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LIBPMV Universrty This is to certify that the thesis entitled HORMONAL CONTROL OF PHOSPHOLIPID SYNTHESIS IN BARLEY ALEURONE LAYERS presented by DON EDWARD KOEHLER has been accepted towards fulfillment of the requirements for PhoD- degree in Biochemistry MW Major professor Date June 15, 1972 0-7 639 ABSTRACT HORMONAL CONTROL OF PHOSPHOLIPID SYNTHESIS IN BARLEY ALEURONE LAYERS By Don Edward Koehler Gibberellic acid (GAS) enhances the rate of phospho- lipid synthesis in barley aleurone layers. Using 32Pi incorporation into chloroform-methanol soluble compounds as an assay for the response, enhancement was shown to start at 4-6 hr after the addition of GA and reached a 32pi maximum after 8-12 hr. The increase in the rate of incorporation was 3-5 fold over the rate in control layers incubated without GA. The GA enhancement of the rate of phospholipid synthe- sis could be inhibited within l—Z hr by cycloheximide, 6- methylpurine, and abscisic acid. The ratio, organic—32p : uptake of 32Pi, was enhanced 50% by treatment with GA. An osmotic stress imposed on the layers by incubating them in mannitol inhibited the rate of phospholipid synthesis but not the increase in the organic- 32P : uptake ratio. Removal of GA from the layers resulted in a decrease in phospholipid synthesis. There was no increase in the rate of phospholipid synthesis when layers were treated with 3', S'-cyclic AMP. Don Edward Koehler The increase in labeling of phospholipids occurred throughout the cell rather than being restricted to a specific cell fraction or organelle. The increase in radioactivity in phospholipids is due to a proportional increase in all phospholipids as shown by TLC. The enhancement of the rate of phospholipid synthesis by GA is thought to be required for the subsequent produc- tion of GA-induced hydrolases. HORMONAL CONTROL OF PHOSPHOLIPID SYNTHESIS IN BARLEY ALEURONE LAYERS By Don Edward Koehler A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1972 ACKNOWLEDGMENTS I wish to thank Dr. J. E. Varner for the inspiration and guidance he provided in the course of these studies as my research supervisor. I would also like to thank those faculty members who served on my guidance and examination committees: Drs. Bandurski, Boezi, Kende, Lang, and Rottman. This work was supported by the U.S. Atomic Energy Commission under contract AT(11-1)-1338 and by a grant (GB-8774) from the National Science Foundation. ii TABLE OF CONTENTS INTRODUCTION . . . . . . . . . . . . . . . . . MATERIALS AND METHODS . . . . . . . . . . . . Preparation of Aleurone Layers . . . . . Incorporation of 32Pi . . . . . Determination of Incorporated Radioactivity Thin- Layer Chromatography . . . . . . . PhOSphorus Analysis . . . . . . . . . Measurement of Radioactivity . . . . RESULTS . . . . . . . . . . . . . . . . . . . Time Course of Phospholipid Synthesis . . . Characterization of 32Pi Incorporation . Effects of Metabolic Inhibitors . . . . . . Hormonal Control . . . . . . . Other Possible Points of Control Additional Biochemical Data . . . . . . . DISCUSSION . . . . . . . . . . . . . . . . . . SUMMARY . . . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . . . . iii Page 11 15 16 16 17 17 28 36 44 59 63 70 76 77 10. 11. LIST OF TABLES Ratio of 32P1 to total uptake for a 24 hr time course . . . . . . . . . . . . . . . . Distribution of labeled phospholipids in different cell fractions . . . . . . . . Particulate nature of supernatant phospholipids Relative distribution of radioactivity in individual phospholipids . . . . . . . . . Cycloheximide inhibition of phospholipid synthesis in control and GA treated aleurone layers . . . . . . . . . . . . . . . . . . Effect of actinomycin D on phospholipid synthesis . . . . . . . . . . . . . Effects of GA and mannitbl on the incorporation of 32Pi into organic phosphates . . . . . I Effects of 3', 5'-cyclic AMP and N6, 02 - dibutyryl cAMP on the rate of phospholipid synthesis . . . . . . . . . . . . . . . . . Incorporation of 14C-acetate by aleurone layers Phospholipid content of aleurone layers Organic phosphate content of aleurone layers iv Page 13 29 31 35 39 43 62 64 65 67 68 10. 11. 12. 13. 14. 15. 16. LIST OF FIGURES Short-term accumulation of 32P in lipids and organic phosphates . . . . . . . . . . . . Short-term chase of aleurone layers . . . . . . Time course of the rate of 32Pi incorporation into phospholipids . . . . . . . . . . . Time course of 32Pi uptake into aleurone layers and incorporation of 32Pi into phosphate- containing organic compounds . . . . . Time course of phospholipid synthesis . Ratio of 32Pi incorporation into organic phos- phates to uptake of 32Pi for the 24 hr time course Schematic diagram of the separation of phospho- lipids by TLC . . . . . . . . . . . Time course of inhibition of phospholipid synthesis by cycloheximide . . . . . . . Inhibition of phospholipid synthesis by 6- methylpurine . . . . . . . . . . The response of phospholipid synthesis to increasing concentrations of GA . . . . . Effect of removal of GA on the rate of phospho- lipid synthesis . . . . . . . . . . . Uptake of 32Pi by aleurone layers undergoing the removal of GA . . . . . . . . . . . Inhibition of GA- enhanced phospholipid synthesis by abscisic acid . . . . . . . . . . . . . Effect of