ABSTRACT ON THE MECHANISM OF HORMONE CONTROLLED ENZYME FORMATION IN BARLEY ALEURONE LAYERS By Tuan-hua David Ho Ribonucleic acid containing polyadenylic acid ( poly A-RNA) is present in barley aleurone layers. The poly A-RNA is polydisperse in size and the 3'-OH end of the molecule is occupied by the poly A segment which is about 80 to 200 nucleotides long. The poly A-RNA becomes labeled with radioactive precursors of RNA during the incubation of isolated aleurone layers with or without gibberellic acid (GAB). However, the rate of synthesis of poly A-RNA is enhanced by GAB. This enhancement begins within 3-# hours after addition of the hormone and reaches a maxium, which is about 50-60% over the control. 10-12 hours after the addition of the hormone. Cordycepin. 3'-deoxyadenosine. inhibits synthesis of total RNA as well as poly A-RNA in barley aleurone layers. However. cordycepin inhibits the hormone-cont- rolled formation of a-amylase only if it is added 12 hours or less after GAB. The rapid accumulation of Tuan-hua David Ho a-amylase after 12 hours of GA3 is due to the gg’ngxg syn- thesis of the enzyme molecule. i.e. accumulation of an a-amylase precursor does not precede the appearance of a-amylase activity. as examined by 13C-amino acid density labeling experiment. Cordycepin has no effect on the turnover of a-amylase. Therefore, it is suggested that d-amylase is translated from stable mRNA which is synthe- sized during the first 12 hours of GA3 treatment and the control mechanism of a-amylase synthesis 12 hours after the addition of GA3 appears to be strictly post-trans- criptional. The accumulation of a-amylase activity after 12 hours of GA3 treatment can be effectively decreased by abscisic acid (ABA). However. the accumulation of a-amy- lase activity is sustained or quickly restored when cordy- cepin is added simultaneously or soem time after ABA. indicating that the response of aleurone layers to ABA depends on the continuous synthesis of a short-lived RNA. Analysis of the newly synthesized proteins by gel electro- phoresis with sodium dodecylsulfate showed that the syn- thesis of a-amylase is decreased in the presence of ABA while the synthesis of most of te other proteins remains unchanged. From the rate of resumption of a-amylase pro- duction in the presence of cordycepin and ABA. it appears that ABA does not have a measurable effect on the stabi- lity of d-amylase mRNA. ON THE MECHANISM OF HORMONE CONTROLLED ENZYME FORMATION IN BARLEY ALEURONE LAYERS By Tuan-hua David Ho A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1976 ACKNOWLEDGMENTS I wish to express my appreciation to Dr. J.E. Varner for his guidance. and patience during this investigation and graduate training. Also I wish to express my sincere thanks to my wife, Berlin, for her understanding. tolerance. and assistance. Without her encouragement. this dissertation would not have been possible. I would like to thank Dr. P. Filner for the many interesting discussions. I thank Drs. J. A. Boezi. D. Delmer. H. J. Kende. A. Lang, R. A. Ronzio. and F. M. Rottman for their helpful suggestions on this manus- cript. The research financial support from the United States Atomic Energy Commission was appreciated. ii TABLE OF CONTENTS Page LIST OF TABLES . . . . . . . . . . . . vi LIST OF FIGURES . . . . . . . . . . . . viii PART I ON THE MECHANISM OF HORMONE CONTROLLED ENZYME FORMATION IN BARLEY ALEURONE LAYERS INTRODUCTION . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . Sources of Seed and Chemicals . . . . . . Preparation of Aleurone Layer , (DVChChN Extraction and Assays of Enzymes . Total RNA Extraction . . , , Isolation of Poly-A RNA . . . . . . . . 1a Isolation of Poly-A segment , , , . , , , 15 Polyacrylamide Gel Electrophoresis for Poly-A . 15 Sucrose Density Gradient Centrifugation for RNA 0 e o e e e e o o e e o 16 Base Composition Analysis of RNA . . . . . 17 Chemical Labeling of the 3'-OH End of the RNA Molecule . . . . . . , . , , 17 Double Labeling of RNA for Checking the Effect of GAB on Poly-A RNA Synthesis . . . . 18 iii Page Induction of Nitrate Reductase and Its Assay . 18 Denity Labeling of Proteins with 13C-Amino ACidS e e o e e e e o e o o e 21 Liquid Scintillation Counting . . . . . . 22 RESULTS 0 e e e e e o e e e o o e 0 2).]