~....~‘-—-~.- THE INTERACTING EFFEfiTS CEF 3-ENDQLEACETIC MID; GIBBERELLIC ACIED APéD 6-BENZYULDENINE ON RE5PIRATION, GROWTH AND NITROGEN ASSIMILATION IN EXCISED EMBRYGS (35‘ PEA (MW L) Thais for the Degree of Ph. D. MICE-“(SAN STA'FE UNIVERSITY Davie! Cameron Mac Lean ”I965 IHESIS LI 3 '7 ‘. R 1' Michigan. Saw University MICHIG III/W III/IIIII/TIII/IIIIII/II/Ii/III/I 312/ m 1/ 9 01591 4074 This is to certify that the thesis entitled THE INTERACTING EFFECTS OF 3-INDOLEACETIC ACID, GIBBERELLIC ACID AND 6-BENZYLADENINE 0N RESPIRATION, GROWTH AND NITROGEN ASSIMIIATION IN EXCISED EMBRYOS 0F PEA (PISUM SATIVUMJ L.) presented by David Cameron MacLean has been accepted towards fulfillment of the requirements for Ph.D. degree in Horticulture / " /A/-) '/'///I/ “+4556: . ILcuH': Major professor Date [ct/H H'I/gc/jfi _/ 0-169 THE IKTERACTING EFFECTS OF S-INDOLKACETIC ACID, GIBBERELLIC ACID AKD 6-BLNZYLADENINB ON RESPIDATION, GROWTH AND FITROGEN ASSIMILATION IN EXCISED EMBRYOS or PEA (PISPM SATIVUM, L.) By David Cameron MacLean 'N ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1965 ABSTRACT THE INTERACTING EFFECTS OF 3-INDOLEACETIC ACID, GIEBERELLIC ACID AND 6-BENZYLADENINE ON RESPIRATION, GROWTH AND NITROGEN ASSIMILATION IN EXCISED EMBRYOS OF PEA (PISUM SATIVUM, L) By David Cameron MacLean Respiration, growth and nitrogen assimilation from the cotyledons in excised pea embryos were in- vestigated following 12 hours inhibition in treating solutions containing auxin (3—indoleacetic acid, 1AA), gibberellin {gibberellic acid, GA) and a kinin (6- benzyladenine, BA) alone or in combination. Preliminary investigations concerned the effect of these growth substances on the respiration rate of fully expanded broccoli leaf discs showed that IAA and GA (10-5 to 10-3 M) did not affect oxygen uptake in broccoli, whereas, BA treatment (leO-Sfl) resulted in a 40 percent reduction of the respiration rate. The presence of IAA and/or GA at the same concentration did not alter the BA-induced inhibition. Oxygen uptake, nitrogen assimilation and growth of pea embryos treated with GA or BA at 5x10"5fl did not differ from the control embryos from O to 72 hours after imbibition. These response were consistently \TI'J inhibited by 1AA treatment (5x10- g) at 24 and 48 hours David Cameron MacLean after imbibition. However, growth of auxin-treated embryos was inhibited to a greater extent than nitrogen assimilation, resulting in a greater nitrogen content per unit growth at these time intervals. A marked stimulation of respiration, nitrogen assimilation and growth was observed in pea embryos treated with 5xio'6 g IAA. A similar, though less pronounced effect was observed for EA 53:10"6 N. GA induced a consistent inhibitory effect on these para- meters at 5x10’7 3. The optimum IAA concentration for stimulation of growth, reapiration and nitrogen assimilation was de— creased from ‘leO-6 to 5x10"-8 N by including 53:10"5 E BA in the imbibing solution, indicating increased efficiency of IAA by BA. The stimulatory effect of auxin (5x10‘6 Q) was blocked by GA or GA plus BA at SxIO-S E. A marked inhibition of all parameters, equal in magnitude to the BA-induced enhancement was observed when 53:10.5 N GA was applied with auxin at 5xlO-8 N. The inhibitory effect of ‘5x10”5 M IAA was dominant when applied alone or with BA and/or GA at various concentrations. No interaction between GA and BA was observed. Growth regulator treatment did not alter the dependency of nitrogen assimilation on respiration. The requirement for a specific concentration David Cameron MacLean ratio of auxins, gibberellins and kinins for growth and associated phenomena was suggested from these studies. Pronounced responses to growth regulator treatments were negated by a ten-fold increase or decrease in concentration. It is postulated that specific concentration ratios of growth regulators, within physiological concentrations, might induce the production of certain nucleic acids which, in turn, may regulate protein synthesis and thereby govern plant growth and development. THE INTE ACTING EFFECTS OF B-INDOLEACETIC ACID, GIBBERELLIC ACID AND 6—BENZYLADENINE ON RESPIRATION, GROWTH AND NITROGEN ASSIMILATION IN EXCISED EMBRYOS OF PEA (PISUM SATIVUM, L) By David Cameron MacLean A THESIS Submitted to Michigan State University in partial fulfillment of the recuirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1965 ACKXOKL EDC Ell EXT S The author is indebted to Dr. David R. Dilley for his encourage— ment and counsel throughout these studies. The advice and assistance offered by Drs. D. H. Dewey, C. J. Pollard, H. H. Sell and S. H. Nittwer whenever requested, and during the preparation of this dissertation are greatfully appreciated. Finally, I would like to thank Dr. R. R. Dedolph for his helpful suggestions in the initial phases of these studies. TABLE OF CONTENTS INTRODUCT I on O O O O O _ O O I O O O O O O O 0 REVIEW 0? LITERATURE . . . . . . . . . . . . AUXINS O O O O O O O O O O O O O O O 0 Structural Requirements for A0t171tyo o o o o o o o o e e o o Auxin and Plant Growth . . . . . . Auxin and Respiratory metabolism . Auxin and Nucleic Acids . . . . . GIBIBRELLINS . . . . . . . . . . . . . Gibberellins and Ilant Growth. . . Effect of Gibberellin (GA) at the Genetic and Enzyme Level. . . . . KININS O O O C O O O O O O O 0 O O O 0 Discovery of Kinetin . . . . . . . Structural Requirements for Kinin Activity. . . . . . . . . . . . . occurrence 0: lining. o o o o o o 0 Effect of Kinins on Plant Growth and Development . . . . . . . . . Effect of Kinins on Metabolic Irocesses . . . . . . . . . . . . Enzyme Activity . . . . . . . nuCICiC A0168 0 e e e o o e e Senescence Inhibition. . . . . INTERACTIONS O O O O 0 O O O O O O 0 ii Iago simmm 10 10 11 13 13 13 14 17 17 19 2] 22 TABLE OF CONTENTS (cont) GrOWtheeeeee Auxins and Gibberellins. Auxine and Kinins. Gibberellins and Transport. . . . . Sequential Responses Regulators. . . . MATERIALS AND METHODS. . . . Treatment Solutions. . Leaf Disc Studies. . . Iea Embryo Studies . . Time Studies . . . . . Interaction Studies. . RESULTS AND DISCUSSION . . . K1711 n8 0 to Growth 0 BROCCOLI LEAP DISC STUDIES DEA ENDRYO STUDIES . . Time Studies . . . Irotein Determinations Effect of Growth Regulator Concentrations. . Interaction Studies. SUMMARY AND CONCLUSIONS . . LITERATURE CITED . . Page 22 22 24 26 27 28 3O 3O 31 32 35 36 38 38 40 4O 46 48 52 64 68 INTRODUCTION Numerous chemicals have been isolated from plants which influence fundamental phases of plant growth and development. Other chemicals, some of which are structurally related to the naturally occurring sub- stances, and which have been synthesized in the labora- tory also possess plant growth regulating activity. Experimentation with these plant growth substances has had a great impact on plant science since Darwin (1880) published his book The EEEEE 9f ygyement in Plants. Research has led to the current agronomic use of some of these chemicals for control of flowering and fruit- ing, promotion or prevention of leaf and fruit abscis- sion, production of seedless fruits, promotion of rooting on cuttings, breaking rest and dormancy of buds and seeds, weed control and other diverse phenom— ena. The naturally occurring growth regulators have been classified, on the basis of biological specificity and chemical structure, into three groups; the auxins, the gibberellins and the kinins. Despite the vast accomplishments of growth regulator research, the basic biological functions of these three classes of com- pounds remains uncertain. In numerous investigations, synergistic, as well 2 as antagonistic reSponses have been reported when two or more naturally occurring growth regulators were applied simultaneously. It is recognized that these opposing responses may, in some cases, be due to tissue type, stage of maturity and the relative concentration of growth regulators used. However, these factors do not fully account for the diverse responses that have been reported. Thus, it seemed desirable to further investigate the interacting effects of plant growth regulators. EXperiments were conducted to ascertain the effects of auxins, gibberellins and kinins on two different plant tissues; one, broccoli leaves, incapable of further growth; and the other, pea embryos, possessing a high growth potential. REVIEW OF LITERATURE Auxins, gibberellins and kinins have been the subject of several recent reviews (Miller, 1961; Galston and Purves, 1960, Phinney and West, 1960a). Therefore, only that portion of the literature concern- ing the basic concepts of these growth substances pertinent to this investigation is reviewed here. AUXINS Charles Darwin (1880) is usually credited with initiating the concept of plant hormone research. He discovered that a stimulus, induced in the tips of Phalaris and £1333 cotyledons by unilateral light, was transmitted to the lower parts where it induced a bending of the shoot towards the light. However, 50 years passed before Went identified this stimulus as auxin (van Overbeek, 1959). Currently, auxins are generally assumed to be ubiquitous in higher plants. Structural Requirements for Activity The chemical structure of most auxins consists of a naphthalene, benzene or indole nucleus. The indole nucleus is characteristic of the naturally occurring auxins to which this review is limited. Al- though many indole compounds occur naturally and exhibit activity in auxin assays, 3-indoleacetic acid (IAA) is 3 4 regarded as the naturally occurring auxin (Kefford, 1963). The activity of the IAA analogs is due to their con- ver31on to 1AA in the assay tissues (Kefford, 1963). To be active, auxins must possess an unsaturated ring and a side chain of certain length terminating in a carbonyl group or a functional group readily converted to a casloxyl group (Galston and Purves, 1960). In some synthetic auxins, namely the phencxy-acids, at least one free ortho position is required for activity (Galston and Purves, 1960). The nature of these requirements for activity has resulted in the acceptance of the general concept that auxin forms a complex with a specific receptor. Foster et al. (1952) applied classical enzymic kinetics to auxin induced coleoptile growth. The auxin induced growth inhibition at high concentra- tions led them to conclude that there was an attachment cf auxin to a receptor, and that this complex is similar to that postulated for a typical enzyme-substrate complex. Although their methods have been challenged by Lockhart (1962} and their interpretations disputed by Truelsen (1961), the concept of an auxin-receptor complex is still generally recognized. It is suSpected that the auxin receptor is a protein. Galston and Purves (1960} list as evidence for this view the low concentration of auxin necessary 5 for activity, suggesting its act on as a cofactor or an enzyme activator, and the auxin-protein complexes that have b6*r reported. However, the IAA-protein complex isolated by Siegel and Galston (1953) from pea roots was claimed co be an artifact of the isolation technique (Andreas and van Ysselstein, 1960). Thus, there is no conclusive evidence that auxin forms a complex with a receptor. Auxin and Plant Growth The literature on the effects of auxin on growth is