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This is to certify that the thesis entitled Activation of phosphorylcholine glyceride transferase by gibberellic acid in barley aleurone cells. presented by Yosef Ben-Tal has been accepted towards fulfillment of the requirements for Ph.D. degreein Botany and Plant Pathology WWW [Wk/v Major professor Date July 12, 1974 0-7639 ,.__.. ____._——- —— -— -- "" ABSTRACT ACTIVATION OF PHOSPHORYLCHOLINE GLYCERIDE TRANSFERASE BY GIBBERELLIC ACID IN BARLEY ALEURONE CELLS By Yosef Ben—Tal This work describes an attempt to understand one of the responses of cereal aleurone tissue elicited by gibberellic acid. 'lhe gibberellic acid-dependent increase in phosphorylcholine glyceride transferase activity in barley aleurone layers was not inhibited by cordycepin nor by amino acid analogs , and was inhibited by cycloheximide only in long term (6 hr or more), but not in short term (1 hr) experiments. 'lhese results indicate that enzyme activation rather than synthesis is involved in the gibberellic acid-dependent increase in phosphorylcholine glyceride transferase activity in this tissue. Mixed hcmogenates of aleurone layers prepared from equal nurbers of gibberellic acid treated half seeds and control half seeds showed as much or more activity than would have been expected if all the half seeds had been treated with gibberellic acid. 'Ihis finding further supports the idea that protein synthesis is not required. It also indicates that the effect is not due to gibberellic acid-dependent conformational change of an inactive form of the enzyme molecule . It seems more likely that gibberellic acid causes a rapid change in the level of some component necessary for the increased phosphoryl- choline glyceride transferase activity. 'lhis may explain that the lack of correlation between the nunber of seeds treated with gibberellic acid Yosef Ben—Tal and the activity Observed is not necessary. ‘When a homogenate prepared.fron11ayers incUbated with gibberellic acid for 15 minutes was mixed with a homogenate from control layers incubated for 8 hours there was a 60% increase of activity over the sum of the activities of the separate components. This more dmmn additive effect was also Observed When the layers were incdbated 'with cyctheximide and gibberellic acid but not with abscisic acid and the hormone for 1 hour. This again shows that the gibberellic acid- dependent increased activity of phosphorylcholine glyceride trans— ferase does not require protein synthesis. Thus, the results indicates that the hormone, gibberellic acid, does not regulate the synthesis of phosphoryICholine glyceride transferase but rather its activation. ACTIVATION OF PHOSPHORYLCHOLINE GLYCERIDE TRANSFERASE BY GIBBERELLIC ACID IN BARLEY ALEURONE CELLS By Yosef Ben-Tal A DISSERTATION submitted to Michigan State University in partial fulfilhment of the requirements for the degree of mC'IOR OF PHILOSOPHY Department of Botany and Plant Pathology 1974 ACKDDMEIXMEN'IS The author wishes to thank Dr . J. E. Varner for his guidance throughout this research. 'Ihe help and advice given by the committee members, Professors H. Kende, C. J. Pollard and R. A. Ronzio are deeply appreciated. I would like to express my sincere thanks to Dr. A. Lang for his guidance and advice throughout my studies and work in the Plant Research laboratory, and last but not least I would like to thank my wife , Maya , for her enormous help and courage which enabled me to complete this work. This work was supported by the U. S. Atomic Energy Commission Contract No. AT(ll-l) ~1338. ii TABLE OF CDN'IEN'IS Page lZN'IRODUCI'IONW...” ...... ..........1 Preparation of aleurone layers . . . . . . . . . . . . . 5 Preparation of the enzyme ......... . . . . . . . 10 Determination of phosphorylcholine glyceride transferase ............... . . . . . . 10 Experiments with the supernatant fraction . . . . . . . . 11 14C-Leucine incorporation into proteins . . . ...... 12 Protein estimation ......... . . . . . . . . . . l3 Amfinoacidanalogs.... ...... .........l3 Nitrate reductase tests ...... . . . . . . ..... 13 Thin layer chromatography .......... . . . . . . 13 RESULTS ................. ..........l8 Validation of the assay procedure ........ . . . . 18 Influence of inhibitors on the gibberellic acid dependent increase in the activity of phosphoryl— choline glyceride transferase . . . ...... . . . . 26 Experiments with mixtures of homogenates from gibberellic acid treated and from control tissues . . . . 31 Some properties of an apparent activation component in the supernatant fraction ............... 35 iii (DN'IEN’IS Page The effect of different incubation times of the tissues on the phosphorylcholine glyceride trans ferase activity ........................ . 42 Experiments for testing possible interpretations ..... 46 Influence of abscisic acid on the tissue response to gibberellic acid ..................... 54 DISCUSSION .......................... 56 BIBLIOGRAPHY ......................... 62 iv LIST OF TABLES Amino acid analogs used in the experiments, and their corresponding amino acids ................ Subcellular distribution and recovery of phosphorylcholine glyceride transferase activity . . . . The effect of gibberellic acid and amino acid analogs on phosphorylcho line glyceride trans ferase activity in barley aleurone cells ................ The effect of amino acid analogs and cordycepin on nitrate-induced formation of nitrate reductase in barley aleurone cells .................. Reversal of the inhibitory effect of amino acid analogs on nitrate reductase formation by the corresponding amino acids ....................... The effect of gibberellic acid and cordycepin a1 phosphorylcholine glyceride transferase activity in barley aleurone layers ................ Observed and expected phosphorylcholine glyceride transferase activities in mixtures of enzyme preparations from gibberellic acid treated and control aleurone cells ................. Observed and expected activities from mixtures of different subcellular fractions of gibberellic acid treated and control tissue . A. Half seeds incubated for 4 hr ........... B. Half seeds incubated with gibberellic acid for 1 hr and the control half seeds incubated for 8 hr The effect of ultracentrifugation of the supernatant on its ability to enhance phosphorylcholine glyceride transferase activity .................. 16 27 28 29 3O 32 36 37 38 Tables Page 10. ll. 12. l3. 14. 15. l6. 17. 18. 19. 20. The effect of boiling the supernatant on its ability to enhance phosphoryld’ioline glyceride trans ferase activity ......................... 39 The effect of dialysis of the supernatant on its ability to enhance phosphorylcholine glyceride transferase activity ................... 40 The effect of cold storage of the supernatant on its ability to enhance phosphorylcholine glyceride transferase activity ................... 41 The effect of different incubation times of the gibberellic acid treated tissue on phosphorylcholine transferase activity in the mixtures ........... 43 The effect of different incubation times of the control tissue on phosphorylcholine glyceride trans ferase activity in the mixtures .............. . . . 44 The earliest response to gibberellic acid observed in barley aleurone sys tern .................. 45 The effect of gibberellic acid addition to a cell free preparation from control tissue on the phosphorylcholine glyceride transferase activity .............. 47 The effect on phosphorylcholine glyceride transferase activity in mixtures made from gibberellic acid-treated and control tissues incubated at 3 C .......... . 49 The effect on phosphorylcholine glyceride transferase activity in mixtures prepared from boiled and unboiled tissues .................... . 50 The effect of cycloheximide on the tissue response to gibberellic acid .................... 51 The effect of cycloheximide on the uptake and incorporation of 14 C-L-leucine in barley aleurone 52 cells ..................... . . . . . . Tables Page 21. The effect of the mixing procedure itself on the activities observed ................ . . . .53 22. The inhibitory effect of abscisic acid on the tissue response to gibberellic acid .............. . 55 LIST OF FIGURES Diagram of the barley seed and preparation of "half seeds" ....................... Flow chart showing the important steps of the preparation of barley aleurone layers and the enzyme assay .......................... Standard curve for protein concentration estimation Relationship between the amount of lecithin formed and the amount of enzyme preparation used ....... Time course of the phosphorylcholine glyceride transferase reaction ................... Phosphorylcholine glyceride trans ferase activity as a function of the time of incubation of the tissue with gibberellic acid .................. Phosphorylcholine glyceride transferase activities of homogenized mixtures of aleurone layers from half seeds which were incubated separately either with or without gibberellic acid ..................... viii .15 .20 22 .24 , 31+ SDS ABBREVIATIONS Amdno acid.analogs Abscisic acid Bovine serum albumin (MC-methyl) cytidine diphosphocholine Cycloheximide Gibberellic acid (32F) orthophosphoric acid Phosphorylcholine cytidyl transferase Phosphorylcholine glyceride transferase Rough endoplasmic reticulum Ribonucleic acid Sodium.dodecyl sulfate Tr'ichloroacetic acid INTRODUCTION Gibberellic acid (GA3) controls the production and secretion of hydrolases in barley (Hordeum vulgare L.) aleurone cells (for reviews see Paleg 1965, Yano & Varner 1971). At least three of these hydrolytic enzymes , a-amylase (Filner & Varner 1967) , protease (Jacobsen & Varner 1967) and ribonuclease (Bennett & Chrispeels 1972) are synthesized in response to added GA3. This synthesis begins after a lag period of 8—10 hr. Early changes such as proliferation of rough endoplasmic reticulum (RER) (Jones 1969) , enhanced incorporation of 329 into phospholipids (Koehler & Varner 1973), increased l4C—choline incorporation into a fraction containing endoplasmic reticulum (Evins & Varner 1972) begin about 4 hr after the 6A3 application. Enhanced activity of phosphorylcholine cytidyl transferase (EC 2. 7.7 .15) and phosphorylcholine glyceride transferase (EC 2. 7.8.2) is observed within 2 hr after GA3 has been added (Johnson & Kende 1971). These facts support the hypothesis that RER and polysome formation are required before enzyme synthesis can occur. It was shown (Valdovinos, Jensen and Sicko 1971, 1972) that proliferation of RER precede formation of cellulase in the abscission layer of tobacco pedicels. In mammary glands of mouse Turldngton and Riddle (1970) showed that increased formation of ribosomes and their organization in polysomes followed insuline treatment. Oka and Topper (1971) treated mammary glands with insulin, hydrocortisone and prolactin, and observed proliferation of RER and casein formation; the latter was detectable 1 only after RER formation. Tata (1970) treated rat liver cells with triiodothyronine and hrman growth hormone and observed an increased 14C-glycerol into phospholipids , incorporation of 32?, 14C—choline and RNA, and protein of the RER. The radioactivity appeared in the RER before it appeared in any other fraction. 'lhus , in all these cases, a specific hormone prompted initiation of RER formation and this was followed by protein synthesis , as was the case in the formation of hydrolytic enzymes in the barley aleurone. O'Malley and Means (1974) were able to demonstrate the complete sequence of events fran the binding of a steroid hormone to its receptor protein, over the synthesis of a specific mRNA, to that of new protein in the chick oviduct. Johnson and Kende (1971) reported that the activity of two of the three enzymes involved in lecithin synthesis , namely, PCCTase and PCG'I‘ase, is stimulated by GA3 within 2 hr of application of the hormone. They also showed that the enhanced activities of both, PCCTase and PCCTase, was inhibited by cycloheximide (CH) and by actinomycin D. However , Chrispeels (1973) demonstrated that higi concentrations of mannitol or of ethylene glycol prevented incorporation of labeled amino acids into proteins in barley aleurone layers , meaning that high osmotic pressure will prevent protein synthesis. Ch the other hand, Koehler, Johnson, Kende and Varner (1972) showed that high concentration of mannitol did not inhibit the GA3-dependent increase in the activities of PCCTase and PCCTase. These results introduced some difficulty in interpreting the effects of CH and actinomycin D. If high osmotic pressure prevents protein synthesis but does not affect the increase in the activities of PCCTase and PCCTase in response to GA3, protein synthesis does not seem to be involved in the GA3-dependent increased activity of the two enzymes. But if so, why did CH and actinomycin D inhibit the GA3-dependent increased activity? The influence of CH and actinomycin D in plant as well as in mammalian cells may be related to effects on metabolic processes other than on protein and RNA synthesis. Such effects include: 1) Inhibition of respiration by actinomycin D (Laszlo, Miller, McCarty 8: Hochstein 1966) , 2) Inhibition of glycolysis by actinomycin D (Honig & Rabinovitz 1965), 3) Inhibition of inorganic ion uptake and oxidative phosphoryla- tion by CH (Ellis & MacDonald 1970) , 4) Inhibition of absorption of orotic acid, uridine and cytidine and their conversion to RNA and cytidylic acid by CH (Ross 1968) and 5) The enhancement of enzyme induction by actinomycin D (Ferrari & Varner 1970; Schwartz 1973). Thus, these drugs are not restricted to a specific inhibition of RNA and protein synthesis but may cause a wide range of changes in metabolic processes which may affect the ability of the cells to function or respond normally. Because the enhanced activities of PCCTase and PCGT‘ase is one of the earliest known responses to GA3 in the barley aleurone system, it is important to establish whether protein synthesis is involved in this response or whether the GA3-dependent increased activity of the two enzymes can be explained on the basis of activation of pre-existing inactive enzyme, and this problem was selected as the objective of this research. It is well established that GA3 in the barley aleurone controls the synthesis of several hydrolases. If such an early response as the increased activities of PCCTase and PCGI‘ase is proven not to require protein synthesis this will be an important fact in any concept concerning the mode of action of CA3 in barley aleurone. In order to simplify the work only one enzyme, PCCTase, was studied. The first approach was to replace CH by a group of amino acid analogs (AAAS) as an inhibitor of synthesis of functional protein and to replace actinomycin D by cordycepin as an inhibitor of RNA synthesis. The rationale for the use of AAAs is , obviously, that enzymes synthe— sized in the presence of such analogs will be nonfunctional due to extensive substitution of the analogs for the normal amino acids , whereas enzymes activities which increase due to activation Should not be affected by the presence of the analogs. Amino acid analogs which are known to be incorporated into proteins were chosen (Dawson, Elliott, Elliott & Jones 1969; Sels 1971) . The use of the AAAs in preliminary experiments indicated that they caused only partial inhibition of 32? incorporation into phospholipids while CH caused complete inhibition. Regarding cordycepin (3' -deoxyadenosine) , it has been shown by Ho (1973) that it is much more effective as a RNA synthesis inhibitor than actinomycin D in barley aleurone layers and that it did not have any additional effects on respiration. Another part of the work was done using mixtures of homogenates from GA.3 treated tissue and from control tissue. Use of such mixtures is a way to decide further whether activation or enzyme synthesis is involved in the increased activity , and may provide some indications about the activation mechanism and the role of GA3 in this mechanism. MATERIAL AND METHODS Preparation of aleurone layers The entire procedure used is shown diagrammatically in figure 1. Barley seeds (Hordeum vulgare L. , cv. Himalaya, harvest of 1969, kindly supplied by the Agronomy Club , Washington State University, Pullman, Washington) were cut across transversly twice so that the complete embryo and a small portion of the opposite tip were removed (Fig. 2). 