138 GENERAL DISCUSSION AND CONCLUSIONS . . . . 139 145 155 ix LIST OF TABLES Table Page 2-1 GLC-MS data obtained with samples from Spinach and with authentic GAS. . . . . . . 62 3-1 Comparison of the effect of exogenous GAZO and LD on stem growth of spinach plants . . 78 4-1 Comparison of GLC-MS data from purified extracts of spinach roots and shoots. . . . 95 4-2 Identification of gibberellin A53 in spinach roots by GLC-SICM . . . . . . . . . . . . . 96 4-3 Comparison of the concentration of GA—like substances in phloem and xylem exudates. . . . . . . . . . . . . . . . . . 99 4-4 Comparison of concentration of GA-like substances in phloem exudate from various species . . . . . . . . . . . . . . . . . . 102 4-5 The effect of EDTA pretreatment on radio- activity in the phloem exudate collection solution. . . . . . . . . . . . . . . . . . 106 4-6 Comparison of concentration of GA—like substances in xylem exudate from various species . . . . . . . . . . . . . . . . . . 110 4-7 The effect of EDTA pretreatment on the amount of radioactivity (3H and C) in the phloem exudate collection solution . . . . . . . . 116 Figure 1-1 1-2 3-1 LIST OF FIGURES Numbering system of gibberellins and kaurenoids (A). Examples of C19- and CZO-GAS O O O O O O O O O O O O O O O O O O Biosynthetic pathway of GAS from mevalonic acid . . . . . . . . . . . . . . . . . . . GA-like activity present in an extract of lyophilized spinach shoots . . . . . . . . Chromatographic behavior of fractions I(A) and II(B) when separately subjected to prepara- tive HPLC. . . . . . . . . . . . . . . . . GA-like activity associated with 0.5% aliquots of individual fractions resulting from analytical reverse phase HPLC of purified fractions I(A) and II(B) . . . . . . . . . . GLC analysis of MeTMS-I. . . . . . . . . . . GLC analysis of MeTMS-II . . . . . .‘. . . . Numbering system of ent-gibberellane (A), and structures of the six GAS identified by GLC-MS in spinach shoot extracts . . . . . Changes in the relative levels of five GAS in spinach shoots as measured by GLC-SICM, and stem length as affected by different dura- tions of LD treatment. . . . . . . . . . . The effect of photoperiod on GA-like activity in root and Shoot extracts . . . . . . . . The effect of photoperiod on the GA-like activity in phloem exudate and leaf extracts Comparison of chromatographic behavior of substances in phloem exudate and five authentic sugars . . . . . . . . . . . . . . xi Page 19 51 53 56 58 60 63 76 91 98 105 Figure Page 4-4 GA-like activity in xylem exudate and in extracts of shoots from plants that were used for obtaining the xylem exudate. . . . 109 4-5 Root systems (top) and their autoradiograms (bottom) from plants allowed tI photo- synthesize in the presence of 4C02 . . . . 111 4-6 Distribution of radioactivity on a chromato- gram of a root extract from 2 plants 24 hr following a foliar application of C3HJ-GA20 115 4-7 GA-like activity in 2200 Spinach roots cultured in Vitro, and the medium in which they were grown . . . . . . . . . . . . . . 119 5-1 Comparison of GA-like substances in spinach leaves and chloroplasts . . . . . . . . . . 132 5-2 Comparison of yields of GA-like substances from chloroplasts of Kolibri wheat using a solution of Triton X-100 in water or aqueous methanol as the extractant. . . . . . . . . 136 6-1 A scheme summarizing the differences in three aspects of the GA status of spinach plants grown under SD or LD conditions . . . . . . 144 A-l The mass spectrum of spinach MeTMS-GA19 . . . 146 A-2 The mass spectrum of spinach MeTMS-GA20 . . . 147 A-3 The mass spectrum of standard MeTMS-GAZO. . . 148 A-4 The mass spectrum of spinach MeTMS-GA29 . . . 149 A-5 The mass Spectrum of standard MeTMS-GA29. . . 150 A-6 The mass spectrum of spinach MeTMS-GAl7 . . . 151 A-7 The mass spectrum of standard MeTMS-GA17. . . 152 A-8 The mass spectrum of spinach MeTMS-GA44 . . . 153 A-9 The mass spectrum of spinach MeTMS-GAS3 . . . 154 xii GA MeTMS-GA C -GA 19 C -GA 20 GGPP CPP GLC-MS GLC-SICM TLC HPLC SD LD EDTA LIST OF ABBREVIATIONS Gibberellin Methyl ester trimethylsilyl ether of gibberellin Gibberellin with 19 carbons Gibberellin with 20 carbons Geranylgeranyl pyrophosphate Copalyl pyrophosphate Abscisic acid Mass to charge ratio Gas liquid chromatography Combined gas liquid chromatography mass spectrometry Combined gas liquid chromatography selected ion current monitoring Thin layer chromatography High performance liquid chromatography Short day(s) Long day(s) Ethylenedinitrilotetraacetic acid xiii Chapter 1 General Introduction and Literature Survey GIBBERELLINS AND THE PHOTOPERIODIC CONTROL OF STEM GROWTH IN THE LONG-DAY ROSETTE PLANT SPINACH BY James David Metzger A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1980 1.1 INTRODUCTION The terrestrial environment is a dynamic, multifaceted system. Plants, like all other organisms which inhabit land environments, must be able to cope with drastic changes in temperature, light parameters, and water availability. Un- like mobile animals which can migrate to survive unfavorable environmental conditions, sessile plants adjust to changing conditions by altering growth and development. In many in- stances, a change in some environmental factor does not act directly in altering growth and development, but instead acts through hormone mediators. There are many examples of such responses. In certain plants, the change from vegetative to reproductive growth is controlled by photoperiod. The Site of perception of daylength is in the leaves, yet the site of the morphological change from the vegetative to the flowering State is in the shoot apex (Zeevaart, 1976). From physiolog- ical experiments it could be shown that an uncharacterized "floral stimulus" is transported from the leaves to the apex where it causes a drastic morphological change (Zeevaart, 1976) . When plants are placed under conditions of water stress, the guard cells in the epidermis become flaccid, and the stomates close, thereby minimizing water loss. Water stress also causes a tremendous increase in the level of the hormone abscisic acid (ABA) in the leaves. Moreover, exogenous ABA has the same effect as water stress on stomatal aperture: rapid closure. This indicates that ABA is a hormone mediator which controls water loss by regulating the stomatal aperture (Raschke, 1975). The preceding two examples can be pictori- alized in the following manner: change in plant .._.___. growth and development change in change in environmental _______. hormone parameter status Long-day (LD) rosette plants such as Rudbeckia bicolor, spinach, and Silene armeria are plants which grow vegetatively under Short day (SD) conditions and have very short stems (little or no internodes). Upon transfer to LD conditions, these plants show a bolting response, i.e. rapid stem elonga— tion and flowering. The evidence indicates that flowering and stem elongation in rosette plants are two closely coupled, but separate developmental events (Zeevaart, 1976). Moreover, evidence presented later will Show that photoperiodic control of stem growth appears to be mediated through some change in the gibberellin (GA) status. The term GA status refers to any qualitative or quantitative changes in GAS that occur, as well turnover rates, compartmentation, and tissue sensitivity to GA. Therefore, photoperiodic control of stem growth repre- sents a useful model system in which to study how environmen- tal factors control plant growth and development through hor- mone mediators. 1.2 GAS: CHEMISTRY AND ANALYTICAL TECHNIQUES 1.2.1. Structure. GAS represent a class of tetracyclic diterpene acids. At present, 56 different GAS are known. The structures of all GAS are based on ent—gibberellane (Fig. la). The familiar trivial nomenclature of individual GAS (e.g. GAl, GA56, etc.) refers only to naturally occurring com- pounds with the ggt-gibberellane skeleton, and whose struc- ture and stereochemistry have been fully elucidated (MacMillan and Takahashi, 1968). The most common structural features of GAS asaclass of compounds include: i) the four rings labeled A, B, C, and D, ii) an exocyclic methylene group at C-l7, and iii) a carboxyl group at C-6. GAS as a group can be broken down into two basic categories: GAS with 20 carbons (Czo-GAS) and GAS that contain 19 carbons (Clg‘GAS). The latter group represents GAS in which C-20 has been removed, and in an overwhelming majority of cases, a 5-membered Y- lactone bridge between C-19 and C-10 has been formed (Fig. lb). C-20 of CZO'GAS can exist in a number of oxidation states e.gq.a methyl, hydroxyl, aldehyde, or carboxyl group. Free hydroxyl groups do not appear at C-20, but instead, exist as a part of a 5- or Six membered lactone bridge with C-19. Both C19- and CZO-GAS also vary in the position and number of hydroxyl groups as substituants on the four rings (Fig. lb). Other minor modifications of the egg-gibberellane skeleton 1,2 A2,3 include A or double bonds, a 2,3 epoxide group, and A16, 17 hydration of the double bond. In this thesis "GA- like substances" will refer to substances of unknown structure, Figure 1-1. (A) Numbering system of gibberellins and their immediate precursors, the kaurenoids. Note that ring B of the gibberellins contains 5 carbon atoms, whereas ring B of the kaurenoids contains 6. (B) Examples of C19- and Czo-GAS. Note various oxidation states of C-20 of Czo-GAS, and different positions that can be hydroxylated. ent-Kaurane ent-Gibberellane but which elicit a response in a bioassay. Only chemically identified compounds will be referred to as "GAS". 1.2.2. Biological Activity. Comparative studies of the various GAS in a number of different bioassays has led to a few generalizations on structure-activity relationships (Reeve and Crozier, 1974). In general, Clg-GAS tend to have greater biological activity than CZO-GAS. However, the posi- tion and degree of hydroxylation is of paramount importance to biological activity. C-BB, C-l3 dihydroxylated Clg-GAS have the highest biological activity in most bioassays, but C-38 or C-l3 monohydroxylated GAS also have fairly high bio- logical activity. Moreover, a 41'2 desaturation appears to somewhat enhance biological activity. On the other hand, C-ZB hydroxylation destroys biological activity of the mole- cule, regardless which other positions are hydroxylated. The stereochemistry of the hydroxylations is also important to biological activity. If the C-38 hydroxyl group of GAl is epimerized to the a-configuration (e i-GAl), biological ac- tivity is greatly diminished. GASl and GA4o are both C-2 monohydroxylated GAS. The B-epimer, GASl' is completely in- active in all bioassays tested, while GA4O, the a-epimer, has some biological activity in a number of bioassays (Sponsel at 31., 1977). The oxidation State of C-20 in Czo-GAS also affects bio- logical activity. If C-20 is an aldehyde or part of a C-19 + C-20 5-lactone bridge, moderate to high biological activity results. A methyl or a carboxyl group at C-20 results in low to zero biological activity (Reeve and Crozier, 1974). 1.2.3. Purification and Quantification. In most initial studies of GA physiology, an attempt is made to correlate levels of the hormone with some physiological event. How- ever, quantitative analysis of GAS poses Special difficulties. In green tissues, GAS occur in extremely minute quantities, usually in the order of a few micrograms per kilogram fresh weight of plant material. Moreover, GAS represent only a small fraction of the many different acidic compounds present in green plant tissue. Therefore, extensive cleanup and puri- fication must be accomplished before effective quantification can take place. Another problem in measuring GA levels is that plants usually contain more than one GA. Since the GAS in a given plant are probably metabolically related, the in- vestigator has an interest in determining the levels of the individual GAS. Consequently, methods for the separation of GAS prior to quantification are necessary. In general, the tissue is first extracted followed by one or more group puri- fication steps (procedures which separate GAS~as a class of compounds from other compounds). Solvent partitioning is the most common group separation technique used for GAS, but charcoal adsorption chromatography (Zeevaart, 1971), silicic acid adsorption chromatography (MacMillan 32 31., 1960), gel filtration chromatography on Sephadex (Crozier 93 31., 1969), anion exchange chromatography (Browning and Saunders, 1977), and chromatography on polyvinylpyrrolidone to remove phenolics (Glenn 33 31., 1972) all have been used for group separation purposes as well. These techniques can also be used in combination to give a purer GA-enriched fraction. Prior to quantification, the partially purified, GA- enriched fraction is usually subjected to a chromatography step which separates GAS and affords further purification. In early work paper chromatography was utilized (see Phinney and West, 1960), but this has been largely supplanted by thin- layer chromatography (TLC) with silica gel (MacMillan and Suter, 1961). Other chromatography techniques that separate GAS have been successfully employed: silica gel partition chromatography using either open bed columns (Powell and Tautvydas, 1967; Durley 33 31., 1972) or high performance liquid chromatography (HPLC) (Reeve 33 31., 1976; Reeve and Crozier, 1978), normal phase partition chromatography on columns of Sephadex LH-20 (MacMillan and Wels, 1973), DEAE- Sephadex chromatography (Grabner 33 31., 1976), and reverse phase HPLC (Jones 33 31., 1980). Gas chromatography (GC) of the GA-methyl esters, the methyl ester trimethylsilyl ethers (Binks 33 31., 1969), or the trimethylsilyl ethers and esters (MacMillan and Pryce, 1973) gives fine resolution forndxtures of GAS, as well as serving as a necessary adjunct to certain quantitative techniques. Quantification of GAS has been done mostly by bioassay. There are three basic types of bioassay used for GAS. The earliest used type was the dwarf seedling bioassays. Most common of these are the dwarf maize (Phinney, 1961), dwarf 10 pea (Brian and Hemming, 1955), and the Tan-ginbozu dwarf rice~ bioassay (Ogawa, 1963; Murakami, 1968). All dwarf seedling bioassays use increased growth as a measure of GA activity. The second class of GA bioassays are the ones which depend on increased elongation of seedling hypocotyls. Of these, there are