ABA on 32P1 incorporation into cellular components . . . . . . . . . . . . . . Progressive inhibition of phospholipid synthesis by increasing concentrations of ABA The effect of mannitol on GA-enhanced phospho- lipid synthesis . . . . . . . . . . . . . . . Page 18 21 24 33 38 42 46 49 51 S4 S6 58 61 ABA ER GA HEPES 32Pi POPOP PPO TCA TLC ABBREVIATIONS Abscisic Acid Endoplasmic reticulum Gibberellic acid (GAS) N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (32p) orthophosphoric acid 1, 4-bis(2-(4~methyl~5-phenyloxazolyl))benzene 2, S—diphenyloxazole Trichloroacetic acid Thin-layer chromatography vi Km INTRODUCTION The seeds of the Gramineae consist of an embryo, the starchy endosperm, and a layer of aleurone cells enclosing the endosperm. In barley, gibberellin-like substances secreted by the embryo during germination cause liquifi- cation of the endosperm (Yomo, 1960; Paleg, 1960). Aleurone layers, whether as part of intact, embryo-less half seeds or isolated from the endosperm, will respond to added gibberellic acid (6A3) by synthesizing and secreting a series of hydrolytic enzymes (Yomo, 1960; Paleg, 1960; Briggs, 1963; Varner, 1964; Chrispeels and Varner, 1967 a). Thus the mobilization of reserves in the endosperm is under control of the germinating embryo via gibberellins (Yomo and Iinuma, 1966; MacLeod and Palmer, 1966; Radley, 1967). The barley aleurone layer consists of non-dividing, protein-rich cells three layers thick. Its response to GA when isolated makes this tissue attractive for studying the hormonal induction of enzyme synthesis. There is an 8-10 hour lag period (Chrispeels and Varner, 1967 a) between the addition of GA to aleurone layers and the initiation of de nave synthesis of a-amylase (Filner and Varner, 1967) and protease (Jacobsen and Varner, 1967). 2 Examination of events occurring during this lag period will aid in the understanding of the effects of GA on the aleurone layer and the processes required for the ultimate synthesis of GA-induced hydrolases. The results of Siekevitz and Palade (1966) on the synthesis and secretion of pancreatic amylase led to their suggestion that synthesis of proteins destined for export from the cell occurs on the endoplasmic reticulum (ER). Tata (1968) found a correlation in the timing of the hor- monal stimulation of RNA, protein, and membrane synthesis in several systems. Thus the study of the aleurone layer in which there is hormonal control of protein synthesis and secretion becomes especially interesting. During the lag period preceeding a—amylase synthesis there are several events controlled by GA which are directly related to protein synthesis. JOnes (1969 b) observed an increase in the amount of rough ER in electron micrographs of aleurone layers treated with GA for 10 hr. Evins and Varner (1971) showed an increased incorporation of choline into a semi-purifed microsomal pellet starting after 4 hours of GA treatment. Two enzymes, phosphorylcholine-cytidyl transferase and phosphorylcholine-glyceride transferase, of the CDP-choline pathway for lecithin synthesis show increased activity within 2 hr of the addition of GA, reaching a maxi- mum after 12 hr of GA treatment (Johnson and Kende, 1971). Finally, enhanced polyribosome formation and an increase in 3 the total number of ribosomes was observed after 4-5 hr and reached a maximum within 10-15 hr of GA treatment (Evins, 1971). Preliminary experiments showed that GA also enhanced 32Pi into chloroform—methanol soluble the incorporation of components of aleurone layers. PhOSphate is a general label for all phospholipids and proved to give consistent results. Thus a detailed study of the control of phospholipid synthe- sis was undertaken to further define processes required for GA-effected hydrolase production. MATERIALS AND METHODS Preparation of Aleurone Layers Aleurone layers were prepared from barley half Seeds (Hordeum vulgare L. cv. Himalaya; seeds from the 1969 harvest supplied by the Agronomy Club, Washington State University, Pullman, WaSh.) essentially following the methods of Chrispeels and Varner (1967a). Half seeds were prepared by making two transverse cuts across a barley seed, removing the embryo half and a small portion of the opposite tip of the seed. Half seeds were sterilized in excess 1% NaOCl for 15-20 min, rinsed 5-8 times with sterile distilled water, and incubated for 3 days on sterile moist sand in a foil-wrapped Petri dish. At this time layers were removed from the starchy endosperm with the aid of two spatulas. In most experiments duplicate samples of 10 layers were shaken in a 25 ml erlenmeyer flask with 2 ml of incubation medium. In some cases 20—30 layers were incubated in a 50 ml flask with 5-6 ml of incu- bation medium. The incubation medium contained 1 mM Na acetate buffer, pH 5.0; 20 mM CaCl 20 ug/ml chlorampheni- 2; col; and 1 uM GA3 where appropriate. Incubations were 5 carried out at 25° on a reciprocal shaker. Sterile con- ditions were maintained during all manipulations. Incorporation of 32Pi Carrier free [32p] H3P0 in 0.1 N HCl (hereafter 32 4 abbreviated Pi) purchased from International Chemical and Nuclear Corp. was used in all experiments. During incubations, aleurone layers were labeled by adding 100-150 uc 32Pi directly to incubation flasks. After 45 min, layers were rinsed in