. Time Course of the GA3 Enhanced a-Amylase Production . . . . . . . . . . . 2h Occurrence and Characterization of Poly A-RNA . 27 Effect of Hormones on Poly A-RNA Synthesis . . 41 Effect of Cordycepin on the Production Of a‘mylase c e o e c e o e e e 47 Effect of Cordycepin on RNA Synthesis . . . 59 De Novo Synthesis of a-Amylase . . . . . . 6h Lack of Cordycepin Effect on the Degradation Of a‘mylase e e e e e e e o e o 76 Lack of Effect of Osmoticum on the Stability 8 of a-Amylase mRNA . . . . . . . . 0 DISCUSSION . . . . . . . . . . . . . 33 PART II THE RESPONSE OF BARLEY ALEURONE LAYERS TO ABSCISIC ACID INTRODUCTION . . . . . . . . . . . . 89 MATERIALS AND METHODS . . . . . . . . . 91 Source of Chemicals and Seed . . . . . . 91 Preparation and Treatment of Aleurone Layers . 91 Extraction and Assays of Enzyme . . . . . 92 iv Page Sodium Dodecylsulfate (SDS) Gel Electrophoresis . . . . . . . . . 92 RESIILTS 0 O O O O O O O O O O O I O 97 Effect of Cordycepin on GA3 Enhanced a-Amylase Formation C O O O O O O O O O O 97 Effect of ABA on d-Amylase Formation . . . . 99 Effect of Cordycepin on the Action of ABA . . 99 DISCUSSION 0 e . . . . o . . . . . . 111 BIBLIOGRAPHY o e c e . o . . . . . . 116 Table 1. 2. 3. 7. 8. 9. 10. 11. 12. 13. LIST OF TABLES Efficiency of Scintillation Counting . . . Base Composition of RNA Isolated from Barley Aleurone Layers . . . . . . . Estimation of the Size of Poly A Segment by the Ratio of AMP to Adenosine . . . . . Localization of Poly A segment on RNA Molucule . . . . . . . . . . . . Retention of RNA by Poly U Filtration . . Effect of ABA on the GA3 Enhanced Poly A- RNA syntheSis e e e e e e e e e 0 Effect of Cordycepin and 2'-Deoxyadenosine on the GAB-Enhanced Formation of d-Amylase in Barley Aleurone Layers . . . . . . Effect of Cordycepin on the GAB-Enhanced Protease Formation . . . . . . . . Comparison of the Effect of Cordycepin Added at Different Times after GA3 on the Produc- tion Of a-Amylase e e e e e e e e 0 Effect of Various Concentrations of Cordy- cepin on the Production of d-Amylase . . . Effect of Bromate and Amino Acids on the Production of d-Amylase in Barley Aleurone Layers . . . . . . . . . . . . Effect of Cordycepin on a-Amylase Production in the Presence of Bromate and Amino Acids. Summary of the 13C-Amino Acids Density Labeling Experiments . . . . . . . vi Page 23 33 38 40 42 #6 .50 56 57 58 65 77 Table 1h. 15. 16. 17. Page Effect of Water Stress on the Sensitivity of d-Amylase Synthesis to Cordycepin Treatment. 82 GA Enhancement of the Production of a-Amy- lage in Different Buffers . . . . . . . 93 Effect of Cordycepin on the GA3 Enhanced Synthesis of d-Amylase . . . . . . . . 98 Lack of Direct Effect of Abscisic Acid on G‘AEylaSe AetiVity e e e e e e e o e 100 vii Figure 1. 2. 3. 7. 8. 9. 10. ll. 12. 13. LIST OF FIGURES Validation of a-Amylase Assay . . . . . Validation of Protease Assay . . . . . Diagram Showing the Double Labeling Pro- cedure Used to Check the Effect of GA3 on Poly A-RNA Synthesis . . . . . . . . Time Course of the GA Enhanced a-Amylase Formation in Barley Aieurone Layers . . . Chromatography of total Barley RNA on Oligo (dT)-Cellu1089 e e e e e e e 0 Size Distribution of Barley Aleurone RNA in a Sucrose Density Gradient . . . . . Radioelectrophoretogram of the Hydrolyzate of the RNase Resistant RNA Segment Isolated from Barley Aleurone Layers . . . . . . Polyacrylamide Gel Electrophoresis of Poly (A) from Barley Aleurone Layers . . . . Effect of GA on Poly A-RNA Synthesis in Barley Aleur ne Layers . . . . . . . Effect of Cordycepin on the Substrate Induc- tion of Nitrate Reductase . . . . . . Inhibition of d-Amylase Formation by Various Concentrations of Cordycepin . . . . . Effect of Cordycepin on a-Amylase Activity . Effect of Cordycepin on the Incorporation of Uridine into RNA . . . . . . . . viii Page 10 12 19 25 28 31 3# 36 4b #8 51 6O Figure 1#. 15. 16. 17. 18. 19. 20. 21. 22. 23. 2h. 25. 