[NVOLVEMENT OF PROEIN SYNTHESES IN AUXEN-ENDUCED ELONGATEON Then: for the Degree of DI'I. D. MECHIGAN STATE UNIVERSiTY Keith K. Schiender 1966 0-169 LIBRA'Hj-L Michigan State University This is to certify that the thesis entitled INVOLVEMENT OF PROTEIN SYNTHESIS IN AUXIN-INDUCED ELONGATION presented by Keith K. Schlender has been accepted towards fulfillment of the requirements for Ph. D. degree in Biochemistry Date Novembe ‘ 1 I R ABSTRACT INVOLVEMENT OF PROTEIN SYNTHESIS IN AUXIN-INDUCED ELONGATION by Keith K. Schlender The exact mechanism of auXin-induced cell elongation is not known. One process which has been implicated in cell enlargement is protein synthesis. The rake of protein syn- thesis in auxin—induced elongation was investigated by employ- ing chloramphenicol. cycloheximide. and gougerotin. In Azgng_and Triticum coleoptiles, auxin-induced elongation and protein synthesis were inhibited by the same concentrations of chloramphenicol. Ag§g5,coleoptiles were inhibited between 5110-4 to 5x10”3 2; concentration. Chloram- phenicol inhibited both protein synthesis and elongation at 51:10"3 fl.in Triticum coleoptiles. In the Alan; coleoptile, preincubation and kinetic experiments supported the view that protein synthesis was necessary for the initiation as well as the continuation of auxin-induced elongatione 1M'C-Leucine and lhc-c-aminoisobutyric acid uptake were inhibited by chloramphenicol. lLPG-au-Aminobutyric acid uptake was also inhibited by chloramphenicol. However, this analog of protein amino acids was not a satisfactory tool for inves- tigating amino acid uptake. ll"C«(v-eliminobutyric acid was Keith K. Schlender rapidly metabolized and its radioactivity incorporated into protein at a rate comparable to that of lac-leucine. Chloramphenicol inhibited the uptake of in C-indole-B-acetic acid, but the inhibition was small and did not contribute to the inhibition of elongation. Chloramphenicol uptake and metabolism were not involved in the high concentrations required for'growth inhibition in éygna, When treated with a SKID-3 fl_solution of chloramphenicol. the internal concentration exceeded 10-3 fl_within 30 minutes. After u hours, the internal concentration, of which 80—90% was unchanged chloramphenicol, equaled the external concentra- tion. The action of Chloramphenicol was not stereospecific in several plant systems. Auxin-induced elongation, 140_ leucine uptake and incorporation into protein, 14C-a-amino- isobutyric acid uptake, buckwheat root elongation, and gib- berellic acid-induced synthesis of a-amylase were inhibited by the four stereoisomers of chloramphenicol. Cycloheximide inhibited auxin-induced elongation in gigga and Triticum coleoptiles. In AIEEE coleoptiles, there was a parallel between the degree of inhibition of elongation and protein synthesis throughout the concentration range of 10"5 to 10"7 :1. Kinetic studies of cycloheximide inhibition of auxin-induced elongation and inhibition of protein syn- thesis indicated a temporal relationship between the two phenomena. The repression of protein synthesis preceeded inhibition of elongation. Keith K. Schlender Gougerotin inhibited auxin-induced elongation in AZEEE coleoptiles, 50% inhibition being reached at 10"6 fl_concen- tration. The compound was an effective inhibitor of protein synthesis in the plant coleoptiles. The relationship between the inhibition of elongation and inhibition of protein synthesis reported in this thesis are consistent with the viewpoint that protein synthesis is an essential requirement for both the initiation and the con- tinuation of auxin-induced elongation. INVOLVEMENT OF PROTEIN SYNTHESIS IN AUXIN-INDUCED ELONGATION By \ IL.- ..(\ I 3 Keith K; Schlender A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Docron OF PHILOSOPHY Department of Biochemistry 1966 ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to Professor H. M. Sell for his guidance, encouragement, and understanding throughout the course of this work. He is also grateful to Professor M. J. Bukovac for his enthusiasm and counsel. His thanks go to Professor H. G. Hansen and Professor N. E. Tolbert for serving on his guidance committee, and to Mrs. Robert Franklin and Mrs. Douglas Randall for their help in preparation of the manuscript. The many discussions and assistance of his fellow graduate students shall always be remembered. His special thanks go to his wife, Shirley, who shared the excitement of new discovery and buffered the moments of disappointment. The support of the National Science Foundation and the National Institutes of Health is appreciated. **************** ii INTRODUCTION . . . . LITERATURE REVIEW Mechanism of A Table of Contents uxin Action . . Effect of Auxin on Enzyme Activity . . Protein Synthesis and Auxin-Induced Elongation 0 Chloramphenicol Inhibition of Protein Synthesis Cycloheximide Inhibition of Protein Synthesis Gougerotin Inhibition of Protein Synthesis PLATEBIALS ANT) I'IETHODS c o c o o 0 Plant Material Avena coleo Triticum co Straight gr Uptake of 140— Fractionation Metabolism of Incubation Thin-layer Incubation ptiles . . leoptiles . owth assay Compounds . of Proteins Chloramphenicol and preparation chromatography and preparation Bioassay of Chloramphenicol . Growth of 0 Determination of protein synthesi a-Amylase Assa Metabolism of d-Aminobutyric Acid . ells . . . . . yo... #5000000. 0 or O O for O Incubation and fractionation . Chromatography of the amino acids Chemicals RESULTS AND DISCUSS ION O O O O O O goo-coco ooooooccmofiooooooo h o to 006000000000 0 O I O r ioa s O 0 000000000). Effect of Auxin on Elongation and Protein Synthesis 0 0030000000 :4 Chloramphenicol Inhibition of Auxin-Induced Elongation and Protein Synthesis . . Effefit of Chloramphenicol on the Uptake of CiIndole- 3-Acetic Acid . Uptake and Metabolism of Chloramphenicol Stereospecificity of Chloramphenicol . iii [1 cocoooooocmcoooooo O . '6 0.0000000050000000 ‘4 O C 0 0k. 0 O O O O O O O 34 #1 60 63 71 Table 93 Contents {Continued} Effect of Cycloheximide on Auxin-Induced Elongation and Protein Synthesis . . . Gougerotin Inhibition in Plants . . . . . Uptake and Metabolism of C-a-Aminobutyr AC 1d- . O O O O O O O O O O O O O O O O SUI’IDIABY O O O O O O O O O O O O O O O O O O 0 BIBLIOGRAPHY O O O O O O O O O O O O O O O O 0 iv 15: 81 9O 93 101 107 Table Mg. 1 10 11 t" l-" U) r,- O freebies Distribution of 14c-Leucine in the Coleop- tile and Primary Leaf of the Avena . . . . Effect of Auxin on thinElongation, Uptake, and Incorporation of C-Leucine into the Protein of the Avena Coleoptile . . . . . Elongation, Uptake, and Incorporation of C-Leucine into the Protein of Triticum Coleoptiles Treated with Chloramphenicol . Auxin-Induced Elongation Avena Coleopt les Pretreated with Chloramphenicol (leO' M) Uptake and Incorporation of luC-Leucine into the Protein of Avena Coleopt les Treated with Chloramphenicol (10' M) . . Uptake and Incorporation of llJ’C-Leucine into the Protein of Avena Coleoptiles Treated with Chloramphenicol (5xlO"3 M) . Incorporation of 14C-d-Aminoisobutyric Acid into the Protein of the Avena . . . . Chloramphenicol (5xio'3 g) Inhibition of the Incorporation of lQC-Leucine into the Proteifi of Avena Coleoptiles Pretreated 'With 1 C-LeuCine o o c o o o o o o o o o o Stereospecificity of Chloramphenicol Inhibition of Auxin-Induced Elongation in the Avena Coleoptile: Concentration Bang 8 O O O O O O O O O O O O O O O O O O Stereospecificity of Chlorigphenicol Inhibition of Elongation, C-Leucine Uptake, and Incorporation into the Protein of Avena . . . . . . . . . . . . . . . . Stereospecificity of Chloramphenicol Inhibition of Buckwheat Root Growth . . . 4O 44 48 50 54 57 59 72 72 75 Table Mg, 12 13 l4 15 16 17 18 19 2O 21 22 23 24 List g§_Tables (Continued) Stereospecificity of Chloramphenicol Inhibition of Elongation in the Triticum Coleoptile: Concentration Range . . . . . Stereogpecificity of Chloramphenicol i x10“ M) Inhibition of Elongation, C-Leucine Uptake, and Incorporation into the Protein of Triticum . . . . . . . Stereogpecificy of Chloramphenicol (5x10' ,M) Inhibition of a-Amylase synth851s o o o o o o o o o o c o o o o 0 Effect of the Isomers of Chloramphenicol on the Activity of ceAmylase . . . . . . . Effect of Chloramphenicol on Protein syntheSiS in E3 0011 o o c o o c o o c o o Stereospecificity of Chloramphenicol (25 ug/ml) Inhibition of Protein Syn- thesis in E, coli . . . . . . . . . . . . Effect of Chloramphenicol Extracts on Protein Synthesis in E, coli . . . . . . . Effectlgf Cycloheximide on the Elonga- tion, C-Leucine Uptake, and Incorpora- tion into the Protein of Avena . . . . . . E£fect of Cycloheximide on the Uptake of l c-c-Aminoisobutyric Acid into the Avena Coleoptile . . . . . . . . . . . . . . . . Gougerotin (10-4 M) Inhibition of Amino Acid Uptake and Protein Synthesis in the Avena Coleoptile . . . . . . . . . . . . . Uptake and Incorporation of luC-a-Amino- butyric Acid into the 70% Ethanol Insoluble Fraction of the Avena Coleoptile Uptake and Incorporation of l”Owl-Amino- butyric Acid into the TCA Insoluble Fraction of the Avena Coleoptile . . . . . Uptake and Incorporation of luC-a-Amino- butyric Acid into the Protein of Several Plant S O O O O O O O O O O O O O 0 O O O 0 vi 76 78 78 79 80 80 84 84 93 97 97 98 List 2: Figures Figgre‘Mg. 1 Kinetics of Auxin-Induced Elongation in the Avena Coleoptile . . . . . . . . . . . 2 Effect of Sucrose and Tween 80 on the Kinetics of Auxin-Induced Elongation in the Avena Coleoptile . . . . . . . . . . . 3 Effect of Chloramphenicol on Auxin-Induced Growth in the Avena Coleoptile: Concentra- tion Range . . . . . . . . . . . . . . . . 4 Kinetics of Auxin-Induced Elongation in Avena Coleoptil s Treated with Chloram- phenicol (5x10' M) . . . . . . . . . . . 5 Effect of Chloramphenicol on the Kinetics of Auxin-Induced Elongation in Avena Coleoptiles Pretreated with Auxin . . . . 6 Uptake of luC-Leucine into Avena Coleopm tiles Treated with Chloramphenicol (5x10‘ ) . 7 Incorporation of luC-Leucine into the Protein of Avena Coleo tiles Treated with Chloramphenicol (5x10‘ M) . . . . . . . . 8 Uptake of luC-a-Aminoisobutyric Acid into Avena Coleoptil s Treated with Chloram- pheniCO]. (5X10- .13.) o o c o o o o o o o o 9 Uptake of 14c-Indoie—3.Acetic Acid into the Avena Coleoptile . . . . . . . . . . . lO Chlifiamphenicol Inhibition of the Uptake of C-Indole-B-Acetic Acid into the Avena COleOptile O O O O O O O O O O O O O O O 0 11 Uptake of luC-Chloramphenicol into the Avena Coleoptile . . . . . . . . . . . . . 