THEBIB .,¥’""“"““‘“ *1 ,. ,,. "”’ LIBRARY “1 Michigan State This is to certify that the thesis entitled Synthesis, Transport and Metabolism of Phosphoryl- choline in Tomato Plants presented by Barry Andrew Martin has been accepted towards fulfillment of the requirements for Doctoral Crop and Soil Science degree in K Date / ' gikf/fl 0-7639 OVERDUE FINES: 25¢ per day per item RETURNING LIBRARY MATERIALS: Place in book return to remove charge from circulation records THE SYNTHESIS, TRANSPORT AND METABOLISM OF PHOSPHORYLCHOLINE IN TOMATO PLANTS By Barry A. Martin A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Crap and Soil Science 1980 ABSTRACT THE SYNTHESIS, TRANSPORT AND METABOLISM OF PHOSPHORYLCHOLINE IN TOMATO PLANTS By Barry A. Martin In extracts made from roots of tomato plants phosphorylcholine was formed at a rate of 9.6 nmoles min.1 (gram fresh weight root)-1. This reaction required magnesium and ATP, and was probably catalyzed by the enzyme choline kinase (EC 2.7.1.32 ATP:choline phosphotransferase). Labelling experiments performed with [32P]-orthophosphate and [1,2-14C]- choline indicated that phosphoryl choline became labelled far more rapidly than did phosPhatidylcholine. Experiments with added [1-14C]— ethanolamine labelled phosphatidylcholine, but not phosphorylcholine. These experiments indicate that phOSphatidylcholine hydrolysis probably does not significantly contribute to the biosynthesis of phosphorylcholine which is transported in the xylem. Phosphorylcholine is the major phosphate ester in the xylem sap of tomatoes, representing 90% of the ester phosphate and up to 30% of the total phosphate. Phosphorylethanolamine was identified as the only other phosphate ester, besides phosphorylcholine, which is found in xylem exudate. Inorganic phosphate in the xylem exudate was labelled with [32P]-orthophosphate added to the nutrient, from a rapidly exchanging Barry A. Martin "root pool" while phosphorylcholine and phosphorylethanolamine were labelled from the more slowly exchanging metabolic pool. Labelling experiments with [1,2-14C1-choline showed that 90% of the choline in the xylem exudate was in phosphorylcholine. When [l-lAC]-ethanolamine was supplied 50% of the ethanolamine in the xylem was phosphorylethanola- mine. The concentration of inorganic phosphate in xylem exudate decreased from 1700 to 170 uM when plants were grown in phosphate free nutrient for 7 days. The concentration of ester phosphate (phosphoryl- choline and phosphorylethanolamine) remained unchanged at 35 t 9 uM. When 1 mM choline was fed through the nutrient solution, the flux of ester phosphate in the xylem exudate increased from 0.4 to 2.2 nmoles min.l (gram fresh weight root).1 while the flux of inorganic phosphate did not change. When 1 mM ethanolamine was supplied the ester phosphate flux increased from 0.4 to 0.9 nmoles min.1 (gram fresh weight root)-1. Nitrogen concentration and triacontanol in the nutrient solution had no effect on phosphorylcholine concentration in the xylem exudate. Defoliation decreased phosphorylcholine labelling in the xylem exudate relative to the labelling of inorganic phosphate. PhosPhorylcholine distribution was examined by supplying tomato shoots with [32Pl-orthoph03phate or [32P]-phosphory1choline. The initial distribution of these compounds in the leaves by xylem transport was essentially the same. [32P]-phosphorylcholine and [32?]-ph03phorylethanolamine supplied to leaves through the xylem were incorporated into phOSpholipids in the leaves with little hydolysis to Pi and the bases. Barry A. Martin Tomato plants homozygous for the pds/pds allele were studied because the phenotype of these plants indicated that they might have a lesion in phosphate utilization. The pds/pds allele was not expressed in callus made from hypocotyls. By grafting experiments the mutation was shown to be expressed only in the shoots of whole plants. Phos- phorylcholine and high nitrogen treatments increased the growth rate of the pds/pds plants, but did not completely restore the phenotope to wild type. Total phenotypic reversion was only achieved by grafting pds/pds shoots onto the shoots of normal plants, and then it only lasted until the pds/pds leaf mass was larger than that of the wild type plant. ACKNOWLEDGMENTS I would like to thank Dr. N. E. Tolbert for his encouragement, support and guidance throughout my graduate career. I would like to thank Dr. Peter Carlson for his advice, support and kind understanding. I also would like to express my gratitude to the other members of my committee Dr. Jon Fobes, Dr. Andrew Hanson, and Dr. M. Wayne Adams, for their support and advice during the last three years. Finally, I would like to give Special thanks to my wife, Jill, whose faith and love has seen me through. ii "What a long, strange trip its been" . . . Robert Hunter iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . LIST OF FIGURES . . . . . . . . . . . . . LIST OF ABBREVIATIONS . . . . . . . . . . . . INTRODUCTION 0 O O O O O C O O O O O O O O 0 CHAPTER 1. THE SYNTHESIS AND METABOLISM OF PHOSPHORYLCHOLINE IN TOMATO PLANTS . . . . . . . . . Introduction . . . . . . . . . . Materials and Methods . . . . . . Plant Growth . . . . . . . . Labelled Compounds . . . . Labelling Plant Material . Extraction and Partioning . . Chromatography . . . . . . . Preparation of Plant Extracts Assays . . . . . . . . . . Enzyme Assays . . . . . . . . Results and Discussion . . . . . Labelling Compounds in Roots with [3 orthophosphate . . . . . . Choline Kinase and Ethanolamine Kinase Labelling Compounds in Roots with [1,2- choline . . . . . . . . . . P-choline Synthesis by Phospholipase Activity . . . . . . . Labelling Compounds in Roots with [1-14C1- ethanolamine . . . . . Labelling Compoggd in Leaves with [3 P]-P-ethanolamine choline and [ Labelling in Whole Plants . Summary . . . . . . . . . . . . . References . . . . . . . . . . iv 2P]- 291-p- 14¢ Page vii ix xi 14 14 15 l7 17 18 20 20 32 CHAPTER 2. 