26. Effect of Cordycepin on RNA Synthesis . . . Densityl Labeling of Barley Aleurone Proteins with 3C-Amino Acids (I) . . . . . . Density Labeling of Barle Aleurone Proteins with 13C-Amino Acids (III . . Densityl abeling of Barley Aleurone Proteins With C-Mino ACidS (III) e e e e e e Densityl Labeling of Barle Aleurone Proteins with 3C-Amino Acids (IVK . . Density1 Labeling of Barley Aleurone Proteins With BC’Amino ACidS (V) e e e e e I Effect of Cordycepin on the Degradation of “-mylase o e e e o e e e e e e 0 Calibration Curve for Molecular Weight Determination on SDS Gel Electrophoresis . . Effect of Midcourse Addition of ABA and Cordycepin on the Synthesis of a-Amylase . . Effect of Cordycepin Added at Different Times on the Synthesis of d-Amylase in the Pre- sence of both GA3 and ABA . . . . . . . Profile of Synthesized Salt-Soluble Proteins on SDS 661 O O O O O O O O O O O 0 Profile of Newly Synthesized Salt-Insoluble Prateins on SDS Ge]. 0 e e e e e e e e Postulated Mechanism of ABA Action . . . . ix Page 62 66 68 7O 72 71+ 78 95 101 104 106 108 113 PART I ON THE MECHANISM OF HORMONE CONTROLLED ENZYME FORMATION IN BARLEY ALEURONE LAYERS INTRODUCTION Mobilization of endosperm reserves during the ger- mination of cereal grains supplies nutrients for the growth of the embryo. This is accomplished by several hydrolases, including d-amylase (E.C.3.2.1.l.) and protease (E.C.3.h). which are synthesized in the aleurone tissue that surrounds the endosperm. Aleurone tissue of barley consists of three layers of homogeneous non-dividing tri- ploid cells. These cells respond to gibberellic acid ( GAB). which is formed in the embryo during the early stage of seed germination, by a series of morphological and bio- chemical changes (66,68). The most prominent among these changes is the increase in a-amylase and protease activities after an eight and ten hour lag period (10,29). The GA3 enhanced activities of d-amylase. protease, and ribonuclease (E.C.3.l.h) have been found to be due to the gg_nqu syn- thesis of the enzyme proteins (3.17.29), and most of the a-amylase and protease are secreted into endosperm after their synthesis (68). Besides the synthesis and secretion of d-amylase. protease.a and ribonuclease. GA3 also enhanced the secretion and to a less extent the synthesis of d-1.3- glucanase (E.C.3.2.1.6) (34) and the release of acid 3 phosphatase (E.C.3.l.3.2) from the cell wall (2). a-Amylase becomes the most predominent protein ( #0%) synthesized in barley aleurone layer after several hours of GA3 treatment (66). Therefore. it is usually used as a marker in studies of the mechanism of the hormone con- trolled formation of enzyme in this system. Inhibitors of RNA synthesis. such as actinomycin D (10.21), 6-methy1- purine (10). or cordycepin (3'-deoxyadenosine) (this study) can block the GA3 enhanced d-amylase synthesis. Incor- poration into salt-soluble RNA is enhanced by GA3 (8). Zwar and Jacobsen (69) demonstrated that the incorporation of radioactive nucleotides into polydisperse RNA was increased in the presence of GAB. and it was reported recently that GA3 enhanced the synthesis of rapidly labeled RNA species in barley aleurone layer (62). These observations tend to support a previous suggestion that the synthesis of a-amylase may depend on the GA3 mediated synthesis of their mRNA (10). During the lag period before the production of hydrolases. there is an extensive proliferation of cellular membranes. princepally endoplasmic reticulum (ER) (32,33), and the formation of membrane bound polyribosomes (13). Concurrrently the incorporation of choline and of 32P into membrane phospholipids is also enhanced by GA3 starting at about 4 hours after the addition of GA3 (38). Furthermore, two enzymes. phosphorylcholine cytidyl transferase (E.C.2. 7.7.15) and phosphorylcholine glyceride transferase A (E.C.2.7.8.2). which are involved in the synthesis of lecithin (phosphatidylcholine). are activated within minutes of GAB addition (“.31). All these events seem to indicate that membrane proliferation is a major GA3 effect before the rapid increase of a-amylase activity. d-Amy- lase is a secretory protein and there is ample evidence for the participation of rough ER in the synthesis and exportation of secreted proteins in eucaryotic cells (53, 60). Thus. a possible post-transcriptional model to account for the CA3 enhanced synthesis of a-amylase has been proposed by Johnson and Kende (31). They reasoned that a-amylase specific mRNA be present and turning-over in the aleurone cell before GA3 treatment and the syn- thesis of d-amylase may soly depend on the availability of proper membrane for the attachment of polysome that carry the d-amylase specific mRNA (31). It is now well established that mRNA (with the exception of histone mRNA) in eucaryotic cells contains a convalently linked polyadenylic acid (poly A) segment (5,22). The occurrence of poly A-RNA in higher plants has been reported in mung bean (2“). rice (#1), corn (61), barley (25.30). 