two that are most often used: the lettuce hypocotyl as- say (Frankland and Wareing, 1960) and the cucumber hypocotyl bioassay (Halevy and Cathey, 1960). These assays have the advantage over the dwarf seedling assays in that they are more rapid, and more easily performed. However, they are not as sensitive, and do not have as broad a Spectrum of response to all the GAS. The third and last of the major types of GA bioassays are the ones which employ the response of cereal aleurone layers to GA. These cells, in response to GA, se- crete hydrolytic enzymes into the endosperm, the most notable of which is a-amylase. One can make use of this fact by determining the amount of reducing sugars produced by de- embryonated seeds (Nicholls and Paleg, 1963), or by measuring the increase in a-amylase activity directly (Jones and Varner, 1967). All bioassays suffer several inherent defects. First of all, not all GAS give the same response in a given bio- assay, and in fact, dose response curves for each GA might have different slopes. Secondly, there are many substances in plant extracts, phenolics and ABA to name a few, which can prove to be inhibitory in the test system. Finally, unless standards of the particular compound in question are 11 available, only relative amounts of GAS can be estimated (Graebe and Ropers, 1978). Because of the disadvantages of bioassays, techniques for physico-chemical estimation of the levels of GAS have been developed. The most commonly used techniques employ some de- tector immediately following gas chromatography. Usually the detectors are either flame ionization detectors or mass spec- trometers (e.g. Davis 33 31., 1968;.Frydman 33 31., 1974). Combined gas chromatography-mass spectrometry(GLC-MS) has the added advantage of providing unequivocal identification of the compound in question (Graebe and Ropers, 1978). One dis- advantage in using GLC with a flame ionization detector for quantification of GAS in plant extracts is that one needs standard compounds (which in many cases are not available) in order to know which peaks are actually GAS. 1.3 GAS AS REGULATORS OF STEM GROWTH In this section the evidence is reviewed that GAS are natural regulators of stem growth. It is now well estab- lished that GAS are natural products from higher plants (Lang, 1970; Graebe and Ropers, 1978). Moreover, GAS have been applied to a large number of Species of angiosperms and gymnosperms and a promotion of stem growth is almost always found (Goodwin, 1978; Pharis and Kuo, 1977). However, even though applied GAS cause a dramatic response in stem growth, this evidence 33; 33 does not permit the conclusion that GAS are involved in the regulation of stem growth. Indeed, 12 responses from exogenous substances may be due to some non- specific "pharmacological" effect (Lang, 1970). Proof of the role of GAS as endogenous regulators of plant growth is pro- vided by several lines of evidence. 1.3.1. Correlations Between Stem Growth and Endogenous GA Content. Stem growth in many Species is positively cor- related with endogenous GA content. The peak of extractable GA-like activity coincides with internode elongation in the barley inflorescence (Nicholls and May, 1964; Nicholls, 1974). Similar correlations have been found in apple shoots (Robitaille and Carlson, 1976), in shoots of Picea abies (Dunberg, 1976), as well as in internodes of rice (Osada 33 31., 1973). Many genetic dwarfs contain little or no extractable GA- like activity. Normal growth can often be obtained by appli- cation of GA. For example, the anl, d3, and d5 mutants of Zea mays (Phinney, 1960), and dwarf mutants of Pharbitis nil (Ogawa, 1962), red clover (Stoddart, 1962), rice (Suge and Murakami, 1968), and Phaseolus vulgaris (Goto and Esashi, 1973) all are plants which lack detectable GA-like activity, but normal stem growth is restored with exogenous GAS. How- ever, some genetic dwarfs contain the same amount of endo- genous GA-like substances as their normal counterparts, and do not respond to applied GA. These "GA-insensitive" mutants include certain cultivars of dwarf rice (Suge and Murakami, 1968), Norin 10 types of wheat (Radley, 1970), and a dwarf variety of Silene armeria (Suttle and Zeevaart, 1978). 13 1.3.2. Growth Retardants. Several synthetic organic compounds have been discovered which retard stem elongation without causing malformations or affecting rate of develop- ment. These compounds are called growth retardants (Cathey, 1964). Use of such compounds as Amo-1618 and CCC have pro- vided further evidence that GAS are involved in the regulation of stem growth. Both of these compounds appear to block GA biosynthesis (see section 1.4.3.). The resulting reduction in the level of extractable GA-like substances is correlated with a reduction of stem growth in a number of plants includ- ing Pharbitis nil (Zeevaart, 1966), Phaseolus vulgaris (Gerhard, 1966), and Cupressus arizonica (Kuo and Pharis, 1973). In all of these cases, exogenous GA was able to re- store normal growth. Clearly, growth retardants can be a powerful tool in in- vestigations on GA physiology. However, as with all inhibi- tor studies, care must be exercised when interpreting experi- mental results. Not all plants are suitable for study of GA physiology by growth retardants (Lang, 1970). There are some cases in which applied GA could not completely reverse the dwarfing effects of the growth retardant, indicating that the compound acted at Sites other than just GA biosynthesis (Lang, 1970; Goodwin, 1978). Indeed, both Amo-1618 and CCC also inhibit sterol biosynthesis in Nicotiana tabacum (Douglas and Paleg, 1974). 1.3.3. Stem Growth in LD Rosette Plants. LD rosette plants represent a class of plants which are physiological 14 dwarfs during a certain phase of their life cycle. This dwarfism is, however, environmentally imposed since exposure to LD causes the plant to assume a caulescent growth habit. Thus, these plants represent good experimental objects to study regulation of stem growth because there is an easily manipulable external factor that will cause stem growth. In many LD rosette plants exogenous GA is able to substitute for LD in inducing the stem elongation response (see Lang, 1965; Zeevaart, 1978). - In a number of instances, application of growth retardants prevents LD induced stem elongation, while exogenous GA can overcome the inhibition, e.g. Silene armeria (Cleland and Zeevaart, 1970), spinach (Zeevaart, 1971), and Agrostemma githago (Jones and Zeevaart, 1980a). This implies that the photoperiod regulates stem growth in LD rosette plants through modulation of the GA status. Most investiga- tors found that LD treatment causes both quantitative and qualitative changes in the content of GA-like substances (see Lang, 1965; Zeevaart, 1978). Moreover, in a few cases, over- all GA turnover was greatly increased with LD treatment. These aspects will be dealt with in greater detail in section 1.5.3. Finally, LD also seem to increase the sensitivity of the tissue to GA (Cleland and Zeevaart, 1970; Zeevaart, 1971; Jones and Zeevaart, 1980a). Thus, photoperiod can affect many aspects of the GA status, all of which could contribute to regulation of stem growth in rosette plants. 1.3.4. Cytology of the GA-response. GA-induced stem elongation must arise from either an increase in the mean 15 Cell length, an increase in cell number, or both. In peas (Griffith, 1957), Pharbitis nil (Okuda, 1959), and watermelon (Liu and Loy, 1976), application of GA results in a substan- tial increase in cell number; there .is little or no increase in the mean cell length. In the case of watermelon, the greater cell division is due to a reduced 8 period in the cell cycle (Liu and Loy, 1976). However, in other plants, GA-promoted growth can be ascribed almost entirely to an in- crease in cell length. Haber and Luippold (1960) found that Triticum seedlings from seeds exposed to gamma radiation to prevent cell division, showed the normal stem elongation re- sponse to exogenous GA, indicating that stem growth was due to cell elongation. In 33333 internodes, the primary locus of response to exogenous GA is the intercalary meristem. In this system, GA halts multiplication, while longitudinal cell elongation is strongly promoted (Kaufman, 1965). In most plants, it seems that both cell division and cell elongation are integral parts of the GA response (Sachs, .1965; Jones, 1973). Application of GA3 to the LD rosette plants Hyoscyamus niger and Samolus parviflorus in non- inductive conditions caused an increase in the frequency of transverse cell divisions within 24 hours of application of GA and an increase in the mean cell length within 72 hours (Sachs 33 31., 1959). Moreover, the cytological response to "LD was very Similar to the GA response and could be Suppressed by growth retardants (Sachs and Kofranek, 1963). Stem growth in both rosette and caulescent plants was due to activity in 16 the subapical meristem, and this is the region which shows the response to exogenous GA (Sachs, 1965). In summary, several lines of evidence Show that GAS are natural regulators of stem growth. First of all, there is an excellent positive correlation between the presence of GAS and the ability of stems to elongate. Extractions of GAS or GA-like substances from many plants Show the highest level of GA-like substances when plants have the most rapid stem elon- gation. Genetic dwarfs which lack detectable GA-like sub- stances will respond to exogenous GA, restoring normal growth. LD rosette plants respond to exogenous GAS in SD with increased stem growth. LD treatment also causes quanti- tative and qualitative changes in GAS, increases GA turnover, and tissue sensitivity to GA. Moreover, the cytological re- sponse to exogenous GA in certain rosette plants grown under non-inductive conditions is Similar to the response elicited when the plant is placed in conditions conducive to stem growth. This is compelling evidence for the role of GAS in regulation of stem growth. 1.4 BIOSYNTHESIS AND METABOLISM OF GAS 1.4.1. Biogynthesis of GAS. In the previous section, evidence was provided Showing the changes in the levels of GAS or GA-like substances that were correlated with Stem growth. This implies that plants have mechanisms by which they are able to regulate GA biosynthesis and metabolism, and that this regulation is of physiological and developmental 17 significance to the plant. However, the metabolic pathway must be known before any study on the nature of the regula- tion of stem growth by GAS can be made. Early structural studies on GA3 suggested that this com- pound might be related biogenetically to diterpenes (Cross 33 31., 1956). Birch 33 31. (1958, 1959) showed that either [2-14CJ-acetate or E2-14CJ-mevalonic acid were converted by cultures of the fungus Gibberella fujikuroi to 6A3, thus pro- viding convincing evidence for the diterpene nature of GAS as well as common features of the biosynthetic pathway shared by isoprenoid compounds in many organisms. In subsequent years, considerable effort was expended by a number of laboratories around the world in elucidating the individual steps leading from mevalonic acid to the gibberellins. From metabolic studies on Gibberella and cell-free preparations from the endosperm of Marah macrocarpus and Cucurbita maxima, a common biosynthetic sequence emerged. Figure 2 shows this sequence. The following is a brief account of the numerous studies which delineated the biosynthetic pathway of GAS. A few years after the initial discovery that mevalonic acid is an early precursor of GA3 in Gibberella cultures, a second metabolite of feeds of [2-14CJ-mevalonic acid was dis- covered. This compound turned out to be kaurene. Because of its Structural Similarities to GA3, it was suggested that kaurene is an intermediate in the GA3 biosynthetic pathway (Cross 33 31., 1963). Later Cross and coworkers (1964) found that refeeding l4C-kaurene to fungal cultures led to the 18 formation of 14 C-GA3, thereby establishing its role as an intermediate in the overall GA biosynthetic pathway. The individual steps leading up to kaurene formation were first studied in cell-free preparations derived from the endo- sperm of 33333 (Graebe 33_31., 1965). The biosynthesis of kaurene shares most of the common features of terpene bio- synthesis in that the molecule is built with successive con- densations of S-carbon isoprene subunits. Geranylgeranyl pyro- phosphate (GGPP), a 20-carbon diterpene (four isoprene units), is the last non-cyclic compound in the pathway (Oster and West, 1968). The cyclization of GGPP to kaurene is a two- step process involving the formation of a Short-lived, but, stable, intermediate, copalyl pyrophosphate (CPP) (Schechter and West, 1969). The enzyme kaurene synthase catalyzes both steps. The first step, the conversion of GGPP to CPP, involves the formation of rings A and B of kaurene ("A" activity of kaurene synthase), while the "B" activity con- verts CPP to kaurene in the second step. The "A" and "B" activities were inseparable by a number of chromatographic procedures and hence were considered part of the same en- zyme or enzyme complex. However, the "A" and "B" activities had different sensitivities to a number of inhibitors and different pH optima, indicating that different sites on the enzyme were responsible for the two activities (Frost and West, 1976). Recently, it was reported that indeed the "A" and "B" activities can be partially separated (West and Duncan, 1979). 19 Figure 1-2. Biosynthetic pathway of GAS from mevalonic acid. This sequence has been found in both higher plants and in the fungus Gibberella fujikuroi (From Graebe and Ropers, 1978; Hedden 33 31., 1978). 