voluminous. Therefore, instead of duplicating what has already been reviewed (van Overbeek, 1,59; Galston and Purves, 1960) only pertinent concepts will be pre- sented. The promotive effect of auxin on cell enlargement is well documented. From 1928 to the present time most research in this area has been carried out using Avena coleoptiles (Went and Thimann, 1937) in which auxin promotes water uptake resulting in growth by extension tr cell enlargement. For this to occur the suction p essure (SP) of the coleoptile cells (or the diffusion pressure deficit} must increase. Since SP is the‘differetce between the osmotic pressure (OR) of the cell ccntents and the wall pressure (WP), either an increase is 0P or a decrease in WP would increase SP. The latter is generally presumed to be the controlling factor. Tagawa and Bonner (1957) demnpstrated that in coleOptiles treated with calcium or magnesium solu- tions, their plasticity and elasticity were markedly 'decrsased. Potassium on the other hand, increased elas- ticity and 1AA increased plasticity. When applied to- gether both elasticity and plasticity were increased. An effect of auxin on cell walls was suggested; They concluded that potassium ions replaced some of the divalent cations binding adjacent pectic chains in the cell wall thereby making it more flexible. Ordin et a1. (1957) suggested that auxin induced the breaking of these calcium bonds by increasing methyl esterification from methionine. Although the mechanism of auxin induced water uptake in coleoptiles is still unknown, these data strongly suggest that the chief auxin effect is a reduction in WP; possibly through an effect on the cell wall constituents. Auxin and Respiratory Metabolism Growth in higher plants is an energy dependent process which, for the most part, relies on the energy produced in aerobic respiration. As early as 1933, Bonner (1933) related auxin induced growth to oxidative metabolism by showing that Avena coleoptiles could not 7 grow under anaerobic conditions. In a later paper Bonner (1949) reported that auxin induced growth was inhibited by cyanide, and that treatment with 2,4- dinitrophenol (DNP). an uncoupling agent, also inhibits coleOptile growth, suggesting that enery-rich compounds produced in oxidative phosphorylation are necessary for growth. Similar results have since been obtained with arsenate, (Bonner, 1950) and other respiratory lnhlb t- ors in several tissue types (Galston and Purves, 1960). From these data Galston ani Purves conclude that auxin promotes water uptake through an energy requiring process which increases the level of phosphate acceptors and thereby stimulates respiration indirectly. 'This is in agreement with the report that auxin stimulates respira- tion only when growth is promoted (Bonner, et al., 1953). Auxin and_jucleip Acids_ .— The interaction of auxin and kinetin on cell divisions in tobacco pith cultures prompted Skocg and Miller (1957) to propose that auxin exerts its effect at the nucleic acid level. Until very recently, however, this view has been, at most, speculative. Nooden and Thimann (1963) measured the effect of 14C-leucine into 1AA on growth and incorporation of protein in etiolated pea stem segments, Avena coleoptiles and artichoke tuber discs. 1AA stimulated growth in 8 all tissues, and an increase in 140 incorporation into protein was observed in the pea segments and tuber discs. No stimulation of protein synthesis occured in 51222 coleoptiles. Using peas, they further studied the effects of specific inhibitors of protein and nucleic acid synthesis. Chloramphenicol, puromycin and Actin- omycinAD all inhibited growth of pea stem sections. The progressive inhibition of elongation paralleled the inhibition of 14C-leueince incorporation at in- creasing chloramphenicol concentrations. They concluded that the primary locus of auxin action is on a nucleic acid that controls the synthesis of a protein necessary for growth. Recently, Nooden and Thimann (1965) described a more thorough investigation in which they found that auxin stimulated growth and 14C -leucine incorporation into protein in all three tissue types. Addition of chloramphenicol completely blocked this effect. The growth of pea stem segments treated with the synthetic auxins, 2,4-D and NAA, were likewise inhibited by chloramphenicol. The stimulation of respiration in artichoke tissue inducedlyrIAA was negated by chloram- phenicol. Furthermore, the same concentrations of chloramphenicol that inhibit these auxin induced res— ponse also inhibit protein synthesis. From these data 9 they therefore concluded that protein synthesis is necessary for auxin induced growth. Thus it seems very likely that auxin exerts its effect at the nucleic acid level and induces the formation of new enzyme(s) which, in turn, affect cell wall plasticity. GIBBERELLINS The gibberellins were first discovered as metabolic products of the fungus Fusarium moniliforme, Sheldon (Giberella fujikuroi) (Yabuta and Sumiki, 1938). Endogenous gibberellins have since been found in 94 species of flowering plants representing 19 families and in gymnosperms, ferns and mosses (van Overbeek, 1962). Gibberellin activity is usually found in seed extracts, and in rapidly growing vegetative parts such as vines (van Overbeek, 1962). Four different gibberellins have been isolated from higher plants and, like the auxins, they are assumed to be ubiquitous (van Overbeek, 1962; Phinney and West 1960a). Gibberellins and Plant Growth Gibberellic acid (GA) has been found to stimulate shoot elongation, promote flower formation and normal and parthenocarpic fruit set and it can replace the long day effect in several photoperiodic phenomena (Phinney and West, 1960a; van Overbeek, 1962; Wittwer and Bukovac, 1958). Thus, reSponses to GA application are many and varied and, in some cases, are probably dependent on the endogenous concentration of ther native growth regulating substances (see later). The responses of the various gibberellins have been thoroughly reviewed else- where (Wittwer and Eukovac, 1958; Bukovac and Wittwer, lO 11 1961; Phinney and West, 1960a, 1960b). Effect of Gibberellin (GA) at the Genetic and Enzyme Level Phinney and co-workers, in a series of studies, showed that the application of GA to any of several dwarf mutants of £33 gays, L. resulted in plants of normal appearance (Phinney and Test, 1960a, 1960b). Dwarfism in these mutants is controlled by a single gene. The ability of GA to overcome this effect suggested that the synthesis of native gibberellins in these mutants is genetically blocked, resulting in a dwarf growth habit. The growth of those genetic dwarfs is proportional to the exogenous GA concentration and is not affected by either kineten or 1AA. Thus, these dwarf mutants of g. ngg have proven useful as a quantitative bioassay for GA (Phinney and West, 1960a, 1960b). Some of the most interesting work on the physiology of GA action is its effect on a specific protein (X- amylase. Other enzymic effects of GA, particularly on the IAA oxidase system, will be reviewed later. The effect on GA on 9(-amylase activity was first reported by Hayashi (1940). Since that time much has been learned of this effect. Paleg (1960, 1961) showed conclusively that GA induces the activation of amylolytic enzymes in barley endOSperm resulting in an increased 12 release of reducing sugars and a concomittant reduction in dry weight. Using isolated aleurone layers, the only living cells of barley endosperm, Varner (1964) and Varner and Chandra (1964) demonstrated that in GA- treated endosperm the major fraction of radioactivity incorporated from 14 C-phenylalanine was recovered in 4 -amylase. Thus the GA-dependent increase in a( - amylase activity is a result of increased synthesis of the enzyme. Furthermore, the duration of sensitivity of the system to Actinomycin-D and p-fluorophenylalanine suggested that GA affects the formation of a specific messenger RNA involved in the d§_ngzg synthesis of the d.—amylase molecule in barley endosperm. The reported effects of GA at the gene, enzyme and nucleic acid level suggest a very basic GA effect. Possibly, GA induces the control of nucleic acid syn- thesis at the gene level thereby affecting the synthesis of specific protein which, in turn, could result in the many reported GA responses. KINIQLS Discovery of Kinetin A cyrstalline substance was isolated by Miller, et a1. (1955) from autoclaved deoxyribonucleic acid (DNA) which markedly promoted cell division in tobacco stem segments cultured on synthetic media. Due to its ability to induce cytokinesis, the active compound, which was later shown to be 6-furfurylaminopurine (Miller, et a1, 1955). was named kinetin. To include substances having similar effects on cytokinesis in tissues which were other wise unreactive, they proposed the generic name kinin. Strugtgggl_fieguirements f0£_Kinin Activit' Following the discovery of kinetin various bio— assays were developed to determine the structural requirements for kinin activity and to find compounds more active than kinetin. They included cell division in tobacco (Rogozinska, et a1. 1964) and carrot (Shantz, 1958) tissue cultures, lettuce seed germination (Skinner, et al., 1957), cell enlargement in radish leaf discs (Kuraishi, 1959), chlorophyl retention in detached zgnthgpg_(09borne and McCalla, 1961; Richmond and Lang 1957) and wheat leaves (Shaw and Srivastava, 1964). With but one exception, the intact purine ring was found to be necessary for activity (Miller, 1961); 13 14 the exception, S—azakinetin promoted cell division in soybean callus culture (Miller, 1960). The furfuryl group in kinetin, which is attached to the amino group in the 6-position, may be replaced by many substitutes and still retain activity. However, only a limited number of 6-substituted purines gave activity equal to or greater than that of kinetin. 