sterile 005 M KH2P04 and incubated an additional 30 min in new 0.05 M KH2P04. At this point, layers were rinsed again in 0.05 M KH2P04 and immediately ground or quick-frozen for homogenization at a later time. It was experimentally verified that freezing the layers did not affect extraction of labeled compounds. The choice of times for the labeling period and the chase was made from the experiments shown in Figures 1 and 2. Figure 1 (lower) is the time course of the accumulation of radioactivity in phospholipids and organic phosphates (these fractions are operationally defined in subsequent paragraphs). After 30 min there is a linear accumulation of 32 P in phospholipids. Therefore the choice of a time longer than 30 min for pulse labeling should give a good measurement of the rate of phospholipid synthesis. The upper graph in Figure 1 shows that the lipid-32F : organic- 32P ratio is also linear after 30 min. This is important because this ratio will also be used to estimate phospho— lipid synthesis. Figure 1. Short term accumulation of 32F in lipids and organic phosphates. Duplicate flasks of aleurone layers which had been preincubated in GA for 12 hr were labeled with 140 uc 32Pi. At the indicated times samples of 10 layers were removed from each flask and the radioactivity in the various components was determined. 3.83. o. \ m..o. x anv I n. unuoEoBO h P b _ p — 00. x n. 3-2.59.0 n. 2-23.. 0 O O O O 3 2 I. 3.22 o. \n..o_ x can; I a 3-22.. so 90 Time (min) 30 8 Early experiments showed that values for ”uptake” of 32Pi were almost independent of time of labeling or phosphate concentration over a wide range of values. That is, there seemed to be an immediate adsorption of large amounts of radioactivity to the layers which could mask significant differences in the amounts of metabolically available phosphate actually inside the cells. Such non-specific binding could be due to calcium phosphate precipitation in the cell walls or the binding of phosphate to other cell wall components. It was found that a short incubation in non-labeled phosphate following a 32Pi pulse seemed to allow the exchange of adsorbed radioactivity with the medium, thereby reducing measured uptake values to meaningful values. The results of such a chase experiment are shown in Figure 2. Incubation periods up to 30 min result in the most rapid loss of uptake radioactivity but still give 32 accumulation of P in lipids. The decrease in lipid radioactivity at 60 min is not representative of most 32p out of results. Usually there is little chase of phospholipids over periods of up to 4 hours. Consequently 45 min and 30 min periods for labeling and chase, respectively, were chosen as standard conditions. These times allow the measurement of the rate of phospho- lipid synthesis without involving excessive time periods during which other unknown effects could take place. A few of the earliest experiments used 30 min for labeling and 15 min for chase. These are noted as they occur. Figure 2. Short—term chase of aleurone layers. Aleurone layers which had been prei cubated for 9 hr in CA were labeled 45 min in 200 uc 3 Pi. They were then rinsed and further incubated in 50 mM KH P04 for the indicated periods at which time the radioactivigy 1n the cellular components was determined. °/o of Maximum cpm IOO C!) O O) O A O N O 10 i—. Lipid Organic Uptake *0 1 1 1 30 60 Time (min) 11 Determination of Incorporated Radioactivity Aleurone layers were homogenized in a mortar with sand and a total of 3.5 ml grinding buffer (0.1 M HBPES, pH 7.55 and 0.45 M sucrose). The homogenate was centrifuged at 4,000 x g for 10 min followed by a centrifugation at 10,000 x g for 15 min. This procedure gave a cleaner final supernatant than could be obtained from just one centrifu- gation as well as providing a crude fractionation of the homogenate. Four determinations were made on the final supernatant; l) aliquots were counted directly for total.uptake; parti- tioned to separate 2) 32 Pi from 3) phosphate-containing organic compounds; and extracted to yield 4) the phospho- lipid fraction. Radioactivity in organic phosphates was assayed by the method of Saha and Good (1970). To 1.0 ml of super- natant was added 5 ml of 10% perchloric acid, 1.0 ml ace- tone, and 1.0 ml of 10% ammonium molybdate. After stirring, 10 ml of n~butanol~benzene (1:1) was added and the two- phase mixture was again vigorously stirred. At this point 1.0 ml of the upper (butanol-benzene) phase was transferred 32Pi. The rest of to a scintillation vial for counting the upper phase was removed by suction, and the lower phase was filtered through Whatman #4 filter paper pre- moistened with water. The filter paper retains any small amounts of the upper phase not removed by suction. An additional 0.1 ml of ammonium molybdate and 10 m1 of 12 butanol-benzene were added to the filtrate. After stirring, all traces of the upper phase were removed by suction. A 1.0 ml aliquot of the lower (aqueous) phase was counted. It was experimentally determined that two butanol-benzene extractions gave complete removal of 32 Pi from the aqueous phase. The procedure outlined above results in the parti- tioning of all inorganic phosphate into the butanol-benzene phase as a phOSphomolybdate complex. Therefore all radio- 32Pi activity in the aqueous phase can be