12 Thin-Layer Chromatography of luC-Chloram- phenicol Extract: Solvent System of Chloroform:Benzene:Ethanol (7:3:1) . . . . vii Figure Mg, 13 14 15 l6 17 18 19 20 21 22 23 List 9: Figures (Continued) Thin-Layer Chromatography of 14C-Chloram- phenicol Extract: Solvent System of Chloroform:Ethyl AcetatezFormic Acid (534:1) o c o c o o c o o o o o c c o o 0 Stereo pecificity of Chloramphenicol i x10“ .fl) Inhibition of the Uptake of C-a—Aminoisobutyric Acid into the Avena Coleoptile . . . . . . . . . . . . . . . Effect of Cycloheximide on Auxin-Induced Elongation in the Avena Coleoptile: Concentration Range . . . . . . . . . . . Effect of Cycloheximide on Auxin-Induced Elongation in the Triticum Coleoptile: Concentration Range . . . . . . . . . . . Effect of Cycloheximide on the Kinetics of Auxin-Induced Elongation in the Avena COleOptiJ—e O O O O O 0 O O O O O O O O O ngect of Cycloheximide on the Uptake of C-IndOle-3-Acetic ACid c o o o o o o 0 Effect of Cycloheximide (10'5 E) on the Uptake of C-Leucine into the Avena COleOptile O . Q C C O C C . O . . C O 0 Effect of Cycloheffimide (10-5 M) on the Incorporation of C-Leucine into the Protein of the Avena Coleoptile . . . . . Effect of Gougerotin on Auxin-Induced Elongation in the Avena Coleoptile: Concentration Range . . . . . . . . . . . Effect of Gougerotin on the Kinetics of Auxin-Induced Elongation in the Avena COleOptile O O O I O O O O O I O O O O O Effiat of Chloramphenicol on the Uptake C of -a-Aminobutyric Acid into the Avena COleOptileoocoooooooooooc viii 69 73 82 82 85 85 88 88 91 91 95 Maria . CAMP . CPM . Cyclo . IAA . TCA . Triticum Abbreviations . . . Avena sativa . . . Chloramphenicol . . . Counts per minute . . . Cycloheximide . . . Indole-B-acetic acid . . . Trichloroacetic acid . . . Triticum vulgare ix INTRODUCTION INTRODUCTION "The biochemist will proudly show the row of vials containing these mysterious hormones mostly in the form of crystal- line powders and will be able to give us the structural formula of most of the substances. The really intriguing prob- len, however, is not what these struc- tures are, but what they do, how they act on the molecular level, and how they pro- duce their actions. There is no answer to this question." Szent-Gyogyi (1960) Over a third of a century has passed since Went (111) first described auxin as an extractable and measurable chemical substance. In the ensuing years great strides have been made in elucidating both the chemical nature and the physiological role of auxins in the growth and development of higher plants (57). Progress on the biochemical mechan- ism of auxin action has not been as rewarding. The basic mechanism of auxin-induced cell elongation still remains unknown. Many early investigations on the changes in protein content and enzyme activity during the elongation process were not successful in determining the role of protein syn- thesis in auxin-induced elongation (19). Further progress was not possible until recent advancements in biochemistry revealed the basic pathway of protein biosynthesis and some of the factors which control it. 3 Selective inhibitors of protein synthesis, which act at specific sites in the biosynthetic pathway, have been of immeasurable value in determining the role of protein syn- thesis in complex physiological systems. In this study, chloramphenicol, cycloheximide, and gougerotin, compounds which are specific inhibitors of protein synthesis in microbial systems, were employed to assess the involvement of protein synthesis in auxin-induced elongation. Concen- tration and kinetic relationships of inhibition of auxin- induced elongation and repression of protein synthesis were compared. In addition, since the success of this approach depends upon the specific inhibition of protein synthesis, a detailed investigation of the uptake, metabolism, and specificity of chloramphenicol suppression in plant systems was undertaken. LITERATURE REVIEW 5' (I) ' i ('3 (3 (I) 11., in LITERATURE REVIEW Mechanism 2: Auxin Action A large volume of experimental evidence has accumulated indicating that, the stimulatory effect of auxin on cell enlargement involves a softening of the cell wall and thus, an increase in the cell wall plasticity (57). Auxin softening of the cell wall was first demonstrated by Heyn in 1932 (41. 42). Later Bonner (7) showed that a striking parallel existed between the concentration of auxin required for plastic bending and the stimulation of elongation. It is important to note in this report, that the plasticity was measured after 60 minutes while the growth measurements were taken 18 hours after treatment. Recently, this phenomenon was investigated by obtaining load-extension curves from a "constant-rate-of-extension" instrument. The latter instrument, Instron Universal Testing Instrument, was originally designed to analyze the effects of chemical modification on the mechanical properties of textile fibers (72). Using this instrument, Olson, Bonner, and Morre (72) studied the mechanical properties of isolated Avena sativa coleoptile cell walls. By the use of isolated cell walls, they eliminated the complications caused by internal tugor stresses. Their results indicated that the difference between the exten- sibility of IAA-treated and non IAA-treated tissue was not dependent upon the presence of an intact protoplast. 5 6 Various chemical and enzymatic treatments helped Olsen, g£,§;. to characterize the portion of the cell wall involved with auxin-induced extensibility. Pronase treatment of the isolated cell wall, which removed 97% of the protein nitrogen, did not effect the extensibility. The latter experiment pro— vided evidence that the extensibility was not a characteristic of the disrupted protoplast, but of the cell wall itself. Hot acid treatment of the cell wall, which removed hemicellulose, did not disrupt the IAA effect on extensibility. Cellulase treatment, which interfered with the cellulose microfibril interaction, had a profound effect on the extensibility. There- fore, the authors concluded that the interaction between the fibrils of cellulose were responsible for the IAA-induced changes in the cell wall properties. They also reasoned that the polymers themselves had been altered, but that the chemical modifications which resulted in the altered mechanical proper- ties were small. Although the changes in the physical properties of the cell wall are now known, the biochemical mechanisms-are not well defined. An early theory (105) suggested that the cell wall rigidity was dependent upon the number of calcium cross linkages between the pectin chains. Auxin treatment was believed to decrease the number of cross linkages by promoting methylation of the carboxyl groups in the pectin molecule. In some expanding tissues auxin does enhance the rate of 1LAC-- methionine incorporation into pectin (74). However, not all tissue which can be induced to elongate by auxin, show a cor- responding increase in methylation (20). Furthermore, Cleland H; i ; M A v. 7 (23), working with Avena coleoptiles, demonstrated with the aid of 14C-methionine and ethionine that auxin-induced elongam tion occurred under experimental conditions where methyl trans- fer was completely eliminated. Further evidence against the involvement of pectin cross linkages was secured by employing radioactive calcium. In preincubation experiments where radioactive calcium was incor- porated into the cell walls, there was no confirmation of auxin= induced loss of cell wall calcium (22). Another method for auxin to affect cell wall properties would be the synthesis of new cell wall material. There have been a number of observations that auxin-induced cell elonga- tion is accompanied by an increase in cell wall material (2). However, there was no detectable increase in cell wall syn- thesis in.Mv§Ma coleoptile sections when elongation was inhib- ited by mannitol, even though isotonic mannitol did not prevent the loosening of the cell wall as measured by the Instron stress~strain analyzer (73). Hence, the increased synthesis which accompanied elongation could be caused by elongation, rather than the cause of elongation. However, other experi- ments employing 1LiC-glucose, and calcium to inhibit elongation, indicated that there was some cell wall synthesis in the absence of growth (2). Cell wall synthesis without growth was called a direct auxin effect. The latter effect seen in the presence of calcium was in the synthesis of matrix polysac- charides and not ancellulose (84). The indirect effect, due to elongation, promoted a-cellulose synthesis (84). 8 An important contribution to this discussion would be to study the plasticity of the tissue prevented from elonga- tion by calcium. If the loosening of the cell wall occurs in the presence of calcium as it did in isotonic mannitol (73) there would be a correlation between direct effects on cell wall synthesis (matrix polysaccharides) and cell wall exten- sibility. Recent evidence indicates that more is involved in cell enlargement than a simple softening of the cell wall followed by a concomitant passive entry of water as suggested by Leopold (57). Cleland (24) studied cell wall loosening in Mzgga coleoptiles in the presence of actinomycin D. After an initial lag period actinomycin D inhibited RNA synthesis (24), protein synthesis (70), and effectively prevented elongation (24, 70). The addition of auxin three hours after actinomycin D treatment induced a considerable increase in cell wall exten- sibility (24). This reaction occurred under the same condi- tions where RNA synthesis was inhibited by 90% and elongation was completely blocked. Cleland concluded that RNA synthesis must be required for some other process such as an adequate supply of water and osmotic solutes. In an independent investigation, Morre (63) investigat- ing the effect of actinomycin D on RNA synthesis, cell elonga- tion, and tissue deformability in pea (giggg sativum L.) and soybean (Glycin NEE) hypocotyds arrived at a similar conclusion. His results revealed clearly that the action of actinomycin D was not simply an inhibition of auxin-induced tissue deform~ ability. Morre postulated that at least two sets of factors 9 (81 and 82) were involved in cell elongation. He designated arbitrarily the first set (Si) as those involved in cell wall loosening. In pea stems 81 was not as sensitive to actinomycin D as 82 since, actinomycin D greatly reduced the ability of the sections to elongate under conditions where tissue deformm ability was adequate to permit cell expansion. In soybean tissue both Si and $2 seemed to be depleted by pretreatment with actinomycin D. Morre suggested that both cell elongation and tissue deformability were dependent upon RNA synthesis. However, these two sets of conditions were independent. Effect 22 Auxin QQDEHZXQG Activity Early studies on the mode of action of auxin were cen- tered around the effect of auxin on both 12.2l12 and 12.2l222 enzyme activity. Most of the ;M_1;tgg studies have been on enzymes and enzyme systems involved in oxidative or respira- tory activities. Auxin in concentrations which promote growth are almost entirely without effect upon ig,zl££g enzymes (8, 19). At high concentrations some enzymes are influenced by auxin, either inhibition or stimulation, but it is difficult to show that these changes in activity have any relationship to auxin-induced cell elongation. The activity of enzymes 1 vivo are usually increased by auxin treatment (57). However, the increase in enzyme activity usually is much slower than the growth response and most likely is secondary, resulting from the increased elonga- tion rather than the cause of auxin-induced growth. 10 Protein Synthesis and Auxin-Induced Elongation The role of protein synthesis in auxin-induced cell elongation is still not completely known. In some tissue there is an increase in protein content during auxin-induced growth. Protein synthesis in artichoke (Helianthus tuberosus) slices was strongly promoted and to a lesser extent protein synthesis was enhanced in potato (Solanum tuberosum) slices during auxin treatment (104). In addition, aged artichoke 14 tuber disks treated with auxin incorporated more C-leucine than did the controls (69, 71). Christiansen and Thimann (16) showed that there was considerable synthesis of protein in pea stem segments in the presence of auxin. However, there was also a considerable synthesis of protein in the controls. When growth was inhibited by various metabolic inhibitors there was a corresponding decrease in protein synthesis. The problem of protein synthesis in pea stem tissue has recently been inves- tigated with the aid of 1LPG-amino acids. The incorporation of 1“Caleucine was enhanced but most of the enhancement may have been due to the increased uptake of the radioactive amino acid (71). Indole-3-acetic acid at concentrations which prom moted weight increases of fresh tissue enhanced 14C-glycine uptake and incorporation into protein (35). Inhibitory levels of IAA decreased uptake and incorporation. Datta and Sen (29) incubated pea internodes for 15 minutes with 1L’C-phenylalanine. After incubation, the subcellular fractions were isolated by differential centrifugation. Auxin strongly increased amino acid incorporation into the nuclear protein fraction. None of the other fractions were affected. 