'me remaining "half seeds" were sterilized in 170 NaOCl for 20 min, rinsed 7-8 times with sterile distilled water, and imbibed on sterile, moist sea sand for 3 days in an aluminum foil wrapped Petri dishes at 25C (Chrispeels 8: Varner 1967). For experimental treatments, 25, 50 or 100 half seeds placed in 50, 125 or 250 ml Erlenmeyer flasks containing 5, 10 or 20 ml of 20 mM Na—succinate buffer (pH 4. 8), respectively, and incubated on a rotatory shaker (120 cycles/min) at 28C for various periods of time, depelding on the particular experimental purpose. According to the particular experiment, the incubation flasks also 6 contained 10' M GAB (Merck), 10‘5 M abscisic acid (ABA) (Sigma Chemical 00.), 10 ug/ml CH (Sigma Chemical 00.), 7x10'3 M AAAs (Sigma Chemical 00., Cyclochemical Corp.), 7x10‘3 -3 M amino acids (Sigma Chemical Co.) , 10 M cordycepin (Sigma Chemical Co.) or various combinations of these chemicals. After incubation the half seeds were washed 3 times with ice—cold deionized and double dis tilled water , and the aleurone layers were stripped from the embryoless half seeds with the aid of 2 spatulas. Groups of 25 layers were collected into 25 ml Erlenmeyer flasks , each 5 Figure 1. Flow chart showing the important steps of the preparation of barley aleurone layers and of the enzyme as say . IMBIBITION OF EMBRYOLESS HALF SEEDS ON WET SAND CA3: INCUBATI ON —-—->NO GA3 h h WASH AND PEEL THE ALEURONE LAYERS v v G R I N D AND F I L T E R ll v CA3 HOMOGENATE CONTROL HOMOGENATE MIXTURE A ii A C E N T R I F U G E 500 x g Discard pellet 0 V! v C.E N T R I F U G E 44000 x g Discard supernatant v R E (léC-methyl) GDP-choline :::::::::: REACTION V R A V S U S P E N D T H E R E E T I M E S V D I O A C T I V I T Y ll PELLETS REACTION V W A S H I N G C O U N T I N G Figure 2. Diagram of the barley seed and preparation of "half seeds". aleurone endo perm let cut layer 2nd cut I remaining ”half seed” acu ellum embryo 10 containing 7 ml of cold water as that used for the washings. The layers were either used immediately or frozen overnight . Freezing of the layers overnight had no significant effect on the enzyme ac tivity . Preparation of the enzyme TWenty five aleurone layers were ground in a mortar and pestle with l g of sterilized sea sand and 1 ml of grinding buffer, consisting of 0.1 M Tris HC1 (pH 7.0), 0.5 M sucrose and 0.3 mMMgClZ. Whenever more than one group of 25 layers had received the same treatment during the incubation the homogenates of these groups were combined. The ground slurry was filtered through 8 layers of cheese cloth and the filtrate will be refered to as the homogenate. Whenever a mixture of 0A3 treated layers and non—GA3 treated layer was made, it was done by combining samples taken from separate homogenates. The homogenates were centrifuged (Sorvall RC-2B) for 10 min at 500xg, and the supernatants transferred to new tubes and centrifuged again for 30 min at 44,000xg. The supernatants were usually discarded, and the pellets used for determination of the PCCTase activity. For this, they were resuspended by gentle stirring with the aid of a glass rod in a resuspension buffer (0.01 M Tris HC1 (pH 7.0) and 0.3 mM MgC12) . For resuspending the pellet derived from 25 layers 0.4 ml of buffer were used. All procedures were conducted at 0—5C. Determination of phosphorylcholine glyceride transferase PCCTase activity in the resuspended pellet from the 44,000xg centrifugation was measured by the anoint of (MC-methyl) cytidine diphOSphocholine (New-England Nuclear, Specific activity 40 mCi/mM) ll (GDP-choline) converted to lecithin. To a test tube usually containing 0.2 ml enzyme preparation and 0.2 ml resuspension buffer, 0.1 ml of reaction mixture (50 moles Ttis HC1 (pH 7.0), 10 moles MgCl2 and 0.025 moles (MC-methyl) GDP-choline 0 .045 uCi) were added . The test tubes were placed in a reciprocal water bath Shaker (145 oscillations /min) at 37C and shaken usrally for 15 min. Up to this point the enzyme preparation and the reaction procedure are generally as described by Johnson and Kende (1971) . The reaction was stopped by adding 8 ml of chloroform : methanol (2:1) to the test tubes and mixing vigorously. Proteins were reloved by spinning for 10 min at 10,000xg. The supernatant was washed 3 times with 10 ml amounts of a mixture of methanol : water : chloroform (48:47:3) . The water contained 0870 NaCl and 0.27., MgCl2 to prevent partitioning of phospholipids into the upper phase (containing mainly water and methanol) . The upper phase was removed by suction and the lower phase (chloroform and chloroform soluble compounds) was washed 3 times. After the washings, 3 ml of the lower phase were transferred to scintillation vials, dried and 9 ml of scintillation fluid, con- sisted of 3780 ml. toluene, 30.9 g 2-(4'-t-buty1phenyl) -5-(4'-biphenyl)— 1,3,4-oxdiazole (butyl-PBD) and 1.89 g 2-(4'-bipheny1) -6-phenyl- benzoxazole (PBBO) (Beckman Instruments), was added. The radio- activity in the vials was counted in a Packard Tri carb, model 3390 scintillation counter . This part of the procedure was generally the same as the method used by Koehler (1972) . Experiments with the supernatant fraction Sore experiments were conducted with the supernatant of the 44,000xg run. Whenever supernatant was used 0.2 ml of it was added 12 to the enzyme preparation instead of the 0.2 ml of the resuspeision buffer. Further centrifugation of the supernatant was done in a Beckman L2-65 ultracentrifuge for 60 min at 106,000xg (rotor 50T) . Boiling was done in an ethylene glycol bath (135C) for 10 min. Dialysis was carried out for 5 hr in a large volume of re- suspension buffer in the cold room at 3C (Dialyzer tubing No. 4465-A2 1.5" from Scientific Apparatus) . l4C-Leucine incogmration into groteins For determination of protein synthesis in the preseice of CH, 4 M 14C_L_ embryoless half seeds were incubated for 1 hr with 2x10- leucine (2.5 uCi) (Schwarz/Mann Radiochemicals, specific activity 312 mCi/mmole), 10‘6 M GA with or without 10 mg/ml CH. Twenty five 3. half seeds incubated in 5 ml. of the proper solution were used per treatment. After incubation the aleurone layers were stripped off the half seeds and the excess labeled leucine was washed out by rinsing the layers in ice-cold 20 mM L-leucine for 30 min. The layers were then ground in mortars with pestiles but instead of using grinding buffer 1 ml of 10% sodium dodecyl sulfate (SDS) (Pierce Chemical Co.) was used. Slurry derived from 25 layers was transferred to scintil- 14C-leucine uptake. Another lation vials , dried and counted for total slurry, also derived from 25 layers, was squeezed through cheese cloth and centrifuged at 500xg to remove all cell debris. The pellet was discarded and 10% trichloroacetic acid (TCA) was added to the super- natant to precipitate the proteins. The solution was centrifuged at at 10,000xg for 10 min. The pellet was collected cn fiberglass filter (AP 200 Millipore Corp.), dried at 90C for 20 min, and counted for l4C-leucine incorporation into proteins. 13 Protein estimation Protein concentrations were estimated by the method of Lowry, Rosenbrough, Farr and Randall (1951) with bovine serum albumin as a standard (Fig. 3) . Amino acid analogs Seven AAAs were chosen (Table 1). According to literature each one of them can be incorporated into proteins (Dawson et al. 1969 , Sels 1971) . Nitrate reductase tests The plant material used in these experiments was the same as the material used for the PCGTase determinations and was prepared and in- cubated in exactly the same way. The incrbation medium was either in 0.05 M KNO3 or water. The enzyme assay was carried out as described by Ferrari and Varner (1969) . Whenever AAAs or cordycepin were used, they were added to the incubation medium before the half seeds were introduced into the flasks. Thin layer chromatography The product of the incubation of labeled GDP-choline with the enzyme preparation was identified by tinin layer chromatography (TLC) . The TLC plates used were precoated with silica gel (Silplate-F-22, Brinkmarm Instruments) and activated by heating at 105C for 20 min. Three hundred ml of the lower phase of the chloroform soluble fraction were applied to the TLC plates, with a sample of L-d-lecithin (Sigma Chemical Co.) as marker. The plates were developed in chloroform : methanol : ammonia (65:35z5). When the liquid front reached a line 1 em from the upper end of the plate, the plate was dried and put for 14 Figure 3. Standard curve for protein concentration estimation. Bovine serum albumin was used as a typical protein. .6-‘- /\.750 .2-1 15 ah. L A l U : r . 