attributed to incorporated into organic compounds (including pyro- or polyphosphates). The exact nature of these organic phos- phates was not determined, but it is assumed they include nucleotides, sugar phosphates, etc. Not included are the phospholipids which partition into the butanol-benzene phase, or RNA and phosphoproteins which are precipitated by the perchloric acid and are removed by the filtration step. In this system, however, these compounds represent only a small percentage of the total radioactivity measured in either the upper or lower phase. Likewise, counting the butanol-benzene phase gives an estimate of the uptake and retention of 32Pi in the aleurone layers. It was found that the ratio,32Pi : uptake,was fairly constant throughout any given experiment (see Table 1). Therefore results are expressed in terms of uptake rather than 32Pi even though both measurements were made. Uptake includes incorporated 13 Table 1. Ratio of 32Pi to total uptake for a 24 hr time course . 32Pi Tlme (hr) 111766—1138 X 100 -GA +GA 4 67 67 8 67 53 12 71 54 18 72 72 24 63 66 Aleurone layers incubated with or without GA were pulse—labeled with 32Pi at the indicated times and radio- activity determinations were made. 14 32p as well as 32Pi and gives a better estimate of the total amount of 32Pi entering the cells. Phospholipids were isolated essentially following the procedures of Folch, 31 El° (1957). Usually 1 ml of super- natant was vigorously extracted with 4-5 ml of chloroform- methanol (2:1). The upper phase and protein at the interface were removed by suction. The lower (chloroform) phase was washed three times with upper phase solvents (chloroform: methanolzwater, 3:48:47) which also contained 0.8% NaCl and 0.2% MgClZ to prevent any further partitioning of phospholipids into the upper phase wash. It was determined that 3 washes were sufficient to break emulsions in the chloroform phase and to remove all non-phospholipid 32p trapped in that manner. Pellets were first extracted with chloroform:methanol (1:1) and centrifuged to remove all debris and precipitated protein. Then sufficient volumes of chloroform and water (containing 0.8% NaCl and 0.2% MgClZ) were added to give a final ratio of chloroformzmethanol:water of 8:4:3 which is needed for best phase separation. After mixing followed by removal of the upper phase, the lower phase was washed as described above. The chloroform phases were either concen- trated for thin-layer chromatography or transferred to a scintillation vial and dried for counting. 15 Thin-Layer Chromatography Phospholipids were separated by thin—layer chroma— tography (TLC) for estimation of radioactivity in individual compounds. Pre-coated 20 cm x 20 cm TLC glass plates with a 0.25 mm silica gel layer (B. Merck, Darmstadt, Germany) were purchased from Brinkmann Instruments, Inc. After activation at 105°C for 20 min, the plates were allowed to cool, and samples were spotted in chloroform: methanol (4:1). The plates were develOped in a 2- dimensional system of Rouser, g: 3;. (1967). In the first direction chloroform-methanol-l4 N NH OH (6S:35:5) 4 was used. After drying, the second dimension was developed in chloroform-acetone-methanol-acetic acid-water (100:40: 20:20:10). Visualization was accomplished first with iodine vapor and then by several specific sprays: the Dragendorf reagent for choline-containing phospholipids (Block, pp al., 1958), the ninhydrin spray for phosphatidyl ethanolamine and phosphatidyl serine; and the molybdenum blue reagent of Dittmer and Lester for all phospholipids (both summarized by Skipski and Barclay, 1969). Identifications were con- firmed by comparing the migration of sample compounds to that of authentic standards (Supelco, Inc.). Individual spots were scraped into scintillation vials for measurement 32 of Pi incorporation. 16 Phosphorus Analysis The chemical analysis of the phosphorus content of phospholipid and organic phosphate fractions was carried out using the method of Bartlett (1959). Suitable aliquots were digested in H SO and the color was developed with 2 4’ ammonium molybdate and Fiske-SubbaRow reagent. Appropriate amounts of an orthophosphate standard were also carried through the procedure for conversion of optical density to umoles Pi. Measurement of Radioactivity The scintillation fluid used for all measurements was a mixture of toluene-Triton X-100 (2:1) which contained 4g PPO and 0.1g POPOP per liter of toluene (Patterson and Greene, 1965). This mixture has the advantage of being able to solubilize phospholipids as well as adequately emulsify aqueous samples. No corrections for quenching were needed when 1.0 m1 acidic aqueous 32P samples were counted in 20 m1 of scintillation fluid. RESULTS Time Course of Phospholipid Synthesis Figure 3 shows a typical time course of the rate of phospholipid synthesis in the presence and absence of GA. Phospholipids were extracted from the 10,000 x g super- natant of an aleurone layer homogenate after pulse labeling at the indicated times as described in Materials and Methods. After 4 hours the rate of incorporation of 32Pi into phospholipids increases rapidly, reaching a maximum 8-12 hours after the addition of GA. The rate of incorporation then decreases and reaches the level of the -GA control by 18-24 hours. Throughout the first 24 