3. PHOSPHAT EXUDAT ABSTRACT Intr Mate E ESTERS AND INORGANIC PHOSPHATE IN XYLEM E OF TOMATO I. LABELLING KINETICS . . . oduction . . . . . . . . . . . . . . . . rials and Methods . . . . . . . . . . . . Reagents . . . . . . . . . . . . . . . . Plant Growth . . . . . . . . . . . . . Radiochemical Labelling . . . . . . . . Separation of Labelled Compounds . . Identification of P-ethanolamine in Xylem Exudate . . . . . . . . . . . . . . . Quantitation of Pi and P-esters in Xylem Exudate . . . . . . . . . . . . . . . . Extraction of Plant Material . . . . . . Results and Discussion . . . . . . . . . . Rate of Labelling the Phosphate Esters . Pulse-Chase Experiments . . . . . . . . Effect of Nutrient thsphate Concentratiiz . C]- Labelling with [1,2-. C]-choline and [l- ethanolamine . . . . . . . . . Summary . . . . . . . . . . . . . . . . . .i. Refe PHOSPHAT EXUDAT rences O O I O O O O O O O I O O O C O O E ESTERS AND INORGANIC PHOSPHATE IN XYLEM E II. FACTORS INFLUENCING THE FLUX AND CONCENTRATIONS OF PHOSPHATE ESTERS AND INORGANIC PHOSPHATE IN XYLEM EXUDATE . . . . . . . . . . ABSTRACT Intr Mate oduction . . . . . . . . . . . . . . rials and Methods . . . . . . . . . . . Reagents . . . . . . . . . . . . . . . ‘ Plant Growth . . . . . . . . . . Collection of Exudate . . . . . . . . Determination of Pi and ester-P . . . Results and Discussion . . . . . . . . . . . Effect of Nutrient Phosphate Concentration Effect of Phosphate Starvation . . . . . Effect of Ethanolamine and Choline in the Nutrient . . . . . . . . . . . . . . . Effect of CCC . . . . . . . . . . . . . . V Page 34 34 36 37 37 37 37 38 38 39 39 39 39 40 41 42 43 51 52 52 54 55 55 55 SS 56 57 S7 58 59 60 CHAPTER Summary . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . 4. P-CHOLINE LABELLING IN TOMATO XYLEM EXUDATE AS AFFECTED BY N03, DEFOLIATION OR TRIACONTANOL TREATMENTS . . . . . . . . . . . . . . . Introduction . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . Results and Discussion . . . . . . . . . References . . . . . . . . . . . . . . . . 5. THE DISTRIBUTION OF P-CHOLINE IN TOMATO PLANTS . Introduction . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . Results and Discussion . . . . . . . . . . Summary . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . 6. EXPERIMENTS ON THE NATURE OF A PRESUMPTIVE MUTANT OF PHOSPHATE METABOLISM (pds/pds) IN TOMATO (LYCOPERSICON ESCULENTUM L.) . . . . . . . . . Introduction . . . . . . . . . . . . . . . . Materials and Methods . . . . . . . . . . . . Results and Discussion . . . . . . . . . . . Effect of P-choline and Choline on Callus Growth 0 O O O O O O O O O O O O O O O The Effect of P-choline on pds/pds Plants . Summary . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . CONCLUDING DISCUSSION . . . . . . . . . . . . . . . . . . APPENDICES . . . . . . . . . . . . . . . . . . . . . . APPENDIX A. The Pi and P-ester Concentrations in Xylem Exudate of Field-Grown Tomato Plants . . . . . . . . B. The Growth Rate of Tomato Plants (Lycopersicon Esculentum) var VF-36 in Standard Nutrient Culture and the Required Rate of Phospha- tidylcholine Synthesis (PC) Necessary to Maintain Growth . . . . . . . . . . . . BIBLIOGRAPHY . . . . . . . . . . . . . . vi Page 61 69 71 71 71 72 77 78 78 78 79 81 85 86 86 87 88 88 88 91 100 101 103 103 104 105 LIST OF TABLES Table Page Chapter 1 l. The Labelling of Compounds iEACultured Tomato Roots After Treatment with [1,2- C]-choline for 10 Days . . . . 22 2. The Effect of Choline nd P-choline on the Labelling of PC and PE by [1 1 C]-ethanolamine . . . . . . . . . . . 23 Chapter 2 l. The Specific Activities of Pi, P-choline and P- ethanolamine in Xylem Exudate Aftga Labelling Roots in Nutrient Solution Containing [ P]-Pi . . . . . . . . . 44 2. The Effect of Phosphate Concentration on the Labelling Rate of Pi and P-choline in Xylem Exudate . . . . . . . . . 45 3. The Distribution of Radioactivity in Tomatoes After Feeding [1,2-14CJ-choline and [l 14C]- ethanolamine . . . . . . . . . . . . . . . . . . . . . . . 46 Chapter 3 l. The Effects of 2 uM Pi, 1 mM Pi and 10 Days of Pi Starvation and Return to Nutrient Containing Pi on the Concentrations and Flux of Pi and P-esters . . . . . 63 2. The Effects of CCC on the Exudation Rate, the Con- centration of Pi and the Concentrations of P-esters in the Xylem Exudate of Tomato Plants . . . . . . . . . . . 64 Chapter 4 1. The Effect of Defoliation on P-choline Labelling in Tomato Xylem Exudate . . . . . . . . . . . . . . . . . . . 74 2. The Effect of N0- Concentration on P-choline Labelling in Xylem Exuda e . . . . . . . . . . . . . . . . . . . . . 75 vii Table Page 3. The Effect of Triacontanol on P-choline Labelling in the Xylem Exudate of Tomatoes . . . . . . . . . . . . 76 Chapter 5 1. The Distribution of [32P]-Pi and [32P]-P-choline to the Leaves of Excised Tomato Shoots . . . . . . . . . . 82 2. The Distribution of [32P]-Pi and [32P]-P-choline in the Leaves of Excised Tomato Plants . . . . . . . . . . 83 3. Labelling of Tomato Leaves by [3 2P]-Pi Fed to the Roots . . . . . . . . . . . . . . . . . . . . . . . . 84 Chapter 6 l. The Effect of Pi Concentration on the Growth of VF-36 and pds/pds Callus . . . . . . . . . . . . . . . . . 92 2. The Effect of P-choline on Callus Induction and Growth . . . . . . . . . . . . . . . . . . . . . . . . . . 93 3. The Growth of pds/pds and VF-36 Plants Grown on Normal and High Nitrogen Medium . . . . . . . . . . . . . . 94 viii LIST OF FIGURES Figure Introduction Known Pathways for P-choline Synthesis in Plant Tissues . . . . . . . . . . . . . . . . . . . Chapter 1 Labelling of compounds in tomato roots from addition Of [3 2P]Pi O O O O O O O O O O O O O O O O O O O O The labelling patigrn in cultured tomato roots after being fed [1, 2 C]-choline . . . . . . . . . The labelling pattern in the aqueozs fraction of cultured tomato roots after [1 C]- -ethanolamine was fed 0 O O O O O I O O O O O O O O O O 0‘ O The labelling of PE and PC after [1 14C]-ethanolamine was fed to cultured tomato roots . . . . . . . . . The labelling2 pattern in tomato leaves after being fed 1 mM [ 2P]-P-choline (lower figure) specific activity 7. 