1.1m Lab; (56). soybean (57). and pea (67). Since poly A can be hybridized with immobilized poly U or oligo dT. mRNA togeher with a certain frac- tions of heterogeneous nuclear RNA can be easily isola- ted from the other RNA species. There is recent evidence 5 which indicates that up to #O% of the mRNA in certain mammalian cells does not contain a poly A segment (#5). However. it has been reported that in higher plant essentially all the translatable messages are poly A containing RNA species (67). In order to elucidate the mechanism of hormonal control of enzyme formation in the barley aleurone cell. that up to ho% of the mRNA in certain mammalian cells does not contain a poly A segment. a detailed study of the relationship between the metabolism of RNA. especi- ally that of mRNA. and the formation of d-amylase be- comes necessary. The approaches used in this study are a l) to determine the effect of hormones on the synthesis of poly A-RNA. 2) to check the stability of a-amylase mRNA by monitoring the synthesis of a-amylase in the presence of specific transcription inhibitors. MATERIALS AND METHODS Sources of Seed and ChemiCals Barley seeds (Hordeum vulgare L.Cv. Himalaya. 1969 crOp) were supplies by Department of Agronomy. Wash- ington State University. Pullman, Wash. in 1972 and stored in the cold room since then. GA3' ABA (mixture of equal amounts of cis-trans and trans-trans isomers, all concen- trations mentioned in this study refer to that of cis- trans isomer only). cordycepin. and azocasein were obtain- ed from Sigma Chemical Co., St. Louis, Mo. Diethyl pyro- carbonate was obtained from Calbiochem., La Jolla. Calif. Potato starch for the d-amylase assay was obtained from Nutritional Biochemical 00., Cleveland. Ohio. 13C-Labeled amino acids mixture (hydrolyzate of alal proteins) was obtained from Merck Inc. Radioactive labeled compounds were obtained from New England Nuclear. Boston. Mass.. Amersham/Searle 00., Arlington Heights, Ill. NCS tissue solubilizer was obtained from Amersham/Searle Co. and a mixture of 9 parts of full strength of solubilizer nd one part of water was used. Poly A and poly U were supplied by Sigma Chem. Co. and Milts Laboratory. Oligo dT cel- lulose (T-2 and T-3) was obtained from Collaborative Re- search, Waltham, Mass. All the other reagents used in 6 this study reagent grade. Preparation ovaleurone Layers The method described by ChriSpeels and Varner (10) was followed. The embryos of dry barley seeds were mechan- ically separated from the endosperm by cutting with a di- secting knife. The embryo-less half of the seeds (half seeds) were surface sterilized by sodium hypochlorite (5 fold dilution of commercial bleach) for 20 min. After a thorough rinse with sterilized deionized water. the half seeds were imbibed on a sand plate moistened with steri- lized water in a gless petri dish. The aleurone layer can be readily peeled from the endosperm with two spatu- las after three days of water imbibition at room tempe- rature. Ten.twenty. or fifty aleurone layers were put in a 25 ml. 50 ml. or 125 ml foil capped sterilized Erlen- myer flask containing 2 ml. 9 ml, or CO ml of 2 mM acetate buffer. pH 5.0. respectively. The concentration of CA3 in this study was 1 uM, and one drop of chloramphenicol (500 ug/hl) was usually added to every 2 ml of buffer in order to prevent bacterial contamination. All solutions used were sterilized either by autoclaving or by filtration through a Millipore filter (VC.pore size: 0.13 u). The flasks containing aleurone layers were shaken in a recip- rocal metabolic shaker (120 oscillations/min) at 25 oC. 8 Extraction and Assays of Enzymes In all the experiments dealing with a-amylase and protease assays, duplicate lO layer samples were used. Af- ter incubation. the medium was decanted and the layers were rinsed with 3 m1 deionized water. The medium and rinse solution were combined (5 ml total). The layers were homogenized