hours of incubation, there is a basal level of phospholipid synthesis in the -GA control tissue which remains relatively unchanged. By 24 hours there may be a slight increase in phospholipid synthesis in the control aleurone layers. In order to follow routinely the uptake and metabolism of 32 Pi, two additional measurements were made in all experiments. Total uptake was monitored by counting an aliquot of the 10,000 x g supernatant. Incorporation of 17 18 Figure 3. Time course of the rate of 32Pi incorporation into phospholipids. Aleurone layers were pulse labeled at the indicated times and 32Pi incorporation into the phospho- lipids of the 10,000 x g supernatant was measured. l9 +GA — p _ ’GA _ O 7 p O 6 — O 5 n-o O O O 4 3 2 .x 58 n. 3.22.. l8 24 l2 TlME(hrs) 8 20 32Pi into organic phosphates was measured as described in Materials and Methods. In this way, effects on 32Pi uptake and metabolism by any of the various treatments can be detected and corrected for when estimating the actual rates of phospholipid synthesis. In Figure 4 it is shown that 32 Pi uptake does vary during the 24 hour time course in both control and GA- treated aleurone layers. The usual pattern is that uptake in hormone-treated layers is equal to or greater than uptake in control layers at early times, but decreases to much lower levels with longer incubation periods. TheSe fluctuations in uptake are directly reflected in the incorporation of 32 Pi into phosphate-containing organic compounds (Figure 4). In addition, the relative rates of 32Pi incorporation into phospholipids are probably being affected in a similar manner. Therefore, it was decided that a more accurate measurement of phospholipid synthesis could be obtained by expressing 32Pi incorporation into phospholipids as a percentage of 32Pi incorporation into the total organic phosphate fraction. In this way, 32Pi incorporation into phospholipids is adjusted for differential rates of labeling of the organic phosphate pools, whether such differences are due to uptake or various metabolic effects. An increase in the ratio of phospholipid radioactivity to organic phosphate radioactivity indiCates a true increase in the rate of phospholipid 21 Figure 4. Time course of 32Pi uptake into aleurone layers and incorporation of 32Pi into phosphate—containing organic compounds. 22 -GA +GA - n h —GA +GA 2.4 18 (hrs) 1 12 04 8 h. ... 3 2 .- nlo_x Emu O _ 0 2 . . . nIo—x Eau m¥-GA 00. x n. 3.6.590 n. 322.. IS IO Time (hrs) 50 Figure 12. Uptake of 32Pi by aleurone layers undergoing the removal of GA. The conditions are the same as for Figure 11. 51 50 - 9A 9 40)— x +GA-)-GA S 3 30- a) x O ‘5. 20- D +GA #0; a IO- 0 l l l 7 IO l3 Time (hrs) 52 Varner, 1967b). It will also inhibit phospholipid synthesis. When ABA is added to GA-treated layers, any further enhance— ment of the rate of phospholipid synthesis is prevented (Figure 13). Other experiments show no effect of ABA on the basal rate of phospholipid synthesis in -GA control layers, whether the ABA is added in mid—course or at 0 time. The addition of ABA causes an increase in 32 Pi uptake in aleurone layers (Figure 14). Increased amounts of radio- activity are then incorporated into organic phosphates and phoSpholipids. However the rate of phospholipid synthesis is lower in ABA-treated layers because there is proportionately less incorporation into phospholipids of these layers than for +GA layers for a given level of organic phosphate radioactivity. It is not clear why hormone fluctuations stimulate 32Pi uptake. In the case of ABA it does not always occur. Sudden changes in hormone levels, whether the addition of ABA or the removal of CA, are not representative of physio- logical conditions. The system may simply be reacting or adjusting to such changes. The effect of increasing concentrations of ABA on the rate of phospholipid synthesis is shown in Figure 15. There is no inhibition of the GA response until the ABA concentra- tion reaches 0.1 micromolar. At 1.0 uM phospholipid synthesis has been reduced to the level of the —GA layers. The inhibition of phospholipid synthesis is sensitive to the 53 Figure 13. Inhibition of GA-enhanced phospholipid synthesis by abscisic acid. Forty aleurone layers per flask were incubated in 0.1 uM GA. After 8 hours, 2 uM ABA was added to one set of layers (0 time on graph). At the indicated times, 10 layers were removed from duplicate flasks and pulse labeled with 32Pi and phospholipids extracted. 54 P xlOO m Organic-3'2 P J}- Lipid-32 N +GA \ + 4 +GA + ABA O 2 4 6 Time (hrs) 55 Figure 14. Effect of ABA on 32Pi incorporation into cellular components. Conditions are as in Figure 13. ABA was added at hour 8. 56 .ob _ x Ego. 9.2.5 A A A B I B I B A l A A A A G M G + A + + + G M G M _ _ P p _ _ _ _ _ _ _ r _ P _ _ _ _ r 4 2 O n(O 2 8 4 O 6 4 2 O .90. x Ego. a mun-2.890 .70. x Ego. a Nn-Eaj l2 I4 IO Time (hrs) 57 Figure 15. Progressive inhibition of phospholipid synthesis by increasing concentrations of ABA. Aleurone layers were incubated for 8 hours with both GA (1.0 uM) and ABA at the indicated concentrations present from the start. The layers were then pulse-labeled with 32Pi and phospholipids ex— tracted. 