6 cpm pmol 1 and 0.1 mM [3 P]— —P- ethanolamine, specific activity 0.5 cpm umol (upper figure) . . . . . . . . . . . . . . . . Whole tomato plants were fed 1 mM [1 14C]- _ ethanolamine specific activity 0.60 cpm umol (upper figure) and 1 mM [1,2 4C]-choline 0.99 cpm pmol (lower figure) . . . . . . Chapter 2 The specific activities of Pi and P-choline plus P-ethanolamine in xylem exudate of tomato plants The amount of [32F] in ghosphate and P-choline after a 10 min pulse with [ 2Pi]-phosphate and a 80 min chase with [ 32P]- -phosphate . . . . . . . . . . ix Page 26 27 28 29 30 31 48 49 Figure Page 3. The amount of radioactivity in P-choline, choline P-ethanolamine and ethanolam ne after adding 100 pM [1,2 l4C]-choline or [1 1 C]-ethanolamine to the nutrient solution at specific activities of 991 and 598 cpm mol'1 respectively . . . . . . . . . . 50 Chapter 3 l. The concentrations of Pi and P-esters in xylem exudate as a function of Pi concentration in the nutrient SOlution O O I O O O O O O O O I O O O O O O O O O O O O 66 2. The effect of Pi starvation on the flux of Pi and P-esters into xylem exudate . . . . . . . . . . . . . . . 67 3. The effect of exogenous choline and ethanolamine on the flux of P-esters into xylem exudate . . . . . . . . . 68 Chapter 6 l. pds/pds plants grown for 70 days in the greenhouse in the summer . . . . . . . . . . . . . . . . . . . . . . 96 2. A pds/pds shoot grafted onto the top of a VF-145 rootstock, the graft was done 40 days before the photograph . . . . . . . . . . . . . . . . . . . . . . . 97 3. A pds/pds shoot grafted onto the top of a VF-l45 plant at the same time as the plant in Figure 2 . . . . . 98 4. pds/pds plants grown in nutrient culture . . . . . . . . . 99 LIST OF ABBREVIATIONS* phosphate derivatives P- phosphatidylcholine PC phosphatidylethanolamine PE phosphatidylserine PS polyvinyl poly pyrolidone PVPP N,N-bis[2—hydroxyethyl] glycine Bicine Gerger—Mneller G-M CCC [2-chloroethyl] trimethylammonium chloride MS Murashige and Skoog' *Non-standard abbreviations. All others follow the Council of Biology Editors, Committee on Form and Style, 1978. CBE Style Manual Ed. 4. American Institute of Biological Sciences, Washington, D.C. (Library of Congress Catalog No. 78-50755; ISBN 0-914340-02-6). xi INTRODUCTION In 1933 organic forms of phosphate were reported to constitute 32 t 4% of the total phosphate in root pressure exudate of field grown corn plants (17). Other investigators subsequently claimed that only inorganic phosphate was in the exudate of roots obtained from plants of many species (9). In 1955 it was found that when [32P]-orth0phosphate was supplied to the roots of barley, bean, willow and tomato plants, two P-esters were labelled in the xylem exudate (3). This work was repeated with barley plants by a group in England, but their chromato- graphy system only resolved one P-ester from P1 (10). ,In 1956 one of the esters was identified as phosphorylcholine (12). It was the major labelled P-ester in the xylem exudate of tomato, where it contained 20% of the 32? and in barley where it was 6% of the total (24). The other P-ester remained unidentified. The above results have not been confirmed by further investi- gation and the physiological significance of P-esters in xylem exudates has not been established. In fact xylem sap obtained by vaCcum ex- traction, from decapitated willow plants, contained either no P-esters, or only a fraction of the total phosphate when compared to root pressure exudate (4). Similarly P-esters could not be detected in xylem sap obtained from decapitated tea plants by mild suction (20). In xylem exudate from pumpkin roots organic phosphate was detected, but the workers were unable to measure its quantity reproducibly (8). Inorganic phosphate transport has been extensively studied in plants (3, 5, 6), but since some workers did not consider that P—esters may be present in the xylem stream, there have been no published reports on P-choline transport or distribution. [32P]-orthophosphate fed to bean plants was reported to circulate throughout the plant, preferentially accumulating in the younger leaves (1), but no attention was given either to the form of phosphorus being translocated or to the forms accumulated in various plant parts. Just as the physiological significance of P-choline in the xylem exudate has remained unresolved, so has its mode of synthesis. P-choline synthesis was first observed in plant extracts as a product of the enzyme choline kinase (ATP: choline phosphotransferase EC 2.7.1.32), partially purified from rape seed (19). The enzyme was shown to have a Km (choline) of 60 uM and a requirement for a 1:1 ATPzMg++ ratio for maximal activity. Choline kinase was investigated as a source of P-choline in tobacco, spinach, squash, barley and wheat plants (23). In barley roots the activity of this enzyme was 0.26 nmoles min.1 (g fresh weight root)-1. The authors felt that this much activity could not account for the flux of P-choline into the xylem exudate of barley, which had previously been observed (25). Since these early publications choline kinase has been reported to be a mitochondrial enzyme in dodder (Cuscuta reflexa) (14). Its activity has also been separated from that of ethanolamine kinase (11). Since the reported activity of choline kinase in plants was too low to be the sole source of P-choline in the xylem exudate in plants, I have re-examined the kinase, as well as alternate sources of P-choline, in xylem exudate. These are summarized in Figure 1. It has been necessary to consider the synthesis of choline as the pre- cursor of P-choline as well as the formation of phosphatidylcholine. It has been established that choline in plants can arise from the decarboxylation of serine and the subsequent methylation of ethanolamine (2), but the methods used did not distinguish between the free serine and ethanolamine and the bound forms of these compounds as intermediates in the biosynthesis of choline. While there is evidence for decarboxylation