11 In other tissues there is no change or a net decrease in protein content during auxin treatment. There was no increase in protein content during the cell elongation of wheat (Triticum sativum) roots (12). Protein nitrogen decreased in corn (Meg Mayg) mesocotyl sections during cell elongation (30). The decrease was not altered by auxin. Insoluble nitrogen did not change in Mzgga mesocotyl tissue during either control or auxin—induced elongation (46). There was a loss of protein content during incubation of excised soybean hypocotyl sections. Although auxin greatly stimulated the fresh weight of the hypocotyl sections, there was only a slight difference in the protein content of the two treatments. Key (54) later indicated that auxin slightly stimulated the incorporation of 1)‘pC-leucine into the TCA insoluble fraction. In 1953, Boroughs and Bonner (9) investigated the effect of auxin on protein synthesis in both corn and oat coleoptiles. Protein levels remained constant in excised sections over a period of 6 hours and were independent of auxin. In addition, auxin did not alter the rate of incor- lLPCuglycine or 14C-leucine into the protein poration of either fraction. This study was confirmed by Thimann and Nooden (71). Another approach to the study of the role of protein synthesis in auxin-induced cell elongation was the use of amino acid analogs. Bonner (6) demonstrated that canavanine, an antagonist of arginine, inhibited auxin-induced growth in Avena. The inhibition was reversed by arginine. In the same study hydroxyproline, an antagonist of proline, suppressed 12 cell elongation and this inhibition was reversed by proline (21). Ethionine, an analog of methionine, repressed elongaa tion and the inhibition was overcome by the addition of methionine (22, 23, 90). The use of canavanine, ethionine, and hydroxyproline as evidence for a requirement of protein synthesis has been criticized by Nooden and Thimann (71). The interpretation of the results is limited because of the participation of arginine, methionine, and hydroxyproline in reactions other than the synthesis of proteins. Indeed, recently, Cleland (25) in a detailed investigation of the mechanism of hydroxy- proline inhibition concluded that hydroxyproline may inhibit elongation by preventing the normal formation of hydroxypro- line-rich cell wall proteins. It is interesting to note that 4-aza1eucine, an analog of leucine which inhibits the growth of bacteria, did not inhibit auxin-induced growth in Mzgga coleoptiles (Unpublished results). However, p-fluorophenyl- alanine did inhibit effectively auxin-induced growth in Mzgga and the inhibition was reversed by phenylalanine (70). Thus far, the most effective approach to the study of the involvement of protein synthesis in auxin-induced growth has been the use of selective inhibitors of protein synthesis. Although the use of inhibitors of protein to determine the participation of protein synthesis in a physiological response is not new (13), Thimann and Nooden were the first to success- fully use this tool in the study of auxin-induced cell elonga- tion (71). Several early attempts were unsuccessful due to the low concentrations of inhibitors employed (49, 92). 13 Thimann and Nooden (71) reasoned from the published data on protein content and auxin-induced growth that auxin may pro- mote the synthesis or turnover of a protein or proteins. This protein may comprise only a small fraction of the total cell protein and thus auxin-induced synthesis of one or even a series of enzymes may not be detected among the total cell proteins. The inhibitors they used in their original study were chloramphenicol and puromycin, inhibitors of protein synthesis (39, 114) and actinomycin D, an inhibitor of DNA- dependent RNA synthesis (47). Their results reported in this communication (71) and two following papers (69, 70) demon- strated a correlation between the concentrations of these three inhibitors required to inhibit auxin-induced growth and protein synthesis. On the evidence that, A. compounds which were known to selectively inhibit protein synthesis also inhibited auxin- induced cell elongation; and that B. a parallel existed between the degree of growth inhibition and the degree of inhibition of protein synthesis, Nooden and Thimann proposed that the locus of auxin action is on a nucleic acid system controlling the synthesis of some essential enzyme or enzymes required for growth. Since these studies were published, there have been several research reports dealing with the interrelationship between auxin-induced elongation and protein synthesis in the presence of various inhibitors which are presumed to be speciu fic inhibitors in plant systems. Key (54) found in soybean tissue that puromycin as well as actinomycin D inhibited both 14 elongation and C-leucine incorporation into the protein 14 fraction. Actinomycin D, puromycin, and chloramphenicol inhibited both auxin-induced growth and control growth in sunflower (Helianthus annuus L) hypocotyls (56). The same three inhibitors also inhibited water uptake in potato disks and leaf cells of MMgeg discolor (62). In these two studies, it was not determined whether the inhibitors being used actually did inhibit protein synthesis in the systems being studied. Penny and Galston (80) reported a detailed study of the kinetics of the inhibition of auxin-induced elongation in green pea stem segments by actinomycin D, ribonuclease, puromycin, chloramphenicol, and p-fluorophenylalanine. Unfor- tunately, they did not relate the kinetics of inhibition of elongation to the kinetics of inhibition of RNA or protein synthesis. Chloramphenicol Inhibition g: Protein Synthesis o=c—03012 H NH OZN c—C—CHZOH OH Chloramphenicol Chloramphenicol was discovered independently in 1947 by two groups. Ehrlich and co-workers (31) isolated the broad spectrum antibiotic from an unidentified Streptomyces found near Carocas, Venezuela while a group at the University of Illinois (14) isolated the same substance from a Streptomyces found near Urbana, Illinois. Rebstock and 15 co-workers (27, 85) characterized and synthesized the compound in 1949. Chloramphenicol inhibits the growth of a wide vari- ety of bacteria at concentrations between 1-100 pg/ml (10). It inhibits the growth of plants (66) and algae (28, 103), but requires concentrations which are 100-1000 times greater than those required for bacteria. The first report on the mode of action of chloramphen- icol was published by Gale and Folkes (39). Their study demonstrated that chloramphenicol preferentially inhibited protein synthesis in intact Staphylococcus aureus and that any changes in RNA and DNA metabolism were of secondary nature. These same observations were soon extended to a number of other bacterial systems (10). Protein synthesis in several microbial cell-free sys» tems was also sensitive to chloramphenicol (110). Detailed studies of cell-free systems established that the activation of amino acids and the transfer of activated amino acyl solublemRNA was not altered by chloramphenicol (67). The exact mechanism of inhibition of protein synthesis is not known, but it is clear that chloramphenicol in some manner prevents the transfer of the amino acyl soluble-RNA to the growing peptide chain. Weisberger and co-workers (109, 110) have suggested that chloramphenicol acts by blocking the attachment of messenger-RNA to the ribosomes, but further work will be necessary before the details of the mechanism will be established. Although, some controversy exists (32, 59). chloramphenw icol is reported to inhibit protein synthesis in plant tissue. 16 Chloramphenicol suppressed the synthesis of phosphatase and amylase in germinating peas (116), a number of enzymes in the chloroplasts of beans (Phaseolus vulgaris) (61), thymidine kinase in the microspores of the lily (Lilium longiflorum) (43), and the gibberellin-induced synthesis of a-amylase in barley (Hordeum vulgare) aleurone layers (106). In addition, there have been numerous reports of chloramphenicol inhibi— tion of l“Ci-amino acid incorporation into protein (32, 45, 50, 51, 69, 71, 77, 78, 79). However, in the latter studies the uptake of the 14 C-amino acids was also repressed and it was difficult to separate the two processes. In this regard, the incorporation of 14C-amino acids in several plant cell- free systems where problems of uptake are eliminated was repressed by chloramphenicol. Inhibition was found in sys- tems from corn (83), tobacco (Nicotiana glutinosa) (100), and wheat (65). The concentration of chloramphenicol necessary for inhibition in both intact cells and in cell-free systems was much greater than the corresponding concentration needed for an analogous microbial system. Since the mechanism of protein synthesis is similar in bacterial and plant systems, the basis for the difference in sensitivity is not clear. Several pose sibilities exist: first, the plant cells may not absorb- chloramphenicol. Vazquez (108) found in several bacteria that there was parallel between the activity of chloramphenicol and its absorption. Uptake of course would not explain the results obtained from the cell-free systems. In the same paper (108) Vazquez noted a relationship between antibiotic activity and 17 the binding of chloramphenicol to the ribosome. Bibosomes obtained from peas did not bind lec-chioramphenicoi as effec- tively as those obtained from M, gglg. A second factor which could be involved in the differ- ence of sensitivity is the metabolism or inactivation of chloramphenicol. Certain strains of bacteria produce an extracellular substance capable of inactivating chlorampheni- col and this ability is believed to be widespread among bac- teria (11). In studies of a variety of animals including man, about 90% of an administered dose was recovered in the urine within 24 hours (91). Of this recovered fraction, less than 10% was free active chloramphenicol. Most of the chloramphen- icol was recovered as the inactive glucuronic acid conjugate. There are no reports of chloramphenicol metabolism in plants. Cycloheximide Inhibition g: Protein Sygthesis CH3 CHOHCH2 NH Cycloheximide Cycloheximide was isolated from a culture of Streptomyces greiseus in 1946 (112). Although this compound did not inhibit the growth of bacteria (112), it did inhibit the growth of fungi (112), algae (75), protozoa (58), animal cells (80), and higher plants (79). 18 In 1958, Kerridge (53) reported that cycloheximide inhibited both protein and DNA synthesis but not RNA synthesis in yeast. Several recent reports (4, 34, 64) have provided evidence that the effect on DNA synthesis was a secondary characteristic of cycloheximide inhibition. Evidence that protein synthesis is the primary site of cycloheximide inhibition has come from investigations on cell- free systems. Cycloheximide inhibited lLFC-amino acid incor- poration into protein by cell-free preparations from yeast (97), mouse tumor cells (4), rat liver (33), and reticulocytes (26). It did not inhibit synthesis in a cell-free system from M, 92;; (34). The site of inhibition was after the formation of amino acyl soluble-RNA (33, 95) and appeared to be at the ribosomal level (96). Cycloheximide inhibits protein synthesis in plant tissue. Varner and co—workers (107) found cycloheximide inhibited both 14C-amino acid incorporation and gibberellin-induced synthesis of aramylase by 90% in barley aleurone layers. Parthier (76, 77) reported cycloheximide inhibited radioactive amino acid incorporation into protein without affecting RNA synthesis in green tobacco leaf disks. The concentration required for inhibition, around 5x10”6 M for 50% inhibition, makes cyclo- heximide the most effective inhibitor of protein synthesis known in plants. 