100 150 200 250 50 pg BSA/2.6 ml SOLUTION 16 Table 1. Amino acid analogs used in the experiments, and their corresponding amino acids. Amino acid analogs Corresponding amino acids 7-Azatryptophan Tryptophan Thioproline Proline o-Fluorophenylalanine Phenylalanine 3,5-diiodotyrosine Tyrosine Ethionine Methionine Methallylglycine Leucine Canavanine Arginine Each one of the amino acids or the AAAS was used at a concentration of 10'3 M. 17 3 min in a closed container with iodine cristals spread on its bottom. Phospholipids appeared as yellow spots on the plate. The location of tine spots was compared with the marker lecithin spot and then the silican gel of each spot was scraped into a scintillation vial and 14 counted for C in a scintillation counter. RESULTS Validation of the assay procedure The amount of 14C-CDP—choline incorporated into lecithin is, under the conditions chosen for the assay , directly proportional to the quantity of enzyme preparation used (Fig. 4). To be sure that the experiments were done within the range of the linear part of the curve, 0.2 ml of enzyme preparation, corresponding to 12.5 layers, was used per test tube. The chloroform-methanol extract of the reaction product was checked on thin layer chromatography plates. Four spots appeared on the plate , but all radioactivity was in one Spot which coincided with the marker lecithin. The time course of the reaction (Fig. 5) shows that during the first 15 min the increase in lecithin formation is almost linear. Therefore 15 min was the reaction time used througlnout the experiments. Figures 4, 5 and 6 confirm Johnson and Kende's report (1971) that GA3 treated aleurone layers have increased phosphorylcholine glyceride transferase activity. The distribution of the enzyme activity was as follows: The 500xg pellet incorporated about 27° of the radioactive GDP-choline in- troduced into the reaction mixture. The 44,000xg pellet incorporated about 8% of the total radioactivity introduced. The supernatant of the 44,000xg spin incorporated about 0.470. Spinning the 44,000xg super— natant at higlner gravity force did not pellet any more enzyme activity (Table 2) . 18 Figure 4. 19 Relationship between the amount of lecithin formed and the amount of enzyme preparation used. The enzyme preparation was made of embryoless half seeds which were incubated with or without gibberellic acid for either 4 or 8 hr. The enzymatic reaction was allowed to continue for 15 min. 0 no GA3, o with GA3, —-——— 4 hr incubation, ---— 8 hr incubation. pmoles lecithin formed/hr/25 aleurone layers 2000" 1.000.. )— 20 : ,L i : - 0.] 0.2 0.3 0.4 Enzyme preparation (ml) 21 Figure 5. Time course of the phosphorylcholine glyceride transferase reaction. The enzyme preparation was made of embryoless half seeds incubated with or without gibberellic acid for 4 or 8 hr. TWO tenth ml of enzyme preparation was used for each reaction. pmoles lecithin/25 layers 6000 4000 2000 l 5 IOI 5 22 Reaction Time 6A3 (8 hr) Mix (|=8) 6A3 (4 hr) Control (8 hr) Control (4 hr) 60 90 (min) Figure 6 . 23 Phosphorylcholine glyceride transferase activity as a function of the time of incrination of the tissue with gibberellic acid. TWenty-five embryoless half seeds were used per treatment. The reaction time was 15 min; 0.2 ml enzyme preparation was used for a reaction mixture. The results are means of 3 separate experiments. formed / hr / 25 layers lecithin pmoles 24 O 3000 -- GA3 O 2000 " O O 1000 .. ‘ k CONTROL ‘ __H e; i e t 1 l 4 6 8 10 12 Incubation time mo GOHumnooaH an e Hmumm mumxma mcousmam Eoum woumamue meancm .munuxHE cowuommp comm cu wooed mumz mcwHQLOImoo Aaanumeloqav mo Ho: mqo.o .oaan onu GH caonm wucmSummuu onu ca woman man: .mucmEHpmexo oomumwwao m m0 momoz N men as can mac wroooee to ccwcecccccm q NNH ac mma Houucoo wxoooqq mo unnumouoasm an tees Heea awmm mac waoooee cc ccaada cm amen Nmoa mafia achccco .mwooose we cdaaca as sea ans caNH mac wroom to ceased ON eon Hem cam Hoeccco wroom to ceased zufl>fluum Hmuou mo N Camacho we\woaoea mpomma mm\moHoEe Eco ucmEummuH coauomum .kuH>Huom omwuommcmuo oefiuooaaw mcflaonoazuonemone mo auo>ooou eon cowunnfluumfle umasaamon5m .N anmH 26 Influence of inhibitors on the gibberellic acid-dependent increase in phosphorylcholine glyceride trans ferase activity Application of AAAS to the half seeds during incubation had no effect on the GA3-dependent increase in the activity of the enzyme (Table 3). To clneck whether the combination of these 7 AAAS at this concentration can indeed prevent the format ion of functional enzymes, the combination was tested for its influence on the formation of nitrate reductase in the presence of its inducer - nitrate. As can be seen in Table 4, the AAAS do prevent the formation of functional nitrate reductase under the same conditions as used for testing PCGTase activity. Another group of experiments (Table 5) showed that the inhibitory effect is not due to a non-specific effect of the AAAS (e.g. ionic strength) because an isoosmotic concentration of the corresponding amino acids did not affect the formation of functional nitrate reductase. Furthermore, the inhibitory effect of the AAAS on the induction of functional nitrate reductase was reversed by adding the corresponding amino acids at twice the concentration of the AAAS (Table 5). If the GA3-dependent increase in activity of PCGTase does not require protein synthesis , it is not likely that RNA synthesis is re— quired. Nevertheless, this possibility was tested by the use of cordycepin. Cordycepin has been shown to be a more effective inhibitor of RNA synthesis in barley aleurone than actinomycin D (Ho, Keates & Varner 1973) . Cordycepin did not inhibit the GA3-induced increase in the PCGTase activity (Table 6), but it did inhibit the formation of functional nitrate reductase under similar conditions (Table 4). Thus, the GA3-dependent increase of PCGTase activity does not require RNA synthesis either . 27 Table 3. The effect of gibberellic acid and amino acid analogs on phosphorylcholine glyceride transferase activity in barley aleurone cells. Treatment during incubation Z of control Standard deviation Control 100 3.9 10”6 M GA3 161 31.4 10’6 M GA3 and 7xlO-3 M AAA'S 167 34.2 Enzyme prepared from aleurone layers after 4 hr incubation of half seeds. The control activity is equal to 1229 pmoles lecithin. Results of 3 separate experiments each one with 2 replications. 28 Table 4. The effect of amino acid analogs and cordycepin on nitrate- induced formation of nitrate reductase in barley aleurone cells. nmoles nitrite Standard Treatment during incubation formed/hr/layer deviation Water 2.82 .05 0.05 M KNO3 10.35 .09 0.05 M KNO3 and 7x10"3 M AAA's 3.54 .05 0.05 M KNO3 and 10_3 M cordycepin 3.57 .06 In vivo assay of aleurone layers prepared from half seeds after 4 hr incubation in the solutions shown. 29 Table 5. Reversal of the inhibitory effect of amino acid analogs on nitrate reductase formation by the corresponding amino acids. nmoles nitrite Treatment during incubation formed/hr/layer Control 9.41 7xlO_3 M AAA's 3.01 14x10-3 M amino acids 10.25 7x10-3 M AAA'S and 14x10”3 M amino acids 9.2 In vivo assay of aleurone layers after 5 hr 0.05 M KNO 3 and 10'6 M GA 3. incubation of half seeds in 30 Table 6. The effect of gibberellic acid and cordycepin on phosphoryl- choline glyceride transferase activity in barley aleurone layers. Standard Treatment during incubation % of control deviation Control 100 4.9 10‘6 M GA 164 11.6 3 10"6 M GA3 and 10—3 M cordycepin 147 16.3 Enzyme was prepared from aleurone layers after 4 hr incubation of half seeds. The control activity was equal to 1162 pmoles lecithin. The results are means of 3 different experiments. 