of free serine and P-serine to ethanolamine and P-ethanolamine in rat liver (16), there has been no report of these reactions in vitro in plant tissues. Decarboxylation of phosphatidylserine to phosphatidylethanolamine has been demonstrated (27) and the phosphatidylethanolamine appeared to be directly methylated to phosphatidylcholine. It has recently been established that the choline, which is oxidized to form betaine in barley leaves during water stress, arises from phosphatidylcholine which is a methylation product of phosphatidylethanolamine (Hitz and Hanson unpublished). This pathway for choline synthesis may also be the major pathway for deungyg synthesis of phosphatidylcholine in plant tissue. The alternate CDP-choline pathway of phosphatidylcholine for- mation which has been demonstrated in soybean seeds (4) and potato tubers (24) in yiyg and in onion leaves (13) in 31539, may be a salvage pathway. The choline used by these enzymes could arise from the hydrolysis of phosphatidylcholine by phospholipase D (phosphatidylcholine phosphatido hydrolase EC 3.1.4.4), which is an almost ubiquitous enzyme in plant tissue (18). P-choline could also be a product of this pathway by phospholipase C (phosphatidylcholine choline-phosphohydrolase EC 3.1.4.3) hydrolysis of phosphatidylcholine. This reaction has been demonstrated in sugar beet, spinach, cabbage and carrot plants (7). By comparison to P-choline synthesis, its metabolism in plants is quite well characterized. P-choline may be hydrolyzed to Pi and choline after its arrival in the leaves. A phosphatase, with an acid pH optimum, has been partially purified from spinach leaves that readily hydrolyzed P-choline (23). While the leaves contained sufficient activity of this enzyme to hydrolyze all of the P-choline arriving from the xylem, the enzyme was 10 times more active with phosphoglyceric acid than with P-choline, indicating that it was not a specific P-choline phosphatase. P-choline has also been reported to be incorporated intact into phosphatidylcholine, via the GDP-choline pathway (13). In this pathway P-choline combines with CTP to form GDP-choline as catalyzed by P-choline cytidyl transferase (CTP: Choline phosphate cytidyl transferase EC 2.7.7.15). The CDP-choline combines with diglycerides to form phos- phatidylcholine. This reaction is catalyzed by choline phosphotrans- ferase (CTP-choline 1,2 diglyceride-choline phosphotransferase EC 2.7.8.2). This enzyme has been partially purified from spinach leaves, and its activity was inseparable from ethanolamine phosphotransferase (Dr. B. Mudd personal communication). The phosphatidylcholine formed by these reactions would be incorporated into membranes, where it represents approximately 50% of the phospholipid in plant tissues (20). The purpose of my investigations was to determine the physio- logical significance of P-choline in xylem exudate. Is P-choline transported in the xylem, from the roots to the leaves? In addition I have attempted to determine which pathway or pathway could provide sufficient P-choline to account for the flux observed into xylem exu- dates and to determine the fate of P-choline after it arrives in the leaves. Part of my research involved in_vivo labelling with 32P or 14C in an effort to deduce a metabolic pathway (A-+ B + C + etc.) based upon the rate and amount of label appearing in various possible inter- mediate products. This method is limited by the fact that each com- ponent measured may be the sum of pools in the tissue. For example, the total label in phosphatidylcholine represents the sum of all the membrane components. Thus if there were a small rapidly turning over pool of phophatidylcholine, as on one membrane component of the root stele, slow labeling of the total phosphatidylcholine of the root would not detect the smaller rapidly metabolized fraction. Since my interpretations of results are based on the simplifying assumption of only one pool of each component in a tissue, there inter- pretations must be considered tentative. .mmSmmHH ucmHm :H mHmoauchm oaHHO£UIm mom mhmBSumm GBOcM .H mustm mcwHOLUIon Al mcHHoxuum . \on17/M.oan< HHV flak new oCHEmHocmLumlm mafifioxv r: a: 4 «I ocHEmHocmsumimQU ma< Amv ANV rmAmmoV me<. oswaonolm ov«uoo>awwm ; mcHEmHocmLuo \/ \\\ ,/ . mcwpmmahmfiumnamosm71 1\\¥5 a: a: is E 2 No n: #6 iv /\ mCHHoon>mHumcamoza IIIIII mwcHEmHocmzumfimmHumcamoxa ocHumm HHHV HA mmov mafiaocu CHAPTER 1 THE SYNTHESIS AND METABOLISM OF PHOSPHORYLCHOLINE IN TOMATO PLANTS Introduction In plants, P-choline may be formed from the phosphorylation of choline, catalyzed by the enzyme choline kinase (ATP: choline phosphotransferase EC 2.7.1.32) (l6, 19, 20). It may also be formed by phospholipase C (phosphatidylcholine choline-phosphohydrolase EC 3.1.4.3), hydrolysis of phosphatidylcholine (PC) (7, 8). PC is assumed to be synthesized by methylation of phosphatidylethanolamine (PE) (25). In Chapter 3 P-choline flux into the xylem exudate of tomato was shown to be 0.4 i 0.1 nmol min.1 (g fresh weight root).1 or higher if choline was added. The reported activity of choline kinase in root tissue, 0.26 nmol min.1 (g fresh weight root)-1 (20), might therefore be too low to account for the flux of P-choline into xylem exudate. Phospholipase C activity might also provide as much P-choline as choline kinase, since it is about one tenth as active as phospholipase D in plant tissue (7, 8), and the latter has a mean activity of 1.0 nmol min.1 (g fresh weight root)-1 (15). P-choline has been recognized as a component of xylem exudates since 1956 (ll, 22), but there have been no reports on how it is utilized after it arrives in the leaves. In onion leaf tissue the 7 entire GDP-choline pathway of choline + P-choline + GDP-choline + PC has been demonstrated in vitro (12). P—choline-cytidyltransferase (CTP: choline phosphate cytidyl transferase EC 2.7.7.15) kinetics and co-factor requirements have been examined with enzyme obtained from spinach leaves (14). While these enzymes have been detected in vitro, it has not been