58 0+GA leOO 0) Organic-32 P A r o-GA Lipid-3'"2 N )- )- O,_1_,;1 L J 1 1 o i0"° :69 IO'8 :0" :66 ABA Cancentratian(M) 59 concentration of ABA over about two orders of magnitude when the ABA is added at 0 time. In summary, the evidence for hormonal control of phospholipid synthesis consists of the concentration dependence of responses to GA and ABA as well as the time course of inhibition by ABA. This inhibition by ABA is rapid and is specific for GA-enhanced phospholipid synthesis. Removal of GA also brings about a decline in the rate of phospholipid synthesis. Other Possible Points of Control Several other approaches to the problem of the control of phospholipid synthesis have also been examined. The first involves the observations of Jones (1969a) that osmotic agents such as mannitol or polyethylene glycol inhibit the induction of a-amylase by GA in barley aleurone layers. Figure 16 shows that mannitol also inhibits the rate of phospholipid synthesis. When layers are incubated in GA with increasing concentrations of mannitol, phospho— lipid synthesis is progressively inhibited until finally it is reduced to the level of the -GA layers. There is no inhibition by mannitol of the basal rate of phospholipid synthesis in -GA control layers. An additional observation is that the enhancement by GA of the organic phosphate—32p : uptake ratio is not inhibited by mannitol (Table 7). This ratio usually shows 60 Figure 16. The effect of mannitol on GA-enhanced phospho— lipid synthesis. Aleurone layers were incubated 8 1/2 hr in the indicated concentrations of mannitol with or without GA. They were then pulse-labeled and phospholipids were extracted. 61 -oI o _ 98) X a.” NELNG- r0”- '0 EE- 0.0 .184" o- 2)— l l l I I 0 0.2 0.4 0.6 0.8 Mannitol Concentration (M) Table 7. Effects of GA and mannitol on the incorporation of 32Pi into organic phosphates. . 32 Mannitol (M) orgamc—L—B x 100 Pi Uptake :31 :EA 0 10.5 16 0 O 2 16.4 0.4 11.5 22.6 0.6 24.9 0.8 14.1 21.4 Aleurone layers were incubated for 8-1/2 hr in the indicated concentrations of mannitol with or without GA. They were then pulse—labeled and phospholipids were extracted. 63 at least a 50% increase with GA treatment. While mannitol has completely inhibited GA-enhanced phospholipid synthesis, the stimulation of the ratio of organic-32F to uptake of 32Pi has not been affected. This means mannitol inhibits phospholipid synthesis and a-amylase formation to similar extents, but leaves untouched another effect of GA, namely the enhancement of the organic phosphate : uptake ratio. Recent work with the barley endosperm system has sug- gested that 3', 5'—CycliC AMP (CAMP) may mediate the action of (Pollard, 1970) or substitute for GA (Galsky and Lippin- Cott, 1969). Phospholipid synthesis, however, is not stimu- lated by CAMP nor by N6, 02 '-dibutyryl CAMP (Table 8). Although experiments were not performed using a sub-optimal concentration of GA as in some of the CAMP work, there is no enhancement of phospholipid synthesis in the absence of GA. In the presence of a saturating level of GA, CAMP appears to inhibit the rate of phospholipid synthesis. Therefore, if CAMP has a role in the enhancement of a-amylase by GA, there is no indication of such a role in its effects on phospholipid synthesis. Additional Biochemical Data In an effort to learn more about the nature of the phospholipid synthesis enhanced by CA, the incorporation 14 of C-acetate by aleurone layers was examined. Table 9 shows there was no effect of GA on the incorporation of 64 Table 8. Effects of 3', 5'-Cyclic AMP and N6, 02'—dibutyryl CAMP on the rate of phospholipid synthesis. Li id—SZP 100 Treatment 0 . X rganic— F -GA +GA Control 4.5 11.8 Cyclic AMP 3.5 5.9 Dibutyryl CAMP 4.2 6.0 Aleurone layers were incubated with or without GA (1 uM) for 8 hr at which time they were pulse—labeled and phospholipids were extracted. The CAMP compounds were present from the beginning of the incubation period at a concentration of 5 mM. 65 Table 9. Incorporation of 14 C—acetate by aleurone layers. -GA +GA (cpm x 10-4/10 layers) Chloroform—methanol soluble 7.1 7.5 TCA insoluble 14.9 14.3 Uptake of l4C—acetate 60.3 73.5 After incubation for 9 hr in medium minus acetate buffer, aleurone layers were labeled with 2 uc of Na acetate—2-14C (85 mC/mmole) per flask for 1 hr. After the regular homogenization procedure, aliquots of the supernatant were counted for uptake, extracted with Chloro— form—methanol, or precipitated with 10% TCA and filtered through a nitrocellulose filter. 66 acetate into compounds soluble in chloroform—methanol or insoluble in TCA. These categories represent lipids, and proteins and membranes, respectively. Thus the stimulation of incorporation of 32Pi into phospholipids is not accomp- anied by a similar increase in fatty acid synthesis from an acetate precursor. Chemical determinations of organic phosphate and phospholipid levels in aleurone layers were also carried out. The 4,000 x g and 10,000 x g pellets and the 10,000 x g supernatant were analyzed for phospholipids separately and the values combined to give the total phospholipid con— tent of the layers (Table 10). When the separate cell fractions were analyzed, the relative distribution of phospholipids in the pellets and the supernatant resembled the distribution of radioactivity in these fractions (Table 2). That is, the 4,000 x g pellet, the 10,000 x g pellet, and the 10,000 x g supernatant contained about 65%, 14%, and 21%, respectively of the total phospholipid phosphorus in the cell. There were no effects of incubation time or GA on this distribution. The phosphorus level of the organic phOSphate fraction obtained from the 10,000 x g supernatant was also determined (Table 11). Here layers incubated in GA show a 50% increase in the chemical level of organic phosphorus at 12 and 18 hours. 