31 Experiments with mixtures of homogenates from gibberellic acid treated and frcmn control tissues The evidence described so far indicated that protein synthesis is not required for the GA3-dependent increase in the activity of PCGTase. Another approach was taken to determine what kind of activation was involved. Samples of homogenates obtained from GA3 treated and from control tissue were carbined in equal amounts or in other proportions, fol- lowed by the regular procedure for obtaining the PCGTase preparation (44,000xg pellet) . The rationale behind this approach was that if protein synthesis during tine incubation with 0A3 was the reason for the enhanced activity, the activity of the mixture should equal the sum of the activities contributed by each of the homogenates . But if GA3 caused enzyme activation which was not based on a conformational change of an inactive molecule then, the observed activity could be different (larger) than that sum. Experiments with mixtures of homogenates from GA3 treated and con- trol tissue are shown in Table 7 and in Figure 7. It can be seen that the PCGTase activity in the mixture is larger than the expected activity, i.e. the sun of the activities of the two separate homo— geates. This result constitutes evidence for the existence of some kind of GA3-dependent activation of PCGTase that becomes apparent when homogenates of GA3 treated and control tissue are mixed. This unknown component is produced in excess because even when the mixture contained only 307.. of homogenate from layers treated with GA3 it still produced the same amount of lecithin i.e. had the same PCGTase activity as if all the layers had been treated with 0A3. 32 . N Nona + mwn .osonm moowuofiom mnu ofl mooom me5 mo wan . N qwma + New mosam> :pouooaxm: .mucoafiuoexo uamumwmwo 0 Mo memos nowumesoafl Mn q umumw oedema moonooam Scum wounaoum mmahuom «Noe mmma mooa mmqa ououxaz meme same mac 2 c-0a mmn Nam Houuooo emuomaxm wo>uomno omuomexm om>womno uaoaumone Camacho wE\Hn\eoSnow aflnuflomfi mmHoEe mumkma mN\HL\noEuom cflnufioma moaoae .mHHmo ocousmam Houucoo wow woodman eflom Ofladmwoenflm 80Hm mSOflumumemue oskmcm mo monouxfle Ge mmflufl>fiuom mmmuwmmomnu cowuooxam moflaonoazuonemonm pmuommxo wow em>uwmeo .m canoe Figure 7. 33 Phosphorylclnoline glyceride transferase activities in mixtures of homogenized aleurone layers from half seeds which were incubated separately eitlner with or without gibberellic acid. Layers from the two sources , at different rations , were homogenized and the mixture tested for plnosphorylcholine glyceride transferase activity. Both gibberellic acid treated end the control layers were incubated for 4 hr. The results are means of 3 different experiments. Shaded areas Show expected values. / Activity expected from the control homogenate. Activity expected from the gibberellic acid \\ treated homogenate. 34 R\\\\\\\\\\> Reg/hm m,///////////A ///////////z._a ‘11 .6/60 A 35 Some prOperties of the apparent activation component in the supernatant fraction Once evidence for a GA3-dependent component which is able to activate PCGTase was found it became important to determine the distribution of this component in the different fractions , and some of its properties. Different fractions from GA3 treated and control tissues were combined and tested for the activity of PCGTase. The activity of mixtures of 44,000xg pellet and 44,000xg supernatant exceed the activity expected by calculation (Table 8) . The increase in activity over the expected level was considerably larger than in mixtures of the two homogenates , probably because the PCGTase activity in the supernatants is quite low. Pellets from 0A3 treated or control tissue was mixed with pellet from either tissue. Thus , even mixtures of supernatant and pellet from control tissue give the same, increased PCGTase activity as mixtures in which at least one component comes from 0A3 treated tissue. It must therefore be assumed that whatever this activiating component is, it exists both in 0A3 treated layers and in control layers. After the £14,000an centrifugation this com— ponent remains in the supernatant and activates the enzymes when the supernatant is added to the pellet. _ Additional experiments showed that further ultracentrifugation (106,000xg) did not precipitate tine activating component (Table 9). Boiling reduced the effectiveness of the supernatant by only 137. (Table 10) . The activating component disappeared during dialysis from the dialyzirng tube in a relative short time (5 hr) (Table 11) . On the other hand, storage of the supernatant in the refrigerator at 3C for 24 hr reduced its effectiveness by 247., and storage for 72 hr by 497. (Table 12). 36 Table 8. Observed and expected activities from mixtures of different subcellular fractions of gibberellic acid treated and control tissue. A. Half seeds incubated for 4 hr. Relative Activity Z increase Fraction over # Fraction used Observed Expected expected 1 Pellet of control 50 2 Pellet of CA3 61 3 Supernatant of control 3 4 Supernatant of CA3 3 Mixture of l and 2 143 111 29 Mixture of 1 and 3 130 53 145 Mixture of l and 4 136 53 156 Mixture of 2 and 3 141 64 120 Mixture of 2 and 4 141 64 120 The PCGTase activity observed in the pellet of the control tissue has been taken arbitrarily as 50%. The expected values are the sums of the separate activities of the fractions involved in a given mixture. The results are means of 3 different experiments. 37 Table 8. B. Half seeds incubated with gibberellic acid for 1 hr and the control half seeds incubated for 8 hr. Relative Activity % increase Fraction over # Fraction used Observed Expected expected 1 Pellet of control 50 2 Pellet of CA3 treated 37 3 Supernatant of control 4 4 Supernatant of CA3 3 treated Mixture of l and 2 132 87 52 Mixture of 1 and 3 116 54 115 Mixture of l and 4 129 53 143 Mixture of 2 and 3 113 41 176 Mixture of 2 and 4 113 40 182 The PCGTase activity observed in the pellet of the control tissue has been taken arbitrarily as 50%. The expected values are the sums of the separate activities of the fractions involved in a given mixture. The results are means of 3 different experiments. 38 Table 9. The effect of ultracentrifugation of the supernatant on its ability to enhance phosphorylcholine glyceride transferase activity. Fraction # Fraction used Relative activity 1 Pellet of 44,000xg 39 2 Supernatant of 44,000xg 2 3 Supernatant of 106,000xg 2 Mixture of l and 2 100 Mixture of l and 3 98 Means of 6 different experiments. The activity of the mixture of the pellet and the supernatant of 44,000xg has been taken arbitrarily as 100%. Part of the 44,000xg supernatant was centrifuged for 1 hr at 106,000xg and 0.2 m1 of the supernatant after ultracentrifugation was mixed with 0.2 ml of the resuspended pellet. Half seeds were incubated 4 hr with or without GA . The pellet was made from control tissue, the 3 supernatant from GA3 treated tissue. 39 Table 10. The effect of boiling the supernatant on its ability to enhance phosphorylcholine glyceride transferase activity. Fraction # Fraction used Relative activity 1 Pellet 39 2 Supernatant 2 3 Boiled supernatant 0 Mixture of l and 2 100 Mixture of 1 and 3 87 Means of 4 different experiments. The activity of the mixture of the pellet and the supernatant of 44,000xg has been taken arbitrarily as 100%. Part of the supernatant was boiled for 10 min and then 0.2 ml of it was mixed with 0.2 ml of the resuspended pellet. Half seeds were incubated 4 hr with or without GA . The pellet was made from 3 control tissue, the supernatant from GA3 treated tissue. Table 11. The effect of dialysis of the supernatant on its ability to enhance phosphorylcholine glyceride transferase activity. Fraction # Fraction used Relative activity 1 Pellet 41 2 Supernatant 3 3 Supernatant after dialysis l Mixture of l and 2 100 Mixture of l and 3 38 Means of 3 different experiments. The activity of the mixture of the pellet and the supernatant has been taken arbitrarily as 100%. Part of the supernatant was put in a dialyzing tube for 5 hr in the cold room at 3C. The dialyzing tube was kept in a large volume of resuspension buffer which was replaced every hour. Half seeds were incubated 4 hr with or without GA . The pellet was made from control 3 tissue, the supernatant from GA3 treated tissue. 