with 5 ml water in a mortar and pestle. Bec- ause a-amylase is fairly stable. the enzyme preparation can be stored at 2-5 OC. but not frozen, for several hours without significant loss of activity. On the other hand, protease is very unstable and it was assayed immediately after extraction. The d-amylase assay was a modification of that of Varner and Mense (65). The starch solution was prepared by boiling 150 mg of potato starch in 100 ml of 50 mM KHZPON’ pH 4.2: 10 mM CaCl2 for l min. After cen- trifugation at 12,000g for 15 min. the upper two thirds of the supernatant was used. The iodine solution was prep- ared by adding 0.3 ml iodine stock solution (600 mg and 6 g KI/1OO ml water) to 100 ml 0.01 N HCl. The assay mix- ture contains 5 to 100 ul of enzyme, 0.5 ml starch solution and 1.0 ml deionized water. After incubation at room tem- perature (22 0C) from I to 5 min the reaction was stopped by adding 1.0 ml iodine solution and the absorbance at 620 nm was measured in a Coleman Junior II A spectrophotometer. The starch solution was diluted with water in order to have an A620 close to 1.0 after reaction with iodine. 9 The amount of enzyme and the length of incubation can be varied in order to give an A620 nm between 0.55 to 0.75. The unit of a-amylase was defined as a change of one absor- bance unit per minute. Alpha-amylase activity is propor- tional to enzyme concentration under these assay conditions (Figure l). Protease was assayed in 20 mM sodium acetate. pH 5.03 lo mM B-mercaptoethanol. The mixture was incubated at 30 °C for one hour and the reaction was stopped by adding 0.5 ml 50% trichloroacetic acid (TCA). After being cooled in an ice bucket for 10 min. the mixture was centri- fuged at 12.000g for lo min. The supernatant was decanted and absorbance at 330 nm was measured. The enzyme unit for protease was defined as one tenth of an absorbance unit per hour. The protease assay also is proportional to en- zyme concentration under the assay conditions used ( Figure 2). Total RNA Extraction The aleurone layers were ground with 100 mM Tris- HCl buffer pH 7.6 containing 1% SDS and 0.1% diethyl pyro- carbonate (1 ml buffer per 10 layers) in a prechilled mor- tar. An equal volume of phenol (redistilled and satu- rated with deionized water): chloroform (1:1) mixture was added to the homogenate ad the whole mixture was stirred vigorously at cold for at least 10 min. After centri- fugation at 12.000g for 10 min. the aqueous phase was 10 Figure l.--Validation of a-Amylase Assay. The concentration of a-Amylase in the medium of a +GA3 sample was assigned as "100". Serial dilutions were then made and the d-amylase activity in each diluted sample was assayed. 11 no)— 40)— ?23 332.78 DI lWION 12 Figure 2.--Validation of Protease Assay. The concentration of protease in the medium of a +GA3 sample was assigned as "100'. Serial dilutions were made and then the protease activity in each diluted sample was assayed. 13 _ _ _ p _ L _ r 8 7 6 5 4 3 2 I. 0 2.23 235... 100 75 50 25 DILUTION 14 decanted and the non-aqueous phase was rextracted by stir- ring with 100 mM Tris-HCl. pH 9.0 (0.8 ml per 10 layers). and the two phases were again separated by centrifugation. All aqueous phases were combined and further extracted with phenolzchloroform mixture. Total nucleic acid was precipitated by adding one tenth volume of l M NaCl and 2.5 volumes of absolute ethanol and storing overnight at -20 oC. The precipitated nucleic acid was collected by centrifugation (12,000g for 30 min) and then dissolved in either deionized water or buffer as indicated in some experiments. RNA was further pufified by DNase digestion. However. barley aleurone cells were found to incorporate little radioactivity from RNA precursors into DNA. Therefore. the DNase digestion step was disocontinued. Isolation of Pgly-A RNA Oligo dT celluloase column (1 x 5 cm) was equili- brated with 10 mM Tris-H01 buffer. pH 7.6. containing 0.5 M KCl (binding buffer). RNA samples were dissolved in binding buffer and applied to the column. After thorough washing with binding buffer and the same con- centration of Tris buffer containing 0.1 M KCl, the bound RNA was then eluted with buffer alone. PreparatiOn of the fiberglass filter with immobilized poly-U and the filtration procedure were modified