67 Table 10. Phospholipid content of aleurone layers. Time (hr) Phospholipid-P (umoles/10 layers) LEA +GA 6 0.67 0.72 12 0.73 0.66 18 0.67 0.67 24 0.70 0.51 Phospholipids were extracted from the 4,000 x g and 10,000 x g pellets and supernatant of aleurone layer hom- genates. The values above are a sum of these three deter— minations. 68 Table 11. Organic phosphate content of aleurone layers. Time Organic—P (pmoles/lO layers) ~21 fl 6 4.6 4.8 12 4.8 7.6 18 3.8 7.0 24 3.8 5.3 Chemical determinations of phosphorus were made on the organic phosphate fraction of the 10,000 x g supernatant of the homogenate of aleurone layers incubated as shown above. 69 This increase is similar to the GA-enhanced incorporation of 32 Pi into organic phosphates of Figure 6, but the time course is slightly different. In Figure 6 and in parallel determinations on material analyzed in Table 11, it was seen that the GA enhancement of organic phosphate radioactivity occurs only during hours 4 to 12. However the increase in the Chemical level of organic phosphorus is not apparent at hour 6, but is present at hours 12 and 18. So the two effects overlap, but do not coincide exactly. Data presented in this section, then, suggest that the . . 32 increase in Pi incorporation into phospholipids does not involve net synthesis of phospholipids, but occurs during turnover of existing lipid or phospholipid components. DISCUSSION In order to study the control of phospholipid synthesis it must first be established that: (a) the radioactivity measured is actually incorporated into phospholipids; and (b) a change in the rate of incorporation of 32Pi into phospholipids is an accurate measurement of phospholipid synthesis and is not merely a reflection of variations in the uptake and/or metabolism of 32P1. In this work, the first point is adequately covered by the extraction and characterization procedures. Chloro- form—methanol extraction followed by a wash of the chloroform phase to break emulsions insures that only 32P in phospho- lipids is counted. Thin—layer chromatography of extracts 32 verified that all P present was incorporated into phospho— lipids. In order to insure that the measured rates of 32Pi incorporation into phospholipids were representative of 13 vivo metabolism, 32Pi uptake and incorporation into organic phosphates were also monitored. Since all 32 P incorporated into phospholipids will have passed through the organic phosphate pool, this pool represents the precursor pool of 32P for phospholipid synthesis. Therefore, the incorporation 70 71 32 of Pi into organic phosphates can be used as an internal 32 standard to estimate the level of P which is available for phospholipid synthesis. Use of the ratio, phospholipid- 32p organic-32P, corrects the level of radioactivity in phospholipids for any differences in labeling of the organic phosphate pools. The result is an accurate measurement of the relative rate of phOSpholipid synthesis. All variations in 32Pi uptake, internal pools of 32Pi, 32 and the rate of incorporation of Pi into organic compounds are taken into account by such a calculation. The enhancement by GA of the rate of phospholipid synthesis can now be added to the series of events which preceed amylase production in barley aleurone layers and which can be directly related to protein synthesis. The response is initiated 4-6 hr after the addition of GA and increases to a maximum after 8-12 hr. This time course is in good agreement with the work of Evins (1971) which showed GA—enhanced polysome formation and an increase in the number of ribosomes over the same time period. Thus there is a concomitant increase in two cellular components having a major role in protein synthesis. The decline in the rate of phospholipid synthesis occurs during the time when the amount of polysomes has also reached a plateau and a linear rate of a—amylase production has been established. The recent work of Collins, g3 31. (1972) showed an increase in the specific activity of 32Pi-labeled CTP 30 72 and 90 min after the addition of GA to wheat aleurone layers. This implies a more rapid turnover of CTP at early incubation times with GA. Since CTP has a fundamental role in phospholipid synthesis, this effect could be related to the enhancement of phospholipid synthesis observed in barley. However, there is a discrepancy in the timing of the events, since the GA effect on the specific activity of CTP has dis- appeared by 2 hr while increases in phospholipid synthesis do not begin until after 4 hr of GA treatment. The importance of phospholipid synthesis for the sub- sequent production of a-amylase and other hydrolases is demonstrated by the metabolic and hormonal control of 32Pi incorporation into phospholipids. Cycloheximide rapidly inhibits any GA—enhanced increase in the rate of phospholipid synthesis without affecting the basal rate in control tissue. The same is true of 6-methylpurine which is known to have no effect