19 Gougerotin Inhibition g: Protein Sygthesis H H O / H C‘—N-'C"C H 3 H \N/ \\IQ H N . //l\ 2 \C=O N \O “.0 i N-— i=0 H—-%-—NH2 H2-C-0H Gougerotin A Gougerotin was isolated from Streptogyces gougerotii by Iwasaki in 1962 (48). The antibiotic inhibited protein synthesis in cell-free systems from M, 22;; (17). mouse liver tissue (98), and reticulocytes (15). In a detailed study of the mode of action, Casjens and Morris (15) demonstrated that gougerotin inhibited the transfer of amino acyl soluble-RNA on to the growing peptide chain but did not affect the release of completed protein chains from the ribosomes. They suggested that gougerotin, which could be considered a structural analog of amino acyl soluble-RNA, interacted with the enzyme which catalyzes the formation of the peptide bond. Since there was no peptide bond formed between gougerotin and the growing pep- tide chain, the polypeptide chain remained attached to the 20 ribosomes. The presence of gougerotin at the active site of the polymerase prevented further synthesis of the peptide chain. There are no reports of the action of gougerotin in plants. MATERIALS AND METHODS MATERIALS AND METHODS Plant Material Mzgga coleoptiles nggg sativa (var. Torch) seeds were soaked in the dark at 26.50 for 2-3 hours in tap water. The seeds were then spread evenly on moist vermiculite in glass trays and allowed to germinate under a dim red light (trays were placed 6 feet below two 60 watt Ruby Red light bulbs) at 26.5° for 24 hours. The seeds were covered with a thin layer of vermiculite and placed in the dark at 26.50. All further operations were made under a green safe-light (68). About 70 hours after planting when the coleoptiles were 2-3 cm in length, 4.5 mm sections were cut 2-3 mm below the tip of the coleoptiles. These sec- tions were floated for 2 hours on a glass-distilled water solution containing 1 mg of MnSOg'HZO per liter. Triticum coleoptiles Triticum vulgare (var. Thatcher) seeds were soaked in tap water for 2 hours in the dark at 26.50. All operations were performed under the green safe-light. The seeds were Spread on moist vermiculite, covered with a thin layer of vermiculite and germinated in total darkness at 26.50. After 70 hours, when the coleoptiles were 2.5-3.5 cm in length, a 4.5 mm section was removed about 3-4 mm below the tip and 22 av O— melt. Au. .3 a\u 23 floated on glass-distilled water for 1 hour. Straight growth assay (68) A pH 5 assay solution was prepared by placing 1 ml of Tween 80, 1.794 g of dipotassium phosphate, and 1.019 g of citric acid monohydrate in a 1 liter volumetric flask and adjusting to volume with glass-distilled water. The buffer solution was stored at 40 until used. Immediately before use, the buffer solution was made to 2% (w/v) sucrose and the appropriate chemicals added. Under the green safe-light 10 coleoptiles, either wheat or oat, were placed in a 6 inch test tube, 2 ml of the assay solution added, and the tubes placed in a revolving drum and turned at l revolution per minute. After the specified time of incubation at 26.5° in the dark, the sections were removed and measured to the near- est 0.1 mm using a photographic enlarger. Uptake 92.1uC-Compounds Using the Avena assay buffer, coleoptiles were incu- bated with the appropriate 1"PC-compound in the dark at 26.50 on the roller-drum. After the incubation period, the coleop- tiles were placed on a wire screen, rinsed with water, and transferred to a 10 ml beaker. The coleoptiles were rinsed for l minute in 8 ml of distilled water, then blotted dry on a paper towel. The coleoptiles were then transferred to a scintillation vial and 15 ml of scintillation fluid was added. The scintillation fluid was prepared from 10 g of 2,5-diphen- yloxazole, 0.1 g of a-napthylphenyloxazole, and 160 g of naphthalene dissolved in 770 ml of xylene, 770 ml of p-dioxane, 24 14 and 462 ml of absolute ethanol. For the studies of C-ccmpounds which were not incorporated into protein, the coleoptiles and scintillation fluid were equilibrated for 12 hours at 40 and l”C-leucine or lz‘LC-cmaminobutyric then counted directly. When acid was employed, the coleoptiles were sonicated (Branson Sonic Power Sonifier) directly in the scintillation fluid before counting. The samples were counted on several different Packard Tri Carb Scintillation Spectrophotometers. The count- ing efficiency of the instruments ranged from 50-70%. However, within any given experiment, the same instrument was used for all of the samples. The data were expressed as cpm per 10 sec- tions or uum per 10 sections. Fractionation g£_Proteins Fifteen coleoptiles were incubated with the buffer used 14 for the straight growth assay along with the C-amino acid. After incubation the coleoptiles were rinsed and blotted as previously described. Five coleoptiles were employed for uptake study and 10 of them were placed in a 5 ml glass vial with a plastic cap and placed on dry ice until the proteins were fractionated. For protein isolation, 2 m1 of ice cold water and 1 ml of ice cold Bovine serum albumin (15 mg/ml) was added to the vial containing the coleoptiles. The tissue was sonicated until completely disrupted (about 60 seconds). The contents of the vial were transferred into a 5 inch test tube and the proteins were precipitated by the addition of 1 ml of 25% (w/v) trichloroacetic acid (TCA). The tubes were placed in an ice bath for 20 minutes and then centrifuged at l500xg 25 for 5 minutes. The precipitate was suspended in 5% TCA, placed in an ice bath for 5 minutes, then centrifuged for 5 minutes at l500xg. The pellet was dissolved in 0.5 ml of l M NaOH, and again adjusted to 5% TCA. After 20 minutes in a ice bath the sample was centrifuged for 10 minutes, the supernatant was removed, the pellet rinsed with 5% TCA, the pellet was dissolved in 0.5 m1 of l M NaOH, and transferred to a scintillation vial. To this preparation was added 15 ml of a scintillation gel con- taining 7 g of 2,5-diphenyloxazole, 150 mg of 1,4-bis-2-(5- phenyloxazolyl)-benzene, 50 g naphthalene, and 36 g of thixo- tropic gel powder dissolved in 200 ml of toluene, 30 ml of absolute ethanol, and 800 ml of p-dioxane (15). Samples were counted on a Packard Tri Carb Scintillation Spectrophotometer. When lac-leucine was added to unlabeled coleoptiles immediately after sonification and the homogenate treated as previously described, all of the radioactivity was removed. The data was expressed as cpm per 10 sections. Since there was no change in protein content during the assay (9), any change in the cpm reflects a change in the specific activity of the protein. Throughout this investigation the primary leaf which does not respond to auxin was not removed. To determine the distribution of the 14C-leucine between the responding coleop- tile and the primary leaf, sections were incubated for 6 hours with 1L’c-ieucine, the primary leaf and the coleoptile separated, and the distribution of the radioactivity measured.. Less than 2% of the total radioactivity taken up was incorporated into the protein of the primary leaf (Table l). ‘i I D kn) 7". § ( k— 11) l Pf" (J) 26 TABLE 1 Distribution of 1Ll'C-Leucine in the Coleoptile and Primary Leaf of the Avena Uptake TCA Insoluble Leaf* 822 119 Coleoptiie* 8,120 4,234 *- Expressed as cpm/10 sections. Metabolism.g£ Chloramphenicol incubation and preparation for chromatography 3 l4 Mgggg coleoptiles were incubated with 5x10- M_ C- chloramphenicol (18,420 cpm/ml) in the dark at 26.5°. After 4 hours the internal concentration was equal to the external concentration. The coleoptiles were placed in a 5 ml vial, 3 m1 of acetone added, the vial covered, and placed in the dark at 4°. After 5 hours the coleoptiles were removed and their radioactivity determined. About 3% of the initial radioactivity remained in the tissue.‘ Extending the extrac- tion period to 24 hours did not remove any further activity. The acetone was removed 12 vacuo and the residue taken up in a small volume of acetone for chromatography. Thin-layer chromatggraphy Since there were no good procedures developed for thin-layer chromatography of chloramphenicol a number of solvent systems were studied. For these studies, small instant thin—layer sheets, 2x6 2/3 cm, were cut from 20x20 27 cm Eastman silica gel chromatogram sheets. The small sheets could be developed in 5-7 minutes and a rapid survey made of many different solvent systems. After development, the chro- matograms were sprayed with a 0.05% solution of Rhodamine B in ethanol. The chloramphenicol was located by viewing the chromatogram under short-wave ultraviolet light. Two solvent systems gave good results. In the first system of chloroform: ethyl acetate:formic acid (5:4:1) the Rf value of chloramphen- icol was 0.75. In the second system of chloroform:benzene: ethanol (7:3:1), the Rf value was 0.39. 14C-Chloramphenicol extracts were chromatographed on 4x20 cm thin-layer sheets. One side of the chromatogram was // spotted with unlabeled chloramphenicol and the other side with the luC-chloramphenicol extract. The chromatogram was developed in one of the above solvent systems for 15 cm. After drying, the chromatogram was cut down the middle to separate the marker spot from the extract. The location of the unlabeled chloramphenicol was determined with Rhodamine B. For location of the radioactive metabolic products of chloram- phenicol, the other half of the chromatogram was cut into 15 equal segments and each one placed in a scintillation vial with 5 ml of scintillation fluid and the radioactivity deter- mined. Incubation and preparation for bioassay The 4 isomers of chloramphenicol at 5x10“3 M.were incu- bated with Avena coleoptiles (8 coleoptiles/ml) for 4 hours in the dark at 26.50. As determined from the quantitative experi- ments with l("C-chloramphenicol, there were about 8 pg of 28 chloramphenicol per coleoptile section. For the extraction, 9 coleoptiles were placed in a 5 m1 vial. Three ml of acetone was added, the vial closed, and placed in the dark at 4°. After 20 hours, the coleoptiles were removed, and 1 ml of acetone extract was transferred into each of three test tubes. The 5 inch test tubes contained about 24 pg of extracted chloramphenicol. The acetone was removed ;p_z§ppp at room temperature and the residue was used directly for the assay of chloramphenicol activity in M, coli. Bioassay p§_Chloramphenicol Growth p§_pp;l§ The culture medium was prepared by dissolving 10 g of tryptone, 10 g of yeast extract, 5 g of KZHP04, and 10 g of glucose in 1 liter of glass-distilled water. The medium was autoclaved before use. Using sterile technique, 8 ml of culture medium in a 6 inch test tube was inoculated with 0.2 m1 of a stock culture of Escherichia pp;;.(Crooks strain). The culture was incubated at 35°. The growth of the culture was followed by determining the optical density at 660 mp (Coleman Jr. Spectrometer). After about 3-4 hours the cell suspension was in the log phase of growth with an optical density between 0.2-0.3. Determination 23 protein synthesis The rate of protein synthesis in the E, coli cell sus- 14 pension was determined by following the incorporation of C- leucine into the protein fraction. One ml of the above cell 29 suspension was transferred to a 5 inch test tube. The cells were incubated for % hour at 350 with the appropriate chemical or plant extract, then luC-leucine (40,000 cpm) was added and the incubation continued for 1 hour. The reaction was stopped by the addition of 2 ml of 10% TCA. The mixture was heated at 800 for 10 minutes, and the insoluble protein was collected on a glass fiber filter disk using a Millipore filter system. The disk was washed twice with 5% TCA, once with ethanol:ether (1:1 v/v), and once with ether. The disk was placed in a scin- tillation vial and counted in 5 ml of scintillation fluid. wager Barley (Hordeum vulgare, var. Himalaya) seeds were cut in half on the equatorial axis and the embryo-half discarded. The tips were removed from the half-seeds and the half-seeds were soaked in Chlorox (5% sodium hypochlorite) diluted five- fold with distilled water for 15 minutes. All remaining steps were performed aseptically. The half-seeds were rinsed in sterile distilled water and transferred to sterile moist sand in a Petri dish. After preincubation for 3 days at room tem- perature in the dark, the seed coat was slit on one side and the endosperm removed from the seed coat and aleurone layers. Ten layers were incubated in a 25 m1 Erlenmeyer flask with 2 m1 of 10"5 M gibberellic acid in a 0.001 11 acetate buffer (pH 4.8) and 0.01 M CaC12 along with the appropriate chemical treatment. After 24 hours incubation in the dark at 21°, the medium was poured off and the layers rinsed with 3 m1 of the acetate buffer. The layers were ground in a mortar with sand 30 and 5 ml 0.2 M NaCl. After centrifugation at 1000xg the medium and extract were assayed separately. c-Amylase activity was measured as described by Shuster and Gifford (94). A starch solution containing 67 mg of solu- ble starch in 100 ml of 0.06 M_KH2P04 was prepared. One ml of this solution was added to enzyme and water to give a final volume of 2.0 ml. After 5 minutes of incubation at 25°, the reaction was stopped by the addition of 1.0 m1 of an iodine- HCL solution prepared from 60 mg of KI and 6 mg of I2 in 100 ml of 0.05 M HCl. Then 5 m1 of water was added, and the optical density (OD) of the resulting solution was measured at 620 mp. The activity of the enzyme was expressed as mg of starch hydro- lyzed per 10 layers per minute. Metabolism p§_d-Aminobutyric Acid Incubation and fractionation Coleoptiles, 160, were incubated with luC-a-aminobutyric acid for 2 hours in the dark at 26.50. Ten coleoptiles were picked at random to determine the total uptake. After counting, 