from that of Sheldon et a1 (59). A 0.15 ml volume of poly-U solution (1 mg/ml 15 10 mM Tris-HCl. pH 7.5) was added to a fiberglass filter (Whatman GF/C. 2.4 cm diameter) supported on a plastic grid. The filter was then dried at 37 oC and irradiated for 3 min on each side at a distance of 20 cm from a 30 Watt Sylvania germicidal lamp. The filter can be stored in the cold for a month. Just before use each filter was rinsed with 50 ml of deionized water to re- move unbound poly-U. The filter was then equilibrated with 10 mM Tris-HCl buffer. pH 7.5. containing 0.12 M NaCl. RNA samples were dissolved in the same buffer and fil- tration was done under unit gravity. The filter was washed ith 20 ml buffer followed by 20 ml 10% TCA. and 10 ml ethanol (95%). After drying. the radioactivity on the filter was measured. Isolation of Poly-A Segaent Bound RNA eluted from the oligo dT cellulose column was made to 10 mM Tris-H01. pH 7.6, 0.1 M NaCl. 1 mM MgC12. After digestion with pancreatic ribonuclease A (2 ug/ml) and T1 RNase (10 units/ml) for 30 min, die- ethylpyocarbonate or SDS was added to inactivate the en- zymes. After phenolschloroform extraction, the RNase resistant fragment was precipitated by ethanol. Pol cr 1 ide Gel ElectropnoresIs for Poly-A The method described by Laemmli (40) was followed. Ten cm sample gels (10%) with 0.1% SDS were used. One l6 hundred ul of RNA solution was applied on each gel. The electrophoresis was run under constant power supply ( 0.5 Watt/tube) until the tracking dye reached the bottom of the gel. The gel was sliced into 1 mm pieces and each piece was digested with 0.5 ml NCS solubilizer at 50 °C for 2 hours before radiioactivity was measured. Sucrose Density gradient Centrifugation for RNA A linear density gradient of 15% to 30% sucrose dissolved in 10 mM Tris-H01. pH 7.6, 1% SDS. 5 mM EDTA was prepared in a nitrocellulose centrifuge tube. The RNA sample was dissolved in the same buffer without sucrose, heated at 65 0C for 10 min. and cooled rapidly in an ice bucket in order to dissociate the aggregated RNA molecules. One to two hundred ul of the solution was applied on the top of the gradient. Centrifugation was carried out at 25 0C in a Beckman L-4 ultracentrifuge equipped with SW-SO L rotor at 47,500 rpm for 7 hours. After centrifugation the tube was punctured and 5 drop fractions were collected. The RNA in each fraction was precipitated by TCA and collected by filtration through nitocellulose filters. After washing with 20 ml 10% TCA the filters were dried and subjected to scintillation counting. 17 Base Composition Analysis of RNA The RNA sample labeled with 32F was hydrolyzed with 0.3 N KOH at 37 °C for 18 to 20 hours. After neutrali- zation with perchloric acid the hydrolysate was subjected to paper electrOphoresis as modified from that of Sebring and Salzman (58). RNA hydrolysate was applied on a 7.5 x 16 in electrophoresis paper and electrophoresis was carried out in pyridine-acetate buffer. pH 3.5, containing 10 mM EDTA at 500 V for about 51/2 hours immersed in 0014 as coolant. After electrophoresis, the pyridine was evaporated and the paper was dried by autoclaving for 3 min. The ultraviolet light-absorbing region on the paper was cut off and radioactivity was determined by scintill- ation counting. Alternatively the radioactivity on the paper could be scanned directly by a Packard strip scanner. Chemical Labeling of the 3’-0H End of tha RNA Molegule The method derived by Randerath et al (51) was followed. To a solution of about 50 ug of RNA in 100 ul of water was added 20 ul of an aqueous solution containing 2 nmoles of NaIOu. The oxidation was allowed to proceed for 2 hours in the dark at room temperature. Then 5 ul of 0.1 N NaBBHu (50 uCi) in 0.1 N KOH was added and the reaction mixture was kept in the dark at room temperature for 2 hours. A drop of l M acetic acid was added at the end of 2 hours to convert excess NaB3Hu into boric acid 18 and tritium gas. The last step was done in a well venti- lated fume hood. The labeled RNA was collected by ethanol precipitation. Double Labeliag of RNA for Checking the Effect of CA3 on Poly-A RNA Syathesis Labeling was carried out by addition of 50 uCi of 3H-adenoisne to a sample (20 layers) containing GAB and 1L‘C-adenosine to two a sample without GAB. and 2 uCi of other samples without 0A3 at specific times. After 2 hours of further incubation. 