6-Benzy1aminopurine (6-benzyladenine) (Osborne and McCalla, 1961; Hamzi and Skoog, 1964; Kuraishi; 1959) and 6-(dimethyla11ylamino)— purine (Hamzi and Skoog, 1964; Rogozinska, et al., 1964) were more effective than kinetin. Miller (1961) regards these active compounds as substituted adenines. Since adenine treatment gave only a slight increase of tobacco callus growth, he suggested that the marked stimulation induced by the active com- pounds might be due to the presence of fat-soluble substituents (the furfuryl, benzyl or dimethylallyl groups) which might permit entry into, or orientation with that portion of the cell in which growth is con— trolled. Qpcurrence of Kinips However, Letham (1963b) purified a substituted adenine from corn and plum fruitlets having kinetin- like activity, suggesting their natural occurrence in 15 plant tissues. Substances possessing kinetin-like activity in various bioassays have been obtained from corn endosperm (Miller, 1956; Letham, 1963a) solid (Shaw and Srivastava, 1964) and liquid (van Overbeek, 1962) endosperm of coconut; apple, quince, pear and plum fruitlets (Bottomley, etal., 1963, Zwar, et al., 1963); and from pea seedlings (Biswas, 1964) and pea blanching water (Skoog, 1965). Furthermore, the alkaloid tricanthine, or 6-amino-3—(t,l’-dimethylallyl)-purine which occurs in at least three Species, showed slight growth promoting activity in tobacco tissue cultures (Rozoginska, et al., 1964). However, when tricanthine was autoclaved prior to addition to the media,growth promotion was ten times greater than that induced by kinetin. Chromatographic studies of autoclaved trican- thine showed that it was probably converted to 6-( f,t'- dimethylallylamino)-purine. They concluded that trican- thine might readily be converted to the active 6-isomer in vivg (Rozoginska, et al., 1964). Effects of Kinins on Plant Growth and Development The primary effect of kinetin is on cell division. In the presence of IAA, kinetin induces mitosis and subSequent karyokinesis in tissue cultures of tobacco pith (Das, et al., 1956), pea root callus (Torrey, 1958) 16 and carrot root explants (Shantz, et al., 1958), however, both IAA and kinetin are without effect when supplied alone. Das, et al., (1956) suggested that a kinetin- like substance was necessary for complete cell division, and probably for DNA replication as well. Wright (1961) has correlated the time of maximum kinetin response with the period of cell division in wheat coleoptiles. Kinins also influence cell enlargement. Kinetin stimulated expansion in etiolated bean leaf discs (Miller, 1956) and several 6-substituted purines, includ- ing kinetin and 6-benzyladenine (BA), induced the same effect in discs from light-grovn radish leaves (Kuraishi, 1959). In both areas the response was due to cell enlargement. Cell elongation in pea stem segments however, was retarded by treatment with kinetin (Brian and Hemming, 1957; Katsumi, 1963). Similarly, kinetin absorbed by the roots from culture solutions supressed the growth of both roots and tops of intact dwarf pea, cucumber and tomato plants (Wittwer and Dedolph, 1963). When applied to individual bean leaves, BA stimulates their expansion, but at the expense of non- treated leaves which very rapidly become senescent (Leopold and Kawase, 1964). It was demonstrated by Mothes and Engelbreet (1961) that treatment of one portion of an excised tobacco leaf with kinetin induces 17 the movement of substances in the leaf to the treated area. They proposed that endogenous kinins are perhaps the natural mobilizing agents in plants. Kinin induced transport will be discussed later in greater detail and is mentioned here only in relation to the work of Osborne and Moss (1963) on the abscission process. When these investigators applied kinetin directly to the abscission zone of bean explants, abscission was delayed. If on the other hand, kinetin was applied elsewhere on the explant, metabolite mobilization towards the site of treatment commenced and the subsequent depletion of the cells of the zone resulted in accelerated abscission. A similar delay in abscission due to kinetin treatment was observed by Chatterjee and LeOpold (1964). Effects of Kinins on Metabolic Processes Enzyme Activity Boothby and Wright (1962) reported that starch degradation in wheat endosperm is promoted in the pre- sence of kinetin. The rate of reducing sugar formation by starch hydrolysis is very much like that induced by GA. They therefore suggested that the effect of kinetin is on amylase activity. In ElEEQ studies showed that the activity of ribonuclease and deoxyribonuclease in bean hypocotyl extracts was stimulated by kinetin 18 (Maciejewska-Potapczyk, 1959). However, Srivastava and Ware (1965) have since proven that the opposite occurs 1 vivo. The reduction of nuclease activity is in —— agreement with the many observations of kinin effects on senescence. The fact that some 6-substituted purines, in the presence of xanthine, induce permanent disorganization of exposed tissues cf planaria, a primative invertebrate, prompted Henderson, et al., (1962) to study the effect of kinins on xanthine metabolism. lg 321333 they demonstrated that kinetin is a potent inhibitor of xanthine oxidase, the enzyme that catalyzes the oxidation of xanthine to uric acid. How this relates to plant metabolism is not clear. Possibly the conversion of accumulated xanthine to purines could allow for increased nucleic acid synthesis. Steinhart, et al., (1964) have recently reported that in the roots of barley seedlings, kinetin and BA induce an increase in the rate of synthesis of tyramine methylpherase (S-adencsylmethionine: tyramine methyltransferase) which catalyzes the conversion of tyramine to N-methyltyramine. The authors conclude that kinins are involved in at least one of the steps in the synthesis of this protein. Kinins also affect respiratory enzymes. Working with tobacco cell suspensions, Bergmann (1963) concluded 19 that the locus of the inhibitory effect of kinetin on respiration was in the glycolytic pathway. Tuli con- firmed this using BA (1964). He further showed that i vitrq, PA competes with adenosine triphosphate (ATP) and adenosine diphosphate (ADP) for the active site on hixokinase and pyruvic kinase, respectively. The absence of an effect on glutamine synthetase indicated a degree cfspecificity for EA on glycolytic kinases. Further, the 14C content in phOSphate esters after 14002 fixation in the light was found to be much less in BA treated broccoli leaves than in the controls, indicating an inhibitory effect on phosphorylation enzymes (Tuli,l964). Nucleic Aci s- As in the case of auxins, the multiplicity of response of Flint tissues to kinin treatment strongly A‘ suggests that the primary effect of kinins is at a very basic level. It was pointed out above that kinins may exert their effect on protein synthesis. Therefore it is possible that the basic effect of kinins might be at the nucleic acid level which, in turn, would affect protein synthesis. There is supporting evidence for this View. Richmond and Lang (1957) first showed that kinetin delayed the loss of protein in detached fignthium leaves. 20 The effect of kinins on protein metabolism has since received considerable attention. Thimann and Laloraya (1960) reported a stimulatory effect on kinetin on protein synthesis in isolated pea stem sections. The most thorough study in this area was performed by Osborne (1962). She found that kinetin sustained both RNA and protein synthesis in detached Egnthium leaves and leaf l4c-orotic acid into RNA discs. The incorporation of and l4C-leucine into protein was markedly higher in kinetin treated leaf discs. Further, treatment with kinetin resulted in a greater incorporation of 140- leucine into protein perunit RNA, even though the protein/RNA ratio remained the same. Osborne (1962), therefore suggested that the kinetin induced increase in the synthesis of protein is brought about by in- creased RNA synthesis. That DNA synthesis was not affected by kinetin was ascribed to the absence of cell division in the tissues studies (Osborne 1962). Very recently Srivastava and Ware (1965) reported that in kinetin treated excised barley leaves the synthesis of both RNA and DNA, as measured by 32F incorporation, was maintained almost as high as in fresh leaves. A concomittant suppression in ribonuclease and deoxyrib- onuelease activity was also noted. These studies (Osborne, 1962; Srivastava and Ware, 1965) leave little doubt that kinetin exerts its effect on protein metabolism 21 throu h nucleis acid S'nthesis. J fienescence Inhibition Senesoence in most higher plant tissues is usually accompanied by chlorophyll degradation and a decline in protein and nucleic acid content. Kinins, particularly BA, can temporarily delay these catabolic processes in many detached plant tissues. Following the discovery of BA as a "senescence inhibitor" in some green leafy tissues (Bessey, 1960; Zink, 1961), many other vegetable and flower crops were screened for this effect (Dedolph, et al., 1961, 1962; MacLean and Dedolph, 1962, 1964; MacLean et al., 1963; Tuli, 1964; Wittwer, et al., 1962). Dedolph, et al., (1961) measured the rate of 002 evolu- tion of treated and nontreated asparagus spears and concluded that the preserving effect of BA was a conse- quence of reSpiratory inhibition. The inhibitory effect of BA on glycolytic kinases ig_zitrg (Tuli, et al., 1964) gave further support to this contention. However, Srivastava and Ware (1965) concluded that the mainte- nance of RNA and DNA synthesis and the supression of the activity of ribonuclease and deoxyribonuclease by kinin treatment preserves the integrity of the ribo- somes. This, in turn, allows for the maintenance of protein synthesis which is necessary for the maintenance of detached plant tissues. . INTERACTIONS In the 1950's when auxins and gibberellins were firmly established as two distinct classes of growth regulators, experiments were conducted concerning the interactions of these substances on plant responses. Likewise, after the discovery of the kinins, they twovere included in growth regulator combination studies. The more significant findings on growth regulator combina- tions, particularly as they pertain to the present study are herein reviewed. Growth Auxins and Gibberellins The concept that auxin is necessary for all plant growth was proposed by Went and Thimann (1937). However, Kefford and Goldacre (1961) have since suggested that all tissues must be predisposed by auxin for other growth hormones to act. An excellent example of the latter suggestion was presented by Cleland (1964). He employed the antiauxin-inhibition method, using p-chlorophenoxy- iso-butyric acid (PCIB) to assess the role of endogenous auxin in the elongation of £2229 leaf sections. His experiments clearly showed that these sections undergo two different types of elongation; (a) endogenous growth, which is accompanied by an increase in both cell number and cell length, and (b) GA induced growth which is due 22 23 to an increase in cell length only. The ability of auxin to overcome the PCIB-induced inhibition of endo- genous growth indicates that endogenous growth requires endogenous auxin. The GA-induced elongation of these leaf sections requires both GA and auxin. GA must be applied exogenously, but the auxin requirement is satisfied by endogenous auxin (Cleland, 1964). Using the same species, Ng and Audus (1964) measured the effect of auxin and GA on the shoot elongation of both inter- node segments and segments including a node. The GA effect was greatly increased and the IAA effect was decreased by the inclusion of a node, suggesting that an endogenous auxin supplied by the node is necessary for the GA response. Thus, in the elongation of Axgga leaf and shoot sections the theory of "predispostion" of the tissues by auxin appears to hold true. However, this is obviously not the only way in which GA interacts with auxin. The lack of a pronounced GA effect on auxin activity in the Aygga test led Galston and Warburg (1959) to suggest that GA activates a system that inhibits the oxidative destruction of IAA. They called this response the "auxin sparing effect" of GA. Halevy (1963) has since shown that the activity of the IAA oxidase system in cucumber seedlings decreases with increasing concentrations of applied GA. 24 Similarly, van der Kerk, et al., (1964) reported that Elema (Ph.D. Thesis, State University of Utrecht, Holland) found considerably less IAA oxidase activity in complete homogenates from GA treated etioloated pea plants than in comparable nontreated homogenates. From these