11+C-a-aminobutyric acid into the protein the incorporation of fraction was estimated by extraction with 70% ethanol (113). The coleoptiles were extracted with hot 70% ethanol for three hours. Extracting with 70% ethanol in a Soxhlet extractor did not remove any further radioactivity. The proteins from 150 coleoptiles were precipitated by the TCA procedure previously described with the exception that carrier protein was not added. After TCA precipitation, the TCA soluble fraction was taken to dryness Mp vacuo and the 31 amino acids dissolved in 4 ml of 0.1 M HCl. The TCA was extracted from the acid solution with ether and the aqueous phase taken to dryness Mp 13939. To remove the excess HCl the residue was taken up in 4 ml of water and the water removed lp_p§ppp. The residue was taken up in 10% solution of 2~propanol for chromatography. The TCA insoluble material was hydrolyzed in 6 M HCl in a sealed glass tube at 1070 for 60 hours. After hydro- lysis the solution was filtered and the HCl solution removed ip,2§ppg. The amino acids were taken up several times in 4 m1 of water and taken to dryness to remove the excess HCl. The residue was taken up in 10% solution of 2-propanol for chromatography. Chromatography p; the amino acids The amino acids were paper chromatographed in two dimen- sions as previously described (5). The chromatogram was developed in the first direction with phenol saturated with water and in the second direction with butanol:propionic acid: water. The amino acids were located by exposure of the dried chromatogram to Kodak No-Screen X-ray film for 2 weeks. The radioactive spots were then counted with a thin-window (Du Pont Mylar film) gas flow counter using a Nuclear Chicago sealer. For preparative chromatography, chromatograms were run in one direction with butanol:propionic acidzwater and the compounds located by direct scan. Chemicals The isomers of chloramphenicol were kindly supplied by 32 Dr. M. Rebstock, Park Davis and Company. Dr. H. Petering of the Upjohn Company supplied the cycloheximide and the gouger- otin was a gift of Dr. A. Mayake, Chemical Industries Ltd., Osaka, Japan. All of the radioisotopes were obtained from New England Nuclear Corporation. The specific activity of the compounds were: leucine, 250 pc/pm; d-aminobutyric acid, 4.1 pc/pm; d-aminoisobutyric acid, 4.0 pc/pm; indole-3-acetic acid, 13.5 pc/pm; and chloramphenicol, 3.08 pc/pm. All other chemi- cals and reagents were obtained from commercial sources. RESULTS AND DISCUSSION RESULTS AND DISCUSSION Effect 9: Auxin pp_Elongation and Protein Synthesis The kinetics of elongation induced by auxin in Avena coleoptiles is shown in Figure l. The auxin used in this experiment and throughout this study was a 10-5 M solution of indole-3-acetic acid. The time course followed the well-known bilinear curve for 513p; coleoptile elongation (93). In the first 8 hours a linear rate of elongation was observed in both the control and the IAA treated sections. The rate of elonga- tion of the auxin treated coleoptiles was about 4 times greater than was the control elongation. Elongation from 8-24 hours continued at a reduced linear rate. The ratio of IAA to con~ trol growth during the second phase was about 2. Throughout this study 2% sucrose and 0.01% Tween 80 were included in the buffer system. Thus, it was important to deter- mine how these additives affected the rate of elongation. Nitsch and Nitsch (68) reported that over a 24 hour period sucrose increased elongation and Tween 80, which was used to facilitate dissolution of the chemical treatments, had little effect on elongation. The effect of the deletion of either sucrose or Tween 80 on the kinetics of elongation is illus- trated in Figure 2. The experiment without Tween 80 did not affect auxin-induced elongation. On the other hand, after a two hour lag period, the deletion of sucrose markedly reduced 34 35 Figure 1 Kinetics of Auxin-Induced Elongation in the Avena Coleoptile 36 3N ON on ”snowy made ma _ Hoapaoo _ _‘»-:l’ (mm) uotquuotg 37 Figure 2 Effect of Sucrose and Tween 80 on the Kinetics of Auxin- Induced Elongation in the Avena Coleoptile 38 :N cm H Ansomv made o «a f \/ omonodm : 44H a _ Cw HOOKS I 1H N :1- (mm) uotquuotg 39 the rate of elongation and all growth was eliminated after 12 hours. Little change was noted in the first 6 hours in the ratio of auxin-induced elongation to control elongation when sugar was excluded from the medium (data not shown). Thus, it appears that sucrose does not directly induce elongation, but rather is a source of energy for auxin-induced elongation. The effect of auxin on the incorporation of 14C-leucine into protein was investigated. The lack of stimulation of protein synthesis during auxin-induced growth has already been reported (9, 71). However, in these reports, the incubation period was 5 and 6 hours while the growth response was much more rapid, there being a marked stimulation during the first hour (Figure l). The effect of auxin on the elongation, uptake, and incorporation of lac-leucine into protein is pre- sented in Table 2. Although the growth rate in the presence of auxin after 1 hour is more than double the control, there was little stimulation of protein synthesis as measured by 14Caleucine incorporation into the TCA insoluble protein fraction. Two hours after incubation the growth rate was 4 times greater than the control, but little effect was noted on protein synthesis. Since protein synthesis is required for auxin-induced elongation as has been postulated, an interpretation of the above results is not directly obvious. Incorporation of 140- leucine into the protein fraction of the controls was rapid, but this incorporation was not enhanced by the addition of IAA under experimental conditions where elongation was increased 294 fold. This does not eliminate the possibility 4O TABLE 2 Effifit of Auxin on the Elongation, Uptake, and Incorporation of C-Leucine into the Protein of the Myppg Coleoptile Egg; 1 Hour 2 Hours MAM Control MAM Control Elongation* 0.5 0.2 1.2 0.3 Uptake** 2,976 3,055 5,711 5,146 TCA Insolub1e** 1,394 1,248 2,928 2,657 :Expressed in mm. Expressed in cpm/10 sections. 41 that auxin may induce the synthesis of some new protein(s)i essential for elongation, but only reflects the overall rate of protein synthesis. Auxin could induce the synthesis of a few specific enzymes and if the amount were small compared to the total protein synthesis, it would not be observed by this method. A second alternative could be a re-direction of protein synthesis. Thus, the lack of stimulation of uC-leucine incorporation into protein does not in itself exclude an essential role for protein synthesis in auxin- induced elongation. QMloraMphenicol Inhip;tion pi Auxin-Induced Mlgpgation and Protein Synthesis The parallel between the concentration of chloramphen- icol required to inhibit auxin-induced elongation and protein synthesis in Myppg coleoptiles, previously reported by Nooden and Thimann (20, 25), was confirmed. The concentration range for inhibition was between 5x10-4 and 5r10"3 A (Figure 3). Investigation of wheat coleoptiles again revealed that a par- allel existed between suppression of protein synthesis and elongation. Triticum coleoptiles required even higher concen- trations than did the szpg. These results are shown in Table 3. Incubation with a 5x10")+ M'solution of chloramphen- icol and auxin for 22 hours increased elongation 25% over that observed for the auxin control. Incubation with a 10'3 M solution of chloramphenicol had little effect while a concen- tration of 5x10'3 M almost completely eliminated all growth over a 22 hour period. Perhaps, the marked stimulation of -4 elongation by the 5x10 M solution was due to the bactericidal 42 Figure 3 Effect of Chloramphenicol on Auxinulnduced Growth in the Avena Coleoptile: Concentration Range 43 SOHDGHPQOOCOU HOOHSOSQEGHOHSO DMIOH N m . ilOH N m MIOH N m H mzdo + ¢¢H mzdo + HOHono ._ ,= . Hoapnoo (mm) HOIQBBUOIE 44 .aoapanassa R can maoapoom oa\amo mm commonaxmss .20apapassd R can as mm dommoamwmt “and was flame oom.m Adv saa.~ on dom.m a twoansaonsH doe Anna mam.m “say mmo.: ANS mom.m gov omn.m : *soaonas Amov m.o Away m.m Asa m.~ gov o.m s ssoasswsoam Ammv 0.0 nouv m.s Ammuv ~.m “ov s.a mm escapswsoam .m m-oawm .m n-0HwH .m suoawm o gem cane SoapQApcoocoo HOOASmSQEmHOHSU Spas condone moaapaooaoo Escapdae mo nfiopoam on» opca oaHoSoAIoia mo SoapmhomsoonH dam .oxmpmb .aoprwcoam m mqm¢8 45 action of chloramphenicol rather than a direct effect on the plant tissue. Chloramphenicol inhibition of elongation, luC-leucine uptake, and incorporation into the protein of Mpgplppg after 4 hours treatment is given in Table 3. At 5x10'4 M, chlor- amphenicol was almost without effect on all 3 parameters. The stimulation of elongation by 5x104 M chloramphenicol after 22 hours was not observed after 4 hours where bacterial contamination was not a problem. There was only a slight inhibition at a concentration of 10"3 M while a 5x10-3 M solution of chloramphenicol inhibited elongation by 65% and protein synthesis by 69%. As in the Myppg coleoptiles, chloramphenicol inhibited the uptake of lL’cnleucine making a direct comparison of the repression of protein synthesis and elongation difficult. To establish further the relationship between protein synthesis and auxin-induced growth, kinetic studies of the inhibition of elongation and protein synthesis in the szpg coleoptile were conducted. As illustrated in Figure 4, there was a 2 hour lag period before a solution of 10"3 M chloram- phenicol inhibited auxin-induced elongation. The elongation from 4724 hours continued at a linear, but at an appreciably reduced rate. Inhibition was obtained within 1 hour when a concentration of 5x10'3 M chloramphenicol was used. There- after, the growth rate declined steadily until after 6 hours when the inhibition was complete. Pretreatment of the tissue with a solution of 5x10'3 M chloramphenicol before addition of auxin indicated the same lag period. Pretreatment for 45 46 Figure 4 Kinetics of Auxin-Induced Elongation in Avena Coleoptiles Treated with Chloramphenicol (52:10"3 M) Figure 5 Effect of Chloramphenicol on the Kinetics of Auxin-Induced Elongation in Avena Coleoptiles Pretreated with Auxin Elongation (mm) Elongation (mm) 1:- N 47 I I I IAA IAA + CAMP (10'3M) . ' IAA + CAMP (5 x 10’3M) . . l l I 6 12 18 24 Time (Hour) 6 T 1 1 I 14.. 2 l l l l 6 12 18 24 Time (Hour) 48 minutes or longer essentially eliminated all auxin—induced elongation (Table 4). As will be seen later in the section on uptake of 1” C-chloramphenicol, the lag period may have been due to the rate of diffusion of chloramphenicol into the cell. In the converse experiment where auxin was supplied to the tissue before chloramphenicol was added, elongation only occurred in the first hour after addition of the inhibitor (Figure 5). TABLE 4 Auxin—Induced Elongation in Avena Coleoptiles Pretreated with Chloramphenicol (5x10-3 M) " Elo ation Time after addition of IAA (Hr) Pretreatment (Hr) 0-2 2-4 4-20 0 0.7 0.1 0.4 1/4 0.7 0.1 0.3 1/3 003 001 0.1 1 1/2 0.2 0.1 0.1 3 001 002 005 6 0.2 0 0.4 No chloramphenicol 1.0 1.1 2.8 The pretreatment experiments supported the hypothesis that protein synthesis was required for the initiation as well as the continuation of auxin-induced elongation. This conclusion is in conflict with the one reached by Cleland (21). Cleland using hydroxyproline as an inhibitor suggested that protein synthesis was required for continuation, but not for initiation of auxin-induced growth in the Mzgpg. The failure to extend the lag period after pretreatment with IAA was 49 evidence that any newly synthesized protein was rapidly being utilized by the cell. The effect of a 10"3 M solution of chloramphenicol on the uptake and incorporation of 14 C-leucine into the protein of Myppg as a function of time is presented in Table 5. These results indicate no clear cut temporal relationship between inhibition of elongation and protein synthesis. Inhibition of protein synthesis varied from 16% to 29% after 1 to 6 hours, respectively. In agreement with the elongation studies, pro- tein synthesis was not completely eliminated, but its rate of synthesis was reduced. The relative inhibition of 1tic-leucine uptake and protein synthesis is confusing” In.certain experi- ments, uptake was inhibited to a greater extent than was pro- tein synthesis. The pattern did not become clear until higher concentrations of chloramphenicol were employed. When a solution of 5x10'3 M chloramphenicol was added, lac-leucine incorporation into protein the inhibition of closely paralleled the inhibition of elongation (Figure 7). The inhibition of protein synthesis slightly preceded inhibi- tion of growth and from 2-6 hours both continued at a dimin- ished rate. The inhibition of lu’C-leucine uptake paralleled the inhibition of elongation (Figure 6). Thus, one was confronted with the difficult problem of assessing whether there was true inhibition of lUC-leucine incorporation into protein or if it was an apparent inhibition due to a decreased level of 14C- leucine in the tissue. To compare the relative inhibition of the two processes concurrently assayed, the data of Figure 6 50 .mnoapoom 0H\aao mm dommoamxmst mm moo.b oss.m mm mmm.aa bmm.ca m an mom.m osm.s AH nmm.m mos.oa s AH mma.a bma.m s Hea.s bms.s N ma mam sso.a Hm smo.m «mo.m H mum mass + emH ¢ 69 Figure 13 Thin-Layer Chromatography of 14C-Chloramphenicol Extract: Solvent System of Chloroform:Ethyl AcetatezFormic Acid (5:4:1) 7O Elf-9 71 Stereospecificity p£,Chloramphenicol Because of the unusually high concentration of chloram- phenicol required for inhibition, the stereospecificity of chloramphenicol action in plant tissue was investigated. Of the four possible stereoisomers only the naturally occurring antibiotic D-threo-chloramphenicol showed any significant activity in intact bacteria (10). In a cell-free system obtained from.