3H-labeled layers were mixed 1"'C-labeled layers (i.e.. 3H-labeled containing GA3 l4C with with -labeled sample without GA : 3H-labeled sample 14 3 without GA3 with C-labeled sample without GAB: see Figure 3) and rinsed extensively with ice cold carrier adenosine solution (10 mMO). RNA was then extracted as described previously. Induction of Nitrate Reductase aad Its Assay Nitrate reductase was induced by incubating aleurone layers with 2 ml sodium acetate buffer pH 5.0. containing 10 mM KNOB. The intact tissue assay of nitrate reductase developed by Ferrari and Varner (16) was used. Ten induced aleurone layers were rinsed twicd with 4 ml of 50 mM KN03, then placed in a 25 ml Erlenmeyer flask with 2 ml of 0.1 M potassium phosphate buffer. pH 7.5. 20 mm KNO and 5% ethanol. The flask was deaerated by bubbling 3. ‘ l9 .enseseeam «zm -4 uses no new no sesame esp geese as see: musceooum mafiaepmq cannon esp wcflzonm Emnwmfiaus.m shaman 20 038.3 ..\u Me 033.“ o:H\mm ooa II n. “flamenco ue>o RV Pauseossnsm nesshem esH Amnvoss Acesvxm ass mm esesem menseseo eeseeassessom so easemenu.s mnm§mm Ez a\_.>< enamosoc< mz< oswmocev< Op mz< no cases see as pseaMem “<1 uses no seam one so consenseem--.m mqm AMP content: ( 25 ) é—-—- 94.1 ———9 (93) FL { poly A { Chain length < X )4 156 —--—9 (number of nucleotides) ( X + 156 ) x 30.2 25X + 156 x 94.1 X 1920 Therefore: Chain length of poly A-RNA: 1920 + 156 = 2076 M.W. of poly A-RNA: 5.6 x 105 Predicted 8 value of poly A-RNA: about 15 S The calculated size of poly A-RNA is in agreement with that obtained by direct sedimentation analysis ( Figure 6). In order to study the localization of poly A seg- ment on the RNA molecule. the 3'-0H end of RNA was selec- tively labeled by NaIOu oxidation followed by NaBBH“ reduction as described under "Materials and Methods“. Barley aleurone poly A-RNA as well as the chemically syn- thesized poly A from Sigma Chemical Co. retained their label after RNase digestion (TABLE 4) indicating that the 3'-0H end of all the RNA is occupied by a poly A segment. However. the results of this experiment do not show that all poly A segment are located adjacent to the 3'-0H 4O .psm59aonp ammoaossonwu shaman :mconpmz use mamanovms: SH panama :mmv we use mo:.m on» pm uoaonma haamoasoso one: moaassm hoammm .epoz m.mm m.osnou 0.0meme apom on» son: .mdc mo soapflcom one ampms maze: 3N amps: cmpmnsosw monpmsm one; mashed econsmam was .covmowosw mm mseo< oeseooho soapeoe< assessoooo so ease newvmsmom ammopomm coossssm:n mo Poommm::.oa mqm_U:u< 33:24-8 was}: 76 hours of GA3 treatment. the rapid increase of a-amylase activity after 12 hours of GAB is due to the gg,ggyg syn- thesis of the enzyme molecule. Furthermore. by also adding a radioactive amino acid during the experiment but after 12 hours of GA3 treatment. it was possible to mea- sure the density shift of the newly synthesized proteins which would represent the maximal density shift (100% g; 3919 synthesis) for that period. By comparing the density shift of d-amylase and that of the total newly synthesized proteins (radioactive peak). one should be able to esti- mate the extent of gg,ngyg synthesis of d-amylase molecule. As shown in TABLE 13. essentially all d-amylase activity that appears after 12 hours is due to the gg_novo synthe- sis. Lack of Cordycepin Effect on the Degradation of g-Agylgse The degradation of d-amylase. measured at the time when its synthesis was shut down by cycloheximide is moderate with a half life about 13 hours (Figure 20). The dissappearance of d-amylase in the medium is much slower than that of d-amylase in the layer: however. disappea- rance from the layer might result from the continuous secretion of d-amylase even though protein synthesis was inhibited (65). Cordycepin does not slow down the degra- dation of d-amylase. Therefore. the insensitiveness of d-amylase production to cordycepin after 12 hours of GAB 77 won.o Ham.H :fimoohupoo opp; “on em op va omp coop Ago mp op ov amp ppm.p :No.o mom.p App pm op may omH coop Au: Np op ov omH moop mam.p mmo.o opm.p An: :N op may onH coop Ape mp op ov omH awe omm.p oso.o upm.p “on pm op ov omp mom.p noo.o oom.p App om op ov omp ooppooopspo m. op x apops ommazsmpo¢owvmm mpsoswnomxm mcwaonmn hpwmson muwo< ocfiso ommomocH Hmpoa azoapmone AueHmanpwmmu hpa>wpo< ommazawpo< ommaha<< -2 x :23 332.2 .3 20 lb 12 106 Figure 24.