studies it is evident that the "auxin sparing effect" of GA is no doubt mediated through the inactiva- tion or inhibition cf the IAA oxidase system. In this way, GA can have an indirect effect on auxin activity. There are, however, a number of discrepencies con— cerning the interacting effects of IAA and GA. Synergistic responses have been observed in the growth of etiolated (Galston and Warburg, 1959) and green pea stem sections (Galston and Kaur, 1961) and in Azgna (Hayashi and Murakami, 1954) and wheat leaf sections (Radley, 1958). Others have reported that the effects of IAA and GA on growth in etiolated pea sections (Kato, 1958) and in gygng coleoptile and first internode sections (Nitsch and Nitsch, 1956) were, at most simply additive. Despite these conflicting responses, even in the same tissue and Species, Galston and Purves (1960) concluded that both auxin and GA are required for growth, but the nature of their interaction is not yet understood. Auxins and Kinins It was mentioned above that neither IAA nor kinetin, 25 when supplied alone, can induce cell division in tissue cultures of tobacco pith, soybean callus (Miller, 1961) and carrot root explants (Steward, et al., 1961). However when applied together, continuous cell division and growth ensues. Hashimoto (1961) showed a similar re- quirement for the presence of IAA and kinetin in combination for the primary thickening of light-grown pea stem segments. Kinetin and 1AA also act synergisti- cally with respect to DNA synthesis in tobacco callus cultures (Skoog and Miller, 1957). Ballantyne (1965) demonstrated that by including the synthetic auxin, 2,4-dichlor0phenoxyacetic acid (2,4-D), the effect of BA on preserving cut flowers of daffodil was markedly enhanced. The same effect might be expected with IAA. Not all responses are synergistic. Kinetin has been shown to inhibit IAA induced elongation in sun- flower hypocotyls (de Ropp, 1956) and pea stem segments (Brian and Hamming, 1957), as well as auxin induced root growth (Raghavan, 1964). Further it has been reported that in pea stem segments (Wickson and Thimann, 1958) and intact pea plants (Sachs and Thimann, 1964) kinetin promotes the elongation of auxin inhibited buds. This, in part, explains the BA effect on the breaking of rest in dormant grape buds (Weaver, 1965). Working with pea stem sections, Katsumi (1965) found that the effect of IAA and kinetin on elongation 26 and fresh weight increase, when applied together, was intermediate to the stimulation and inhibition observed when IAA and kinetin were applied alone, respectively. To resolve these differences, Fox (1964) conducted studies which showed that in the growth of tobacco and soybean tissue cultures, the antagonism between IAA and kinetin is such that the inhibitory levels of one can be overcome by providing more of the other to the medium. Thus it seems valid to conclude that auxins and kinins, either directly or indirectly, interact in plant growth. Qibberellins and Kinins Interacficns between GA and kinins are limited in number. Skinner,et al., (1958) have demonstrated a GA- kinetin synergism in seed germination of three different species. The fresh weight jrcrease of radish leaf discs treated with GA plus kinetin, relative to either alone, were only additive, and for disc expansion, less than additive (Kuraishi and Hashimoto, 1957). In irtact dwarf pea plants, hittwer and Dedolph (1963) showed that kinetin reduces the top/root ratio and induces flowering at a lower node. GA has the opposite effect, but when applied together these res- ponses w re intermediate. 27 Transport When Mothes and Engelbrect (1961) applied kinetin to localized areas of tobacco leaves, the movement of amino acids and other metabolites towards the treated region was detected. They found auxin to be ineffective in this "kinetin-induced directed transport." More recently, however, Seth and Wareing (1964) observed definite growth regulator interactions in the transport of 32F in bean internodes. Although kinetin was not effective alone, in combination with 1AA a very marked enhancement of transport occurred. A synergistic effect on transport, of even greater magnitude, was observed when IAA was applied in combination with GA, even though GA applied alone was ineffective. These obser- vations led Seth and Wareing (1964) to conclude that these three growth regulators act through the same system and that they are in some way involved in the movement of metabolites toward growth centers. The basipetal transport ofl4C-1abeled BA through bean petioles is increased when IAA is also added (Osborne and Black, 1964). The lack of an 1AA effect in tobacco leaves (Nothes and Engelbrect, 1961) was probably due to the presence of endogenous auxin in nonolimiting amounts. 28 Seguential Responses to Growth Regulatgrs One of the most significant worhato date concerning plant responses to growth regulators, is, perhaps that of Wright (1961). He treated wheat coleoptiles with 1AA, GA or kinetin at 12 hour intervals from 18 to 78 hours after sowing and determined their growth at each interval. Over the same period of time, the cell number and cell volume of comparable coleoptiles were also determined. Wright found that coleoptile growth consists of two phases, a relatively short initial phase of rapid cell division, followed by a slower, almost linear, phase of cell enlargement. Growth reSponses showed the coleOptiles to be most sensitive to applied GA at or before 18 hours after sowing, with a steady decline thereafter. Kinetin gave an equally high response at 18 hours, but a still greater response at 30 hours, followed by a decline in activity. The GA and kinetin responses both approached the control level at 66 hours after sowing. No positive response to IAA was observed until 30—42 hours, with the maximum activity at 54 hours after sowing. In light of these data, Wright (1961) concluded that: l) the GA effect is correlated with the period of expansion just prior to division; 2) when most cells are about to divide the coleoptile is most sensitive to kinetin; and 3) the lack of an initial IAA 29 response indicates that auxin is associated with the later, steady-state period of cell elongation. He fur- ther postulated that there may be an Optimum concentra- tion ratio of these three growth regulators for each stage of growth (Wright, 1961). MATERIALS AND METHODS Treatment Solutions 3-Indoleacetic acid (IAA), gibberellic acid (GA) and 6-benzy1adenine (6—benzy1aminopurine) (BA) were used as representative members of the auxins, gibberellins and kinins, respectively. Stock solutions containing 1.5x10m4 fl IAA (Eastman Organic Chemicals), GA (K-salt of gibberellio acid; Merck and Company) or BA (Shell Chemical Company) were made up in pH 6.7 phosphate buffer. The buffer solution contained 0.067 E KH2P0 and 0.067 m K2HPO 4 4 (6:A v/v) made up in distilled-deionized water. IAA and GA were first dissolved in a minimal amount of 5% (w/v) KaHCOB; BA was dissolved in hot buffer solution (pa 90°C). Dilutions to obtain other concentrations were made using the buffer, or in the case of combination treatments, with another buffered stock solution. Thus, all treatment solutions were of the same pH and buffer concentrations. Treatment with the buffer alone served as the control. Solutions containing higher concentra- tions of the growth regulators (leOn4 or 5x10-3 fl) were prepared when required. Between experiments the treatment solutions were stored at Ea 5°C. Solutions used were never more than one week old. 30 31 Leaf Disc Studies Leaf discs (10 mm diam.) were removed from interveinal portions of fully expanded broccoli (Brassica gleracea var. Italics cv. 'Spartan Early') leaves of similar physiological age. The discs were washed by gentle shaking in distilled water for two hours. This procedure insured complete randomization of discs from all leaves. The leaf discs were rinsed with distilled water, placed in 50 ml of the treating solutions and gently shaken on a wrist-action shaker for 16 hours at 20°C. Once treatment commenced the leaf tissue was maintained in the dark except for brief exposure to weak green light when necessary for tissue manipulation. Following treatment the discs were blotted and stored at 20° C under conditions of high relative humidity to prevent drying. Thirty-two hours later (48 hours after initial contact with the treatment solutions) 0.5 gm of discs from each treatment were placed into each of four Warburg respirometers. Oxygen uptake was measured manometrically for three hours in the dark at 30° C. All leaf disc experiments were repeated at least three times. Thus, the data presented are the averages for four Warburg flasks in each of three or more discrete trials (Tables I, II). 32 The initial experiments were performed to determine tide effective growth regulator concentrations, and sxibsequent factorial experiments were conducted to study ‘tlie interacting effects of these compounds on oxygen uptake. gee Embgo Studies Seeds of Alaska peas (Eisum sativum L., cv. Alaska), prrovided by the Rogers Brothers Company, Idaho Falls, Iciaho (Lot No. 423), were used in all experiments. The seeeds were randomly divided into replicates of 10 seeds eeich and soaked in treatment solutions for 12 hours (11ereafter referred to as the imbibition period). The iinbibed seeds were transferred to 9 cm Petri dishes ((Dne dish per replicate) containing a Whatman No. 1 filter paper disc moistened with the treatment solution arua held for 48 hours, unless otherwise Specified. IInhibition and subsequent storage were carried out in tile dark at 20° C. Following the storage period, five of the original 1C) seeds of a replicate having typical appearance were Selected for embryo excision. The excised embryos were pldaced in 20 ml Warburg.re5pirometer flasks containing WEiter in the side arms to maintain a high relative knAmidity, and 0.2 ml 10% (w/v) KOH in the center well 33 Table I. Oxygen consumption, as percent of control, by broccoli leaf discs as affected by various concentrations of growth regulators. Molar Concentration Treatment 10"5 2x10"5 ‘leO—5 10-4 10:3 Kinetin 93.2 75.1 67.9 50.7 -- BA 8508 6009 5503 4708 -"' IAA 104.5 108.0 99.4 94.9 102.6 GA 98.7 -- 97.0 94.2 92.8 34 Table II. Rate of Oxygen consumption by broccoli leaf discs as affected by growth regulator treat- ments Treatment (5:10-33) Oxygen uptake as percent of control 1AA GA BA IAA + GA IAA + BA GA + BA IAA + GA + BA ~— er T—w **Significantly different from the (Duncan, 1955). 