20 Hydroxylaum Hhvalonic Acid Oxidation of c-zo l l aviation of Gig-GA. \ I .n—uu. 00: 3.3 Dimothylallyl tJ-Isopencenyl Pyrophosphato Pyropho-phato Y Gorlnyl Pyrophoophatc rile: Pornonyl Pyropho-phato l Geranylsnranyl Pytophoophntc Copalyl Pyrophosphato lactone 21 Most of the essential features of the pathway from mevalonic acid to kaurene have been confirmed in a number of cell-free systems: immature seeds of Cucurbita maxima (Graebe, 1969) and Pisum sativum (Anderson and Moore, 1976), pea shoots (Coolbaugh 33 31., 1973), expanding cotyledons of Ricinus communis seedlings, and germinating tomato seeds (Yafin and Schechter, 1975). Moreover, the enzymes involved in the conversion of mevalonate to kaurene appear to be solu- ble, i.e. they occur in the 105,000x3 supernatant of cell extracts. The conversion of kaurene to the first compound with the 333fgibberellane skeleton, GAlz-aldehyde, involves the se- quential oxidation of C-19 from a methyl to a carboxyl group, hydroxylation of C-7, and finally contraction of ring B from a six to a five—membered ring to form the 333-gibbere11ane skeleton. Again, the elucidation of this part of the GA bio- synthetic pathway relied on studies using the cell-free sys- tem derived from 33333 endosperm along with complementary studies in Gibberella. In Marah, the C-19 methyl group of kaurene is first oxidized to a hydroxyl group to form kaurenol (Graebe 33 31., 1965). The hydroxyl group is oxi- dized to an aldehyde (kaurenal) which in turn is converted to kaurenoic acid (Dennis and West, 1967). C-7 of kaurenoic acid is then hydroxylated to form the last compound in the GA biosynthetic pathway that lacks the 333-gibberellane skeleton, 73-hydroxykaurenoic acid (Lew and West, 1971). Contraction of ring B occurs at this point beginning with 22 the abstraction of a hydrogen from the 68 position followed by the extrusion of C-7 from the ring to form GA -aldehyde 12 (West and Fall, 1972; Graebe 33 31., 1975). Subsequently, the sequence of kaurene to GAlz-aldehyde has been shown to operate in cell-free systems derived from immature seeds of Cucurbita maxima (Graebe 33 31., 1972) and Pisum sativum (Ropers 33 31., 1978) . ‘ Whereas the enzymes responsible for the conversion of mevalonic acid to kaurene are soluble enzymes, the oxidative enzymes responsible for the conversion of kaurene to GAlz- aldehyde are microsomal (Dennis and West, 1967; Lew and West, 1971; West, 1973). Moreover, they require molecular 02,NADPH, and have other properties that indicate these enzymes are mixed function oxidases which involve the participation of a species of cytochrome P-450 (Murphy and West, 1969; West, 1973). 1.4.2. Interconversion of GAS. GAlz-aldehyde, the first compound in the biosynthetic sequence to have the 3335 gibberellane skeleton, is first converted to Czo-GAS, which are in turn converted to Clg-GAS. Clg-GAS are generally be- lieved to be responsible for biological activity (Graebe and Ropers, 1978). There are two main centers of GA metabolism in the formation of the various GAS from GAlz-aldehyde: the C-20 methyl group can be oxidized to a variety of oxidation states to form other CZO-GAS, or it can be removed to form - and C -GAS can be hydroxylated in any C -GAs. Both C 19 20 19 number of combinations, so all GAS can be derived from a 23 common precursor, GAlZ-aldehyde. One of the early events in the conversion of GAlz- aldehyde is the oxidation of the C-7 aldehyde to a carboxyl group. In the cell-free systems derived from immature seeds of Cucurbita and Pisum, GA -aldehyde is converted to GA 12 12 (Graebe 33 31., 1974; Ropers and Graebe, 1978). In Gibber- 3113, GAlz—aldehyde is first hydroxylated at C-3 to form GAl4-a1dehyde just prior to the oxidation of the C-7 aldehyde group (Hedden 33 31., 1974). The exact pathway leading to the removal of C-20 and the formation of the C-l9-tC-10 lactone bridge is unclear. Cell- free preparations from Cucurbita endosperm are able to con- vert GAlz-aldehyde to GA4 (a C-3 monohydroxylated Gig-GA). This system is also able to sequentially oxidize C—20 from a methyl to a carboxyl group (Graebe 33 31., 1974a,b). However, the oxidation state of C-20 at which CZO-GAS are converted to Clg-GAS is still not known. In fact, no Czo-GA with an oxi- dation State higher than a methyl group has ever been shown conclusively to be converted to a Clg-GA (Graebe and Ropers, 1978). It is known that both oxygen atoms of the Y-lactone bridge of C 9-GAS are derived from the C-19 carboxyl group of 1 CZO-GAS (Bearder 33 31., 1976). Moreover, the loss of C-20 results in the evolution of 14CO2 when [l4C-203-kaurene is fed to cultures of Gibberella (Dockerill and Hanson, 1978). Apparently, the conversion of CZO-GAS to Clg-GAS involves a nucleophilic attack by the C-19 carboxyl group on an electro— philic center at C-lO generated by the removal of C-20 24 (Bearder and Sponsel, 1977). While these facts eliminate many possibilities, the exact path of conversion remains un- known (Hedden 33 31., 1978). As pointed out earlier in section 1.2.1.,the position and degree of hydroxylation of the 333-gibberellane skeleton controls, in a large part, the degree of biological activity of the molecule. Hydroxylation reactions can occur soon after the formation of GA -aldehyde, e.g. the formation of GA - 12 14 aldehyde in Gibberella (Hedden 33 31., 1974), GA53 in Pisum (Ropers 33 31., 1978), and GA36 in Cucurbita (Graebe 33 31., 1974a). Hydroxylations can also occur after the formation of C -GAS: the C-28 hydroxylation of GA20 to form GA29 in in- 19 tact immature seeds of Pisum (Frydman and MacMillan, 1975), or the formation of GA3 from GA7 in Gibberella (Bearder 33 31” 1975). Unlike the microsomal mixed-function oxidases which con- vert kaurene to GAlz-aldehyde, the enzymes responsible for oxidation of C-20 in the Cucurbita endosperm are soluble and 3 require Fe+ for activity (Graebe 33 31., 1974a,b). Likewise, a cell-free preparation from germinating bean seeds which converts GAl to GA8 by a C-28 hydroxylation is also a soluble enzyme that requires Fe+3 and NADPH (Nadeau and Rappaport, 1972; Patterson and Rappaport, 1974) . Thus, while the conver- sion of kaurene to GAlz-aldehyde shares the common property of increasing the oxidation state of the substrate with the metabolism of GA -aldehyde to other GAS, they occur in dif- 12 ferent parts of the cell. This is probably a reflection of 25 the solubility properties of the different substrates in question. Kaurene and the intermediates of its oxidation to GAlz-aldehyde are not very water soluble, and probably need to be in a hydrophobic environment if an enzymatic reaction is to occur. On the other hand, the products of GAlz-aldehyme metabolism become increasingly polar in nature, and the en- zymes are correspondingly water soluble (Hedden 33 31., 1978). Catabolism of Clg-GAS is not well understood. Some plant material, particularly mature seeds, contain "bound" or con- jugated GAS. For the most part, such GAS are glucosyl esters or ethers (Graebe and Ropers, 1978; Hedden 33 31., 1978). While conjugated GAS could simply represent biologically in- active end-products of metabolism, it has been suggested that they serve as storage (Lang, 1970) or transport forms (Sembdner, 1968). C -GAS can be metabolized further in 19 other ways. Pea seeds will catabolize GA29 by oxidation of the C-2 hydroxyl group and opening of the lactone ring to form the C a,B-unsaturated ketone (Sponsel and MacMillan, 19 1978; Durley 33 31., 1979). Somatic cell cultures of carrot 1’10 unsaturated, non-lactonic are able to convert GAl to a A counterpart of GAl (Noma 33 31., 1979). Apparently, a very early catabolic process of Cl9-GAS is the destruction of the lactone bridge and concomitant formation of the C-19 acid. 1.4.3. Mechanism of Action of Growth Retardants. Cer- tain synthetic compounds are known to reduce growth in a num- ber of plants, and are therefore called growth retardants. The dwarfing activity of these compounds suggests that they 26 act through some alteration in the GA status. Early work had Shown that both CCC and Amo-l618 inhibited GA3 production by Gibberella (Kende 33 31., 1963). Later it was found that these compounds also caused a reduction in extractable GA-like substances in a number of higher plants (see Graebe and Ropers, 1978). It now appears that Amo-1618 and CCC, both quaternary ammonium compounds, as well as the phosphons (phosphonium salts) prevent the cyclization of GGPP to kaurene in both Gibberella (Fall and West, 1971) and Marah (Frost and West, 1977). However, CCC was nearly ineffective in Marah 2M) were used. Moreover, the unless high concentrations (10- "A" activity of kaurene synthase is much more sensitive to the inhibitors than the "B" activity. Other quaternary ammo- nium compounds are also effective at this step, e.g. N,N,N- trimethyl-l'-methyl-(2',6',6'-trimethy1cyclohex-2'-en-1'-yl) pro-Z-enylammonium iodide (Hedden 33 31., 1977). Interest- ingly, some of these quaternary ammonium compounds also in- hibit the cyclization of squalene to cholesterol in enzyme preparations from rat liver, indicating a similar mechanism of action of animal squalene epoxidase and plant kaurene synthase (Douglas and Paleg, 1972; Ono and Bloch, 1975). Other growth retardants inhibit GA biosynthesis at dif- ferent steps in the GA biosynthetic pathway. Ancymidol has been shown to block the oxidation of kaurene, kaurenol and kaurenal in cell-free preparations from the endosperm of Marah (Coolbaugh and Hamilton, 1976). It has been suggested that ancymidol interacts directly with the cytochrome P-450 27 of the mixed function oxidases that is responsible for the oxidation of the kaurenoids (Coolbaugh 33 31., 1978). 1-a1kylimidazoles appear to inhibit GA biosynthesis at the same locus as ancymidol (Wada, 1978). It should be pointed out, however, that not all growth retardants act by blocking GA biosynthesis. Compounds such as B-995 (Alar) apparently dwarf plants through some other mechanism than reduction of endogenous GA levels (Lang, 1970). 1.4.4. Dwarfism. In section 1.3.1., several examples of genetic dwarfs which lack GA-like substances were cited. However, the molecular mechanism underlying dwarfism is not well understood. The most intensely studied plant has been the dwarf pea. Dwarfism in this case is not due to a lack of GAS, and so for the purposes of this literature review will not be considered further (see Goodwin, 1978). Perhaps the best understood of the GA-deficient dwarfs is the d-S maize mutant. Apparently, the lesion lies at kaurene synthase. Cell-free preparations from normal maize coleoptiles are able to convert mevalonic acid to kaurene. Similar preparations from the d-S dwarf convert mevalonic acid and GGPP to iso- kaurene (kaur-lS-ene) instead of kaurene (kaur-l6-ene). Iso- kaurene cannot be converted to GAS (Hedden and Phinney, 1979). Waito-C,a dwarf variety of rice, contains the same amount of extractable GA-like substances as normal rice varieties. However, this variety responds only to C-38 hydroxylated GAS such as GAl and GA3. The major GA in rice (both normal and Waito-C) appears to be GAlg,a C20, C-13 28 monohydroxylated GA, which is inactive in promoting stem growth in Waito-C. Thus, it is likely that this mutant lacks the ability to convert GA to C-Bfi hydroxylated GAS 19 (Murakami, 1971). These examples illustrate the potential use of mutants in elucidating metabolic pathways and mode of action of hormones. The foregoing discussion should help to answer questions about the bewildering number of GAS known to exist. If one accepts the view that most GAS are precursors, intermediates, or deactivation products of a few biologically important ones, GA physiology is a bit easier to comprehend. 1.5 CONTROL OF GA BIOSYNTHESIS AND METABOLISM 1.5.1. Control of Enzymgs. The level of a particular substance is determined by the sum of its rates of synthesis and conversion. Unfortunately, little is known about regula- tion of the individual enzymes and reactions that occur in GA biosynthesis and metabolism. However, one possible control point that has been studied is the conversion of GGPP to kaurene by kaurene synthase. Since metabolism of GGPP is a branch point to a number of different compounds such as the phytol chain of chlorophyll and carotenoids, the cyclization of GGPP to kaurene is seen as a likely candidate for a major regulatory step in GA biosynthesis (Simcox 33 31., 1975). Coolbaugh and Moore (1969) found the rate of 333-kaurene synthesis from mevalonic acid in different stages of pea seed development was correlated with the amount of 29 extractable GA—like substances. Also, the change in the rate of kaurene synthesis was correlated with the change in the rate of growth of pea seedlings (Eckland and Moore, 1974). However, in both of these cases, it was assumed, but not shown, that the enzyme under regulation was kaurene synthase. It is possible that other enzymes in the pathway from mevalonic acid to kaurene are under regulation as well. In the endosperm of Marah macrocarpus, kaurene synthesis from mevalonic acid is regulated by the energy charge. However, the enzyme found to be most subject to regulation was pyro- phosphomevalonate decarboxylase, not kaurene synthase (Knotz 33 31., 1977). A number of C-28 hydroxylated GAS are known to exist and all haVe little or no biological activity. Therefore, con- versions from active GAS to C-28 hydroxylated GAS may serve as a mechanism by which plants can control the level of the active GA by regulating the rate of its deactivation. Pre- sumably, this kind of deactivation is irreversible. In pea seeds, the C-28 hydroxylation reaction occurs only at a cer- tain developmental stage, indicating a regulatory role for the conversion (Sponsel and MacMillan, 1977). Conjugates of GAS, most common of which are the glucosyl ethers or esters, are generally thought to be biologically inactive (Graebe and Ropers, 1978; Hedden 33 31., 1978). Since a number of plant enzymes are able to hydrolyse GA conjugates, particularly the glucosyl esters, to form free GAS (Knofel 33 31., 1974), it is possible that the relative 30 rates of conjugate formation and hydrolysis control the levels of the active substance. Mature bean seeds have a higher content of glucosyl esters than immature seeds. The situation is reversed for the levels of free GAS (Hiraga 33 31., 1972; 1974). Moreover, when the seeds germinate, the level of the conjugates declines while free GAS increase (Yamane 33 31., 1975). Exogenous [BHJ-GA3-glucosyl ester is much more rapidly hydrolysed in germinating seeds than in l4-day-old seedlings (Liebisch, 1974). This suggests a storage role for conjugates in maturing seeds to be utilized after hydrolysis during germination. However, GA conjugates are not prevalent as natural components of green plant tissue, so it seems unlikely that conjugation as a mechanism of regu- lation of GA levels is of widespread importance. 