41 Table 12. The effect of cold storage of the supernatant on its ability to enhance phosphorylcholine glyceride transferase activity. Fraction # Fraction used Relative activity 1 Pellet 39 2 Supernatant 2 3 24 hr old supernatant 1 4 72 hr old supernatant 2 Mixture of #1 and #2 100 Mixture of #1 and #3 76 Mixture of #1 and #4 59 Means of 3 different experiments. The activity of the mixture of the pellet and the fresh supernatant has been taken arbitrarily as 100%. Supernatants from previous days were kept in the refrigerator at 3C for 1 or 3 days. 0.2 ml of these supernatants were added to 0.2 ml of a resuspended pellet. Half seeds were incubated 4 hr with or without GA . The pellet was made from control tissue, the supernatant 3 from GA3 treated tissue. 42 The effect of different incubation times of the tissue on the phosphorylcholine glyceride trans ferase ac tivi g Johnson and Kende (1971) showed that the GA3-dependent increase in the activity is observed within 2 hr and reaches a maximum at 8 hr; at the maximum the activity is approximately 3 times greater than that of the control tissue. The activity of the control tissue remains unchanged for at least 12 hr. These observations were confirmed (Fig. 6) and it was shown that even at zero time, in other words with- out any incubation, the same level of activity can be found. Most of the experiments reported up to this point were conducted with half seeds which were incubated 4 hr. In order to determine how soon the response to GA3 is observable, the incubation time for the GA3 treated tissue was decreased. The results (Table 13) indicate that when this was done the PCGTase activity in the mixture is also somewhat decreased. But when homogenate from tissue that had been incubated with CIA3 for 15 min or one hour was mixed with homogenate from control tissue incubated for 4 hours and the percent increase over the expected valtes was calculated , it was found that the activity in the mixtures actually increased as incubation time of the 0A3 treated tissue was decreased. In contrast, when the incubation time of control tissue was extended, it caused an increased PCGTase activity of both the observed and the calculated activity over the expected values of the mixture without a change in the activity of the control tissue (Table 14). The conclusion from these experiments is that there are two different components - one in each tissue - which are involved in the activation process of PCGTase. The first one, already mentioned, existes in the supernatant of both kinds of tissue, and is capable of activating the inactive form of the enzyme in the pellet of the control 43 .muou ixfia oo>am m CH eo>ao>ofl muoaame onu ca mmaufl>fluom oumuwemm one mo menu new mum mosam> emuomexm OLE .%Hm>fiuomewmu Mg m can u: q mp3 mumm vacuum and umHHm can ca mououxfia one Ham How mvoom mam: Houuaoo osu How maflu coaumn50ow one .auw>fiuum Now on :ome me pom vacuum onu CH an m Houwm pow wudmfiwumexm mo pom umpfim map me Gowumnooofi H: q umuwm mommfiu Houuooo onu ca omauow ofifiuflomfi mo unseen one .muaoawumexo unmuomwflp m mo mono: a Ana «ma om awe w mm oma mma cm a mm Boa «we mm H mm «ma mNH om em a oe woe omH mm H em om wee we mm.o wouummxm empowexm eo>uomeo Houuooo mo mounuxfla men muaoaeroo oumumemm mafia cowumnsooH ommouoaa N mo mmflua>wuom mfiH onu mo mmHuH>Huo< .mmuduxHE onu ca >uw>wuom ommuwwmamuu mpfiuwoaaw oCHHOLOHmponemoca so mommwu nmummuu meow oaaamuonnaw who Mo moEHu coaunnsooa uomumwwfle mo oommmm one .mH oHan I>Huom wumumamm msu mo mECm mLu mum mmCHm> vmuomQXm mSH mwmmm HHCL wmummuu um mammHu HopuCou man CH vaMow CHLuHomH mo uCaoEm MCH .muauxHE Cm>Hw m CH Um>Ho>CH mCOHumeamHm msu mo mmHuH <0 wCu mo mBHu COHumfisoCH wCH .HL H was mmusuxHE mnu HHm CH @mms .kuH>Huom Nom mm memu mCB COHCCQCUCH CC q .mquEHHmaxm quHmwwHw m Mo mCmmz «a MOH oom oq m mm NOH qu om q NH HOH mHH qq mm H wmuommxm wmuumaxm wm>wmmao HouuCoo m<© ACLV Hm>o mmmmuuCH N meCuxHE mnu mo mmHuH>Huum mLH mquCanou mumummmm msu mo meuH>Huo< mEHu COHuwnsoCH |H%uosmmonm Co .mmusuxHE wCu CH >uH>Huum mmmummmCmCu meHmo%Hw mCHHoso mammHu HouuCoo msu mo mmaHu COHumpCoCH quumHMHm mo pummmm 0:8 .qH mHan 45 .mpnuxHE Cm>Hw m CH wm>Ho>CH mCOHumHmCmua mnu mo mmHuH>Huum wuwummmm mnu mo mECm msu mum mmCHm> wmuomaxm may .Nom mm mewu Cmmn mm: CoHumnCoCH Hg w Cmumm mammHu HouuCoo may mo wmahow CHCuHomH mo quoEm mfiH .mquEHuwaxm quumMMHv m mo mCmmz mq mad woa m cam C C0 musuxflz mm Hoa «ma N Ham H C0 mysuxflz me C; H mummno mEHH quEummuH umnasz HQN/O mwmmHoCH N zuH>Huum m>HumHmm COHumnsoCH quEummuH .Emumzm mCouanm hmHCmn mnu CH wm>ummno vHom UHHHmHmnnHw Cu meommwu umeHHmm mCH .mH mHan 46 tissue . However , there is no evidence that the GAB—dependent activiation of PCGTase is indeed the same as the activation by the component from the supernatant. The second component is in the control tissue. This component seems to be activated or used only after the control tissue was combined either with GA3 treated tissue or with supernatant. This second component is time dependent , longer incubation time resulting in more of this component available for the PCGTase system. 'lhe relationships between these two components will be discussed more thoroughly in the discussion. However, it is clear from the results that the second component is the one which controls the level of activiqr but it has to be "switched on" by the GA3-dependent component. 'lhis ”switching on" of the system by CA3 sears to occur rather quick as only 15 min of incubation with GA3 were enough to bring the system to its maximum level (Table 15). Experiments for testing possible interpretations At this stage of the research several experiments were conducted to test different possible explanations for the observations so far described. First, in order to check whether the GA3 molecule itself is capable of activating the enzyme , GA3 was added directly to homogenate prepared from control tissue. There was no increase of the PCGTase activity over homogenate without GA3 (Table 16) . Second, the effect of incubating the tissue at a low temperature with and without GA3 was tested. Control and GAB treated half seeds were incubated in the cold room at 3C for 8 hr or for 15 min. Their activities and the activities of their mixtures were compared with the activities of half seeds incubated at the regular temperature (28 C). 47 Table 16. The effect of gibberellic acid addition to a cell free preparation from control tissue on the phosphoryleholine glyceride transferase activity. Treatment Relative activity % increase Treatment over #. expected Observed Expected 1 Control 50 2 6A3 58 3 GA added to the control 3 homogenate 53 Mixture of #1 and #2 151 108 40 Mixture of #1 and #3 104 103 1 Means of 2 different experiments. GA3 was added to the homogenate after the slurry was squeezed through the cheese cloth. The activity observed in the control has been taken arbitrarily as 50%. The expected values are the sums of the separate activities of the preparations involved in a given mixture. 48 The results show (Table 17) that the GAB—dependent increase in activity does not occur in extracts from tissues incubated at low temperature . To test whether specific configurations of proteins are necessary for the mixing effect, both homogenates were boiled and mixtures were prepared from boiled control and unboiled GA3 treated tissue, or conversely, boiled (3A3 treated tissue and unboiled control. 