100.9 95.3 58.7** 101.2 62.3** 57.8** 61.1** — '0 control at 1% level 35 for CO2 absorption. Each Warburg flask was considered a replicate. By reducing the number of embryos to five, those injured during excision could be discarded and readily replaced and oxygen uptake determinations could continue for at least three hours. ReSpiration, as indexed by oxygen consumption was determined manometrically over a three hour period in the dark at 30° C. The embryos were then weighed, by replicates, to the nearest mg and their nitrogen content was determined accoding to the method of Umbreit, et al.,(1964). In some instances protein nitrogen was determined after precipitation with trich- loroacetic acid. The effect of growth regulator treatment on growth, nitrogen assimilation from the cotyledons and the reapiration rates of pea embryos was thus determined. The experimental design was such that replicates were maintained separately from tne commencement of imbibition, through the s-orage period and throughout the respira- tion,growth and nitrogen determinations. Time Studies The respiration rate, fresh weight and nitrogen content of treated embryos were determined immediately or at 24, 48, or 72 hours after the inhibition period 36 to ascertain the effect of growth regulator treatment with time. Treatments in these time studies consisted of IAA, GA or BA at leo'5 5. Three discrete experiments were conducted in which each treatment was replicated three times. Interaction Studies After the effect of concentration from 5x10"8 through 5x10“5 3 of each growth regulator was deter- mined, treatment with various growth regulator combin- ations was applied. In these experiments the effect of an increasing concentration of one growth regulator, in the presence of the other two, applied either separately or together, at 5x10‘5 M was tested. For example, if IAA was applied in increasing concentrations (5x10'8 to 5xio'4 p), then GA, BA and GA plus BA were also applied at 5x10 5‘3. Thus in all eXperiments, when the concentration of one growth regulator was varied the other two, alone and in combination, were held constant at 5x10'5 fl. Growth, nitrogen content and the respiration rate of each replicate was deter- mined as in the time studies. Manometer limitations necessitated replication by days or runs. A control was included each time, and .due to the nature of the experimental design, certain 37 treatments overlapped. As a result, the data for the controls were based on 21 observations; six observations were obtained for each treatment in which IAA, GA and BA were applied alone or in combination at 5x10"5 3. Data for the remaining combinations were based on at least th— ree observations each. RESULTS AND DISCUSSION BROCCOLI LEAF DISC STUDIES The effects of various concentrations of IAA, GA, kinetin and BA on oxygen uptake by broccoli leaf discs are presented in Table I. Treatment with kinetin and BA resulted in increasing inhibition of oxygen consump- tion with increasing concentration. BA has been shown to be more active than kinetin in several different bioassays (Kuraishi, 1959; Osborne and McCalla, 1961; Strong, 1958; Wittwer and Dedolph, 1963). Similarly, these studies showed that inhibition by BA at a given concentration was consistently greater than the kinetin- induced inhibition. Therefore, in all further experi- ments, BA rather than kinetin was used for kinin treat- ments. Neither GA nor IAA appreciably affected oxygen consumption in broccoli leaf discs at the concentrations used (10"5 to 10‘3 g). The results of factorial design treatments with the three growth regulators at 5x10"5 E are presented in Table II. The absence of an interacting effect was clearly demonstrated. Oxygen uptake as compared to the controls was consistently suppressed by treatment with BA. This BA-induced respiration inhibition was previously observed in broccoli (MacLean and Dedolph, 38 39 1964; MacLean,et al., 1963; Tuli, 1964). IAA and GA, on the other hand, did not significantly affect overall respiration when applied either singly or in combina— tion with each other. Furthermore, the inhibitory effect of BA was not altered by treatment with either IAA, GA or IAA plus GA. The data for oxygen uptake (Table II) were not confounded by tissue changes. Fully expanded leaves were used, and the number of 10 mm discs per gram fresh weight was constant for each eXperiment. Oxygen uptake measurements were taken 48 hours after treatment application. The only noticable tissue difference were in the BA treated discs which were somewhat greener than the controls or other treated tissue at the time of oxygen uptake determinations. The absence of a GA-effect on oxygen uptake is in agreement with the data of Weller,et al., (1957) on beans. They found that although oxygen uptake was increased by GA on a plant part basis, there was but little effect when eXpressed on a fresh weight basis. The increased respiratory activity was evidently a function of growth. Auxin-induced respiration increases have been correlated with growth (French and Beavers, 1953). and it has been suggested (Bonner and Bandurski, 1952) that 4O auxin may couple the respiratory and growth processes by making the energy formed in respiration available only to these tissues capable of growth. Thus, the absence of an appreciable effect of auxin on oxygen consumption in broccoli leaf discs may be, as in the case of GA, due to the fact that the tissues were fully expanded and incapable of further growth. PEA mmmgp STUDIES Time Studies The effect of treatment with 5x10’5 3 IAA, GA and BA on the rate of oxygen consumption by excised pea embryos during a 72-hour period following imbibition in the treating solution is presented in Figure 1. There was no effect of BA throughout the 72 hour period. GA did not influence oxygen uptake until after 48 hours, whereupon the respiration rate of GA treated embryos continued to increase in a linear manner, while the rates for the controls and BA treated embryos leveled off. The auxin treated tissue respired at a reduced rate throughout the first 48 hours. At 72 hours, however, their respiration rate was as high as the controls (fig. 1). The non-treated embryos grew in a linear manner after the first 24 hours (fig. 2). (The radicle generally emerged through the tests 18-24 hours after 41 Figure l The respiration rate of control and IAA-, GA- and BA-treated pea embryos as a function of time. ul cum/5 memos 20° ' O CONTROL A 5xl0‘5 g IAA v {mo-0 3 GA 175- o “.04 M BA I50 |25 IOO 75 50 25 1 l o 24 48 HOURS AFTER IZ'HR IMBIBITION PERIOD 72 42 the imbibition period.) Neither GA nor BA affected em- bryo growth, IAA treatment, on the other hand, resulted in suppressed growth for 48 hours. 7 Although the general response patterns were the same for both oxygen uptake (fig. 1) and growth (fig. 2), differences were apparent. Both oxygen uptake and fresh weight determinations were made on the same embryos. Metabolic changes would necessarily preceed visible differences. Thus, a lag period between altered respiratory metabolism and subsequent effects on growth could account for the lack of the IAA and GA induced differences in oxygen uptake at 72 hours to be re- flected in growth. The increase with time in nitrogen content of the growth regulator treated embryos was generally similar to that of growth and oxygen uptake when expressed on a per embryo basis (fig. 3). The nitrogen content of IAA—treated embryos was significantly less than the control embryos at 72 hours. Otherwise, all treatments at all times, were not substantially different than the controls. However, when the nitrogen content was expressed on a per unit growth basis (mg N/gm fresh weight) a different response was observed (fig. 3). Although the GA and BA treated embryos were not different than the controls, the IAA treated embryos were. 43 Initially, the nitrogen content of the auxin treated embryos was less than the others, but at 24 and 48 hours after imbibition it was greater. These data suggest that at zero-time the exogenous IAA coupled with .endogenous auxin results in a superoptimal concentration which impairs nitrogen assimilation from the cotyledons before growth commences. However, at 24 hours, when growth was evident, this was overcome and the nitrogen content per unit growth of the IAA-treated embryos was greater than that of the others. At 24 and 48 hours after imbibition, nitrogen assimilation in auxin treated embryos was proportionately greater than growth (fig. 3). Thus other factors than nitrogen were limiting growth. In seedling growth, the nitrogen content generally parallels fresh weight increases resulting in a relative- ly constant ratio of nitrogen per unit growth. These data (fig. 3) indicate that GA and BA had no effect on this ratio, which became constant within 48 hours. Treatment with IAA delayed the time at which this constant ratio was reached by 24 hours. Throughout these time studies the only consistent marked deviation from the controls was that induced by IAA. If it is assumed, as Wright (1961) suggested, that specific concentration ratios of auxins, gibberellins and kinins are required for the growth and development 44 Figure 2 Growth of control and IAA-, GA- and BA- treated pea embryos as a function of time. Figure 3 Nitrogen content of control and IAA-, GA- and BA- treated pea embryos as a function of time. Nitrogen content expressed on a plant part basis (N/S embryos) and on a per unit growth basis (N/ unit fresh weight). 260 " 8 5 _ 0 common. 3 A 5 1: I0" I» v 5:: IO" 0 \ 0 51l0" '5 IDOL- 2 III a 3 .. Ill 2 II. E ., IOO - l I p. 3 O C O IO 35 SOLID SYMBOLS - mgN/gm. fr. wt. ' OPEN SYMBOLS - poll/5 EMBRYOS 3 no mo memos (x I02) 6 O 24 4O 72 HOURS AFTER I2-I‘IR IMBIBITION PERIOD In N/gm FRESH WEIGHT 45 of plant tissues, then the IAA induced inhibition of growth and reSpiration in these time studies would suggest that auxin was the predominate endogenous growth regulator during the first few days of embryo growth. On the other hand, the absence of a pronounced eff- ect of GA and BA on pea embryos could suggest that the endogenous concentration is so low that even with exogenous applications of leO_5 E the total gibberellin or kinin concentration was still below the threshold level for activity. The validity of this is doubtful since both GA (Fhinney and West, 1260b) and BA (Miller, 1961) are active at very low concentrations in several p. 1 y. ' ivg systems. ——— Another possibility suggested by the lack of a GA or BA effect is that neither is required during these early stages of growth. However, if the efficiency of plant systems is considered, and if it is realized that germinated seeds are a rich souce of endogenous gibberellins (van Overbeek, 1962) and kinins (Biswas, 1964; van der Kerk, et al., 1964), then such a supposition is very unlikely. The data from these time studies seem best ex- plained in terms of the ratio of these three groups of plant growth regulators. Raghavan and Torrey (1964) suggested a rapid sequence of changing hormone inter- actions during the early growth of Capsella embryos. 46 Thus, in these time studies, addition of auxin may have resulted in a ratio within the embryos, in which auxin was exceedingly dominant, not necessarily in terms of actual concentration, but in effective concentration. The resultant superOptimal auxin concentration therefore inhibited growth and respiration. Furthermore, treat- ment with GA and BA during this period when endogenous auxin was dominant, had no effect. Even though the actual relative concentration of GA or BA was increased by application of 5x10‘5 3, the relative effective concentration was not similarly altered and the effect of endogenous auxin was predominate. Erotein Determinations To better interpret the nitrogen content data, the total and protein nitrogen content of comparable embryos as affected by growth regulator treatment at ‘ixlO-5 E was determined 48 hours after the imbibition period (Table III). Treatment differences were not statistically significant regardless of the basis used to express the data. However, these data indicate that the nitrogen content of excised pea embryos is a reasonably valid estimate of protein content since the majority of nitrogen is in the form of protein nitrogen (64-80%). 