1.5.2. Compartmentation. The availability of a GA to an active site could possibly be controlled by compartmentation. This means that absolute tissue levels would be irrelevant. The regulated release of sequestered molecules from an organ- elle or some compartment could be responsible for control of physiologically relevant levels of GA that have access to the active site. Presently, chloroplasts seem the most likely candidate to fill such a role. First of all, chloroplasts from a number of plants are known to contain GA-like sub- stances: Brassica oleracea and Hordeum vulgare (Stoddart, 1968); Pisum sativum (Railton and Reid, 1974); Triticum aestivum (Browning and Saunders, 1976). Secondly, there is some evidence to suggest that plastids and sonicated 31 chloroplasts have the ability to synthesize kaurene from GGPP (Simcox 33 31., 1975; Coolbaugh and Moore, 1976). Moreover, chloroplast preparations from Hordeum are able to convert kaurenol to 78-hydroxykaurenoic acid (Murphy and Briggs, 1975). Thus, chloroplasts may have the ability to synthesize GAS from mevalonic acid. Etioplasts derived from leaves of Hordeum (Evans and Smith, 1976) or Triticum (Cooke 33 31., 1975) Significantly'in- creased:hiGA content when given short exposures to red light. Within 20 minutes, the GAS produced in etioplasts were se- creted into the medium. The regulated release from etioplasts could modulate the effective levels of GAS and therefore con- trol biological responses (Cooke 33 31., 1975). 1.5.3. Photoperiodic Control of GA Metabolism. In a.num- ber of rosette plants, application of GA will substitute for the inductive photoperiod in eliciting stem growth, indicat- ing that photoperiod controls stem growth in these species through regulation of some aspect of the GA status. In the most simple case, the plants in the unfavorable photoperiod lack. GAS. Indeed,there are several examples in which anal- ysis by bioassay has demonstrated a correlative increase in the level of GA-like substances when plants are subjected to photoperiodic conditions that cause stem growth: Hyoscyamus 31333 (Lang, 1960); Rudbeckia bicolor (Harada and Nitsch, 1959); Nicotiana sylvestris (Grigorieva 33 31., 1971). However, the relationship between absolute levels of GAS and stem growth in other photoperiodically sensitive species is 32 not so clear cut. The LD rosette plant Agrostemma githago Shows a large transient increase in the level of GAS when the plants are subjected to LD. Although photoperiodically con- trolled stem growth in this Species is mediated through regu- lation of the GA status, the absolute level of GAS does not correlate with stem growth. LD treatmentgreatly increases overall GA turnover (Jones and Zeevaart, 1980b); In spinach there is no change in the total level of endogenous GA-like substances when plants are transferred from SD to LD condi- tions. However, there iS a balance between a decline in one GA-like substance and a corresponding increase in another during LD treatment. Also, there is an increase in overall GA turnover under LD conditions (Zeevaart, 1971; 1974) as found in Agrostemma. Silene armeria, a LD rosette plant re- lated to Agrostemma, also Shows increased GA turnover in L0 (Cleland and Zeevaart, 1970; Van den Ende and Zeevaart, 1971). Moreover, all three of these plants Showed greater sensitivity to exogenous GA in LB than SD in eliciting stem growth. Thus, :greater GA turnover combined with higher tissue sensitivity to GA in LB might regulate Stem growth in plants rather than absolute levels of GA (Jones and Zeevaart, 1980aqhn Zeevaart, l97l; Cleland and Zeevaart, 1970). Peas, like other monocarpic annuals, die following fruiting. Preceding death are a series of processes termed _ apical senescence that lead to death of the apex and ulti- mately to the death of the plant. There exists a genetic line of peas termed G2 in which apical senescence following 33 the reproductive phase is dependent on photoperiod. If fruiting GZ plants are kept in LD, apical senescence and death of the plant will ensue. However, if the same plants are maintained under SD conditions, apical growth continues unabated for some time (Proebsting 33 33., 1976). Moreover, G2 plants produce a graft-transmissible substance which de- lays apical senescence in photoperiodically insensitive lines of peas. Apparently, this graft-transmissible substance is gibberellin (Proebsting, 33 33., 1977). Analysis by bioassay andGLC—MS of the G2 line Showed that plants under SD condi- tions contained higher levels of two GAS than plants in LD. Moreover, the apparent rate of GA interconversion is also higher in SD than LD (Ingram and Browning, 1979; Proebsting 33 33., 1978). Light, not necessarily duration of the light period, is also known to affect the rate of GA metabolism. Short bursts of red light cause suspensions of wheat or barley etioplasts to increase in the level of extractable GA-like substances (Cooke and Saunders, 1975; Evans and Smith, 1976). Exposure to light decreases metabolism ofI§HPGA20 to GAl in tobacco callus cultures. Since greater growth of the callus is ob— tained in the dark, it was suggested that light regulation of growth in tobacco callus was due, in part,to control of the conversion of GA20 to GAl (Lance 33 33., 1976). Likewise, dark-grown seedlings of Pisum sativum metabolized [3Hj-GA5 faster than light-grown seedlings. Moreover, the dark-grown seedlings had a greater response to exogenous GA5 (Musgrave 34 and Kende, 1970). In Phaseolus coccineus, metabolism of [BHJ-GA4 was correlated with growth and the level of extract- able, endogenous GA-like substances. Light caused greater metabolism of exogenous [3HJ-GA4, lower levels of extractable GA-like substances, and a reduction in growth (Brown 33 33., 1975). Cell-free preparations from light-grown pea seedlings had a greater capacity to synthesize kaurene from mevalonic acid than did corresponding cell-free extracts from dark grown seedlings (Eckland and Moore, 1974). While it is obvious that photoperiod or light can sub- stantially alter GA metabolism, it is .also unclear as to what mechanisms are involved in such control. AS mentioned earlier, the conversion of mevalonic acid to kaurene in cell- free extracts of Marah macrocarpus endosperm is regulated by the adenylate energy charge of the system, which a higher energy charge favoring synthesis of kaurene (Knotz 33 33., 1977). Since variations of the energy charge in green plant cells can occur during changes between light and dark conditions (Santarius and Heber, 1965), it is possible that one aspect of light regulation of GA biosynthesis and metabolism is con- trol by light over the level of adenylates. Although control of the energy charge might explain some aspects of light regulation of GA metabolism, photoperiodic effects on GA metabolism are not so easily explained. Many photoperiodic effects on growth have been attributed to phytochrome control of GA metabolism (Vince-Prue, 1975), but exact mechanisms remain almost totally unknown. Leaf 35 unrolling in etiolated cereal leaves is controlled by the phytochrome status of the leaf. Exogenous GA3 can substitute for red light in causing the leaves to unroll. Moreover, etiolated wheat leaves Show an increase in the level of extractable GA-like substances 15 minutes following a brief exposure to red light (Beevers 33 33., 1970). AS stated in section 1.5.2., the increases in GA-like activity are con- fined to the etioplast fraction where phytochrome apparently regulates the release of GAS from the etioplast (Cooke 33 33., 1975; Evans and Smith, 1976). Phytochrome has been detected in the envelope membranes of barley etioplasts. It has been suggested that phytochrome in the Pfr form acts to increase the permeability of the envelope membrane to GAS, thereby decreasing the internal GA concentration in the etioplasts. This depletion of GAS within the etioplast could activate further GA biosynthesis through some feedback mechanism (Evans and Smith, 1976; Evans, 1975). Thus, in this model, the action of phytochrome on GA metabolism is indirect; the primary effect of phytochrome in the Pfr form is the re- distribution of GAS between compartments. Whether or not this model can be expanded to cover photoperiodic effects on GA metabolism in other Species remains to be seen. In any event, much more preliminary work must be done before a uni- fied theory on the mechanism of photoperiodic control of GA metabolism can be made. Endogenous GAS must be identified and the metabolic relationships between GAS and GA precursors Should be definitively established before one can reasonably 36 hope to understand how various reactions in a pathway are regulated. 1.6 GAS: ROOT-SHOOT INTERRELATIONSHIPS The growth and development of roots and shoots are separate, but highly coordinated events. Tightly coupled development of the various organs suggests that there exist means of communication between organs. Hormones transported between organs could act as integrating factors that enable one organ to signal another when to initiate or terminate a certain phase of development. Went (1938) observed that root excision or flooding roots of tomato (Went, 1943) caused an inhibition of stem growth. Because the inhibition was not due to mineral or water uptake problems, it was suggested that the roots produced a hormone-like factor which is trans- ported from the roots to the Shoot where it controls stem growth. This factor was termed "caulocaline" (Went, 1943). Because Stem growth appears to be regulated by some aspect of the GA status (Jones, 1973; Goodwin, l978),it has been sug- gested that at least part of caulocaline is a GA (Phillips, 1964; Reid and Crozier, 1971). If GA is identical to, or part of,caulocaline. then three criteria have to be met: i) GAS Should be produced in the roots, and this production Should be correlated with observed effects of flooding, etc. on stem growth; ii) transport of GAS from the root to the shoot in the xylem Should reflect changes in production of GAS in the roots; iii) exogenous GA Should at least partly 37 be able to overcome the inhibition of stem growth by root excision or flooding. Roots have been Shown by indirect means to have the apparent capacity to synthesize GAS. First of all, Butcher (1963) has Shown that a clone of excised tomato roots main- tained in culture for five years contained GA-like substances. This implied that the GAS had been synthesized by the roots Since carry-over from the mother plant after so many sub- cultures is unlikely. Secondly, through use of a diffusion technique, the Sites of GA synthesis in Helianthus were de- duced to be the root and Shoot tips (Jones and Phillips, 1966). Today, it is generally believed that root and Shoot tips are Sites of GA synthesis in other plants as well (Lang, 1970; Graebe and Ropers, 1978). However, besides certain immature seeds, no definitive biochemical data (i.e. location of Specific enzymes) yet exist that indicate where in the plant GAS are made. Studies using radio-labeled GAS applied to roots have Shown that GAS will move from the roots to the shoots (par- ticularly to the mature leaves) in the transpiration stream (Davies and Rappaport, 1975). Moreover, GA—like substances have been detected in xylem exudate in a number of Species (e.g. Carr 33 33., 1964; Phillips and Jones, 1964; Sitton 33 33., 1967; Sweet 33 33., 1974). Since the Sites of GA synthesis are not definitively known, the origin of GAS in the xylem is not clear. Nevertheless, there exists some evidence, albeit weak, which indicates that GAS transported 38 from the roots in the xylem regulate stem growth. Applica- tion of GA3 to the base of the stem of de-rooted soybean seedlings completely overcame the inhibition of stem growth caused by the treatment (Holm and Key, 1969). Exogenous GA3 was partially able to reverse the Stunting effects caused by waterlogging tomato plants (Reid 33 33., 1969; Reid and Crozier, 1971). In this particular case, waterlogging was associated with a precipitous decline in the level of GA-like substances found in the xylem exudate. In addition, the level of extractable GA-like substances found in the roots and the Shoots was also sensitive to waterlogging. This led the authors to conclude that root-produced GAS, sensitive to O2 deprivation, controlled stem growth (Reid 33 33., 1969; Reid and Crozier, 1971). However, there are other equally viable explanations. Since the ultimate origin of GAS in the xylem is unknown, it is possible that GAS produced in the Shoot are transported down to the roots via the phloem and then exported back to the Shoot in the xylem. Such recircu- lation patterns have been observed for other substances as well (see Ziegler, 1975). Girdled Citrus Sinensus trees accumulatedsakiike substances in the lateral shoots and bark above the ring while the roots Showed a significant decline in GA-like activity. This suggests that in reality root and xylem GAS are substantially derived from the Shoot (Wallerstein 33 33., 1973). Likewise, the reduced stem growth in waterlogged tomato plants may not even be due to a reduction in the levels of GAS. The stems of waterlogged 39 plants produce copious amounts of ethylene, a plant hormone known to reduce stem growth (Bradford and Dilley, 1978). Apparently O2 deprivation of the roots stimulates the pro- duction of the ethylene precursor, l—aminocyclopropane-l- carboxylic acid in the roots. This substance is then trans- ported up to the Shoot in the transpiration stream where it is converted to ethylene (Bradford and Yang, 1980). Inci- dently, this provides a perfect example of a chemical Signal produced in one organ, transported to another, where it ini- tiates a sequence of events that modifies growth and develop- ment i.e. a hormone. In conclusion, the evidence supporting a role for root- produced GAS in the regulation of shoot growth is equivocal at best, and points out the absolute necessity of determining the location of enzymes involved in GA biosynthesis and metabolism. Studies using exogenous GAS indicate that GAS travel with the flow of assimilates from source leaves to Sinks such as Shoot tips and roots (Zweig 33 33., 1961; McComb, 1964). GA-like substances have been detected in the phloem sap of a number of Species (Kluge 33 33., 1964; Hall and Baker, 1972; Hoad, 1973). It is not known whether GAS transported in the phloem regulate growth processes in the sink end of the transport system. In general, exogenous supplies of GAS to roots on intact plants have little or no effect on root growth (Goodwin, 1978). GAS have been reported to increase the elongation of isolated root tips in a number of Species 40 including maize (Mertz, 1966), pea (Pecket, 1960), and tomato (Butcher and Street, 1960). However, in all these cases, growth proceeds well in the absence of externally supplied GAS; this is evidence that the cultured root tip is self- sufficient for GAS (Goodwin, 1978). This, coupled with the fact that root tips are purported Sites of GA biosynthesis, (Jones and Phillips, 1966) indicates that Shoot-produced GAS are not important in the regulation of root growth. Considerable radial transport of assimilates from the phloem to nearby tissues can occur (Peel, 1967). GAS behave Similarly and could conceivably regulate physiological pro- cesses such as cambial division and subsequent expansion, aS well as maintenance metabolism of cortical cells (Bowen and Wareing, 1969). GAS traveling with the flow of assimilates also accumulate at the Shoot tip, a Site where GAS are known to affect stem growth. It is not known if GAS transported from the mature leaves to the Shoot tip in the phloem actu- ally regulate Stem growth Since the Shoot tip is supposed to be a site of GA synthesis (Jones and Phillips, 1966). In conclusion, GAS are known to be transported over long distances in the phloem and in the xylem. However, the pre- cise physiological role of long-distance GA transport is obscure at best. Some investigators feel that GAS are pro- duced at, or near the Site of utilization (see Graebe and Ropers, 1978). The presence of GAS in the two transport Streams might only be coincidental, and conceivably, have little or no physiological importance. 