§, 92;; the L-erythro and L-threo isomers were inactive. However, Jyung, Wittwer, and Bukovac (51) observed that the L-threo isomer repressed protein synthesis in iso- lated cells from tobacco. The inhibition of auxin-induced elongation in Mggpg by the four isomers of chloramphenicol is given in Table 9. All four isomers were effective inhibitors of auxin-induced 4 elongation to about the same degree from.5x10' to 5x10"3 M concentration. Furthermore, all of the isomers effectively 1”Culeucine into protein (Table 10). inhibited incorporation of As shown for D-threo-chloramphenicol activity the 3 non-anti- biotic structures markedly inhibited the uptake of 1”CH-leucine. In addition, all four isomers were very strong inhibitors of lL"C-c.-aminoisobutyric acid uptake (Figure 14). To establish whether this inhibition was a general phenomenon in plants or unique to the Mggpg, several other plant systems were investigated. Since the L-threo isomer was reported to repress root growth in higher plants (88), all four isomers were tested for activity in the buckwheat (Fagopypum esculentum) assay (89). D-Threo, L—threo, and L-erythro-chloramphenicol were very effective inhibitors of 72 .coapanfisna & one mnoapoom oa\aao ca commoanwmt .modpdpasna R can aa aa commonnxmut .mnsom : Hon mm: noapensonHt as “one emcee Acme amend Anne Nmm.a none oNn.H Ace Nas.s .weoanaaonsa .¢.o.a ANmV Shane “any NNm.e “one mom.n nose smN.m Ace omm.m stressed: Away m.a none eta Away m.H Acne H.H Ace m.N sesoapowsoam .Immmmwmmum .Immmmummum cowsaun oceanic Honbsoo dzobd no :aoponm on» ouna :oapmhoaaooQH one .oxdpnb endowed load .aoupmwaoam mm aofipdnaan A: maoava Hoodnosgadhoaso mo hpaoamaoommoouopm ,. oH mamas .zoapapaan R we dommmnmwmss .mnson em you an: soaanSoaHe we on mm as Wm: Ioawm 0: 03 5m 03 EM! IOHNH a- N N new 2 atoaam oaspamnq oanpwamIQ ooasaiq oonsaia nodpmhpcoocoo owzmm aoapcnpmoosoo .oaapmooaoo samba esp Ca coapmmcoam doosdaHI2HNS< no :oapwnaszH Hooanosaamhoaso mo mpfiodmaooav ooaoum m mqmda 73 Figure 14 Stereospecificity of Chloramphenicol (51:10"3 M) Inhibition of the Uptake of 1L"C-c.-Aminoisobutyric Acid into the Avena Coleoptile Control L-Erythro-chloramphenicol D-Erythro-chloramphenicol L-Threo-chloramphenicol D-Threo-chloramphenicol \J‘l-F'KJONH o o 74 Time (Hour) 1 ' T T .. -M W? ‘ q... N .1 to (1% _. I I \, I. \ 1 l L - N n o in H H suctqoas 01/;0! x was ‘75. root growth (Table 11). D-Erythro-chloramphenicol also inhib- ited root gorwth, but to a lesser extent. On a concentration basis, root growth was more sensitive to chloramphenicol than was coleoptile growth. TABLE 11 Stereospecificity of Chloramphenicol Inhibition of Buckwheat Root Growth Concentration D-Threo L-Threo D-Erythro L-Erythro 10'”6 .Ii 9* 2 -4 6 10'5 21. 30 15 5 7 10’” M 46 22 13 15 10'3 Ii 57 53 18 49 * . Expressed as % inhibition. The activity of the isomers on auxin-induced elonga- tion in wheat coleoptiles is shown in Table 12. At 5x10'3 M concentration all of the isomers strongly inhibited elonga- tion. These data are further evidence that the stimulation of growth by a 5xlO'# M_solution of chloramphenicol during the 22 hour assay was due to its interference with bacterial growth. L-Threo, D-erythro, and L-erythro-chloramphenicol, which do not inhibit bacterial growth (10), slightly inhibited elongation. In Table 13, the effect of the stereoisomers on elongation, uptake and incorporation of 1”Ca-leucine into the protein fraction of Triticum is presented. The isomers effec- tively inhibited elongation, uptake, and protein synthesis. 76 .Sodpanfissa R use mGOHpoom 0H\Emo Ga Ummmohmxmsss .aoapananafi R one as :H dowmoaaxmst .mhdos 3 you was noHpMDSOQHa Acme oom.a Acme omo.a Arne NAN none was xov mom.N swsoapsaowsH .4.o.a Anne soeaN “one Hmm.N game oes.a “any mmN.N gov omm.m essences: Anne H.H gone mad none m.o Anne m.o gov o.N teacheswsoam onsewnmuq onsoawmun consanq cones- Howesoo asoapdha mo :Hoponm may opna nodpmnoanoonH one .oxdpms cndoaoqno . .nodpmwnoam no moapanassH A: ntoawmv Hooaaosmamnoaso mo hpdoamaoommooaomm ma mqmda .onpanaszd & me commonawmta .mndos NN you was zoflpcDSOSHt me an as No .a muoawm 0H NH ma on .a m-oawa N m m .amN- .a SIOme chaphhmtu thphhmwm ooHSEIA omhzeln nodpmhpzoonoo owcmm zoapenpzoozoo ”oaapaooaoo adoHpHaB map Ca soapmwcoam mo SoapanannH Hoowsosmawhoano mo mpaoamaooamoohopm NH mqm<8 77 On the basis of luC-leucine incorporation into the TCA insoluble protein fraction. it appeared that chloramphenicol repression of protein synthesis was not stereospecific in plants. To obtain a more direct assay of protein synthesis in a plant system, the ability of the isomers to inhibit gibberellio acid-induced synthesis of a-amylase in barley aleurone layers was investigated. Varner (106) previously reported that D-threo-ohloramphenicol inhibited gibberellic acid-induced a-amylase synthesis. The chloramphenicol isomers at 51:10”3 fl_concentration were incubated for 2“ hours with barley aleurone layers and a 10'5 fl solution of gibberellic acid. After incubation, d-amylase activity in the medium and in the tissue was examined separately. Total activity of the combined medium and extract ranged from 73% inhibition with the least active D-erythro to 83% inhibition for the most active L-threo isomer (Table 1a). The release of the enzyme; was also inhibited. The chloramphenicol isomers at the high- est concentration found in any of the assays did not repress the activity of a-amylase (Table 15). The possibility existed that plant tissue had the capacity to racemize L-threo, D-erythro, and L—erythro isomers into D-threo-chloramphenicol and only the latter isomer was active p§;,§§, To test this hypothesis a bioassay for D-threo- chloramphenicol activity was utilized. A cell suspension of E, ggli_was preincubated with the appropriate chemical or plant extract. After the preincubation period. Inc-leucine was added and the inhibition of its incorporation into hot TCA insoluble protein was measured. The sensitivity of the g, 92;; to chloramphenicol is presented in Table 16. 78 .mnomma oonSoHs 0H non ouazda pom dmuhaohohs season mo w: a“ commonaxm .Haxma mm as mamsomH** .3. ~H~.m mos.w mum.w om~.m No:.m **apd>fipo¢ oanpuumaq oesnsnm-o omesauq omaseuo Honpsoo ommahadno no apabapod on» no Hooanosnadaoaso Mo whoaomH on» no uoommm * ma mamas .maomma oonSon hon opsnaa Mom douhaoadzg :oacpm mo w: ma commoaaxM# mm mm mm mm o nodpdpdnsH m mn~.m oom.m aom.m omn.: ama.am *Hspoe omw.m oo:.m mmm.a mew.m mo:.m *pomopxm mm:.m oo:.m moo.a mum.a mmm.ma *asaemz mmmmwmmum caspanm-o omega-q omesauo Hopscoo mamoSpshm mmmahadnd no zodpapHSCH «a mnoaxmv Hooficosaamaoaso mo hpaoamaoommomhopm 3H mqmda 79 TABLE 16 * Effect of Chloramphenicol on Protein Synthesis in E, coli Concentration pg/ml __0 i2 ii a: TCA Insoluble (CPM) 132 9 4 2 *gfletreated 1/2 hour with inhibitor. Incubated 1 hour with Caleucine. At a concentration as low as 10 pg/ml, protein syn- thesis was inhibited by over 90%. D-Threo-chlorampheniool, 25 pg/ml, completely inhibited protein synthesis while the non-antibiotic isomers had little effect (Table 17). A1§g§_coleoptiles were incubated A hours with the chloramphenicol isomers and then extracted with acetone. An E, ggli_oell suspension was preincubated with a plant extract equivalent to 2b pg/ml of the chloramphenicol isomer. The results given in Table 18 demonstrated conclusively that the plant tissue did not racemize the "inactive" chloramphen- icol isomers into the "active" D-threo-chloramphenicol. In conclusion, chloramphenicol inhibition of plant systems was not stereospecific. This lack of specificity appears to be a general phenomenon since auxin-induced elonga- 14C_ tion, root growth, 1Ll'C-o.-aminoisobutyric acid uptake. leucine uptake and incorporation into protein, and d-amylase synthesis were all inhibited. Although there were some minor differences in the degree of inhibition of the various physio- logical responses, all four isomers inhibited in the same order of magnitude and over the same concentration range. The 80 .oGHoSoHIU Spas 950: H oomeSonH .powapxo Spas Mao: N\H dopooapoam * 3H mum own mom m omm m- xzmov oHnsHomsH <09 oasoaomuq ooSpahm-o ooase-q ooesauo Hobosoo ooenauo w: mm mpomhpxm oomwaB “Hoe .m ca mamoSpchm :Hopoam so mpowapxm Hooasosaamhoaso mo uoommm ma mqm¢8 .osdosoauooa soda too: a oooonsosH .hopdoasna spas boos «\H ooooohponmr :ma Had mma NI mmfi AEmUV mHQSHomQH 408 opsoahmuq ohawaomso oohneuq ooeseuo o Haoo .m ea mamoSpnhm campoam mo nofipanHSQH AHa\wm mmv HOOHQoSQEmHOHSU mo mpaofiwwoommooaopm NH mqmda 81 activity was not due to a racemization to the D-threo isomer. Effect gprycloheximide 2n Auxin-Induced Elongation and Protein Synthesis Further evidence on the involvement of protein synthesis in auxin-induced cell enlargement was obtained by investigating the effect of cycloheximide on elongation and protein synthesis. The concentration range of cycloheximide inhibition of auxin- induced and control elongation in Avena and Triticum is given in Figure 15 and 16. A marked inhibition was noted in both auxin-induced and control elongation. The most striking prop- erty of cycloheximide activity was its extremely low concentra- tion required for inhibition. In both A122; and Triticum, 50% inhibition was obtained with a concentration of about 2x10.6 fl, Cycloheximide was more active than was any inhibitor previously reported. Elongation, 14C-leucine uptake, and 1LPG-leucine incor- poration into protein were all inhibited over the same concen- tration range in the Alena (Table 19). In all cases studied, the suppression of lfi C-leucine uptake was smaller than the inhibition of protein synthesis, indicating a direct repres- sion of protein synthesis as well as inhibition of amino acid uptake. As shown in Table 20, the uptake of ll"C-a.-aminoiso- butyric acid was also inhibited by a 10.5 fl_concentration of cycloheximide. Time course studies with cycloheximide at 21:10-6 fl_and 10‘5 m concentration presented in Figure 17, showed 50% and 40% inhibition after two hours incubation. With a 10-5 fl 82 Figure 15 Effect of Cycloheximide on Auxin-Induced Elongation in the Avena Coleoptile: Concentration Range Figure 16 Effect of Cycloheximide on Auxin-Induced Elongation in the Triticum Coleoptile: Concentration Range Elongation (mm) Elongation (mm) IAA + Cyclo Control " Cyclo 5 l l l i 7 io-6 io-5 10'“ 10-1 Cycloheximide Concentration Control ‘-_-__--—_——‘- Cyclo I l l . 10-6 10'5 10'“ 10‘3 Cycloheximide Concentration 82 Figure 15 Effect of Cycloheximide on Auxinclnduced Elongation in the Avena Coleoptile: Concentration Range Figure 16 Effect of Cycloheximide on Auxin-Induced Elongation in the Triticum Coleoptile: Concentration Range Elongation (mm) Elongation (mm) km 0\ U Control Cyclo l l l l 10-6* 10-5 10'“ io-J Cycloheximide Concentration _—_—_~—_-___d— J l J - - 10-6 10'5 10'“ 10" Cycloheximide Concentration 8n .msoapoom oa\amo mo oommmaaxM* mm on :m sodpdnds HER som.: mon.m mao.a oaoao + seH amm.oa mmH.o *:HH.