47 Table III. Relationship between protein nitrogen and total nitrogen in excised pea embryos 48 hours after a 12-hour imbibition period. (IAA, GA and BA at 5x10-5 g). __. Chemical Treatment Control IAA GA BA ‘pg N/S embryos Total 1003 977 845 907 Protein 664 694 646 722 mg N/gm fr wt Total 7.99 8.80 7.48 8.89 Protein 5.15 6.60 5.73 7.17 Protein N as % of total per 5 embryos per gm fr wt 66.2 71.0 76.4 79.6 64.4 75.0 76.6 80.6 48 Effect of Growth Regulator Concentrations Prior to conducting studies on the interacting effects of IAA, GA and BA it was necessary to ascertain the effect of these growth regulators when applied alone. The pattern of response to concentration for nitrogen content, fresh weight increase and respiration rate of excised pea embryos was similar for each growth regulator (figs. 4, 5, 6). A marked stimulation of these three 6 M IAA (fig. 4). A ten- parameters occurred at 5x10— fold increase in concentration induced a drastic reduction of these reaponses, indicating that at 5x10"5 5, the total auxin concentration (exogenous plus endogenous) was superOptimal for growth, nitrogen assimilation from the cotyledons and normal respiration. Responses to IAA concentrations below ‘5x10-6 3 were lower, but not necessarily inhibitory. GA treated embryos exhibited no pronounced responses to concentrations from 5x10"8 to 5x10"5 3 (fig. 5). Nevertheless, leO-7 H GA did result in a consistent reduction of growth, nitrogen content and reSpiration rate. The responses to BA at 5x10-6 M were slightly stimulatory relative to the response induced by a ten- fold higher or lower concentration (fig. 6). However, at leO’6 E BA, none of these responses were signifi- cant. 49 Figure 4 Effect of IAA concentration on oxygen uptake, nitrogen content and fresh weight of excised pea embryos 48 hours after a 12-hour imbibition period. Points are the averages of three experiments, each of which con- sisted of three replicates. Control values were; 189.0,ul, 1343‘pg, and 177.2 mg for oxygen uptake, nitrogen assimilation and fresh weight, respectively. €25 moimzm n \ 2 as m. s 1|4 — 200 " . . O 0 8 6 «983 n \ m... \ No 3 I40 b IBO-f — L . O 0 6 4 35:12“; :85. I0'5 (x5) I 1 10'7 I0'6 IAA CONCENTRATION (molar) I0"8 Izo-fiv 50 Figure 5 Effect of GA concentration on oxygen uptake, nitrogen content and fresh weight of excised pea embryos 48 hours after a 12 hour imbibition period. Points are the averages of three experiments, each of which con- ' sisted of three replicates. Control values were; 189.0‘pl, l343,ug, and 177.2 mg for oxygen uptake, nitrogen assimilation and fresh weight, reSpectively. qI4 face moEmzm m \ 2 as 2 200- . — 0 0 m 6 moimzu n \ m: x No .4. I40- - I90- -%0 I70- _ o .b 3.5 Emmi :mmmu I30 L701 IO'5IX5) I0'5 GA CONCENTRATION (molar) IO'7 IO‘8 51 Figure 6 Effect of BA concentration on oxygen uptake, nitrogen content and fresh weight of excised pea embryos 48 hours after a 12 hour imbibition period. Points are the averages of three experiments, each of which consisted of three replicates. Control values were; l89-O‘pl, 13433pg, and 177.2 mg for oxygen uptake, nitrogen assimilation and fresh weight, respectively. “No.5 moEmzm n \ 2 as 1I4 2 ‘IO - u - FR.W'IZ 2I0 - . _ 0 O 9 7 mo>mmzm m x m... x No .4. I50- _ O B zoos Iso- .95 tam; rmmmu I40fi/L IO'5IX5) IO'7 I0'6 IO'B BA CONCENTRATION (molar) Interaction Siudies Figure 7 depicts the interacting effects of GA, BA and GA plus BA, all at 5x10"5 E, with increasing IAA concentrations on respiration and nitrogen assimilation. 'For instance, the BA labeled curve describes the response observed when embryo were treated with 5xlO-5 3 BA alone (on the ordinate) and in combination with IAA concen- 8 to 5x10-4 3. Similar curves are trations from 5x10- presented for 5x10.5 fl GA and GA plus BA over the same auxin concentrations. The most pronounced responses in these experiments where the IAA concentration was varied occurred at the low auxin concentration (‘5x10-8 g). The rate of oxygen uptake and nitrogen assimilation were markedly stimulated by BA and inhibited by GA at the low auxin concentration. The stimulation and inhibition were of the same magnitude. Obviously, exogenous IAA at 5x10‘8 fl did not result in a superOptimal auxin concentration. The optimal IAA concentration in the absence of added BA or GA was ‘jxlO-6 fl (fig. 4). Furthermore, in the absence of exogenous IAA or when the IAA concentration was increased ten-fold to leO”7 E, these stimulatory and inhibitory effects were absent, indicating a requirement for a specific concentration ratio of these growth regulators for optimum growth. When embryos were subjected to a combination of GA plus 53 BA over increasing IAA concentrations the response paralleled that obtained with GA alone, suggesting that at low auxin concentrations the endogenous effective concentration of GA was greater than that of BA. At 5x10‘7 3, IAA apparently became predominant and increas- ing auxin concentration progressively inhibited oxygen uptake and nitrogen assimilation. The almost toxic effect of 511110"4 E IAA on oxygen uptake was not as pronounced for nitrogen content.‘ This was probably due to the relatively high nitrogen content of the embryos prior to treatment. The data for oxygen uptake (figs. 7, 10, 11) were eXpressed on a per 5 embryo basis (5 embryos per treat— ment replicate) throughout the interaction studies. This was justified by the highly significant correlation (r = 0.864) between oxygen uptake on a per 5 embryo versus oxygen uptake on a per mg nitrogen basis for all combinations (fig. 8). The growth response of embryos to combination growth regulator applications very closely paralleled the respiratory response. Significant correlations were observed between growth and oxygen uptake regard- less of the units used (fig. 9). Thus in all growth regulator interactions, the general patterns of response for fresh weight increase, nitrogen assimilation and 54 Figure 7 Effect of increasing IAA concentrations in the presence of leO'5 E GA, BA and GA plus BA on the rate of oxygen consumption (tOp) and nitrogen content (bottom) of excised pea embryos. / MI 5 embryos pl 0‘ pg NIB ombryoclxmzl I70 I30 0 O V +OA . (1' BA (- 00” TROL *F—LH l 1 , mo“? mo" axle-5 our“ 1 {+BA A l l f axle“ woo-7 um“ um" OXIO" IAA CONCENTRATION I NOLAR) 55 Figure 8 Correlation between the rate of oxygen consumption per I unit nitrogen and the oxygen uptake rate on a per plant basis. (Y = 45.4 + 0.726 x) I70 8 u! OZIHR/mq , N l I I so , I30 u-I OleR /5 EMBRYOS I I70 56 oxygen uptake, for a given treatment combination, were similar. The data for experiments in which the GA concen- tration varied and the concentration of IAA, BA and IAA plus BA were held constant at 5x10"5 g are presented in figure 10. The concentration of IAA in combination treatments was 5x10”5 E. This concentration was based on preliminary determinations of oxygen uptake of embryos in the treating solutions. Later, when deter- minations were made in a moist chamber, it was shown that ‘leOm6 fl rather than 5x10".5 m IAA was most effective. There was no marked GA effect in the range of concen- trations tested (fig. 10). Nitrogen assimilation was slightly stimulated in the IAA and IAA plus BA treated embryos in the presence of a low GA concentration (‘3x10"8 fl). However, this increase was still well below the control level. The effect of IAA at 5x10"5 E was dominant over the BA effect and the effect of increasing GA concentrations (fig. 10). However, when BA was the variable (fig. 11), a marked increased in oxygen uptake and nitrogen assimilation occurred at 5x10"6 5 in the presence of GA at 5x10_5 g. At BA concentrations above or below fl, he stimulatory effect we absent. a stimula- AS I -6 Q. tory response was observed at 5213 g BA in the absence 57 Figure Correlations between growth and consumption when expressed on a (left; Y = 2.10 + 0.887x) or on basis (right; Y = 22.67 + 0.923 O J the rate of oxygen plant part basis a per unit nitrogen x). z semi No .4. mo>mmzm nxmrlo .1 om. oi Om. 0.0. .o on. on. on. 0: cm .0 q u q u V q q q - AJQ‘O O 3‘ o -2. 9 no 0 m omH fl no 3 3 SH M B 9 H u 1 on. C O L mow. of added GA (fig. 6) which suggests that GA was not interacting with BA in promoting this stimulation (fig. 11). The curve for IAA plus GA, over increasing BA concentrations very closely followed the curve for IAA (fig. 11), demonstrating once more the dominant, inhibitory effect of leO-5 §_IAA on excised pea embryos. The dominant features of the interaction studies were: 1) The downward shift observed in the optimal IAA concentration induced by BA. 1n the absence of BA the optimum IAA concentration was 5x10-6 H, while in the presence of 5x10”5 3 BA the optimum IAA concentration 8 fl. 2) The dominant effect of was reduced to 5x10- IAA when applied alone or in combination with GA or BA in the presence of increasing GA or BA concentrations. 3) The dominant effect of BA in the presence of GA. 4) The inability of various growth regulator combina- tions to alter the dependency of nitrogen assimilation on reapiration. An explanation of some of these dominant features will be attempted here. The significant effect of BA on reducing the effective concentration of IAA (figs. 4, 7) suggested that the presence of BA either increased the sensitivity of the embryos to IAA or enhanced the activity of IAA per se. The former 59 Figure 10 Effect of increasing GA concentrations in the presence of leO-5 p IAA, BA and IAA plus BA on the rate of oxygen consumption (tep) and nitrogen content (bottom) of excised pea embryos. I70 .. i . I30 comma; ° + - g o A+ IAA ‘ \ A——"""' \ 3 so 0 \% \N 0 +IAA+BA 0 \ '- c4 K 50 - 4 . j i 01.]; 1 L l 4 J moo-3 , 5XIO'7 oXIo-G 5XIO'5 moo-4 CONTROL 4 IO-/ p! III/5 embryo: 1X10 2) o L \:Z\ \e [>> 0 / V... U D e O a 0 + /IAA +BA 0L"; 1 l l l j woo" woo" SXIO" SXIO’5 our“ GA CONCENTRATION (MOLAR) 60 Figure 11 Effect of increasing BA concentrations in the presence of 5xio'5 Li IAA, GA and IAA plus GA on the rate of oxygen consumption (tOp) and nitrogen content (bottom) of excised pea embryos. pl 02/ III/5 embryos p0 III/5 embryos 0002) I70 /+GA I 30 . ,_ CONTROL wt \HAA +IAA+GA 50 ~ 0 ’L 1 1 L l 9 ' ' two"a 5XIO'7 :mo‘6 5xI0‘5 oXIo" O P , +GA ‘ '° ’ CONTROL\ ‘ -u— — — — —— — -— —— —— ——15 I. a 0 0~ / 9 ) \O 8 . QNA 0 fi‘ ‘ + IAA A\ a \ K * MMGA \ i o ‘2 “IO" 5on" "IO" 5XI0'5 5100“ BA CONCENTRATION (MOLARI 61 possibility appcsre to bc the most likely. Nooden and Thimann (1965) dcmonctrctcd that continued protcin synthesis was necessary for growth. Further, Srivastava and Wire (1965) showed thc kinins to bc involved in sustaining nuclcic ccid cynthccic. It is poeciblc therefore, that the increased sensitivity of the embryos to IAA by BA was s concequcncc of BA-induccd reduction of nuclcscc activity and/or incrccscd protein synthesis. Convcrccly, GA had an inhibitory cffcct on IAA activity. At the low auxin concentration (5110"8 5), GA induced an inhibitory response equal in magnitude to the BA-induccd