41 1.7 STATEMENT OF PURPOSE In the beginning of this chapter, it was proposed that photoperiodic control of stem growth in LD rosette plants offers a useful model system in which to study environmental control of growth and development in plants. Early work by Zeevaart (1971; 1974) has established that photoperiodic control of stem growth in Spinach is mediated by a change in the GA "status" of the plant. This work also demonstrated that photoperiod affected two related aspects of the GA status in Spinach: the relative levels of the various GA- 1ike substances and the overall GA turnover rate. This thesis is an account of further investigations on the role of photoperiodically-induced changes in GA metabolism in the regulation of stem growth in Spinach. Specifically, I was interested in four areas: 1) Chemical identification of the GA-like' substances present in Spinach plants. 2) The quantitative relationships between the GAS as a function of photoperiod. 3) Nature and physiological significance of long- distance GA transport between roots and Shoots of Spinach. 4) Preliminary information on the nature of subcellular distribution of GAS in Spinach leaves. Chapter 2 The Identification of Six Endogenous Gibberellins in Spinach Shoots 42 43 2 . 1 INTRODUCTION Long-day rosette plants respond to the transfer from SD to LD with increased stem elongation and subsequent flower formation. It has been demonstrated in Silene armeria (Cleland and Zeevaart, 1970), Agrostemma githago (Jones and Zeevaart, 1980a), and spinach (Zeevaart, 1971) that photo- periodic control of stem growth is mediated through regula- tion of the GA status (see 1.5.3.). In Spinach, LD treatment of plants previously maintained under SD did not increase the total level of GA-like substances, but levels of individual GA-like substances did vary. Moreover, overall GA turnover was enhanced considerably in LD. This indicates a profound change in GA metabolism had occurred (Zeevaart, 1971; 1974). It is assumed that this change in GA metabolism is of physio- logical Significance in the photoperiodic control of Stem growth in Spinach. However, before the mechanism of photo- periodic regulation of GA metabolism and its relation to stem growth can be fully investigated, the chemical identity and quantitative relationships of the endogenous GAS should be known. This chapter represents an account of the identifica; tion of Six endogenous GAS in Spinach Shoots. 44 2.2 MATERIALS AND METHODS 2.2.1. Plant Material. Seeds of spinach, Spinacia oleracealncv.Savoy Hybrid 612 (Harris Seed Co., Rochester, New York) were sown in the field in early August, 1978. Whole shoots were harvested 1.5 months after sowing. The harvested Shoots were washed with distilled water, and the yellow, senescing leaves discarded. The remaining plant ma- terial was frozen in liquid N lyophilized, and stored at 2! -15°C prior to extraction. Approximately 2,500 plants yielded 2.5 kg of dried plant material. 2.2.2. Bioassay. The presence of GA-like substances was detected with the d-5 corn bioassay. Homozygous seeds (Zeevaart, 1966) were germinated in moist vermiculite in a humid, dark incubator at 27°C. After Six days the seedlings were transferred to plastic boxes (20 x 10 x 8 cm) containing half-strength Hoagland's solution. Fractions from extracts were dissolved in 0.5 ml of 0.1% Tween 20 in water. The solution was then distributed equally on each of four seed- lings. The first leaf, at this stage, had not yet unrolled, and so formed a convenient~cup with a capacity of about 0.15 ml. After application of the test solutions, the plants were then placed in a growth chamber at 27°C with 16 hours of light daily from fluorescent lamps (Gro-lux, Sylvania) and 4OVVincandescent bulbs (total irradiance = 28v§m-2). One week after treatment, the sum of the lengths of the first two leaf sheaths was determined and the data converted to percentages of an untreated control. A standard curve 45 (lng to lug) was prepared using GA3 (Sigma). 2.2.3. Extraction and Purification Procedures. Freeze- dried spinach Shoots, in 200 g lots, were homogenized with 10 liters of ice-cold 80% aqueous methanol (20 ml per gram dry weight) in a Waring Blender. The extract was filtered, and the residue was stirred overnight at room temperature in four liters of 100% methanol. After a second filtration, the two extracts were combined, and the methanol removed under reduced pressure in a rotary evaporator. An equal volume of 1 M phosphate buffer (pH 8.2) was then added to the remaining aqueous residue, and the resulting mixture was partitioned three times with petroleum ether. The aqueous phase was adjusted to pH 2.5 with GhJHCl and purified on a charcoal-celite column as described by Zeevaart (1971) , ex- cept that two grams of charcoal were used for every 10 g of dry plant material extracted. Elution of GAS was achieved with 80% aqueous acetone. The acetone was removed under re- duced pressure in a rotary evaporator. The remaining aqueous residue was adjusted to pH 2.5 with 6biHC1 followed by par- titioning 4 times against equal volumes of ethyl acetate. The acidic ethyl acetate fraction obtained after char- coal chromatography was purified by silicic acid adsorption chromatography as described by Zeevaart (1971), except that 60% ethyl acetate in chloroform was used as the elution mixture. Two grams of silicic acid (Mallinkrodt, 100 mesh) were used for every gram of lyophilized plant material extracted. 46 The eluate from the silicic acid adsorption column was fractionated via preparative reverse phase HPLC using a Waters Model 5000A liquid chromatograph equipped with four stainless steel columns (each 60 cm x 0.65 cm i.d.) packed with Bondapak Clg/Porasil B (Waters Associates). GAS were eluted from the column with a linear gradient of 95% ethanol in 1% aqueous acetic acid (BO-100% in 25 minutes) controlled by a Waters 660 solvent programmer. Dried samples were re- dissolved in 5 ml of 30% aqueous ethanol, and the solution filtered through a 0.45 um HA Millipore filter. The filtered sample was loaded onto the column via a Waters 06K Universal Injector with a 5 ml loop. The gradient was started 1 min after injection; the solvent flow rate was 9.9 ml min-1. Fractions were collected every minute from the time of in- jection and corresponding fractions from 12 HPLC runs were combined and dried. The remaining residues were redissolved in 2 ml of ethanol and 1.0% of each of the combined fractions was tested for the presence of GA-like substances with the d-5 corn bioassay as described in section 2.2.2. Fractions which contained biological activity, or fractions in which authentic GAS eluted (Jones 33 33., 1980) were further puri- fied by silicic acid partition chromatography. In this pro- cedure the method of Powell and Tautvydas (1967) was followed, except that the stationary phase consisted of water with the pH adjusted to 3.0 by the addition of a few drops of tri- .fluoracetic acid. Elution of GAS was achieved with a gradi- ent of increasing concentration in 5% increments of ethyl 47 acetate in hexane. Both solvents were saturated with water at pH 3.0 before mixing. I Fractions which contained biological activity, or eluted at the same step as authentic GAS, were subjected to final purification using analytical reverse phase HPLC with a u- Bondapak C column (30 cm x 0.4 cm i.d.). Elution of GAS 18 from this column was achieved with either a 30-100% linear gradient of methanol in 1% aqueous acetic acid (grad A), or a 10-70% linear gradient (grad B). In both cases the gradi- ent was completed in 30 min and the flow rate was 2 ml min-l. The gradient was started 2.5 minutes after injection, and fractions were collected every minute from the time of in- jection. Other procedures were identical to those described for preparative HPLC. Grad B was used only for eluting poly- hydroxylated GAS (e.g. GAl’ GA29, GAB; see Jones 33 33., 1980). 2.2.4. 333. Preparative TLC was carried out on 20 x 20 cm glass plates coated with silica gel H (EM Reagents). Par- tially purified acidic extracts were applied as a narrow band 12 cm long. Authentic GAS were spotted 2 cm to the side of this band. The thin layer plates were developed to 15 cm from the origin in chloroform-ethyl acetate-acetic acid (60:40:5, v/V). The resulting chromatogram was divided into 10 equal zones, and the Silica gel was scraped off into test tubes. The Silica gel was then eluted twice with water- saturated ethyl acetate and once with acetone. The combined eluates from each of 10 zones were assayed for the presence of GA-like substances using the d-S corn bioassay. The side 48 of the plate which was Spotted with the authentic GAS was left intact and Sprayed with a sulfuric acid-ethanol mixture (5:95, v/v). The plate was heated at 100°C for 10 min to visualize the reference GAS. 2.2.5. Derivatization. Appropriate fractions from ana- lytical reverse phase HPLC were methylated with ethereal diazomethane. The trimethylsilyl ethers of the methyl esters were prepared by adding 100 pl of a solution containing pyridine-hexamethyldisilazane-trimethylchlorosilazane (9:3:1, v/v) to dry methylated samples in Reacti-vials (Pierce Chemi- cal Co.). 2.2.6. 333. Derivatized samples were chromatographed on a Hewlett-Packard 402B gas chromatograph equipped with a U- Shaped glass column (183 cm x 0.3 cm i.d.) packed with 4% SE- 33 on Gaschrom Q, 80/100 mesh. All runs were isothermal at either 215 CO or 225 CO. The carrier gas was He, and the flow rate was 40 ml min-l. 2.2.7. GLC-MS. Derivatized samples were chromatographed on a Hewlett-Packard 5840-A gas chromatograph with a glass column packed with 2% SP-2100 on 100/120 Supelcoport. The column temperature was programmed from 170 to 280°C at 10°C min“1 with a 2 min isothermal hold at the beginning of the program and a 5 min isothermal hold at the end. The flow 'rate of the carrier gas (He) was 25 ml min-l. The GLC was connected to a Hewlett-Packard 5985 mass Spectrometer by a jet separator, and mass Spectra were collected every 4.5 second. The ionizing potential was 70eV. 49 2.3 RESULTS 2.3.1. Characterization of GA-like Substances in Spinach Shoots. A sample of field-grown spinach, 20 g dry weight, was extracted and subjected to charcoal adsorption chromatography, silicic acid adsorption chromatography, and TLC. Figure 2-1 Shows the biological activity associated with 10 equal strips of the chromatogram. Two zones of biological activity are apparent. The zone at Rf 0.2, called fraction I, co-chromato- graphed with GA , while the less polar zone at Rf 0.4-0.5 1 (fraction II) co-chromatographed with GAZO‘ These results indicate that the pattern of GA-like activity from field- grown spinach is similar to that from plants grown in growth chambers (Zeevaart, 1971; 1974). When I and II were first separated by TLC and then subjected to preparative reverse phase HPLC, fraction I no longer behaved chromatographically like GA (Fig. 2-2A), while fraction II still co-chromatographed l with GA (Fig. 2-2B). 20 2.3.2. Identification of GAS found in Spinach Shoots. In order to identify the GAS present in fractions I and II, it was necessary to extract and purify large amounts of plant material. After charcoal and silicic acid adsorption chroma- tography, followed by preparative reverse phase HPLC, I and II were separated by silicic acid particion chromatography. Fractions I and II were eluted with 55% and 40% ethyl acetate in hexane, respectively. Final purification of both I and II was achieved by analytical reverse phase HPLC. Small aliquots (0.5%) of each fraction were assayed for the 50 Figure 2—1. GA-like activity present in an extract of lyophilized Spinach Shoots (20 g). The partially purified acidic extract was fractionated by TLC and the resulting chromatogram divided into ten equal strips. Each strip was eluted and the eluate was assayed for the presence of GA- like substances by the d-5 corn bioassay. 