determined whether the P-choline which arrives in leaves from roots, is incorporated into lipids, stored, or hydrolyzed to phosphate and choline. While it appeared doubtful that much P- choline could be stored, since its reported concentration is 20 pH (1), it is possible that P-choline was hydrolyzed, since leaves have an acid phosphatase capable of using P-choline as a substrate (20). In this investigation I have considered how the P-choline, which is found in the xylem exudate of tomato plants, might be syn- thesized in the roots and how it might be utilized in the leaves. Materials and Methods Plant Growth. Tomato (Lycopersicon esculentum L.) cv. VF-36 seeds were sterilized for 20 min in 1.0% NaOCl and 0.1% SDS, rinsed in sterile glass-distilled water for 15 min and placed on a slightly modified Murashige and Skoog culture medium (13). The medium contained 20.6 mM NH NO 18.8 mM KNO3, 3.0 mM CaCl 4 3, 1.5 mM MgSOA, 1.25 mM 2’ RH P0 0.1 mM H B0 0.1 mM MnSO , 37.0 pM 2nsoa, 5.0 pM KI, 1.0 pH 2 4’ 3 3’ 4 NaMoOa, 1.5 pM CuSO4, 2.2 pM CoClZ, 5.5 mM myo-inositol, 3.0 pM thia- mine, 4.9 pM pyridoxine, 8.1 pM nicotinic acid, 88.0 mM sucrose and 0.9% Difco bacto agar. After 10 days at 25°C day (70 uE sec.1 m-Z) and 20°C night, the seedlings were transferred to Hoagland's nutrient solution and grown as described in Chapter 2, or 1 cm root tips were excised for culture. Three cultured roots were grown in 125 m1 erlenmeyer flasks with 50 ml of the medium described above, minus agar at pH 5.9. The flasks were rotated continuously at 125 rpm to provide aeration. Roots were subcultured every seventh day, by removing 1 cm of root tip and placing it in fresh medium. Labelled Compounds. The [32P]-orthophosphate was from Amersham and 14 4 [1,2 C]-choline and [l1 C]-ethanolamine were from Research Products. [32P]-P-choline and [32P]-P-ethanolamine were made and purified as follows. Choline or ethanolamine was incubated with [32P]-ATP from New England Nuclear and choline kinase from Sigma. The mixture also contained 50 mM Bicine pH 8.5, 10 mM MgCl2 and 1 mM dithiothreitol. After the reaction was judged to be complete, the mixture was boiled for 3 min and centrifuged at 15,000 X g for 20 min. The supernatant was applied to a l X 20 cm QAE-sephadex column, and the column was washed with water to remove the buffer, excess choline or ethanolamine and the salts. The column was developed with a 0—l.0 M KOH gradient. P-choline and P-ethanolamine were eluted with 0.1 and 0.3 M KOH respectively. The labelled fractions were reduced in volume by rotary evaporation, the mixture was neutralized by addition of HCl and the compounds were assayed for purity by 2-dimensional chromatography, as described in this paper, and by absorption at 260 nM. The specific activity of the products was 100-500 Ci mMol-l, and they were diluted with unlabelled P-choline and P-ethanolamine to the specific activities 10 used in the in vivo labelling experiments, which are stated in the figure legends. Labelling Plant Material. The labelled compounds were fed at a concen- tration of 1 mM except when indicated. The specific activities varied 3 to 3.5 Ci mol-l, depending on the isotope and the from 0.9 X 10- duration of the experiment. Specific activities are indicated in the figure legends. Cultured roots weighing 50—100 mg were labelled in 13 X 100 mm tubes containing 4 ml of root culture medium, which was vigorously aerated during the experimental period. After labelling, the roots were washed in fresh medium and blotted dry. Whole plants (19 or 20 days old) were labelled by adding the radioactive compounds to the nutrient solution, which was continuously aerated. After labelling, the roots were washed, blotted dry and weighed. Leaf tissue was labelled directly, by cutting the stem immediately beneath a leaf with a razor blade and placing the stem in a 1:5 dilution of xylem exudate, which contained the radioactive tracer. Extraction and Partioning. Plant material was extracted using a modi- fied Folch procedure developed by Dr. A. D. Hanson and Dr. B. Hitz (unpublished). After labelling, roots were cut into 1 cm segments and leaves were cut into 1 cm2 pieces, and placed into 13 X 100 cm tubes. Liquid nitrogen was poured in and around the tubes and the frozen material was pulverized with a glass rod. Two ml of isopropanol was added and the tubes were vortexed and incubated at 50°C for 15 min, then centrifuged at 1500 X g for 5 min. The samples were flushed with ll nitrogen gas and were stored at -20°C overnight. The next day the isopropanol was removed and placed in a conical centrifuge tube, and 2 m1 of CHClB:Me0H (2:1) was added to the plant material. The material was vortexed and centrifuged for 30 min. The supernatant was removed and combined with the isopropanol extract. The plant material was extracted once more with CHC13:Mc0H centrifuged, and the supernatant was added to the others to make a total volume of 6 ml. The combined supernatants were partioned once against 1.2 ml of 20 mM CaCl2 in water. The upper aqueous phase was removed and added back to the plant material. The organic phase was then partioned twice against 1.2 m1 of 20 mM CaC12, which had been saturated with CHCl3:MeOH:isopropanol (2:1:0.6). After partitioning, the upper phases were added back to the plant material. The organic fraction, which contained the lipids was cooled to -20°C for at least 3 hours and the remainder of the upper aqueous phase was removed. A small amount of anhydrous NaZSO4 was then added to com- plete the drying. The liquid was filtered through glass wool and the solvents were evaporated at 35°C, under a stream of nitrogen gas. The combined aqueous phases, with plant material was heated at 50°C for 10 min, vortexed, centrifuged and the supernatant was filtered through glass wool. The extract was evaporated to dryness under a stream of nitrogen gas at 45°C. Chromatography. The organic phase was taken up in 0.1 ml of CHC13:MeOH (2:1) and applied to the origin of a 0.25 mm Brinkman silica TLC plate. The plate was developed