stimulation, indicating that in some manner GA interfcrce with auxin activity. Ng and Audus (1964) studied growth regulator interactions in 513;; and concludcd that GA and IAA probably not at the ccmc growth promoting centers and possibly compctc for them. Thc possibility of this occurring in pen embryos is not overruled. However, since the 5110"5 5 GA plus 5110"8 g IAA response (fig. 7) was lccs than that which occurred when cash was applied alone at thcec same concentrations, the observcd GA-IAA intcrccticn in pcn embryos suggests more than c simple competition for sotivc cites. Thc absence of a pronounced response to GA con- centretions, either alone (fig. 5) or in combination with 62 IAA, BA or both (fig. 10) is difficult to interpret. During the first few days after germination, embryo growth is chiefly a result of cell enlargement, a process to. which GA has been closely linked (Phinney and West, 1960b). however, since germinated seeds are a rich source of endogenous gibberellins (van Overbeek, 1962), the possibility exists that any exogenous GA results in a superoptimal concentration which, in turn, inhibits growth. the maximum concentration response to treatment with BA occurred at 5x10"6 5 (fig. 6). In the presence of 5x10'5 LI GA this concentration response was not altered, indicating that GA did not influence the BA induced stimulation at this concentration. However, in the presence of IAA or IAA plus GA at 5110‘s 5 the BA response was absent. Thus, when the auxin concentration was low relative to BA, during the first few days of embryo growth after imbibition, it appears that growth and associated phenomena were stimulated. Whereas when the IAA concentration was low relative to GA the opposite response occurred. Whenever IAA at 5x10"5 5 or BA at 5x10" :3 was applied, inhibition occurred regardless of other combinations. Exogenous IAA or BA at these concentrations probably results in a total auxin or kinin concentration which is superoptimal. 63 The various responses to different growth regulator combinations and concentrations herein reported, are probably not a consequence of differential uptake; By imbibing-dry pea seeds inwthe treating solutions for 12 hours it is very probably that all cells received chem- ical treatnent. Further, the treating solutions were the only sauce of moisture after the imbibition period. In broccoli leaf discs the duration of exposure to the growth regulators (16 hours) probably assured their uptake. SUMMARY AND CONCLUSIONS In the preliminary studies on fully expanded broccoli leaf discs IAA and GA were ineffective on oxygen uptake. The only response to growth regulator treatment occurred when BA was included in the treatment solution. the inhibitory effect of BA either alone or in combination with IAA and/or GA, on oxygen uptake was probably the result of delayed senescence through maintenance of RNA and DNA synthesis, as suggested by Srivastava and Ware (1965). The effects of IAA, GA and BA treatments on excised pea embryos were assessed using three parameters: 1) growth, as fresh weight increase; 2) nitrogen assimilation from the cotyledons; and 3) the respiration rate, as indexed by oxygen uptake. In all treatments these three parameters were similarly affected by any given treatment. Significant correlations between growth and respiration were obtained for embryos receiving combination growth regulator treatments. Further, various growth regulator treat- ment combinations did not dissociate the similarity between growth, nitrogen assimilation and respiration. Interactions between IAA and BA and between IAA and GA were observed. However, no such interaction between GA and BA could be detected. The IAA concentra- 64 65 tion which induced maximum stimulation was reduced from 53:10“6 E to 51:10“8 5 by 5x10'5 35 BA. The IAA response at 5x10”6 5 was blocked when 5:10'5 5 GA was included in the treatment. Further, this GA concentration resulted in a marked inhibition of growth,nitrogen assimilation and oxygen uptake when applied in combination with 5n 52:10"8 5 IAA. The stimulatory effect of BA at sno‘ was not appreciably affected by ‘5x10"5 5 GA. A ten- fold increase or decrease in growth regulator concen- tration usually removes these responses, suggesting the requirement for a specific concentration ratio of these . three groups of plant growth regulators, and perhaps others as yet undiscovered, for plant growth and development. A possible mechanism for the hormonal control of plant growth and development can be formulated from the results presented in this dissertation and the data of other investigators. Wright (1961) inferred that during the early growth of wheat coleoptiles, different growth regulator ratios occur. A sequential shift in the ratio occurs with growth in which gibbere- llin, kinetin and auxin dominate the ratio in that order. It was also demonstrated that the inhibitory effects of one growth regulator could be overcome by increasing the concentration of another (Fox, 1964). Thus, there is precedent for the governing effect of specific growth 66 regulator ratios. The multiplicity of effects-induced by IAA, GA or BA treatments suggest that their primary effect is very basis. The manner in which these basic effects are expressed may be influenced by the tissue type and environmental factors, resulting in a great variety of responses. The basic effect may be at the nucleic acid level. The evidence for auxin (Nooden and Thimann, 1963, 1964) gibberellin (Phinney and West, 1960b; Varner, 1964; Varner and Chandra, 1964) and kinin (Osborne, 1962; Srivastava and Ware, 1964) effects at the nucleic acid level have already been presented. . An intricate balance between these three growth regulator groups probably exists. The balance may be such that the addition of a reasonable concentration of one growth regulator would affect the relative effective concentration of all three. The number of possible concentration combinations is almost limitless The response to GA concentrations was minor rela- tive to IAA and BA concentrations. Further, the absence of the pronounced inhibition at 5:10" 11 GA that occurred-in the presence of IAA of BA at 5x10“ 5 suggests that, if such a balance exists, GA must be less effective on the balance than corresponding additions of BA or IAA. 'Purther, superoptimal IAA or BA concentrations 67 (5x10.4 g) probably disrupt the entire balance, whereas the inhibitory effect of GA at the same concentration is much less. Therefore, if the action of these growth regulators is assumed to be at the nucleic acid level, one ratio may induce the synthesis of enzymes governing different (responses. The type of tissue, stage of maturity and the environmental conditions prevailing at the time a specific concentration ratio occurs could further influence the responses to the basic effect. With such a scheme, the many responses ascribed to the hormonal control of plant growth and development can be rationalized. However, this hypothesis can be tested only after a tissue is found that can be starved of endogenous growth regulators and still retain the capacity for growth when they are added back. LITERATURE CITED Andreas, W. A., and H.W.H. van Yeselstein, 1960. Studies on 3-indoleacetic acid metabolism. IV. 3-Indolea- cetic acid uptake and metabolism by pea roots and epicotyls. Plant Physiol. 35: 225-232. Ballantyne, D. J. 1965. Senescence of daffodil (Narcissus pseudonarcissus) cut flowers treated with benziL- .a on no an auxin. Nature 205: 819. Bergmann, L. 1963. Action of 6-furfurylamino purine on the respiration of cell suspensions of Nicotiana tobacum. Naturforsch, 11: 942-946. Bessey, P.H. 1960. Effects of a new senescence inhibitor on lettuce storage. Univ. Ariz. Exp. Sta. 189: 5-8. Biswas, P.K. 1964. Identification of a kinetin-like substance from pea seedlings. Pisum sativum. Bature 204: 297-298. Bonner, J. 1933. The action of the plant growth hormone. Jour. Gen. Physiol. 17: 63-76. Bonner, J. 1949. Limiting factors and growth inhibitors in the growth of the Avena coleoptile. Amer. Jour. 30". 56: 323-332o Bonner, J. 1950. Arsenate as a selective inhibitor of growth substance action. Plant thsioi. 25: 181-184. ' Bonner, J., and R. S. Bandurski. 1952. Studies of the physiology, pharmacology and biochemistry of the auxins. Ann. Rev. Plant Physiol. 3: 59-86. Bonner, J.,R. S. Bandurski, and A. Millard. 1953. Linkage of respiration to auxin-induced water uptake. Physiol. Plantarum 6: 511-522. Boothby, D., and S.T.C. Wright. 1962. Effects of kinetin and other plant growth regulators on starch degradation. Nature 196: 389-390. lotto-lg, '., I.P. Kefford, J.A. Zwar and P.L. Goldacre. 1 63. Kinin activity from plant extracts. 1. Biological assay and sources of activity. Aust. Jour. Biol. Sci. 16: 395-406. 68 69 Brian, P., and H. Hemming. 1957. Effects of gibberellio acid and kinetin on the growth of pea stem sections. Naturwiss. 22: 594. Bukovac, ng., and 8.3. Wittwer, 1961. Biological evaluation of gibberellins A1, A , A , and A4 and some of their derivatives. P. 505-523. In R. M. Klein (Bd.), Plant Growth Regulation. Iota State Univ. Press, Ames, Iowa. Chattorjeo, S. K., and A. C. Leopold. 1964. Kinetin and gibberellin actions on abscission process. Plant Physiol. 39: 334-337. Cleland, B. 1964. The role of endogenous auxin in the elongation of Avena leaf sections. Physiol. Plantarum 17: 1263135. 1 Darwin, 0. 1880. The Power of Movement in Plants, J. Murray, London, 592 pp. Das, R.K., K. Patau and P. Skoog. 1956. Initiation of mitosis and cell division by kinetin and IAA in excised tobacco pith tissue. Physiol. Plantarum Dedolph, R.R., S. H. Wittwer and V. Tuli. 1961 Senescence inhibition and respiration. Science 134: 1705. Dedolph, B.R., S. H. Wit wer, V. Tuli and D. Gilbert. 1962. Effect of N -bcnzylaminopurine on respiration and storage behavior of broccoli (Brassica oleracea var. Italica). Plant Physiol. de Ropp, R. 1956. Kinetin and auxin activity. Plant Physiol. 31: 253-254. Duncan, D.B. 1955. Multiple-range and multiple-P tests. Biometrics 11: 1-42. Poster, B.J., D.B. Renae and J. Bonner. 1952. Auxin- induced growth inhibition, a natural consequence of two-point attachment. Proc. Natl. Acad. Sci. 38: 1014-1022. Fox, J.B. 1964. Indoloacetic acid—kinetin antagonism in certain tissue culture systems. Plant Cell Physiol. 5 : 251-254. 70 French, R.C. and H. Beevers, 1953. Respiratory and growth responses induced by growth regulators and allied compounds. Amer. Jour. Bot. 40: 660-666. Galston, A.W., and R. Kaur. 1961. Comparative studies on the growth and light sensitivity of green and etiolated pea stem sections. p. 687-705 In W.D. McElroy (Ed.), Light and Life, Johns (Hopkins Press, Baltimore. Galston, A.W., and W.K. Purves, 1960. The mechanism of auxin action. Ann. Rev. Plant Physiol. 11: Galston, A.W., and H. Warburg. 1959. An analysis of auxin-gibberellin interaction in pea stem tissue. Plant Physiol. 34: 16-22. Halevy, A.H. 1963. Interaction of growth-retarding compounds and gibberellic acid on indoleacetic acid oxidase and peroxidase of cucumber seedlings. Plant Physiol. 