51 GAZO Ir——l GA. l-—l ""1 J L 200 P I80 " ISO " I40 ' 6:80 .6 E853 LO 0.6 0.8 0.4 0.2 52 Figure 2—2. Chromatographic behavior of fractions I(A) and II(B) when separately subjected to preparative reverse phase HPLC as detected by the d-5 corn bioassay. An acidic extract from 20 g dry weight of plant material was prepared, and I and II separated by TLC. Standard GAl and GAZQ were run separately for comparison of the chromatographic behavior. Percent of Control 53 GA. GA“ L M ‘ zoo - 4 I l80 - , I I60 - I40 t I40 " l20’ l—L—1_r—1 L no l2 l4 l6 l8 Fraction No. 20 22 24 26 54 presence of GA-like substances by the d-S corn bioassay. Both I and II were contained in Single fractions. Figure 2-3 Shows that II co-chromatographed exactly with GA20(B), whereas I(A) co-chromatographed with none of the available GA standards (see Jones 33 33., 1980). The MeTMS-derivatives of the fractions resulting from analytical HPLC that contained I and II were prepared. Gas chromatography of the derivatized fractions indicated that both I and II were contained in fractions that had only one major peak (Fig.2-4A,B). Thus, reverse phase HPLC, used in conjunction with other chromatography techniques, is extremeky useful in purifying small quantities of substances from plant material. Authentic MeTMS-GA had the same retention time 20 as the main peak of MeTMS-II (Fig. 2-4B). GLC-MS analysis of this peak showed that its mass Spectrum was identical to that of authentic MeTMS-GA (Table 2—1). None of the other 20 minor peaks from this fraction had fragmentation patterns recognizable as any of the known GAS. When the derivatized fraction containing I was analyzed by GLC-MS, the mass Spec- trum of the large peak seen in GLC analysis (Fig. 2-4A) closely resembled the published mass spectrum (Binks 33 33., 1969) of MeTMS-GA19 (Table 2—1). Unfortunately, a reference sample of GA19 (Fig. 2-5) was not available for further con- firmation. However, on the basis of the similarities between the published spectrum (Binks 33 33., 1969) and the mass spectrum obtained from I, it is concluded that I is comprised of GA19. 55 Figure 2-3. GA activity as measured by the d-5 corn bioassay associated with 0.5% aliquots of individual frac- tions resulting from analytical reverse phase HPLC of puri- fied fractions I(A) and II(B) using grad A. 56 ‘If‘l 6 We. 2 A n B 4 M... .2 H2 2 2 2 l 0 so 2 2 m1 - r - m rm m - . mmH Ti .6. I .M M P. B .m .D . p p p p p _ 70%. p p . . b n 0 0 0 O O 0 O O O m m m m 2 0 8 B m B m .8200 to E853 Fraction No. 57 Figure 2-4A. GLC analysis of MeTMS-I. Derivatized samples were chromatographed on a U-Shaped glass column (183 cm x 0.3 cm i.d.) packed with 4% SE-33 on 80/100 mesh gaschrom Q. All runs with MeTMS-I were isothermal at 225°C. Detector Response 58 MeTMSI illllJllllLlll 0 246810 :2 mm 59 Figure 2-4B. GLC analysis of MeTMS-II. Derivatized samples were chromatographed on a U-shaped glass column (183 x 0.3 i.d.) packed with 4% SE-33 on 80/100 mesh gas- chrom Q. All runs with MeTMS-II were isothermal at 215°C. Authentic MeTMS-GA20 was also run for comparison. 60 MeTMSH Me ms GA 20 J, Detector Response K llllLlllllLlllL 02468l012 I4 min. 61 Table 2-1. GLC-MS data obtained with samples from spinach and with authentic GAS. In cases where no authentic GAS were availabe, published data from mass spectral analysis are presented: MeTMS-GAlg (Binks 33 33., 1969); MeTMS-GA44 (Frydman 33 33., 1974); MeTMS-GA53 (Bearder 33 33., 1975). Fraction numbers refer to fractions eluted from analytical HPLC system. 62 mnsvwm eras an mourn »: anon unannnsa .a\n «apnea. non: .aws. tun: Hmpanpwo .m~.z+.w~. SSQAQ. .m..woo. So~auav uQSAaH. uom.qn. nou..q. HH Hc.¢ .Ho.:+.~oo. .ou.-. uuwAQu. umoAHQ. NooAHo. noqauu. zoezmravuo no.9 guo.z+.woo. .ou.~q. uqm.qu. umoAHm. ~caAHO. ~ou.~ov unnonwoa Hm w~.. ao~.3+.~u. .ac.-. gu~awu. SQHAm. wouAHu. nomawoo. zoezmrn>wq HH.. aw~.x+.wa. .ao.~m. au~.~w. .oHAHa. uquANm. ~oa.~oc. unannpos p. -.a moalx+.pso. .u~.-. ssslpm. ..s.o. ~oeluu. ~cs..o. soazmro>~o w~.a mom.z+.~oo. sow.~m. SQQAm. .aq.~a. noaaua. anASH. mnann»o= no Hu.w au~.z+.uu. SHQAo. uqu.~q. umHAo. ~uo..~. ~cu.uw. noqawoo. :me:mrn>.. su~.:+.mu. awq.~o. uuu.-. ~m~.w. anAum. nooASQ. ~oqawco. mnmonwos up Hp.» 5.9.:+.uw. .HwAHo. awa.~u. use.... nomAao. ~oq.~oo. xoezmnn> .bo.z+.u.. Spra. .wa.m. umo.-. noQAom. ~cqawco. mu 63 Figure 2-5. Numbering system of ent-gibberellane (A), and structures of the Six GAS identified by GLC-MS in Spinach Shoot extracts (B). 64 65 Since GA19 and GA20 are both C-l3 hydroxylated GAS, it would seem logical that other C-13 hydroxylated GAS are pre- sent in Spinach shoots. If these GAS were biologically in- active, or were present in minute quantities, they would escape detection in the bioassay. Fractions resulting from preparative reverse phase HPLC which would contain various C-13 hydroxylated GAS (see Jones 33 33., 1980), if present in spinach Shoot extracts, were purified by silicic acid parti- tion chromatography. Final purification was achieved by analytical reverse phase HPLC. Corresponding fractions from the spinach extract where various C-13 hydroxylated GAS are known to run in this system (Jones 33 33., 1980) were deriva— tized and analyzed by GLC-MS. Fraction l9 (grad A) and fraction 14 (grad B) contained substances that had retention times and mass Spectra identical to those of authentic MeTMS- GAl7 and MeTMS-GA29, respectively (Table 2-1). Fractions 1? and 21 (grad A) contained compounds which had mass Spectra Similar to those published for MeTMS-GA 4 (Frydman 33 33., 4 1974) and MeTMS-GA53 (Bearder 33 33., 1975), respectively (Table 2-1). Both GA and GA (Fig. 2-5) occurred in such 44 53 low quantities that they would have escaped detection in the bioassay. No GAl, GAB’ GAS' or GA8 was detected in extracts of Spinach Shoots. Bar graphs from the mass Spectra of these compounds and available reference compounds can be found in the Appendix. 66 2.4 DISCUSSION The above results demonstrate the presence of at least Six GAS in spinach shoots: GA53, GA44, GAlg, GA17, GAZO’ and GA29 (Fig. 2-5). All six GAS have in common a C-13 that is hydroxylated. Only GA29 has an additional hydroxyl at the C-28 position. Four of the six GAS are Czo-GAS: GA 3, GA 5 44' GA and GA17 with C-20 as a methyl, 6-lactone, aldehyde, 19' or carboxyl group, respectively. Both GA20 and GA29 are C19- GAS of which C-20 has been removed and a C-19 + C-lO lactone bridge has been formed. The same six GAS present in Spinach shoots have recently been identified in immature seeds of Vicia faba (Sponsel 33 33., 1979), whereas GA GA44, GA 53’ 19’ have been found in young tassels of Zea mays GA and GA 17’ 20 (Phinney, 1979) and shoots of Agrostemma githago (Jones and Zeevaart, 1980b). Thus, this combination of C-13 hydrox- ylated GAS appears to occur commonly in higher plants and suggests a biogenetic relationship between these various GAS. If one assumes that the six GAS found in spinach Shoots are related metabolically in a pathway, one can postulate a sequence based on the sequential oxidation of C-20 from a methyl to a carboxyl group, its subsequent removal with the formation of a C-19 + C-lO lactone bridge, and finally C-ZB hydroxylation. This would indicate the existence of the following pathway: GA53 + GA44 (in the open lactone or hydroxy-diacid form) + GA19 + GA17 + GA20 + GA29. However, there is little direct evidence from other plant systems that indicates the existence of this pathway in GA metabolism. 67 Conversion of GA20 to GA29 has been observed in a variety of higher plant systems including Phaseolus vulgaris seeds (Yamane 33 33., 1977), immature seeds of Pisum sativum (Frydman 33 33., 1974), and leaves of Bryophyllum daigremon- tianum (Durley 33 33., 1975). This type of reaction may serve as a deactivation process,since GA29 and other C-ZB hydroxylated GAS have little or no biological activity (Hedden 33 33., 1978). In a cell-free system derived from Cucurbita maxima endosperm, C-20 of GA12 (a non-hydroxylated analog of GASB) was sequentially oxidized from a methyl (GAlz) to an aldehyde (GA24, GA36), and finally to a carboxyl group (GA13, GAZS’ GA43) (Graebe and Hedden, 1974). However, no CZO-GA with a higher oxidation state than a methyl group at C-20 has been found to act as an intermediate in the con- version of CZO-GAS to Clg-GAS. Thus, the identity of the direct precursor of Clg-GAS remains unknown. Zeevaart (1971; 1974) observed that the levels of two GA-like substances in Spinach shoots, here called I and II, changed in relation to photoperiod. The biological activity associated with these two GA-like substances can now be attri— and GA . The other GAS found in Spinach 19 20 shoots are either present in quantities too low for detection buted to GA by bioassay, or are biologically inactive in the d-5 corn bioassay. It appears from the earlier bioassay results Zeevaart 1971; 1974), that the level of GA19 declines with LD treatment, while the level of GA20 increases during the same period. Moreover, the bioassay results indicated that 68 the total GA level remained fairly constant during LD treat- ment. Since GA19 and GA20 elicit similar responses in the d-S corn bioassay (Crozier 33 33., 1970), the bioassay data Should reflect absolute differences in the levels of both GAS. Taken together, these results could be interpreted as a precursor-product relationship between GA19 and GAZO' In the next chapter, quantitative analysis by GLC-SICM of photo- periodically-induced changes in the levels of the Spinach GAS will provide further circumstantial evidence favoring this hypothesis. Chapter 3 The Effect of Photoperiod on the Levels of Endogengus Gibberellins in Spinach Shoots 69 70 3.1 INTRODUCTION In the previous chapter, the identification of six C-13 hydroxylated GAS (Fig. 2-5) in spinach shoots was described. Moreover, the bioassay results of Zeevaart (1971; 1974) com- bined with the identification of the two GAS responsible for GA-like activity in spinach shoot extracts indicate that the level of GA19 declines with LD treatment, whereas the GA20 level increases during the same period. Bioassays have, however, several limitations, most important of which is the difference in sensitivity to various GAS (see section 1.3). Thus, important quantitative information is lost because the d-S corn bioassay is not sensitive to GA29 (Crozier 33 33., 1970) or GA (Yokota 33 33., 1971). This set the stage for 17 an analysis of photoperiodically-induced changes in the levels of the GAS found in Spinach Shoots by GLC-SICM. 3.2 MATERIALS AND METHODS 3.2.1. Plant Material and Photoperiodic Treatment. Spinach seeds (see 2.2.1.) were sown on vermiculite. After ten days, the seedlings were transferred to 340-ml plastic cups containing a gravel-vermiculite mixture (1:2), and were watered twice daily with half-Strength Hoagland's solution. The plants were maintained under SD conditions until ready 71 for experimentation, approximately six weeks after sowing. SD treatments consisted of an 8 hr period of light from fluorescent and incandescent lamps (total irradiance = 33VVm72), followed by 16 hours of darkness. LD treatment consisted of the same 8 hr illumination as in the SD treat- ment, followed by 16 hours of low intensity illumination from 2). LD treat- incandescent bulbs (total irradiance = 0.7th- ments were staggered in such a way that all plants were har- vested at the same time. Each treatment consisted of ten plants. At the end of an experiment, the stem length of each plant was determined. The Shoots were then cut off at the soil level, frozen in liquid N2, lyophilized, and stored at -15°C prior to extraction. 3.2.2. Extraction and Purification Procedures. The extraction and purification procedures were identical to those described in 2.2.2. Methanolic extracts were reduced to a small aqueous residue and purified by charcoal adsorp- tion chromatography and silicic acid adsorption chromatog- raphy. The eluate resulting from silicic acid adsorption chromatography was fractionated by preparative reverse phase HPLC as described earlier. Fractions known to contain GA44, GA GA and GA20 (all monohydroxylated GAS) were combined 19’ 17' and subjected to analytical reverse phase HPLC as described before (2.2.3.) for monohydroxylated GAS (grad A). The fractions containing these four GAS were then combined. The fraction from preparative HPLC that contained GA29 was also purified with analytical reverse phase HPLC, using the 72 gradient system described previously for dihydroxylated GAS (grad B, see 2.2.3.). The resulting final two fractions were then methylated with ethereal diazomethane. The trimethylsilyl ethers of the methyl esters were prepared by the addition of 20 ul of a solution containing pyridine-hexamethyldiSilazane-trimethyl- chlorosilazane (9:3:1, v/v) to methylated samples dried in a capillary. 3.2.3. GLC-SICM. GLC-SICM was performed using a Hewlett? Packard 5985 mass Spectrometer that was interfaced by a glass jet separator with a Hewlett-Packard 5840-A gas chromatograph. The four monohydroxylated GAS were chromatographed using a glass column (183 x 0.2 cm, i.d.) packed with 2% SP-2401 on 100/120 mesh Supelcoport. Samples were injected (2 ul) onto the column at 180°C. Following a 2 min isothermal hold, the 1 temperature was programmed 10°C min- until the column tem- perature was 205°C, whereupon the rate was Slowed to 1°C mind: When the temperature reached 215°C, the rate was increased to 20°C min-1 until the column reached the maximum temperature of 255°C. GA29 was chromatographed on 2% SP-2100 with column ' conditions identical to those described in 2.2.7. For each GA, three fragments with the following m/e values were monitored: GA44-432, 373, and 207; GA19—462, 434, and 374; GA -418, 419, and 375; GA -492, 460, and 208; 20 17 GA -506, 507, and 207. The dwell time for each fragment 29 monitored was 200 msec. The relative level of each GA was calculated from the SICM response of the molecular ion of 73 each compound, except for GA19' in which case the base peak (m/e 434) was used. The ratios of the SICM response of the three fragments monitored for each GA were checked in every sample to ensure that interfering compounds did not affect the SICM response. Each sample was analyzed twice, and the average of the two readings was used in the subsequent cal- culations. No pair of readings ever differed by more than 5%. Other parameters of the mass spectrometer were the same as described in 2.2.7. 3.2.4. Application of GAZQ. GAZO (a gift from Drs. N. Murofushi and N. Takahashi, Department of Agricultural Cheimistry, University of Tokyo, Japan) was dissolved (0.4,qg/ 1) in an aqueous solution of 0.1% Tween 20 and 5% ethanol. Fifty/41 were applied to the Shoot tips of spinach plants maintained under SD. Ten such applications, for a total of 100;!g GA20 were made on alternate days. The stem of each plant was measured one week following the last application. Five plants were used for both treatment and control. 