with acetone:petroleum ether (3:1) and dried. The plate was then developed, in the same direction with 12 CHC13:MeOH:glacial acetic acid:water (85:15:10:3.5). Labelled zones were located by autoradiography. The aqueous phase was dissolved in 0.1 m1 of water and 20 pl was applied to the origin of either a 0.25 or 0.5 mm Brinkman cellulose TLC plate. The plates were developed in a 2 dimensional system, with the first solvent being 80% phenol:water and the second direction being butanol:propionic acid:water (2:1:l.3). When additional resolution was required, the compounds were eluted with water and reapplied to a TLC plate (cellulose) which was developed with MeOH:NH40H:H2) (60:10:30). Labelled compounds were identified by co- chromatography with the authentic compounds, some of which were obtained from Sigma, and some of which were provided by Dr. A. D. Hanson. Radio- activity was counted by scraping the labelled material into 20 ml scintillation vials which contained 1 m1 of water and 9 ml of a cocktail consisting of 1 liter Triton X-100, 2 liters toluene, 12 g PPO and 0.15 g POPOP. The mixture was vortexed, cooled to 4°C and counted in a Packard Tricarb liquid scintillation counter. Preparation of Plant Extracts for Enzyme Assays. Tomato roots were cut into 1 cm segments and placed in a buffer containing 50 mM Bicine pH 9.0, 1 mM Na-EDTA, 1 mM MgC12, 10 mM mercaptoethanol, and 2% PVPP. The roots were initially macerated with an electric knife, and then homogenized in a Waring blender at low speed for 60 sec. The homogenate was filtered through 2 layers of miracloth and the filtrate was centrifuged at 100,000 X g for 1 hour. The experiments designed to detect choline kinase in a particulate fraction were done as described elsewhere (19). 13 Preparation of extracts for assay of phospholipase C activity was performed similarly, except the grinding buffer was 50 mM Tris pH 7.0, 10 mM mercaptoethanol, and 2% PVPP. The filtrate was centrifuged at 1500 X g for 10 min. Enzyme Assays. Choline kinase was assayed by a radiochemical assay (23) in a total volume of 0.1 ml. The reaction mixture contained 50 mM Bicine pH 9.0, 8.0 mM MgCl 8.0 mM ATP and 1.4 mM [1,214C]-choline 2. (0.5 Ci mol-l). The reaction was initiated by addition of plant extract, run for 15 min at 30°C, and stopped by boiling for 3 min. The mixture was centrifuged at 15,000 X g for 5 min and 50 ul of the supernatant was applied to the origin of a 0.25 mm cellulose TLC plate. The reaction products were separated by developing the plate in butanol: propionic acid:water as described earlier. Phospholipase C was assayed in a total volume of 1.0 ml which contained 50 mM Tris pH 7.0, 50 mM CaCl 0.02% Triton X-100 and 0.1 M 2. [1,214C]-choline labelled phosphatidylcholine (0.5 Ci mol-l). The labelled PC was obtained from cultured roots which were labelled with [1,2-14C]-choline for 10 days. To assure dispersion of PC the buffer was sonicated immediately before the plant extract was added to initi- ate the reaction. The reaction was run at 30°C and samples were removed every 5 min. The samples were added to MeOH:CHC1 to stop the 3 reaction. The mixture was made biphasic by addition of CHCl3 and water. 14 The aqueous phase containing the [1,2- C]-choline was removed and chromatographed in the butanol:propionic acid:water system. 14 Results and Discussion Labelling Compounds in Roots with [32Pj-orthophosphate. In Figure l the rates of [32P]-Pi incorporation into P-choline, CUP-choline, PC and PE by cultured tomato roots are presented. The amount of label in P-choline plus GDP-choline, in the first 2 hours of labelling, is higher than is found in PC. In addition, the combined pools of P- choline and CDP-choline are 20 fold lower than the PC pool (Table 1). Thus the specific activity of the phosphate group of the esters is much higher than that of PC. These data indicate that the phosphate group of P-choline is much more rapidly labelled than the lipid pools. P-choline is probably labelled by ATP, which has a half time for labelling in plant tissues, of l to 2 min (2). PC is labelled at a much slower rate. The rates of labelling PC and PE are similar, but since the PE pool is only about one third as large in tomato plants (18), the PE may turn over more rapidly than the PC pool. Choline Kinase and Ethanolamine Kinase. The activity of choline kinase was assayed in extracts made from 5 lots of tomato roots. A mean 1 SD activity of 9.6 i 2 nmol min.1 (g fresh weight root)"1 was observed. This level of activity is sufficient to account for a flux of about 0.4 nmol min.1 (g fresh weight root) of P-choline into the xylem exudate of tomato plants (Chapter 3), and the synthesis of PC, which was about 0.45 nmol min”1 (g fresh weight root).1 when 1 mM choline was provided to tomato root (Figure 2). This and other rates of phospholipid syn- thesis were calculated by the formula [cpm (g fresh weight)-1] (spe- cific activity of supplied tracer)-1 (time)-l. The physiological choline concentration was measured at 54 to 90 pM (Tables 1 and 2). 15 Therefore choline kinase was functioning near its Km choline value which is 60 to 80 uM (16, 20). A 4 fold stimulation of P-choline flux into xylem exudate was observed when 1 mM choline was supplied to the roots of whole plants (Chapter 3), which indicates that choline phosphorylation was not normally limited by ATP, but may be limited by the supply of choline in vivo. In experiments designed to detect choline kinase in a particulate fraction (19), at least 95% of the choline kinase in tomato roots was in the cytoplasmic fraction. The value of 9.6 nmol min.1 (g fresh weight root)"1 of choline kinase is about 40 times more activity in tomato root extracts than had previously been detected in barley roots (20). The reasons for this difference are unknown, however our inclusion of mercaptoethanol and PVPP into the grinding buffer may have stabilized the activity of choline kinase in tomato root extracts. The difference could also be due to interspecies variation. The previous conclusion