~ ¢4 mo :Hoponm oSp opzfi ScapegoaaoocH dew .oxmpab ozaodoquoda .zoapowmoam on» no moaafixosoaomo mo poommm 0H mqm¢B 85 Figure 17 Effect of Cycloheximide on the Kinetics of Auxin-Induced Elongation in the Avena Coleoptile Figure 18 Effect of Cycloheximide on the Uptake of lec-Indoie-3-Aoetio Acid Elongation (mm) 86 IAA IAA + Cyclo (2 x 10'6M) t—{T V l l IAA + C clo 10'5M y ( yr) O\F—- 12 is Time (Hour) 24 CPM x 103/10 Sections IAA + Cyclo (10'5g) *‘r 3 ‘ 4 Time (Hour) 87 solution of cycloheximide elongation proceeded at a reduced linear rate from 2-12 hours and thereafter elongation was eliminated. The inhibitory action of a 2x10"6 M'solution of cycloheximide was lost with time.’ During the first 6 hours of incubation the elongation was linear and was considerably less than the control. However, the elongation from 6-2# hours closely paralleled the nontreated A3223. The strong inhibition of control elongation was an indication that cycloheximide inhibition was not mediated through the repression of IAA uptake. Direct evidence for this observation is illustrated in Figure 18. Only after u and 6 hours was there any suppression of uptake and the inhibition was small compared to the repression of elongation. The time course of l”C-leucine uptake and incorpora- tion into protein with a 10-5 fl_solution of cycloheximide was similar to the time course of elongation (Figure 19 and 20). On a per cent basis cycloheximide more effectively inhibited protein synthesis than elongation in the first 2 hours. From 2-6 hours, both processes were inhibited to about the same extent. A short lag appeared between the start of cyclo- heximide inhibition of protein synthesis and the inhibition of elongation. The correlation between the concentration of cyclo- heximide required to inhibit elongation and protein synthesis provides further evidence that protein synthesis is a require- ment for auxin-induced elongation. In addition, the lag between the start of inhibition of protein synthesis and the inhibition of elongation is an important observation. These 88 Figure 19 IA Effect of Cycloheximide (10'5 m) on the Uptake of C-Leucine into the Avena Coleoptile Figure 20 Effect of Cycloheximide (10’5 E) on the Incorporation of lI‘Lce-sIseucine into the Protein of the Avena Coleoptile P H O U\ U! . CPM x 103/10 Sections CPM x 103/10 Sections 89 l l l l I l IAA ' IAA + Cyclo (10‘5g) I l l I g I i 2 3 1+ 5 6 Time (Hour) l f l 1 1 l IAA IAA + Cyclo (io’sg) A v . f ,. 1; l l 1 -1 i 2 3 1i 5 6 Time (Hour) 90 results indicate that the inhibition of protein synthesis was the cause of the inhibition of elongation, and not a reflec- tion of growth inhibition. Gougerotin Inhibition ig_P1ants In cooperation with Mr. Allen Burkett, NSF Undergradu- ate Fellow, the activity of the new antibiotic gougerotin was investigated. Gougerotin is a specific inhibitor of protein synthesis in both bacteria and animal cells (l5, 17, 98). No report in the literature has demonstrated the biological activity of this compound in plant systems. Hence, several experiments were devised to determine whether this antibiotic was active in plant systems. Gougerotin was an effective inhibitor of auxin-induced elongation in the Alena coleoptile (Figure 21). A gougerotin 5 concentration of 10- fl.was required for 50% inhibition. The time course of 10"“ 14; inhibition is shown in Figure 22. Within 2 hours, gougerotin repressed elongation by 30%; and elongation continued at a much reduced level through 10 hours. A 10'“ M solution of gougerotin inhibited the uptake and incorporation of l)‘I'leeucine into protein by 38% and 50% respectively (Table 21). It also inhibited the uptake of lL‘C-omaminoisobutyric acid. These preliminary studies indicate that gougerotin does repress elongation and protein synthesis in plant tissue. The concentration required for inhibition was about 10 times greater than those reported for a cell-free system from E. coli (17), but comparable to those reported for animal 91 Figure 21 Effect of Gougerotin on Auxin-Induced Elongation in the Avena Coleoptile: Concentration Range Figure 22 -1+ Effect of 10 fl Gougerotin on the Kinetics of Auxin-Induced Elongation in the Avena Coleoptile Elongation (mm) Elongation (mm) \h .r: u: N H 92 l - 10'7 10'5 10'3 10"“ Gougerotin Concentration + Gougerotin (10' g) l 'l I 1 67 12 18. 2“ Time (Hour) 93 systems (15, 98). Since the mode of action of gougerotin is known in detail (15), this antibiotic should prove to be a valuable tool for additional study of auxin-induced growth and other plant responses requiring protein synthesis. TABLE 21 Gougerotin (104+ fl) Inhibition of Amino Apid Uptake and Protein Synthesis in the Avena Coleoptile l“Cum-Aminoisobutyric Elongation Acid Uptake % Inhibition 42 33 1Ll'C-Leucine l“'C-Leucine Uptake TCA Insoluble % Inhibition 38 50 * Incubation was for 4 hours. Uptake and Metabolism.p§, 1L’C-ct-sAminobutyric Acid While working with chloramphenicol, an attempt was made to separate inhibition of amino acid uptake from protein synthesis. In this study as reported in another section, lL"C--c.--aminoisobutyric acid was used. However, before this amino acid was employed, several studies were made using lac-a.- aminobutyric acid. In these investigations it was assumed, as others had assumed, (60) that 1“Cum-aminobutyric acid was luC-a- not incorporated into protein. When the uptake of aminobutyric acid was followed in the presence of chloram- phenicol, uptake was strongly inhibited within 1 hour and 9h thereafter it was taken up at a much reduced level (Figure 23). To test the possibility that some of the radioactivity might be incorporated into the protein fraction, coleoptiles were incubated for l and 2 hours with 1”Cori-aminobutyric acid, then total and 70% ethanol insoluble radioactivity were deter- mined. The radioactivity from lLI'C-«i-aminobutyric acid was readily incorporated into the 70% ethanol insoluble fraction (Table 22). To confirm this observation, coleoptiles were incubated with l”Cad-aminobutyric acid either in the presence or absence of chloramphenicol (51:10“3 E). After A hours incubation total uptake and incorporation into TCA insoluble protein were determined. By this procedure 25% of the radio- activity taken up in the absence of chloramphenicol was incorporated into the protein fraction (Table 23). In the chloramphenicol treated coleoptiles, uptake was greatly reduced, but lh% of the radioactivity was transferred to the protein fraction. A number of plants were surveyed for their ability to incorporate l”Cu-w.-an'1inobutyric acid into protein. For this study 4.5 mm sections were removed from either the coleoptile or hypocotyl of 4 day old etiolated seedlings. After incuba- tion for # hours in luC-a-aminobutyric acid, the total uptake and incorporation into the 70% insoluble fraction was deter- mined (Table Zh). Although there was a considerable varia- tion, all of the plants tested showed significant incorpora- tion into the protein fraction. The amount ranged from 28% of the total for cat coleoptiles to 6.#% for barley coleop- tiles 0 95 Figure 23 1h Effect of Chloramphenicol on the Uptake of C-d-Aminobutyric Acid into the Avena Coleoptile 96 Ansomv mafia sucthoes oI/COI I WJO 97 .mmbapoom oH\sao we commonanm*** .. macawm pm Hooaaosaaoaoanott .ww so: a you no: soapmnsoaHt mam bmmum {moo.e esssmbiow azam Hoabsoo remade Howssoo oHosHomsH «as _i||mmmmmmlmmmmmlln , oaapaooaoo oaobd 0:» mo nodpomnm mapsdomnH dog can cpna odo¢ oahhpsnoadfi¢taioa mo scapegoanoonH one onpAD H MN Manda .maoapoom oa\amo mm commonanmt mma.s mmm.bH m amm.a smao.m a mannaomzH oxcpmo mafia Hosanna men oHHpaooHoo muobm map mo Coapomam oHQSHomQH HocmSpm Rom can opsfi odod canavanoadadudu03H mo noprHoQHooaH one madam: NN mam<9 .maoauoom oa\aao mm comonANM** nonson : you was zoameSoaHt 98 mom.a ems.a bob.~ capsaoosH . Hosanna non nma.oa Hmm.a omm.a causes econ; com poo mm: mmo.m oom.a mmm capsaonsH Hosanna non mam.~ sa~.oa mom.m seesw.na canoe: maapaoq nonaaoso Shoo .mflmflmmwu opswam .1. Homebom mo cacaoam map opaa odod oaampsnoadadndloaa no nodpmaoaaoocH one oxmpmb 3N mqmde 99 The incorporation of radioactivity into the protein fraction could have been due to a direct incorporation of l“Gnu-aminobutyric acid as reported previously for other nonprotein amino acids (38, 87, 115). Another alternative would be the rapid metabolism of luC-a-aminobutyric acid into some other amino acid and its subsequent incorporation into protein. Therefore, Apppa,coleoptiles were incubated for 2 hours with 1“ C-a-aminobutyric acid and the amino acid fraction (TCA soluble. ether insoluble) and the protein fraction (after hydrolysis) separated by paper chromatography. There were 3 radioactive spots in the amino acid fraction. The major spot, 53% of the total radioactivity, cochromato- graphed with l# C-a-aminobutyric acid. The two other spots were not rigorously identified. However, the upper spot (21%) cochromatographed with leucine and isoleucine. The middle spot (26%) cochromatographed with valine and methio- nine. Chromatography of the protein hydrolyzate indicated only 2 radioactive spots. There was no radioactivity in the region of a-aminobutyric acid giving conclusive proof that a-aminobutyric acid was not incorporated into protein.ppp,§p, The 2 radioactive spots chromatographed with leucine-iso- leucine (13%) and valine-methionine (87%). Oxidation with H202 before chromatography did not convert the latter amino acids to oxidized methionine. On the basis of comparative biochemistry, the upper spot was tentatively identified as isoleucine. a-Aminobutyric acid was an effective precursor of isoleucine in E, coli (1), Neurosppra crassa (no). and a lOO plant tissue culture (D. K. Dougall, Unpublished data), Presumedly, a-aminobutyric acid is transaminated to a-keto- butyric acid which is a normal precursor of isoleucine. The rigorous identification of these amino acids will require further work. However, it is significant that; A. 1”Ch-aminobutyric acid is rapidly metabolized, B. its metabolites are incorporated into protein, and C. it cannot be used to separate factors which affect uptake of amino acids from factors which affect protein synthesis. These results emphasize that in all individual cases where amino acid analogs are used, their possible incorporation into protein should be examined. SUMMARY SUMMARY Kinetic analysis of auxin-induced elongation in Apppg coleoptiles revealed a rapid rate of elongation from 1-8 hours followed by a reduced rate from 8-2u hours. In the first linear phase, the rate of auxin-induced elongation was 4 times the rate of control elongation. The ratio of IAA to control elongation during the second phase was 2. After an initial lag period, the deletion of sucrose from the assay medium reduced the rate of elongation. Tween 80 did not affect the kinetics of elongation. Sucrose appeared to serve as a source of energy rather than directly affecting elongation. Under experimental conditions where elongation was stimulated by auxin 2-4 fold, the incorporation of 1uC-leucine into the protein fraction was not enhanced. Chloramphenicol inhibited auxin-induced elongation, lLPG-leucine uptake, and protein synthesis in the Avena coleop- tile. The concentration range for these parameters was 5x10'u to 5x10'3 M, Higher concentrations were required for inhibi- tion in Triticum coleoptiles. Both elongation and protein were markedly inhibited by a solution of 5x10""3 M,chloramphen- icol. At lower concentrations (10"3 and 5x10-“ fl) elongation was stimulated. The stimulation appeared to be due to the bac- tericidal action of the lower concentrations of chloramphenicol. 