'lhe observed PCGTase activities were similar to the sum of the separate activities, i.e. similar to the expected activities (Table 18). The contribution of the boiled homogenate to the activity of the mixture in either case was zero and there was no "mixing effect" when boiled homogenate of either kind was a component of the mixture. To test if there is any effect of CH on the GA3-dependent activation, half seeds were treated with both GA3 and CH for 1 hr, and the homogenate was mixed with homogenate from control tissue incubated for 8 hr. The activity observed in this mixture was similar to the activity obtained in an analogous mixture of control tissue and tissue treated with GA3 but no CH (Table 19) . To make Sure that the CH is able to penetrate into the tissue and is effective in 14C_L_ preventing protein synthesis , the uptake and incorporation of leucine into barley aleurone layers within 1 hr in the presence of GA3 with or without CH was measured. The results are shown in Table 20; they indicate that CH, while not suppressing the "mixing effect", does interfere with protein synthesis in this system within 1 hr. Another possibility which was tested was the influence of the mixing procedure itself on the enzyme activity. It can be envisaged that short incubation releases some inhibitor and that this and not an action of GA3 is the reason for the "mixing effect". Homogenates prepared from control tissue which had been incubated for different 49 mooumamm oCu mo mECm oCu ouo mmon> wouoomko oCH H: w “comm mommHo HouoCoo who mo wmehom CHsoHowH mo uCCoEm 05H .opouxHB Co>Hw m CH wm>Ho>CH mCOHuonCon osu mo moHuH>Hoom .huH>Huom Non mm Coxou Coon was 0 mm um COHumpCoCH .muCoEHHomxm quHomme m mo mCmoz m mm mm m CCm H mo ououtz w wm 00H 5 CCC H mo muoutz we mm qu 0 CCC H m0 ousutz we m £5 2 mac C we 3 fie 2 mas o 8 m S w mac m «2 mm .3. w mac s we m CH8 mH HouoCou m Ho m CC w HouuCoo m cm mm CL w HouuCoo H pouoomxo wooooaxm wo>uomno muoomuoaaoe oEHH oCmEumoHH HonECz Ho>o ommoHoCH N moH>Huom m>HumHom COHuthoCH COHomnooCH uCoEumoCH .u m om wouwnooCH moommHo HopuCoo ow commouu Scum moms mououxHE CH moH>Hoom ommuomemHu owHHoosz oCHHosoH%Honmmo:m Co uoowwo oCH .mH oHan 50 .oHCoKHE Co>Hw m CH po>Ho>CH mCOHumeCmHa mLo mo moHuH>Hooo womuwoow osu mo mfism oso who moCHw> omuooaxm mza .Nom mo memu Como mm: HouuCco ono mo on>Hoom one .posoHHow mmz onovwooua HmHCmop wan pom COHommCMHCuCoo CoCu ow CHE OH How pmHHon me oumCowofioC Como mo onom .moCoaHuoaxo uCommmme N mo mCmmz o N N a saw N C0 upsuxfiz s OH NH s sum H to whamxflz NH- Ho mm m saw N C0 mysuxfiz Nm OCH NsH m saw C so mpauxfiz 0 mac smfifiom q es mac m H. HOHHCOU COHflom N om Coacaou C wouoomxo wouooaxm po>ummno quEummHH HonECz Scum I!» 11! ooCoHoHMHw N on>Hoom o>HumHom uCoEumoHH .mmomCoonOL meHOQCC CCm uwHHon Scum woummoum mououxHB CH on>Hoom ommuommCouo mpHumosz oCHHomoH%H0£Cmona Co ooommm oLH .wH oHnt 51 .ouauxHE Co>Hw m CH wo>Ho>CH chHuoummoua ono mo moHuH>Huom muouomom onu Ho meow oLu who moon> wmuoomxo one .uuma wCooom emu CH NOOH mo COHumnooCH H: o Houmm odmmHo HouuCoo Eouw zuH>Huoo osu paw .oHLou ofio Ho upon umuHm ozu CH Nom HHH>Huom mmmeccm meH mo memu Coon mom COHuonCoCH MC w Hmumo mommHu HouoCoo EOHM mCOHomConHQ CH .muCoEHHomxo uCoumMMHw q mo upon @Cooom osu .5 mo Coma m mH upon umHHw oLH mm- mHH C; 0 mo Hs\msOH sum muompo oEHH uCoEummHH HoHECz Eoum ooConwHHw N zuH>Hooo m>HumHom COHuonooCH uCoEuomHH .oHom oHHHonQQHw oo omCoamoH mommHo o5“ Co owHEonLOHowo Ho ooommo oge .mH mHamH 52 Table 20. The effect of cycloheximide on the uptake and incorporation of l[*C-L-leucine in barley aleurone cells. Relative activity Treatment Uptake Incorporation Control 100 52 10 ug/ml CH 102 11 Means of 2 different experiments. Half seeds were incubated for 1 hr with GA3 radioactive leucine and the medium indicated in the table. The radioactivity recovered from the uptake measurements of the control was taken as 100% activity. 53 amp mo meow ecu mum moCHm> wouoomxo oCH Houmm mommHo HouuCoo osu Scum ooEHow CHnuHooH mo uCooEm one .oHouxHE Co>Hw m CH wm>Ho>CH mCOHumHoCoHC osu mo moHuH>Hoom oumummom .hoH>Huom Non mm meCC Coon mm: COHumpsoCH H: w .mquBHHoaxm quHoMMHo N m0 mme2 ml mm qm m ow H m0 ousutz o mOH mOH q wCo H mo oHCutz N+ «OH OCH m ow H mo musutz me CC H Homho oEHB oCoBuonH Boum ouCoHommHv N muH>Huom o>HumHmm CcHumnCoCH uCoEumoHH .wo>meno moHoH>Hoom oCo Co HHomuH oHCoooOHa wCHxHE ono mo uoomwo oHH .HN oHan 54 periods of time were mixed together and their PCCTase activity was measm‘ed. As can be seen in Table 21, there is no difference between the regular control of 8 hr incubation and the various combinations of the mixed controls. Influence of abscisic acid on the tissue response to gibberellic acid Johnson and Kende (1971) showed that the GA3-dependent increase in PCGTase activity was inhibited by ABA. This phenomenon was confirmed and it was fomd that the inc1bation of tissue with both GA3 and ABA for 1 hr not only prevented the GAB-dependent increase in PCGTase activity in the 44,000xg pellet, but also prevented the expression of the "mixing effect" in mixtures of homogenates from tissue treated with (3A3 and ABA and homogenate prepared from control tissue (Table 22) . 55 r 1|" I NH N .OHH>HOOH NOO mm Coxmo mm3 upon wCoomm ono CH CcHuonCoCH H: w Houmm ow “puma umHHm map CH CH q Hmumm oCmmHu HouoCoo map CH pmfiuow CHCuHooH mo uCCoEm may moHuH>Huom oumnmoom mno mo mBCm oCu mum mmCHm> wouoomxo one .oHCuNHE Cm>Hw m CH wo>Ho>CH mCOHumHmCoHC wsu mo .moCoaHHoCxo uCoHoHMHw m we mCmoz O OHH OHH H OOH O Ho OHOHOHC OH HHH NNH H qu H H0 OHOOtz HH HHH OOH O Ham N H0 musutz HO HOH NON N Ham H CO OHOHOHz HO HO H OOH z OuOH OOH OHO H NO C; O OOH z OIOH O HO CO H OHO N Om H; O HOHHOOO H O NO NOH H Ham O Ho OHOHtz H- NO OO H OOH H CO OHOOtz Om NNH NOH O OOH N No musutz OO NNH OOH N OOH H CO OHOOtz NH H; H OOH z O-OH Oam OHO H OO H: H «OH 2 OuOH O NH CO H OHO N OO H; H HOHOOOO H wouoomxo Couoomxm oo>Homno oEHH uCoEomoHH HocECz Scum ooCoHomew N >oH>Huom o>HumHom COHumcooCH oCoEumoHH .uHom oHHHwHonnHw 0o omCoamou mommHu one Co pHom onHomnw mo ooowmo NHOCHLHLCH.oLH .NN oHan DISCUSSION a-Amylase is synthesized in response to GA3 in barley aleurone layers (Filner & Varner 1967) . 'Jhis synthesis begins 8-10 hr after the application of the hormone. During this lag period several processes such as increased polyscme formation (Evins & Varner 1972) , increased RER formation (Jones 19 69) , increased incorporation of 14C-choline into a membrane fraction (Evins 6: Varner 1971) take place in the tissue. These processes are indications of the build-up of the machinary which is required for enzyme synthesis. But as these processes do not occur until 4 hr or more after treatment with GA3 it cannot be postulated that they are regulated by GA3 in a direct manner . Proliferation of RER requires lecithin, and the synthesis of lecithin is catalyzed by PCCTase and by PCGTase. Both these enzymes are enhanced by GA3 within 2 hr after application (Johnson & Kende 19 71) , and the later processes described above may be an indirect result of the CA3 effect on these two enzymes. As demonstrated in this thesis, at least one of these enzymes, PCGTase, is activated rather than de novo synthesized in response to GA3. The fact that no inhibition is caused by cordycepin (Table 6) rules out the possibility that newly synthesized RNAs are involved in the GAB-dependent increase of PCGTase activity. The analogs experiments (Table 3) ruled out the possibility that enzyme synthesis is involved in the GA3-dependent increased activity of PCGTase. Absence of de novo synthesis of the enzyme is strengthened by the fact that a mixture of homogenates prepared from tissue treated with both CA and CH and 3 56 57 from control tiSSLe showed the same level of activity as a mixture of GA3 (no CH) treated and control tissue (Table 19) . The mixing experiments indicated that fine increase in activity involves some kind of an activating component, rather than a con- formational change of an inactive molecule , because in the latter case the resulting activity should be proportional to fine amount in the homogenate mixture of tissue that had been incubated with GA3 and not to exceed the activity of homogenates made only from tissue which had been incubated with 6A3. An activity exceeding the activity