3.3 RESULTS Using GLC-SICM, a standard calibration curve was con- so that the absolute amount of GA Structed for GA present 20 20 in the plant material could be determined. This curve was linear over a range from 10 to 200 ng. The lower limit of sensitivity was 1 ng, which is about the same limit of sensi- tivity as the d-S corn bioassay (Phinney and West, 1960). In contrast, at least 100 ng was necessary to get a good 74 spectrum using repetitive scanning GLC-MS. Because authentic samples of the other GAS present in Spinach were either not available, or available in quantities too small to permit accurate weighing, standard calibration curves for these GAS could not be constructed. In these cases, only changes in the relative levels could be expressed. The largest SICM response for a given GA from the series of photoperiodic treatments was normalized to a unit dry weight basis and then arbitrarily assigned a value of 100. Normalized values from the other treatments were then expressed in proportion to the highest SICM value. In order to provide for an estimate of losses incurred during the purification procedures, a Spike of 50,000 dpm of [3Hl-GA was added to a separate methanolic extract of l spinach shoots, and the extract purified as described in section 3.2.2. Typically, 50-60% of the radioactivity was recovered after analytical reverse phase HPLC. Similar re- sults were obtained by Jones and Zeevaart (1980b) using the same purification procedures with extracts of shoots from Agrostemma githago. These estimated losses were not used as correction factors in any of the quantitative determinations made in this chapter. Figure 3-1 shows the changes in the relative levels of 5 GAS as well as stem height as a function of LD treatment. GA occurred in quantities too small to be measured. It is 53 clear from this figure that the relative level of GA19 de- clined 5-fold with LD treatment, whereas the levels of both 75 Figure 3-1. Changes in the relative levels of five GAS in spinach as measured by GLC-SICM, and stem length as affected by different durations of LD treatment. The highest concentration (SICM response/unit dry weight) of each GA was arbitrarily assigned a value of 100, and the other concentra- tions expressed in proportion to this value. The SICM re- sponse of the molecular ion was used in all calculations except for GA19, in which case the base peak was used. Ten plants were used in each treatment. 76 Relative SICM Response per Unit Dry Weight - m/e4l8 8 8 8 8 M434 3 as a; o 0 mm 8 5 00 N O “”0150 00000 0>~o 50 a: o 6 8w: e h A 0 rule Relative SICM Response per Unit Dry Weiq .b o m/ N O .00 m a: o o 492 N O .00 a as m o o o m/e 432 N O I x ..\ x.\\.\ {x F p p n _ p bl b lsfib _ p p p p r x x xix I x/\ /x\ I x/ \X I llxllxllx {\ p r p p P . _ L «V? T b a .l p p p .. mSB rose; x 0?; \ x I X\J /XL\ I x x\ «Fin n P 13“ p p p P \M p n p b n — p n OmeQ.O.N_b Omem_O.N3 2o. On PC On PC 77 GA20 amd GA29 increased dramatically during the same period. The levels of GAl7 and GA44 remained fairly constant through- out photoperiodic treatment. In absolute terms, the level of GA20 increased from 0.8,ug/100 g dry weight (30 ng/plant) to 5.5/kg/100 g dry weight (200 ng/plant), a nearly 7-fold in- and GA 19 20 with LD treatment as measured by GLC-SICM confirm earlier crease. The realtive changes in the levels of GA work on Spinach by Zeevaart(197l; 1974), using the d-S corn bioassay for quantitating the levels of GA-like substances. However, GA29, which reportedly has very little biological activity in the d-S corn bioassay (Yokota 33 33., 1971), would have been overlooked in the earlier work. In Chapter 2, it was shown that the biological activity associated with spinach shoot extracts is due almost entirely to the combined effected of GA and GA which give Similar responses in the 19 20 d-S corn bioassay (Crozier 33 33., 1970). This indicates that in absolute terms, GA19 occurs in a Similar, but in- verse, range of quantities as GAZO' GAZO' when applied to plants maintained under SD, was able to substitute (at least partially) for LD in eliciting stem growth (Table 3-1). Zeevaart (1974) found that exogen- ous GA20 could completely replace LD treatment in causing increased petiole elongation and changing leaf orientation (position). Moreover, GA20 was more active than GA3 (Zeevaart, 1974). 78 Table 3-1. Comparison of the effect of exogenous GAzo and LD on stem growth of Spinach plants. GA20 was applied to the tips of plants, on alternate days, 20 ug per plant, for a total of 100 pg of GAZO per plant. Stems of the plants were measured 7 days following the last GAzo application. Other plants given 17 LD. Five plants per treatment. Treatment Stem Length (mm) SD 0 so + GAZO 75 i 61) 1? LD 123 t 15 1) Standard error of the mean. 79 3.4 DISCUSSION In Chapter 2 the following metabolic pathway, based on structural considerations, was proposed to occur in spinach: GA —uGA -—>GA —>GA —>GA -9GA (Fig. 2-5). The 53 44 19 17 20 29 decline in the level of GA19 with the concomitant rise in the level of GA20 with LD treatment is indicative of a precursor-product relationship between the two GAS. More- over, the co-occurrence of GA19 and GA20 in a number of disparate species, including Agrostemma githqgo (Jones and Zeevaart, 1980b), Pharbitis nil (Jones 33 33., 1980), Phaseolus coccineus(Graebe and Ropers, 1978), Pisum sativum (Ingram and Browning, 1979), and 333.3313 (Phinney, 1979), is also circumstantial evidence supporting the notion that GA19, a CZO-GA, is eventually converted to GAZO' a Clg-GA. However, as yet no definitive biochemical evidence is avail- able that proves such a conversion takes place (Graebe and Ropers, 1978; Hedden 33 33., 1978). The initiation in the rise of the level of GA29 lagged Slightly behind the increase in the amount of GA20 (Fig. 3-1), and was also observed in a second experiment (results not shown). This is consistent with the idea that GA is con- 20 verted to GA29. Indeed, this conversion has been demonstrated in a number of systems, including Pisum sativum (Frydman 33 al., 1974), Bryophyllum daigremontianum (Durley 33 33., 1975), and Phaseolus vulgaris (Yam3ne 33 33., 1977). Thus, it is probable that such a conversion is also a natural process in spinach. Since C-Zflhydroxylated GAS, such as GA are 29' 80 usually inactive in eliciting GA responses, conversions of the type proposed above are generally thought to be inactiva- tion steps (see 1.3.1.). Stem growth is preceded by a precipitous rise in the level of GAZO‘ Since exogenous GA?-0 is able to cause stem growth in spinach plants maintained under SD, a major factor in the control of stem growth in spinach could be the avail- 20. The level of GA20 is controlled through a balance of production and metabolism. If GA19 is ability of endogenous GA a precursor of GA the data presented in Fig. 3-1 indicate 20’ that the step(s) GA19-—>Gm20 is under photoperiodic regula- tion. This suggests that one major aspect of photoperiodic control of stem growth in spinach is effected through regulation of this conversion. The fact that GA19 is at its highest level during SD could mean that it is itself inactive in promoting stem growth, and must be converted to biologi- cally active GAZO' Consequently, GA19 could serve in spinach as a "pool" gibberellin, a role postulated for GA19 in rice (Kuroguchi 33_33., 1979). Obviously, the exact metabolic relationship between the endogenous GAS must be ascertained before a clear picture of the mechanism of photoperiodic control of stem growth can be made. Chapter 4 Comparison of the Levels of Endogenous Gibberellins in Roots and Shoots of Spinach in Relation to Photoperiod 81 82 4.1 INTRODUCTION In Chapter 3 it was suggested that photoperiod controls stem growth in Spinach by regulating the level of GA How- 20' ever, the mechanism of regulation of this particular aspect of GA metabolism is not known. 3 priori, the levels of indi- vidual GAS in a particular organ could be regulated in at least two ways. Most obvious would be a direct control of the organ's enzymes responsible for GA biosynthesis and meta- bolism. It is also possible that transport of GAS or GA precursors and intermediates could play a Significant role in regulating developmental events (see section 1.5). In fact, GAS are known to be present in the phloem and in the xylem, and appear to be transported in tissues over long distances (King, 1976; Graebe and Ropers, 1978). Went (1943) originally proposed that substances produced in the root exert a hormonal control over Shoot growth. Later, it was suggested by other investigators that these substances are, at least in part, GAS (Reid 33 33., 1969; Reid and Crozier, 1971). However, the conclusions reached by these authors are disputed by others (see 1.5; Graebe and Ropers, 1978). Thus, although GAS appear to be transported in the plant over long distances, the physiological importance of such movement remains unclear. 83 This chapter is a preliminary study on the possible role of root-Shoot interactionsin.GA-mediated growth responses in spinach. First of all, the GA content of roots and shoots in relation to photoperiod was analyzed, with special attention to the distribution between roots and Shoots of the six GAS previously identified in spinach shoots (Chapter 2). Secondly, as an indication of the movement of GAS between roots and Shoots, the GA content of both phloem and xylem exudate was examined. Finally, in an effort to ascertain the Sites of GA production, the GA content of excised root tips grown in culture was determined. 4.2 MATERIALS AND METHODS 4.2.1. Plant Culture and Photoperiodic Treatments. Spinach seeds (2.2.1.) were sown on vermiculite. After 10 days the seedlings were transferred to 37 x 30 x 22 cm trays outfitted with a cover to hold 20 seedlings. The trays were filled with half-strength Hoagland's solution, which was con- tinuously aerated. During the course of an experiment, the medium was changed once a week. The plants were maintained under SD (3.2.1.) until ready for experimentation, approxi— mately six weeks after sowing. At the end of an experiment, whole plants were harvested and divided into roots and shoots. Both parts were frozen in liquid N2, lyophilized, and stored at -15°C prior to extraction. 4.2.2. Effect of Photoperiod on Levels of Extractable GA-like Substances from Roots and Shoots. The details of the 84 extraction and purification procedures have been described in 2.2.2. Methanolic extracts of roots or Shoots were puri- fied by charcoal adsorption chromatography and silicic acid adsorption chromatography. The eluate resulting from silicic acid adsorption chromatography was fractionated by preparative TLC. The chromatogram was divided into 10 equal zones and each zone analyzed for the presence of GA-like substances using the d-5 corn bioassay (2.2.4.). 4.2.3. Identification of GAS in Root Extracts. Metha- nolic extracts of the roots from 500 plants (ca 100 g dry weight) were purified as described before and then fraction- ated via preparative reverse phase HPLC (2.2.2.). Fractions known to contain Spinach shoot GAS in this system were puri- fied further by analytical reverse phase HPLC (2.2.2.). Appropriate fractions resulting from analytical HPLC were methylated with ethereal diazomethane. The trimethylsilyl ethers of the methyl esters were prepared by adding 50 ul of a solution containing pyridine-hexamethyldisilazane- trimethylchlorosilazane (9:3:1, v/v) to dry samples in Reactivials (Pierce Chemical Co.). The derivatized samples were then subjected to GLC-MS under the same conditions described before (2.2.7.). 4.2.4. GA Content of Phloem Exudate. Phloem exudate was collected from detached spinach leaves using the method of King and Zeevaart (1974). Leaves from 100 plants were detached and placed into beakers containing a 20 mM solution of EDTA (pH 7.0). The beakers contained enough solution so 85 that only the cut surface and a few mm of the petioles were exposed to the EDTA solution. After 2 hr the treated ends of the petioles were rinsed with distilled H20, and phloem exu- date was then collected in double-distilled H20 over the next 10-12 hr. The phloem exudate was frozen, lyophilized, and the dry weight of the residue determined. The residue was taken up in 50 ml of 0.1 M phosphate buffer that was previ- ously adjusted to pH 2.5 with€5N HCl and partitioned 4 times with equal volumes of ethyl acetate. The acidic ethyl ace- tate fraction was concentrated, subjected to preparative TLC, and the GA content determined as described earlier. 4.2.5. GA Content of Xylem Exudate. Spinach plants were individually grown hydroponically in 4 liter bottles that were wrapped in aluminum foil to keep the root system in dark- ness. Plants were maintained under SD for 4 weeks and then given 7 LD. The plants were decapitated and latex tubes placed over the cut ends. Xylem exudate was collected via the latex tubes in flasks packed in ice. Collection of the xylem exudate continued for 12 hr. The xylem exudate from 100 plants was pooled and the pH of the exudate adjusted to 2.5 withiSN HCl. The acidified xylem exudate was partitioned 4 times with equal volumes of ethyl acetate. The GA content of the acidic ethyl acetate fraction was determined after fractionation by TLC as described before. 4.2.6. GA Content of Spinach Roots Cultured in Vitro. Spinach seeds were surface-sterilized by treatment with water saturated with bromine for 7 min. The seeds were then rinsed 86 thoroughly 4 times with sterile distilled water. Following the last rinse, the seeds were sown on sterile 1% agar in Petri dishes, and then placed in the dark at 27°C. After 7 days 10 root tips, 0.5 cm long, were excised and placed in a 125 ml Erlenmeyer flask containing 50 ml of modified White's medium (White, 1943). The only modification of the original formulation was replacement of the Fe2(SO4)3 with 500 mg of Sequestrene (Geigy Industrial Chemicals) in the stock solu- tion. The root tips were allowed to grow for 2 weeks in the dark at 27°C. During that period, the roots grew to a length of 15-20 cm. A total of 2200 cultured roots were lyophilized (dry weight = 4.1 g), extracted, and analyzed for the presence of GA-like substances as described in section 4.2.2. The medium in which the roots were cultured was also analyzed for the presence of GA-like substances. Approximately 12 liters of culture medium in 500 m1 batches were acidified to pH 2.5 with H PO 3 4’ with Bondapak ClB/Porasil B (Waters Associates). GAS were and then pumped through a column (1 x 25 cm) packed eluted with 250 ml of 95% ethanol. Fifteen m1 of phosphate buffer (pH 8.2) were added to the eluate and the ethanol removed under reduced pressure. The pH of the aqueous resi- due was reduced to 2.5, and the solution was partitioned 4 times with equal volumes of ethyl acetate. The acidic ethyl acetate fractions from 24 Similar runs were combined, re- duced to a small volume,‘and fractionated by TLC as described earlier. The resulting chromatogram was then analyzed for 87 the presence of GA-like substances as described earlier (4.2.2.). [14 4.2.7. Cj-Labeling of Assimilates. Blades of intact Spinach leaves were enclosed in a 17 x 31 x 2 cm clear Plexi- glas chamber. l4C02, generated by reacting 2 mg of Ba l4CO3 (New England Nuclear, 59.7 mCi/mM) and 3 mg of non-radioactfln: BaCO3 with a few ml of 20% lactic acid, was circulated over the enclosed blades for 8 min. During that time the blades were irradiated by light from two 250 W flood lamps filtered through 5 cm of water. 