--Profile of Newly Synthesized Salt- Soluble Proteins on SDS Gel. Aleurone layers (30 to 40) were labeled with 3H-leucine (l5 uCi/ml) for 2 hours (18 to 20 hours after GAB). Salt-inso- luble proteins were extracted as des- cribed under Materials and Methods. One mm thick gel pieces were sliced and digested in 0.5 m1 NCS solubilizer (9 parts of full strength NCS solubilizer and 1 part of distilled water) at 50 °C for two hours. Ten milliliter of toluene based scintillation fluid (6 g PPO and 75 mg POPOP/1iter)were used in each sample. 107 A. 6A3 ONLY +—- A _ 3 mum-m 2 To. a 23 2O 50 4O 30 60 MW B, (SA3 mo ABA _ 2 no.3. ”I ‘ o. w va 20 3O 50 4O 60 MWKIO 108 Figure 25.--Profile of Newly Synthesized Salt- Insoluble Proteins on SDS Gel. Experimental methods were the same as described in Figure 24. 2O 15 cmx nod/sues 3 IO CPMXIO 109 1 A, as..." 1 L l J 1 so so 40 so so MWXIO—a B. 6A3 ANDABA 1 1 1 1 1 so so 40 so no wa10' 110 The major predominant salt-insoluble protein (Figure 25) has a molecular weight of about 52.000 daltons. which is slightly larger than that of d-amylase. Whether this protein is a precursor of d-amylase or an a-amylase covalently linked to cell wall or certain membrane fractions is unknown. DISCUSSION Although the synthesis of d-amylase after 12 hours of GA3 treatment is no longer subject to transcriptional control. the inhibitory effect of ABA on a-amylase production at this same sage apparently depends on the continuous synthesis of short-lived RNA (regulator-RNA in Figure 4). I propose that this regulator-RNA. or its translation product. can decrease the rate of translation of a-amylase mRNA without influencing protein synthesis in general. The mRNA of d-amylase is stable for at least 12 hours after exposure of the tissue to GAB. and its stability is main- tained in the presence of ABA. Ihle and Dure (27). working with precociously germinating cotton embryos. obtained evidence that the translation of carboxypeptidase mRNA was inhibited by ABA. Because they found that actinomycin D prevented the ABA inhibition. they prOposed that a suppressor molecule and had to be formed to bring about the ABA inhibition (27). The action of ABA thus appears to be similar in the two systems. the precociously germinating cotton embryos where gibberellins probably have no regulatory role. and the mobilization of reserve nutrient in the germinating 111 112 barley seed where ABA prevents the GA3 enhanced d-amylase production in the aleurone cells. Therefore. it seems reasonable to suggest that some ABA effects depend on trans- cription although there is no evidence for a requirement of transcription in some fast effects of ABA such as that of preventing the activation of phosphorylcholine glyceride transferase (4) and that of stomatal closure (52). There are two alternative sites of action for ABA (Figure26): 1) ABA might derepress the regulator-RNA and cause the synthesis of regulatory-RNA and/or regulator- protein. or 2) the regulator-RNA is under continuous turnover. ABA activates it or works with it to prevent,the translation of d-amylase mRNA. In human reticulocytes a new species of low molecular weight RNA has been reported to be able to preferentially stimulate the synthesis of one of the globin chain (19.20). The mechanism proposed here is by no means the only mode of action of ABA one might propose in barley aleurone cells. It has been shown that ABA prevents the GA3 enhanced poly A-RNA synthesis (Part I) indicating besides the translational control ABA can also stress its effect on the transcriptional machinery before 12 hr of GAB. Because of the many convenient properties of barley aleurone tissue and because of the ability of ABA to modify the tissue's response to GAB. as reported in this study. it is feelt that aleurone tissue is an important system 113 Figure 26.-~Postulated Mechanism of ABA Action. 114 AMYLASE GENE REGULATOR GENE 7 AMYLASE mRNA REGULATOR RNA FABA (STABLE) (SHORT-LIVED) +-------- l 7 AMYLASE REGULA'IOR PROTEIN 115 for the further study of how ABA modifies a tissue's response to GAB. 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