102 103 Azppg coleoptile elongation was inhibited within the first hour when they were treated with a 5x10'3 fl_solution of chloramphenicol. When treated with a 10"3 M solution, there was a 2 hour lag period before inhibition. Repression of protein synthesis by chloramphenicol (5x10-3 3) followed a time course similar to inhibition of elongation. A direct measure of protein synthesis was difficult to 10 obtain because of the simultaneous inhibition of C-leucine uptake. lL"C--<1--Aminoisobutyric acid uptake was also inhibited by chloramphenicol. The latter amino acid was not incorpor- ated into protein and was not metabolized. In the absence of chloramphenicol it was accumulated against a gradient. Chloramphenicol prevented any accumulation of luC-a-aminoiso- butyric acid. Chloramphenicol also repressed the uptake of IAA, but the inhibition was slight and it was not a principal contributor to the inhibition of elongation. Pretreatment of Azppa coleoptiles with 1“'Cnleucine provided direct evidence that protein synthesis as well as amino acid uptake was being inhibited under experimental con- ditions where elongation was inhibited. l"(C-chloramphenicol into Avena coleop- The uptake of tiles was by diffusion. The internal concentration approached that of the external concentration within 4 hours, but the external concentration was not exceeded with continued incu- bation. The entry of chloramphenicol into the tissue accounted for the lag period before inhibition was observed. However, penetration was not a factor in the low sensitivity of the Avena coleoptiles to chloramphenicol. 10h As determined by thin-layer chromatography and biolog- ical assay, chloramphenicol.was not rapidly metabolized by the Appp§,tissue to an inactive form. After A hours incuba- tion of Apppg coleoptiles in a solution of lac-chloramphen- icol, 80-90% of the extracted radioactivity cochromatographed with authentic chloramphenicol. In addition, the extract still maintained its biological activity in E, 221;. Chloramphenicol inhibition was not stereospecific in the plant systems investigated. L-Threo, D-erythro, and L-erythro-chloramphenicol were effective inhibitors of auxin- induced elongation in Agppg and Triticum coleoptiles, ll"C- leucine uptake and incorporation into the protein of Apppp, and Triticum coleoptiles, 1LPG-cz-aminoisobutyric acid uptake into Apppg coleoptiles, buckwheat root elongation, and gib- berellic acid-induced synthesis of a-amylase in barley aleurone layers. Although there was some variation in the assays, all three isomers had activity similar to the anti- biotic, D-threo-chloramphenicol. The non-specific activity of chloramphenicol in plant tissue was not a result of the non-antibiotic isomers being . racemized to D-threo-chloramphenicol. Cycloheximide inhibited auxin-induced growth in Apppg and Triticum coleoptiles. With a solution of 2x10.6 M, elongation was inhibited by 50%. Solutions of 10-5, 105'6 and 10"7 fl_were equally effective in inhibiting auxin-induced elongation and protein synthesis in Apppp.coleoptiles. 14C- Leucine and th-d-aminoisobutyric acid were inhibited to a lesser degree. 105 In kinetic studies, auxin-induced elongation and protein synthesis were repressed in the first hour and both continued at a much reduced rate throughout the 6 hour incubation. Cyclo- heximide inhibition of protein synthesis appeared to proceed suppression of elongation. Gougerotin, a specific inhibitor of protein synthesis in bacteria and animal cells, inhibited auxin-induced elonga- tion and protein synthesis in.Apgp§ coleoptiles. .A 10-5.5 solution inhibited elongation by 50%. This was comparable to the concentration required for animal systems. Gougerotin should be a valuable tool for additional study of the role of protein synthesis in auxin-induced elongation. l“Ch-aminobutyric acid was rapidly taken up into the Apppg_coleoptile. The radioactivity was incorporated into the lfi protein fraction as readily as C-leucine. Six other plants including cucumber, wheat, pea, lentils, barley, and corn all incorporated radioactivity from 1lI'C-ct-aminobutyric acid into their protein fraction. lu'C-Labeled protein obtained from Avena coleoptiles 14C incubated with ~a-aminobutyric acid was hydrolyzed and the resulting amino acids separated by paper chromatography. 1“C- d—aminobutyric acid was not incorporated into protein, but 2 of its metabolites were incorporated. Hence, 14C-a-aminobu- tyric acid cannot be used to separate factors which affect amino acid uptake from factors which affect protein synthesis. In conclusion, the relationship between the repression of auxin-induced elongation and the inhibition of protein synthesis by chloramphenicol, cycloheximide, and gougerotin 106 support the hypothesis that protein synthesis plays an essen- tial role in auxin-induced elongation. Complete proof of this hypothesis must await the isolation and characterization of the enzymatic activity associated with the newly synthesized protein(s). BIBLIOGRAPHY 9. 10. ll. 12. 13. 14. 15c 16. 1?. BIBLIOGRAPHY Abelson, P. E., J. Biol. Chem. ggé, 335 (195h). Baker, D. B. and P. M. Ray, Plant Physiol.'fig, 345 (1965). Bennett, L. E., Jr., Smither, D. and C. T. Ward, Biochim. Biophys. Acta 82, 60 (1964). Bennett, L. E., Jr., Ward, V. L. and H. W. Brockmann, Biochim. Biophys. Acta 1 3, #78 (1965). Benson, A..A., Bassham, J. A., Calvin, M. Goodale, T. C., Haas, V. A. and W. Stepka, J. Am. Chem. Soc. 22, 1710 (1950)- Bomer. Jo. Am. J. Batany fig 323 (1949). Bonner, J., 2. Schweiz. Forstv. 0, 101 (1960). Bonner, J. and R. Bandurski, Ann. Rev. Plant Physiol. 3, 59 (1952)- Boroughs, H. and J. Bonner, Arch. Biochem. Biophys. fié, 279 (1953)- Brock, T. D., Bact. Rev. g5, 32 (1961). Burchall, J. J., Ferone, R. and G. H. Hitchings, Ann. Rev. Burstrom, H., Physiol. Plantnfi, 199 (1951). Carlo, N., Marks, J. and J. Varner, Nature 180, 1142 (1957). Carter, H. E.,.Gottlieb, D. and H. W. Anderson, Science 1 z, 113 (1998). Casgens, S. R. and.A. J. Morris, Biochim. Biophys. Acta 108, 77 (1965). '— Christiansen, G. S. and K. V. Thimann, Arch. Biochem. Biophys. £8. 117 (1950). Clark, J. M., Jr. and J. K. Gunther, Biochim. Biophys. Acta 6. 636 (1963). 108 18. 190 20. 21. 22. 23. 24. 250 26. 27. 28. 29. 30. 31. 32. 33. 34. 36. 37. 38. 39. 109 Clark-Walker, G. D. and A. W. Linnane, Biochem. Biophys. Res. Comm. 23, 8 (1966). Cleland, R., in Enc clo edia of Plant Ph siolo , W. Ruhland, ed.,-S3%1Hggr:VerlagT—BEEITE¥_1931§IVo1. 14, p. 754. Cleland, R., Nature 18 , 44 (1960). Cleland, E., Nature 299, 908 (1963). Cleland, R., Plant Physiol. 33, 585 (1960). Cleland, R., Plant Physiol. 38, 12 (1963). Cleland, 3.. Plant Physiol. fig. 595 (1965). Cleland, R., Plant Physiol. 41, XLVI (1966). Colombo, E., Felicetti, L. and C. Baglioni, Biochim. Biophys. Acta 11 , 109 (1966). Controulis, J., Rebstock, M. C. and H. M. Crooks, Jr., J. Am. Chem. Soc. 2;, 2463 (1949). Casjens, S. H. and A. J. Morris, Biochim. Biophys. Acta 129.. 677 (1965). Datta, A. and S. P. Sen, Biochim. Biophys. Acta 102, 352 (1965). Davison, R. M., Thesis, University of London, King's College, London (1957). Ehrlich. Jo. Bartz, Q. B... 83111511. R. M., JOSIF. Do A. and P. R. Burkholder, Science 106, 417 (19 7). Ellis, H. J., Phytochem. 3, 221 (1964). Ennis, H. L. and M. Lubin, Fed. Proc. pg, 269 (1964). Ennis, H. L. and M. Lubin, Science 34g, 1774 (1964). Fang, 23)C. and Te Chang Yu, Plant Physiol. 49, 299 19 . Ford, J. H., Klomparens, W. and C.IL. Hamner, Plant Disease Heptr. 42, 680 (1958). Ford, J. H. and B. E. Leach, J. Am. Chem. Soc. 29, 1223 (1948). Fowden, L., J. Exp. Botany 14, 387 (1963). Gale, E. F. and J. P. Folkes, Biochem.J. 53, 493 (1953). 110 40. Herrmann, R. L. and J. L. Fairley, J. Biol. Chem. 222, 1109 (1957). 41. Heyn, A. N. J., Bot. ReV. Q, 515 (1940). 42. Heyn, A. N. J., Compt. Bend. Acad. Sci. 222, 1848 (1932). 43. Hotta, Y. and H. Stern, Proc. Nat. Acad. Sci. U. 3. 5+2. 648 (1963). 44. Huang, M., Biggs, D. R., Clark-Walker, G. D. and A. W. Linnane, Biochim. Biophys. Acta 114, 434 (1966). 45. Hudock, G. A., McLeod, G. G., Moravkova-Kiely, J. and R. P. Levine, Plant Physiol. 32, 898 (1964). 46. Hurst, H. M., Thesis, University of London, King's College, London (1958). 47. Hurwitz, J., Furth, J. J., Malamy, M. and M. Alexander, Proc. Nat. Acad. Sci. U. s. 52. 1222 (1962). 48. Iwasaki, H., Yakugaku Zasshi §2, 1393 (1962). 49. Iyengar,)M. B. S. and R. L. Starkey, Science 112. 357 1953 . 50. Jacobyé B. and J. F. Sutcliffe, J. Exp. Botany 33, 335 19 2 . 51. Jyung, W. H., Wittwer, S. H. and M. J. Bukovac, Nature 221. 921 (1965). 52. Kepps, A. and G. N. Cohen, in The Bacteria, Gunsalus, I. C. and R. Y. Stanier, ed., Academic Press, New York, 1962, Vol. 4, p. 179. 53. Kerridge, D., J. Gen. Microbiol. 22, 497 (1958). 54. Key, J. L., Plant Physiol.,32, 365 (1964). 55. Key, J. L. and J. B. Hanson, Plant Physiol. 6, 145 (1961). 57. Leopold, A., in The Hormones, Pincus, G., Thimann, K. and E. Astwood, ed., Academic Press, New York, 1964, Vol 4, p. 1. 58. Loefer, J. B. and T. s. Matney, Physiol 2001. 25, 272 (1952). 59. Mann, J. D., BioSci. p. 464 (1965). 60. Mann, J. D., Jordan, L. S., and B. E. Day, Plant Physiol. 32. 84o (1965) . 61. 62. 63. 64. 650 66. 67. 68. 69. 700 71. 720 73. 71+. 75- 76. 77. 78. 79. 8o. 81. 82. 83. 111 Margulies, M. M., Plant Physiol. 31, 473 (1962). Mitra, R. and S. P. Sen, Nature 20 , 861 (1965). Morre, D. J., Plant Physiol. 22, 615 (1965). Morris, 1., Nature 2;;, 1190 (1966). Morton, R. K. and J. K. Raison, Biochem. J. 2;, 528 (1964). Netien, G. and 0. Scotty, Bull. Mens. Soc. Linneenne Lyon 25. 86 (1955). Newton, B. A., Ann Rev. Microbiol. ;2, 209 (1965). Nitsch, J. P. and C. Nitsch, Plant Physiol. 3;, 94 (1956). Noodené L. D. and K. V. Thimann, Plant Physiol. 22, 193 (19 5). Noodené6L. D. and K. V. Thimann, Plant Physiol. 2;, 157 19 . Nooden, L. D. and K. V. Thimann, Proc. Nat. Acad. Sci. U. S. Olson,6A3 C., Bonner, J. and D. J. Morre, Planta 55, 126 (19 5 . Olson, A. C. and R. Cleland, Plant Physiol. 32, V (1964). Ordin, L., Cleland, R. and J. Bonner, Plant Physiol. 32, 216 (1957). Palmer, 0. and T. E. Maloney, Ohio J. Sci. 55, 1 (1955). Parthier, B., Flora ;55, 344 (1965). Partheir, B., Nature.22§, 783 (1965). Parthier, B., Malaviya, B. and K. Mothes, Plant Cell Physiol. 5, 401 (1964). Peaud-Lenol, C. and C. De Gournay-Margerie, Phytochem. ;, 267 (1962). Penny, P. and A. W. Galston, Am. J. Botany, 53, 1 (1966). Poole, R. J. and K. V. Thimann, Plant Physiol. 32, 98 (1964). Pramer, D., Exp. Cell Res. ;5, 70 (1959). Rabson, R. and G. D. Novelli, Proc. Nat. Acad. Sci. U. S. 5+5, net. (1960). 84. 85. 86. 87. 88. 89. 90. 91. 92. 93. 91+. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 112 Ray, P. M. and D. B. Baker, Plant Physiol. 22, 353 (1965). Rebstock, M. C., Crooks, H. M., Jr., Controulis, J., and Q. R. Bartz, J. Am. Chem. Soc. 1, 2458 (1949). Rendi, R. and S. Ochoa, J. Biol. Chem. 232, 3711 (1962). Richmond, M. H., Bact. Rev. 25, 398 (1962). Ronnike, F., Physiol. Plant, ;;, 421 (1958). Schlender, K. K., Bukovac, M. J. and H. M. Sell, Phytochem. 5.9 1.33 (1965). ‘ Schrank, A. R., Arch. Biochem. Biophys. 5;, 348 (1956). Sellers, T. F., Jr., LeMaistre, C. A. and A. P. Richardson, in Pharmaco;ogy in Medicine, V. A. Drill, ed., McGraw- Hill Book Co., New York, 1958, p. 1143. Sen, G. A. and S. P. Sen, Nature 122, 1290 (1961). Shibaoka (19645. Shuster, L. and a. H. Gifford, Arch. Biochem. Biophys. 25, 534 (1962). Siegel, M. R. and H. D. Sisler, Biochim. Biophys..Acta 8 , 83 (1964). Siegel, M. R. and H. D. Sisler, Biochim. Biophys. Acta 10 , 558 (1965). Siegel, M. R. and H. D. Sisler, Nature 200, 675 (1963). H. and I. Hurusawa, Plant Cell Physiol. 5, 273 Sinohara, H. and H. H. Sky-Peck, Biochem. Biophys, Res. COM. A._8_. 98 (1965). So, A. G. and E. W. Davie, Biochem.,2, 132 (1963). Spencer, D. and S. G. Wildman, Biochem..3, 954 (1964). Sutcliffe, J. F., Nature ;22, 294 (1960). Sypherd, P. 3., Strauss, N. and H. P. Treflers, Biochem. Biophys. Res. Comm.,2, 477 (1962). Taylor, F. J., Nature 2_2, 783 (1965). Thimann, K. v. and G. M. Loas, Plant Physiol. 2, 274 (1957). van Oberbeek, J., Bot. Rev. 5, 655 (1939). ' Varner, J. E., Plant Physiol. 32, 413 (1964). 107. 108. 109. 110. 111. 112. 113. 114 O 115. 116. 113 Varner, J. E., Ram Chandra, G. and M. J. Chrispeels, J. of Cellular Comp. Physiol. éé, 55 (1965), Suppl. I. Vazquez, D., Nature 2_3, 257 (1964). Weisberger, A. S. and S. Wolfe, Fed. Proc. 23, 976 (1964). Weisberger, A. 8., Wolfe, S. and S. Armentrout, J. Exp. Med. 120, 161 (1964). Went, F. W., Rec. Trav. Botan. Neerl. 25, 1 (1928). Whiffen, A. J., Bohonas, J. N. and R. L. Emerson, J. Bact. 52, 610 (1946). Wollgiehm, V. R. and B. Parthier, Flora 1 4, 325 (1964). Yarmolinsky, M. B. and G. L. de la Haba, Proc. Nat. Acad. Sci. U. S. 55. 1721 (1959). Yoshida, A., Biochim. Biophys. Acta 2;, 98 (1960). Young, J. L. and J. E. Varner, Arch. Biochem. Biophys. 22, 71 (1959).