4.2.8. Transport of [3Hj-GA20 and [14CJ-Labeled Assimi- lates. Two plants were grown hydroponically under SD for 4 weeks as described in 4.2.5., and then given 7 LD. Each plant 5 received a foliar application of 4.0 x 10 dpm of [2,3-3H]- GA 0 (a gift from Dr. R. P. Pharis, University of Calgary, 2 Alberta, Canada) dissolved in an aqueous solution of 0.05% Tween 20 and 10% ethanol. The [2,3-3HJ-GA20 was diluted with cold GA from an original Specific activity of 3.3 Ci/mmol 20 to 25 mCi/mmol. After 24 hr the roots were harvested, frozen in liquid N2, and lyophilized. The freeze-dried root systems were extracted, purified and fractionated by TLC as described in 4.2.2. The resulting chromatogram was divided into 10 zones, and each zone eluted. The resulting eluates were dried on cellulose powder in Packard Combustocones. Each of these was combusted in a Packard model 306 Tri-Carb sample oxidizer for 45 sec, and then counted for 5 min in vials containing 15 m1 of Packard Monophase-40 and 2 ml of Permafluor V using 88 a Packard model 3255 Tri-Carb liquid Scintillation spectrome- ter. The counting efficiency was determined, and the data converted to dpm. In other experiments Simultaneous transport of exogenous C14C3-sucrose and [3HJ-GA20 out of Spinach leaves was followed. A small circle, 1 cm in diameter, on a leaf from a plant sub- jected to 10 LD, was lightly abraded with Silicon carbide powder (400 grit, Sargent-Welch). The abraded area was then bounded with a wall of softened lanolin. Fifty ul of a 5% ethanol and 0.5% Tween solution in water containing 0.5 uCi of C14CJ-sucrose (New England Nuclear, 658 mCi/mM) and 0.5 uCi of C3HJ-GA20 (3.3 Ci/mM) were applied in the lanolin well. To prevent evaporation, the well was covered with a small square of polyethylene plastic film. Two leaves of the same Size (blade ca 7 cm long) from a plant were Similarly treated, and placed under high intensity light (33VVm-2) in a growth chamber. After 1 hr the 2 treated leaves were detached, and the cut end of the petiole from one of the leaves was treated with a 20 mM EDTA solution (see 4.2.4.) and the other treated with double-distilled H20 (pH 7.0). The leaves in the treat- ment solutions were placed in a dark, humid incubator at 270 for 1 hr. The cut end of the petiole from each leaf was rinsed with double-distilled H20, and then placed into a scintillation vial containing 2 mls of H20, and allowed to exude in the dark at 27°C for 16 hr. The H20 in the scintil- lation vial was evaporated with a stream of N2, and 10 ml of Formula I scintillation cocktail (King and Zeevaart, 1974) 89 added. 14C and 3H were counted Simultaneously using a Packard model 3255 Tri-Carb liquid scintillation spectrometer. In a similar experiment, 2 leaves from plants subjected to 10 LD were allowed to photosynthesize in the presence of 14CO2 as described in section 4.2.7. Following labeling, the leaves were excised and treated with an EDTA solution or double-distilled H20 as described above. ‘After pre- treatment, the leaves were allowed to exude into a scintil- lation vial containing 2 ml of H20 for 8 hr. The H20 was evaporated, and the residue counted as described before. 4.2.9. Carbohydrate and Protein Analysis of Phloem Exudate. Total carbohydrate was determined by a phenol- sulfuric acid test (Aminoff 33 33., 1970). Fifty ug of dried phloem exudate were dissolved in 2 ml of double-distilled water. Fifty ul of 80% phenol were then added, followed by 5 ml of H 804. After the solution had cooled, absorption at 2 488 nm was measured. Quantitative determinations were made after interpolation of the reading on a sucrose standard curve. The characterization of individual component sugars in the phloem exudate was performed by comparison of the Rf in TLC of various authentic sugars and compounds in the phloem exudate. Ten ug of the phloem exudate residue in 10 ul of water were Spotted on a 20 x 20 glass plate coated with a 0.25 mm thick layer of Silica Gel 60 (EM Reagents), along with 10 ug of 5 reference sugars: glucose, fructose, sucrose, raffinose, and stachyose. The plate was then developed 4 90 times to 15 cm from the origin in ethyl acetatezacetic acid: water (60:30:12, v/v). The compounds were visualized by Spraying the chromatogram with a mixture of acetic acid: sulfuric acid:peanisaldehyde (50:1:0.5, v/v) and heating for a few minutes at 100°C. Protein determinations were made by the method described by Bradford (1976) employing Coomassie Brilliant Blue G-250 (Sigma). 4.2.10. Autoradiogr3phy. Three plants were grown hydro- ponically in SD as described in 4.2.5. Following 10 LD, 2 leaves from each plant were labeled with 14 CO2 (4.2.7.) and placed back into the growth chamber. After 2, 4, or 6 hr, a root system was detached, pressed between 2 pieces of card- board, and frozen in liquid N2. The root systems were then lyophilized. Kodak Industrial M-54 X-ray film was exposed to the freeze-dried root systems for 7 days. Development of the film was by standard procedures. 4.3 RESULTS 4.3.1. Effect of Photoperiod on Levels of GAS in Shoots and Roots. The approximate dry weight ratio of shoot mate- rial to root material from hydroponically grown plants was 5:1. Therefore, it was necessary to extract the roots from 80 plants as compared to Shoots from 12 plants in order to have comparable amounts of dry plant material. Figure 4-1 Shows the GA content of extracts from roots and shoots as affected by different durations of LD treatment. In 91 Figure 4-1. The effect of photoperiod on GA-like acti- vity in roots and Shoots. Partially purified acidic extracts were fractionated by TLC, and the resulting chromatograms divided into 10 equal zones. Each zone was assayed for the presence of GA-like substances using the d-5 corn bioassay. Roots from 80 plants (20 g dry weight), shoots from 12 plants (15 g dry weight). °/o of Control 92 c’/o Of COI'lil’Ol 50. mo woos 1.0 moo; Bro zoom 50 1 Bo .mo .00 I I .IIJ IRHL U V\ \% \\ 1 who NNO N00: .00: .I. 50.. 30 E .mo .8 I IJIUJ I. m L3 I In IUD \\ V\ mo mzooa .210 mzoolm 3 r0 macaw 1 I TI’I ELI -LILILlr . L. I LIL) hILLLLlrLLL o o.» ow om om Straw 0.».8 ow om 6:0 o.~ 0.3 on om 8 ml . 3 ml \\ 93 agreement with previous work by Zeevaart (1971), extracts of Shoots showed the presence of two GA-like substances which change in level with LD treatment. The level of a polar GA (Rf 0.1-0.3), which has been identified as GA19, declined with LD treatment, whereas the level of GA 0 (Rf 0.5) in- 2 creased during the same period (Chapter 2, 3). Roots, on the other hand, contained only one zone with GA-like activity with chromatographic properties identical to that of the shoot GA19. The level of this substance(s) remained constant with different durations of the LD treatment. The root ex- tracts Showed no GA-like activity in the zone where GA20 chromatographs regardless of photoperiodic treatment. It should also be noted that the Shoots had a higher GA content than roots whether expressed on a per unit weight basis (3 times) or per plant basis (20 times). 4.3.2. Identification of Root GAS. In view of the above results, it was of interest to see which of the six GAS pre- viously identified in spinach Shoots (Chapter 2) were present in root extracts. Therefore, it was necessary to extract and purify roots from ca 500 plants. Various fractions were derivatized and then analyzed by GLC-MS. Similar prepara- tions from Shoots of the same plants were used for analysis by GLC-MS. Full repetitive scans of various root fractions indicated the presence of three substances which had identi- cal retention times and similar mass Spectra to three GAS previously found in shoots (Chapter 2): GA44, GA and 19’ GA29 (Table 4-1). Neither GA GA nor GA20 (Fig. 2-5), 53’ 17’ 94 all of which were found in the shoot extracts, was detected in root extracts by repetitive scanning mass Spectrometry. However, the lower limit of detection by this technique was 100 ng of GA20 per injection. Presumably, this figure would be Similar for other GAS as well. It is possible that GA53, GA17, and GA20 were present in minute quantities and there- fore escaped detection by GLC-MS with repetitive scanning. A more sensitive, albeit less definitive, detection technique was GLC-SICM which increased sensitivity by 100 times to 1 ng GA20 per injection. Using this technique, a compound was detected with the same retention time and with percentages for 4 m/e values Similar to those of MeTMS-GA53 (Table 4-2). From these data it can be concluded that GA53 is present in trace amounts in the roots. No evidence was obtained by GLC-SICM for the presence of either GA20 or GAl7 in root extracts. 4.3.3. GA Content of Phloem Exudate. Figure 4-2 Shows the GA content of phloem exudate from plants subjected to either SD or 10 LD. It is clear that the pattern of GAS detected by bioassay was Similar to that in extracts from leaves. The concentration of GA-like substances can be calculated, assuming that sugar constitutes about 15% (w/v) of the phloem contents, and that sugar comprises almost all of the dry material (Pate, 1976; Ziegler, 1975). The volume of phloem exudate can then be calculated from the dry weight of the exudate (Table 4-3). The total GA-like substances found in the phloem exudate can be determined by interpolation emopm SIP. noapwfiwmo: Om mbOIZm mono mHOB osnwmwmm manmonm Om mpwomo: Hoonm mom wroonm. 95 Beam 0m momwm H: Smmm momonnca Aa\m noonmsn nosomsnnmnwo: 80 3H :0 ovuImQSMwIma5wIpwwm mcomwmsomm w: wrwoma mxcmmno mHoa wImnsMm owoweoe> meoe> somcfimm oosoummmm A H IHE wuswam>wsvam Eon“ mumpsxo anmx cw mwosmumnom mxHaIdo mo 20wumuusoocoo mo SOmHHmmEou .olv manna 111 Figure 4-5. Root systems (top) and their autoradiograms (bottom) from plants allowed to photosynthesize in the pres- ence of 14C02. Roots were harvested 2, 4, or 6 hr after labeling, frozen, and lyophilized. X-ray film was exposed to the freeze-dried root systems for 7 days before development. Plants subjected to 10 LD prior to use. 112 113 shoot to the roots. This movement was fairly rapid, since radioactivity was detected in the root systems detached 2 hr after labeling. Figure 4-6 Shows the distribution of radioactivity on a chromatogram of a root extract 24 hr following a foliar application of [3HJ-GA20. Two distinct zones are apparent: a polar zone at Rf 0.1-0.2, and one which has the same Rf as authentic GA20 (Rf 0.4-0.5), and probably represents un- metabolized E3H3-GA Although the total radioactivity 20' found in the roots was a small percentage of the total [BHJ- GA20 applied to the plant (0.35%), it illustrates that exo- genous GA20 does move from the root to the shoot. In order to test whether exogenous [3HJ-GA20 moves in the phloem, a double-label experiment was performed to com- pare the mobility of simultaneously applied [14CJ-Sucrose and [3Hj-GA2 One hour after application, 2 treated leaves 0' were detached, and the cut end of the petiole of one leaf was treated with a 20 mM EDTA solution, while the other was placed in water. Table 4-7 shows that EDTA pre-treatment was necessary for significant amounts of 3H or 14C to be present in the phloem exudate. Note that the ratio of 3H:14C remained relatively constant. This indicates that exogenous GAZO moved from the Shoot to the roots with sucrose and other assimilates in the phloem. Thus, it is likely that at least part of the endogenous GA20 present in the phloem moved to the root system. 114 Figure 4- 6. Distribution of radioactivity on a chromato- gram of a root extract from 2 plants 24 hr following a foliar application of 4 x 105 dpm of [2, 3-3 H] -GA20 to each plant. 115 _ _ _ __,_ L0 04 06 CH3 02 I000” _ _ , 0 0 o o e 4 :53 >t>Cowo ruouwanwaawu n: HnuHchswn3 H0 NO n>~cawoc~ mama 0>woawo€v + 93 2.53 men: mrozo>a~02 Il‘ 03b enougntmmpm n: «canmelIlllIV 0> rn n: ~mm »: Hmmoou no:I mam «roan utmx.w~n: a>~0unosI ~o no o>~o «was. ~e no n>~c noun. 0) chzoHm rat o> ncn=o ncnsam roe nfiumca mmsuwnwm. :wmr anumcm anamwnwm. aNmmcmmnn. Heavy APPENDIX APPENDIX This Appendix contains the bar graphs of the mass spectra with background subtraction of the GAS found in extracts of spinach Shoots, along with the Similar graphs of appropriate reference compounds (if available). 145 w 146 Figure A-l Spinach MeTMS-GA19 20 40 60 80 100 420 120 «e 140 160 480 147 Figure A-2 Spinach MeTMS-GA20 148 00. . 1 Figure A-3 8°* Standard MeTMS-GA . 20 69. I 40, 80. 20 40 60 80 100 120 140 160 IOOJ 801 60. 40. 80. W 243 260 W lee, I 80, ' 60. 40, 20. I 340 see 380 400 423 449 460 I 480 r00. 36. 63 404 eel 10sz1 80. 60. 4o: 20. Figure A-4 Spinach MeTMS-GA 180 EQQ Rafi 100. 340 360 -SBQ_____529 29 380 £46 149 80 140 400 100 ififlL____339 430 580 120 «a 600 140 13306 460 4 433 160 seal 3 ‘6. 4'8 3‘3 0‘ I. 3 150 Figure A-5 Standard MeTMS-GA29 100 180 140 160 151 Spinach MeTMS-GA19 130 r“! 160 100 L°°< Figure A-6 HQ A 152 Figure A-7 Standard MeTMS-GAl7 1 If “8 3.4.88, J A w L $3.3 .6 A A .3 35.3. T“ v fl v—v 153 Figure A-8 Spinach MeTMS-GA44 120 140 160 480 154 Figure A-9 30“ Spinach MeTMS-GA53 40“ 30" 80 40 60 80 100 120 140 166 3.00., P LBW am 266 2W 225 60. 340 360 380 403 429 «a 460 480 REFERENCES REFERENCES Amininoff, D., W. Binkley, R. Schaffer, and R. Mowry. 1970. In W. Pigman and P. Horton, eds., The Carbohydrates: Chemistry and Biochemistry. Academic Press, New York, pp. 740-808. Anderson, J.D. and T.C. Moore. 1967. Biosynthesis of (-)- kaurene in cell-free extracts of immature pea seeds. 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