level in homogenates of GA3 treated tissue was also found when these homogenates were mixed with the supernatant of the 44,000xg centrifugation. In this case, the increase could be up to 15070 over the expected activity. This activation was obtained with sxpernatants from bofin GA3 treated and control tissue. From fine results so far obtained on fine activation of PCGTase by the supernatant it is not possible to say whefiner fine effect of the com- ponant in the supernatant is the same as fine GA3-dependent effect. One connection that may be envisaged would be that the GA3 during incnbation changes the properties of the membrane and therefore allows the com- ponent frcm fine supernatant to penetrate into or associate wifin the membrane. In a cell free system this change of membrane properties would not be necessary because the activating component from the snper- natant is introduced into fine reaction mixture and is available for enzyme activation directly, i . e. without changes in membrane properties. However , at finis point finere is no evidence either to support or to reject finis or other suggestions. It is clear that fine component in fine supernatant is different from fine component in the pellet which accumlates with time (Table 14) . 58 The component in fine supernatant is apparently a small molecule because it is lost in dialysis. It is probably neither a protein be- cause it does not lose its activity after boiling, nor an inorganic ion as it loses its activity while being stored. It might be either an organic acid or a fatty acid. For example, Sribney and Lyman (1973) reported finat PCCTase extracted fran chicken or rat liver was stimulated 4 fold by addition of oleic acid into fine reaction mixture. 'llne response to fine GA3 is rapid and can be observed in mixing experiments as soon as 15 min after fine tissue that had been treated with GA3 (Table 15) . Further characterization of this rapid response would seem to be a promnising approach to an understanding of fine tissue response to 6A3. The increase in PCGTase activity in mixtures with such tissue over fine expected value is at least as high as if fine 6A3 treatment of fine treated tissue had been continued for 1 hr, and much bigner finan when this incubation lasted for 4 hr or more (Table 13). In order to synthesize lecithin, diglycerides are needed as sub- strate. It is possible that a diglyceride is fine component which is accumlated in fine control tissue during incubation and that this accurulation does not occur already during the inhibition of the half seeds which precedes fine incubation of fine isolated aleurone layers , but it has been shown by Pollard (1970, 1971) and by Collins, Jemer and Paleg (1972) finat preincubation is necessary to get the rapid GA3 responses that they observed. This means finat some events do occur during incubation of aleurone layers in buffer finat do not occur during inhibition of half seeds in water. When two preparations are combined , we combine the activated PCGTase from fine GA3 treated tissue , fine activating component from fine GA3 treated tissue, the inaCtive form of the enzyme from the 59 control tissue, and the accumulated substrate from the control tissue. The combination of finese carponents results in the mixing effect. One might speculate that bofin fine enzyme and the substrate are membrane oriented because when either one of fine preparations involved in fine mixture was boiled, it lost its PCGTase activity completely. This is obvious in the case of the enzyme, but if fine control tissue is supplying only soluble substrate fine latter should not be affected by boiling. However , if fine diglycerides were also membrane bound or membrane oriented boiling would make them unavailable. When GA was added to the tissue either during or after grinding 3 it did not cause any increase in PCGTase activity (Table 16). Furfinermore, incubationwith GA3 for 8 hr in the cold also did not result in an increase of PCGTase activity over that in control tissue (Table 17) , nor was fine "mixing effect" observable when fine tissue was incubated with GA3 in fine cold rafiner finan at room temperature (Table 17) . 'llnus, if it is‘ fine GA3 molecule itself which activates fine enzyme, this activation requires metabolic canditions. Several models for fine mechanism of activation of PCGTase by GA3 which account for fine results so far obtained can be proposed. Thus, it may be assured finat the aleurone cells of fine barley seeds contain free, small organic molecules , probably organic or fatty acids . 'llnere are also molecules of an inactive or less active form of PCGTase, a menbrane bournd enzyme. 'llne enzyme is activated by combining with fine small molecule just mentioned. This reaction between fine enzyme and fine activating factor can occur only in fine presence of GA3 . 'llne substrates for PCGTase are GDP-choline and diglycerides. 'llne diglycerides have to be oriented in somne manner toward fine enzyme and fine membrane before fine reaction can take place. This orientation is a time 60 dependent process that is also dependent on some metabolic event(s) . When half seeds are incubated with GA3 fine enzyme is activated and the level of lecithin formed is determined by the duration of fine incubation, because the length of fine incrbation time will control fine amount of the time dependent component (diglycerides) which will be available for the reaction. In a mixture the arount of lecithin fenred is a function of the activated enzyme contributed by the CA3 treated tissue and of fine amount of available, i.e. membrane oriented, diglycerides contributed by the control tissue . It is premnature to try to fit fine relationship of fine supernatant component and the GA3-dependent effect in finis model because fine identity of neither one is established, and the entire model is only one among several which could explain fine present data on activation of PCGTase by (3A3. Another assumption that can be made is that GA3 causes a release of Ca-H' from fine membrane and finat finis results in activation of the enzyme. It is known (Johnson 1973) that when Ca” is added to the incrbation medium finere was no increase in PCGTase activity in aleurone tiSSLe even finough GA3 is present. Jones (1973) reported that GA3 allowed movement of K+ and Mg++ through fine plasmnalemmna of barley aleurone cells. It is possible that one of finese ions is responsible for fine enzyme activation. Much more information is needed before it will be possiblr to determine if any of finese models is indeed a real one. Johnson and Kende (1971) showed finat in long term incubation of fine aleurone layers with GA3 and ABA fine latter is able to cause the same level of inhibition as caused by (11. This finding was confirmed. But while CH does not inhibit fine "mixing effect" when tissues are 61 incubated with CH and GA3 for only 1 hr, ABA caused 68% inhibition under similar conditions (Table 22) . 'llnese results indicate that ABA is interfering wifin an effect of CA3 which does not depend on RNA or newly protein synthesized. 'Ihis interference can be further elucidated by incubating aleurone layers with ABA , (1-1 and GA3 simmltaneOusly. 'Ihe ABA inhibition, which can be detected within 1 hr (perhaps less), may provide a tool to study further the responses of the tissue to fine two hormones. Investigations of the inhibitory effect of ABA within the first 15 min of incubation wifin GA3 may show how close fineir sites of action are and perhaps in what way finey affect each other. BIBLIOGRAPHY Bennett, P.A. and M.J. Chrispeels. 1972. De novo synthesis of ribonuclease and B-l,3-g}‘ucanase by aleurone cells of barley. Plant Physiol. 49 :445- 7 Chrispeels, M.J . 1973. Mechanism of osmotic regulation of hydrolase synfinesis in aleurone cells of barley : Inhibition of protein synthesis. Biochem. Biophys. Res. Comm. 53:99—104 Chrispeels, M.J. and J .E. Varner. 1967. Gibberellic acid enhanced synthesis and fine release of a-amylase and ribonuclease by isolated barley aleurone layers . 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