on the rate of phospholipid synthesis in -GA layers (R.A.B. Keates, unpublished data) although it inhibits any GA—induced increases in this rate. The role of abscisic acid as an antagonist of GA action has been documented for lettuce seed germination (Khan, 1968), germination of hazel seeds (Ross and Bradbeer, 1971), and in the aleurone layer system for inhibition of a—amylase production (Chrispeels and Varner, 1966), polysome formation (Evins and Varner, 1972), and production of lecithin- synthesizing enzymes (Johnson and Kende, 1971). Abscisic 73 acid also causes a rapid and Specific inhibition of the GA enhancement of the rate of phospholipid synthesis. This inhibition is dependent on ABA concentration. Complete inhibition is obtained at 1 uM ABA which means that phospho- lipid synthesis is more sensitive to ABA than seed germina— tion and is as sensitive as other processes in aleurone layers cited above. Additional evidence for hormonal control of phospho— lipid synthesis is the requirement for the continued presence of GA. Removal of GA in mid—course results in a decrease in the rate of phospholipid synthesis. Moreover, this rate also depends on the concentration of GA in the incubation medium. Furthermore, attempts at mimicking the GA stimulation of phospholipid synthesis failed. Cyclic AMP was not effective. Addition of amino acids to the incubation medium did not substitute for GA (unpublished results). Likewise, the addition of a series of compounds (glucose, amino acids, MgClZ, MnClZ, KCl, CaClZ, glycerol, and inositol) which would be expected to be mobilized by early GA action did not stimulate phospholipid synthesis. The inhibition of phospholipid synthesis by mannitol has several interesting aspects. The inhibition closely resembles the degree of inhibition of a—amylase production by mannitol and is specific for the GA—induced increase in the rate of phospholipid synthesis. 74 An additional effect of GA, the increase in the ratio, 32 32 organic- P Pi uptake, is not affected by mannitol. 32Pi metabolism This means that the relative increase in caused by GA is an early event not subject to regulation by osmotic stress. This is important because it is an example of a GA-induced process which can be physiologically separ- ated from later GA-induced processes. It has also been found (Koehler, 23 31., 1972) that mannitol does not inhibit to a significant extent the GA— induced increases in phosphorylcholine-cytidyl transferase (30% inhibition) or in phosphorylcholine-glyceride trans- ferase (no inhibition). This Clearly orders some of the events preceeding hydrolase induction. The increases in phospholipid synthesis and a-amylase production follow the increases in the activities of the lecithin-synthesizing enzymes. Between these events is a metabolic step which is sensitive to osmotic stress. It has been proposed (Jones and Armstrong, 1971) that osmotic regulation is of physiological importance in con— trolling hydrolase production in germinating barley seeds. The effect of osmotic stress on d-amylase production may be via the inhibition of phospholipid synthesis. Alternatively, both phospholipid synthesis and hydrolase production may be inhibited because osmotic stress prevents the mobilization of precursors necessary for these processes (Koehler, e: 31., 1972; Jones, 1969 a). 75 There was no indication of a specific stimulation of phospholipid synthesis associated with the microsomal fraction of the aleurone cells. Phospholipid radioactivity had the same enhancement by GA in the 4,000 x g pellet, the 10,000 x g pellet and the 10,000 x g supernatant (Table 2). There is the possibility of some cross contamination of membranes in the cell fractions. Aleurone cells have very thick cell walls and the severe grinding procedures needed to insure all breakage could disrupt organelles. Neverthe- less, the GA enhancement of phospholipid synthesis seems to be a general phenomenon throughout the cell. The increased incorporation of radioactivity cannot be attributed to any specific type of membrane or to any individual phospholipid. The absence of an increase in lipid phosphorus during GA treatment and the lack of a GA stimulation of acetate incorporation into lipids suggest that there is a GA— enhanced turnover rather than a net synthesis of phospho— lipids. GA may cause the mobilization of phospholipids from storage to support membrane synthesis. If the mechanism of mobilization results in a partial degradation of the phos— pholipid molecule, 32P could be incorporated into phospho— lipids during resynthesis. This would account for the lack of an increase in total lipid phosphorus or in acetate in— corporation. The storage site of phospholipids could be the spherosomes which are present (Jones, 1969 C) in aleurone cells. SUMMARY Evidence for the hormonal control of phospholipid synthesis in the barley aleurone layer has been presented. 32Pi incorporation into phospho- The enhancement by GA of lipids occurs throughout the cell. Inhibitors, whether metabolic, osmotic, or hormonal, which inhibit the synthesis of a-amylase have a similar effect on the rate of phOSpho- lipid synthesis. In all Cases, the inhibition is specific for the GA enhancement of the basal rate of phospholipid synthesis. 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