that there was inadequate choline kinase in roots to form P-choline in xylem exudate therefore seems in error. An activity of 12 nmol min-1 (g fresh weight root).1 of ethanolamine kinase in tomato roots was measured. This is approximately the same level of activity as has been observed in spinach leaves (10), where ethanolamine kinase was separated from choline kinase activity. However from the tomato root no effort has been made to separate the two activities, which could be due to the same enzyme. Labelling Compounds in Roots withLl,2-14C]-choline. The rates of labelling PC, P-choline and CDP-choline, in cultured roots, which were 14 supplied with 1 mM [1,2- C]-choline are shown in Figure 2. P—choline 16 was labelled before PC, which lends support to the hypothesis that tomato roots have the capacity to phosphorylate choline at a high rate. Choline was incorporated into PC at a linear rate of 0.45 nmol min-l (g fresh weight root)-1 during the period shown. The labelling kinetics of P-choline, CDP-choline and PC suggest that this incorporation could occur, at least in part, by the CDP-choline pathway (9). The lower rate of 0.11 nmol min-1 (g fresh weight root)”1 of PC synthesis when [32P]-Pi was supplied indicates that the concentration of choline in tomato roots may limit PC synthesis by this pathway, since a higher rate was observed with added choline. A less likely possibility is that the gamma phosphate of ATP was not in equilibrium with the added [32P]-Pi, which would lead to an underestimation of the rate of PC synthesis by 32F incorporation rates, during the latter time periods shown in Figure 1. A linear rate of PE synthesis, 0.11 nmol min-l (g fresh weight 14C]-choline was added to the root root)-1, was also observed when [1,2- culture medium, but no labelled ethanolamine or P-ethanolamine was observed. Since PC was labelled much faster, such data suggest that PC may be demethylated to PE in tomato roots. This rate of PE formation is about five times as slow as the rate of PC formation from added [1-140]-ethanolamine which we have observed (Figure 4). It has been suggested that the CDP-choline pathway is the major source of PC synthesis in the cotyledons of germinating seeds (4, 6) and in potato tuber (21). In tomato roots this pathway also seems to contribute to net PC synthesis, but its contribution to dg_novo syn— thesis of PC is difficult to ascertain. l7 P-choline Synthesis by Phospholipase Activity. When [14C]-labelled phosphatidylcholine was added to extracts of tomato roots, choline was liberated at a rate of 0.08 to 0.10 nmol min”1 (g fresh weight root)-1, which is about the same rate as has been reported in barley roots (16). No P-choline was found as a hydrolysis product. The choline formation was indicative of phospholipase D activity. While our reaction conditions were not identical to those used previously to assay for phospholipase C activity in plant extracts, they were what is considered optimal for the yeast enzyme. [32P]-P-choline was included in one of the controls and no [32P]-Pi was formed, which ruled out the possibility of hydrolysis of P-choline to choline. These results seem to eliminate the possibility of P—choline formation in tomato root extracts by phospholipase C activity. Labelling Compounds in Roots with [1-14C1-ethanolamine. In Figures 3 and 4 data are presented on the labelling rates of PE, PC, CDP- ethanolamine, P-ethanolamine and choline after [1-14C]-ethanolamine was supplied to cultured tomato roots. The labelling kinetics suggest that at least some of the PE in tomato roots could be made by the CDP- ethanolamine pathway. The labelling rate of PC was about half that of PE during the first 10 to 20 min of the experiments. This indicates that if the PC were made by methylation of PE, the newly-made PE must not mix with the entire PE pool of the root before methylation, since the labelling kinetics do not indicate a precursor-product relation- ship. At the end of 3 hours of labelling, the PC pool in the root had about the same specific activity as choline. The P-choline and the CDP-choline pools had no detectable label. These experiments suggest 18 that choline is either made as a hydrolysis product of PC, or by the methylation of ethanolamine. Our failure to detect labelled P-choline may have been due to its small pool size. There is nothing in our data however to suggest that any large portion of its synthesis occurs by hydrolysis of PC. Table 2 contains the results of competition experiments, in which cultured roots were given [l-lAC]-ethanolamine with and without 1 mM choline or P-choline. While addition of both choline and P- choline resulted in a 30% reduction in the labelling of PE, the quantity of label in PC was reduced by 65%. The choline concentration of the root tissue, as measured by isotope dilution (17), indicated that the choline treatment increased the choline pool by about 40%, but addition of P-choline had no significant effect on the choline pool. One explanation for these results is that some of the PC was formed by the CDP-choline pathway after ethanolamine was methylated to choline. Since the rate of PC synthesis from added 1 mM [1--14 C]-ethanolamine was never in excess of .050 nmol min.l (g fresh weight root)-1, PC synthesis may be rate limited by the methylation of ethanolamine or its bound forms. These experiments also suggest that choline may be synthesized in tomato roots by the methylation of both free and bound forms of ethanolamine, but it is impossible to assess the contribution of each reaction. Labelling Compound in Leaves with [32PJ-P-choline and [32P1-P- ethanolamine. Young expanding tomato leaves were supplied with 1 mM [32P]-P-choline, and the labelling of CDP-choline and PC are presented in the lower portion of Figure 5. P-choline was incorporated into PC 19 of tomato leaves at rates as high as 0.27 nmol min.1 (g fresh weight leaf)-1, in the same manner as in rat liver (9). In these experiments no labelled PE was observed. Hydrolysis of P-choline to Pi during the time course