38: 731-737. Hamzi, l.Q., and P. Skoog, 1964. Kinetin-like growth- romoting activity of l-substitutcd adenines l-benzyl-6-aminopurine and l- (X',X’dimethylallyl) ;2-a;inopurine). Proc. latl. Acad. Sci. 51: -8 . Hashimoto, T. 1961. Synergistic effect of indoleacetic acid and kinetin on the primary thickening of pea stem segments. Bot. Hag. (Tokyo) 74: 110-117. Hayashi, T. 1940. Biochemical studies on Bakanae fungus of rice. VI. Effect of gibberellin on the activity of amylase in germinated cereal grains. Bull. Agr. Soc. Japan, 16: 531-538. Hayashi, T., and Y. Hurakami. 1954. The biochemistry of Bakanae fungus, Part 32. The physiological action 3? gibgerellin. VII. Journ. Agr. Chem. Soc. Japan 8: 54 '545o Henderson, T.R., C.G. Skinner and R.E. Bakin. 1962. Kinetin and kinetin analogues as substrates and gghigggoggsof xanthine oxidase. Plant Physiol. 71 Kato, J. 1958, Studies on the physiological effects of gibberellin, II. Physiol. Plantarum. 11: 10-14. Katsumi, H. 1963. Physiological effects of kinetin. Bffect of kinetin on the elongation, water uptake and oxygen uptake of etiolated pea stem sections. Physiol. Plantarum 16: 66-72. Kefford, H.P. 1963. Natural plant growth regulators. Science 142 : 1495-1505. Kofford, N.P., and P.L. Goldacre. 1961. The chan ing concept of auxin. Amer. Jour. Bot. 48: 643- 50. Kuraishi, S. 1959. Effect of kinetin analogues on leaf grozth. Sci. Papers Coll. Gen. Bduc. Univ. Tokyo. 9: 7-104o Kurashi, S., and T. Hashimoto. 1957. Promotion of leaf growth and acceleration of stem elongation by gibberellin. Bot. Mag. (Tokyo) 70: 86-92. Leopold. A.C. and H. Kawase. 1964. Benzyladenine effects on bean leaf growth and senescence. Amer. Journ. late 51: 29‘-298o Letham, D.S. 1963a. Zoatin: A factor inducing cell division isolated from Zea mays. Life Sci. 8: 569-573. Letham, D.S. 1963b. Isolation of a kinin from plum fruitlets and other tissues. In,J.P. Nitsch (Ed.), Natural Plant Growth Regulators. Gif, France. Lockhart, J.A. 1962. Kinetic studies of certain anti- gibberellins. Plant Physiol. 37: 759-764. Haciejewska-Potapczyk, W. 1959. Influence of kinetin, IAA, and gibberellic acid on nuclease activity of been hypocotyls. lature 184: 557-558. HacLean D.C., and R.R. Dedolph. 1962. Effects of '5 -benzylaminopurine on postharvest respiration of C santhemum morifolium and Dianthus ca 0 llus. Bot. Eaz. 124: 20-21. MacLean, D.C., and R.R. Dedolph. 1964. Phytokinins and senescence in Broccoli. Amer. Journ. Bot. 51: 618-621o 72 HacLean, D.C., R.R. Dedolph and B.H. Wittwer. 1963. Respiratory responses of broccoli (Brassica oleracea var. Italica) following pro-and posthar- vest treatments witE H5 -benzyladenine. Proc. Amer. SOCo lorto Sci. 83: 48‘-487o Killer, 0.0. 1956. Similarity of some kinetin and red light effects. Plant Physiol. 31: 318-319. Miller, C.G. 1961. Kinetin and related compounds in plant growth. Ann. Rev.P1ant Physiol. 12: 395-408. Miller, 0.0., P. Skoog, R.R. Van Saltza and 3.x. Strong. 1955. Kinetin, a cell division factor from DIA. Jour. Amer. Chem. Soc. 77: 1392-1397. Miller, 0.0., P. Skoog, P.8. 0kumara, R.R. Von Saltza and P.H. Strong. 1955. Structure and synthesis of kinetin. Jour. Amer. Chem. Soc. 77: 2662-2666. Hiller, 0.0., P. Skoog, P.S. 0kumara, R.R. Von Saltza, and P. H. Strong. 1956. Isolation, structure and synthesis of kinetin, substance promoting cell division. Jour. Amer. Chem. Soc. 78: 1375—1381. Hothes, K., and L. Engelbrect. 1961. Kinetin induced directed transport of substances in excised leaves. Phytochem. 1: 58-62. Ng, E.K., and L.J. Audue. 1964. Growth-regulator interactions in the growth of the shoot system of Avena £33333 seedlings. Jour. Exptl. Bot. 156 67-95. Kitsch, J.P. and C. Nitsch. 1956. Studies on the growth of coleOptile and first internode sections. A new, sensitive straight-growth test for auxins. Plant Physiol. 31: 94-111. Nooden, L.D., and K.V. Thimann. 1963. Evidence for a requirement for protein synthesis for auxin- induced cell enlargement. Proc. Natl. Acad. Sci. 50: 194-200. Nooden, L.D., and K.V. Thimann. 1965. Inhibition of protein synthesis and of auxin-induced growth by chloramphenicol. Plant Physiol. 40: 193-201. 73 Ordin, L., R. Cleland and J. Bonner. 1957. Methyl esterification of cell wall constituents under the influence of auxin. Plant Physiol. 32: 216-220. Osborne, D.J. 1962. Effect of kinetin on protein and nucleic acid metabolism in Xanthium leaves during senescence. Plant Physiol. 37: 595-602. Osborne, D.J., and M.K. Black. 1964. Polar transport of a kinin, benzyladenine. Nature 201: 97. Osborne, D.J., and D.R. McCalla. 1961. Rapid bioassay for kinetin and kinins using senescing leaf tissue. Plant Physiol. 36: 219-221. Osborne, D.J. and S.E. Moss. 1963. Effect of kinetin on senescence and abscission in explants of Phaseolus vulgaris. Nature 200: 1299-1301. Paleg, L. 1960. Physiological effects of gibberellic acid. II. On starch hydrolyzing enzymes of barley endosperm. Plant Physiol. 35: 902-906. Paleg, L. 1961. Physiological effects of gibberellic acid. III. Observations on its mode of action on barley endosperm. Plant Physiol. 36: 829-837. Phinney, B.0., and C.A. West. 1960a. Gibberellins as native plant growth regulators. Ann. Rev. Plant Physiol. II: 411-436. Phinney, B.0., and C.A. West. 1960b. Gibberellins and the growth of flowering plants. p. 71-92. In D. Rudnick (Ed.), Developing Cell Systems and-their gontrol. XVIII Growth Symp., Ronald Press, New ork. Radley, M. 1958. The distribution of substances similar to gibberellic acid in higher plants. Ann. Bot. 22: 297-307. Raghavan, V. 1964. Interaction of growth substances in growth and organ initiation in embryos of Capsella. Plant Physiol. 39: 816-821. Raghavan, V., and J.G. Torrey, 1964. Effects of certain growth substances on the growth and morphogenesis o immature embryos of Capsella in culture. Plant Physiol. 39: 691-699. 74 'Richmond, A.E., and A. Lang. 1957. Effect of kinetin on protein content and survival of detached Xanthium leaves. Science 125: 650-651. Rogozinska, J.R., J. P. Helgeson and P. Skoog. 1964. Tests forkinetin-like growth promoting activities of tricanthine and its isomer, 6-( , -dimethy1a- 11y1amino)-purine. Physiol. plantarum 17: 165-176. Sachs, T., and K.V. Thimann. 1964. Release of lateral buds from apical dominance. Nature 201: 939-940. Seth, A., and P.P. Wareing. 1964. Interaction of auxins, gibberellins and kinins in hormone-directed transport. Life Sci. 3: 1483-1486. Shantz, R.R., K. Hears and P.C. Steward. 1958. Compar- ison between the growth promoting effects on carrot tissue of coconut milk and of kinetin and of certain of its analogues. Plant Physiol. 33: xvi (Suppl.) Shantz, R.R., and F.C. Steward. 1952. Coconut milk factor: The growth promoting substances in goconut milk. Jour.. Amer. Chem. Soc. 74: 6133- 135. Shaw. H., and B.I.S. Srivastava. 1964. Purine-like substances from coconut endosperm and their effect on senescence in excised cereal leaves. Plant Physiol. 39: 528-532. Siegel, S.M., and A.W. Galston. 1953. Experimental coupling of indoleacetic acid to pea root protein in vivo and in vitro. Proc. Natl. Acad. Sci. 33:1'171'1-1118'7 _ Skinner, C.G., J.R. Claybrook, P. Talbert and W. Shive. 1957. Effect of 6-(substituted) thio and amino- purines on germination of lettuce seed. Plant Physiol. 32:32-36 Skinner, C.G., P.D. Talbert and W. Shivc. 1958. Effect of 6-(substituted) purines and gibberellin on the rate of seed germination. Plant Physiol. 33: 190-194. Skoog, P. 1965. Personal communication. 75 Skoog, F., and 0.0. Miller. 1957. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exptl. Biol. 11: 118-131. Srivastava, B.I.S., and G. were. 1965. The effect of kinetin on nucleic acids and nucleases of excised barley leaves. Plant Physiol. 40: 62-64. Steinhart, C.E., J.D. Mann and S. H. Mudd. 1964. Alkaloids and plant metabolism. VII. The kinetin-produced elevation in tyramine methylpherase levels. Plant Physiol. 39: 1030-1038. Steward, F.C., E.M. Shantz, J.E. Pollard, M.O. Mapes and J. Mitre. 1961. Growth induction in explanted cells and tissues: Metabolic and morphogenic manifestations. In Molecular and Cellular Structure. Growth Soc. Symp. 19: 193-246. Strong, P.M. 1958. Kinetin and kinins. p. 98-157. in P.M. Strong. Topics in Microbial Chemistry. John Wiley, New York. Tagawa, T., and J. Bonner. 1957. Mechanical properties of the Avena coleoptile as related to auxin and ionic interactions. Plant Physiol. 32: 207-212. Thimann, K.V., and M.M. LaLoraya. 1960. Changes in nitrogen in pea stem sections under the action of kinetin. Physiol. Plantarum 13: 165-178. Torrey, J.G. 1958. Differential mitotic response of diploid and polyploid nuclei to auxin and kinetin treatment. Science 128: 1148-1149. Torrey, J.G. 1961. Kinetin as a trigger for mitosis in mature endomitotic plant cells, Exptl. Cell. Res. Truelsen, T.A. 1961. Growth-promoting and growth- inhibiting effects of high indole-3-acetic acid concentrations. Physiol. Plantarum 14: 520-532. Tuli, V. 1964. Mode of action of N6-benzy1adenine in the . inhibition of respiration in higher plants with special reference to broccoli (Brassica oleracea var. Italica cv. Spartan Early). PE. 5. esis Michigan State University. 76 Umbreit, W.W., R.R. Burris and J.P. Stauffer. 1964. Manometric Techniques (4"I Edition). Burgess Publishing Co., Minneapolis, Minn. 305 pp. van der Kerk, G.J.M., G.W. van Eyk and J.A. Weber, 1964. Plant growth regulators and their interrelation- ships. Chemisch Weekblad 60: 185-194. van Overbeek, J. 1959. Auxins. Bot. Rev. 25: 269-350. van Overbeek, J. 1962. Endogenous regulators of fruit growth. p. 37-58. In Proc. Plant Sci. Symp. Campbell Soup Co. Varner, J.R. 1964. Gibberellic acid controlled synthesis of a.-amylase in barley endosperm. Plant Physiol. Varner, J.R., and G.R. Chandra. 1964. Hormonal control of enzyme synthesis in barley endosperm. Proc. Natl. Acad. Sci. 52: 100-106. Weaver, R.J. 1963. Use of kinin in breaking rest in buds of Vitis vinifera. Nature 198: 207-208. Weller, L.E., 8.1. Wittwer, M.J. Bukovac and H.M. Sell. 1957. The effect of gibberellic acid on enzyme activity and oxygen uptake in bean plants (Phaseolug vulgaris). Plant Physiol. 32: 371-372. Went, P.W. and K.V. Thimann. 1937. Phytohormones. McMillan Co., New York. 294 pp. Wickson, M., and K.V. Thimann. 1958. The antagonism of auxin and kinetin in apical dominance. Physiol. Plantarum 11: 62-74. Wittwer, 3.1., and M.J. Bukovac. 1958. The effects of gigbggellin on economic crops. Econ. Bot. 12: 1" 5o Wittwer, 8.1., and R.R. Dedolph. 1963. Some effects of kinetin on the growth and flowering of intact green plants. Amer. Jour. Bot. 50: 330-336. Wittwer, 8.2., R.R. Dedolph, V. Tuli and D. Gilbert. 1962. Respiration and storage deterioration in celery (Apium graveolens L.) as affected by postharvest treatments with N5 -benzylaminopurine. Proc. Amer. Soc. Hort. Sci. 80: 408-416. 77 Wright, S.T.C. 1961. A sequential growth response to gibberellic acid, kinetin and indolyl-3-acetic acid in the wheat coleoptile (Triticum zplgarg,L.) Nature 190: 699-700. Yabuta, T., and Y. Sumiki. 1938. Communication to the editor. Jour. Agr. Chem. Soc. Japan. 14 (In Japanese). Zink, F.W. 1961. N6-benzy1adenine. a senescence inhibitor for green vegetables. Jour. Agr. Food Chem. 9: 304-307. Zwar, J.A., W. Bottomly and N.P. Kefford. 1963. Kinin activity from plant extracts. II. Partial purifi- cation of kinins in apple extract. Aust. Jour. Biol. Sci. 16: 407-415. "mlfiiillfl'fllliflWATWTTTW