of these experiments was not detectable, even though P- choline was supplied at a 20 fold higher concentration than measured in normal xylem exudate (Chapter 3). P-choline was apparently incor- porated rapidly into the PC pools. In the lower portion of Figure 5 data are presented which show the incorporation of [32P]-P-ethanolamine into PC and PE of tomato leaves. P-ethanolamine was incorporated into PC and PE at about the same rate. Since no P-choline was found in the aqueous fraction, PC may arise by methylation of PE, after the P-ethanolamine was incor- porated into PE. When the ratio of labelled PE to PC formed in these experiments, from P-ethanolamine, is compared with labelling in roots, from ethanolamine (Figure 4), it appears that a greater fraction of the PE synthesized from P-ethanolamine in leaves is methylated to PC than when PE is made from ethanolamine in roots. This may have occurred for several reasons. The rate of PE synthesis in leaves may have been limited by P-ethanolamine uptake. Since ethanolamine may be incor- porated into PE by base exchange (23), this PE may not be as readily methylated to PC as that formed by the incorporation of P-ethanolamine. Roots and leaves may have different levels of the enzymes necessary to methylate PE to PC. Since this question was not central to the role of P-choline in the xylem exudate, these possibilities have not been further tested. 20 Labelling;in Whole Plants. When whole plants were fed [1,2-14C]- choline or [1-14C]-ethanolamine through their roots, these compounds were transported to the leaves intact, or as their respective phos- phate esters. When labelled choline was supplied, 90% of the label in xylem exudate was in P-choline, but when labelled ethanolamine was supplied only 50% of the label was in P-ethanolamine (Chapter 2). In Figure 6 data is presented for the labelling patterns in tomato leaves after these compounds were supplied to the roots of whole plants. A similar labelling pattern was seen as in the cultured root experiments, with a few exceptions. Mere label was found in choline when choline was supplied. This may have occurred because the P- choline was used for P-choline synthesis and the choline pool filled up. This did not occur when labelled ethanolamine was fed. The labelling in whole plants of PE and PC more closely resembled the dis- tribution in leaves (Figure 5) than in roots (Figure 4). Perhaps there is a significant difference in the metabolism of ethanolamine in leaves and roots. The data indicate that P-choline is used for PC syn- thesis in leaves though a small amount of hydrolysis to Pi and choline may occur. Summary The in 3133 labelling experiments suggest that P-choline is made by the phosphorylation of choline, catalyzed by the enzyme choline kinase, in the roots of tomato plants. After P-choline arrives in the leaves most of it is incorporated into PC although a little hydrolysis to Pi and choline may occur. Ethanolamine and P-ethanolamine are both incorporated into PE and PC in tomato plants and there seems to be 21 some difference in the metabolism of these compounds between leaves and roots. The experiments as a whole suggest that a possible role for P— choline transport in plants, is the distribution of available choline for phospholipid synthesis throughout the plant. 22 o.oOH ¢.om o.¢ H «.QH mm o.mom o.om¢ m.o H m.wn um m.Hm H.m~ m.c H ¢.q mcHHoaUImno «.0 m.o Ho. H m.o osHHosolm o.mw ~.on H.< H «.mH oaHHozo z: Hlaua nmouw my moHoa: IAuoou u3 nmoum mv «OH x Emu vcsonaou may cH vmnHuomom mm vauHucwvH mam monomuuxo whoa mvcaomaou one . GO.“ U006 m finer—HOE . Hos: Emu mc.H mm? muH>Huom HI UHMHuoam muH mom :1 ON mos ucmBHuonxo onu mo wchanmn ecu um :OHumuuaoucoo oaHHosu one name .mmae oH new weHHeao HIN H_ nqu uaoaumoue Houm< muoom oumaoa mmuaufizo cw mvasoaaoo mo wcHHHmnmA 03H .H oHan 23 H mH H o.HHH as Hm e.oH H.~ H ~.e~ e.o H m.~ eeHHoeu-m as H H.H H H.HH He em m.~H H.e H m.H~ e.o H H.~ meHHoao as H mH H o.~e ooH ooH H.e~ m.H H e.om ~.o H e.a HoHHeoo AueHHoaov me on on mm on ucoaumoufi IAuoou us amoum wv moans: Houucoo Mo Hmuoe cow 3 mm w Emu N m< Ho N Hnau H a He v oH x m .aOHuomw mos mammH .AmHv ouonammam umnHuomov mm mmaHahouom mm3 QOHumuucmucou ocHHonu one moonuoa osu :« cmnHuummm mm vmucsoo mom moHMHucovH mama mmcaoaaou oSu mam vmuomuuxo u may .ocHEMHocmsuo voHHoan ecu wchmm ouomon mason N pom mucoaummuu maHHonolm mam ocHHOSU anHmAMHas :uHa moumnsocH noon on: muoou 63H . Hoe: Emu mm.H mo huH>Huom UHmHumam m mom :5 H mo GOHumuucoucou m an muses N How vaHamam mos maHamaocmnum vaHman may -_o . mafia—NH OGNSUN «H H_ an me Ham on He meHHHeeea was so eeHHoaene eee eeHHoeo Ho Hummus may .N mHees 24 Figure Legends Figure l. Labelling of compounds in tomato roots from addition of [32P]-Pi. [32 P]-Pi was added at a final concentration of 1 mM and a specific activity of 20 cpm nmol-1. o———-o P-choline + GDP-choline; A'°"A phosphatidylcholine; o-—--o phosphatidylethanolamine. Figure 2. The labelling pattern in cultured tomato roots after being fed [1,2 14C]-choline. Choline was fed at a concentration of 1 mM and a specific activity of 572 cpm nmol”l in the upper figure and 1626 cpm nmol-1 in the lower figure. PC o—-—-o; P-choline o----o; choline A——-A; and GDP-choline 3' ' ' '3. Figure 3. The labelling pattern in the aqueous fraction of cultured tomato roots after [1 l4C]-ethanolamine was fed. The concentration was 1 mM and the specific activity was 1210 cpm nmolIl. Choline‘j"°*:; P-ethanolamine o-—--o; ethanolamine o————o; GDP-ethanolamine A----A. Figure 4. The labelling of PE and PC after [1 14C]-ethanolamine was fed to cultured tomato roots. The concentration was 1 mM and the specific activities were 60 cpm nmol.1 in the upper figure and 1320 cpm nmol-1 in the lower figure. PE o——-—o; PC o----o. Figure 5. The labelling pattern in tomato leaves after being fed 1 mM [32P]-P-choline (lower figure) specific activity 7.6 cpm pmol-1 and 0.1 mM [32P]-P-ethanolamine, specific activity 0.5 cpm pmol.1 (upper figure). PC o———-o; PE o----o; P-choline A'°"A; and CDP-choline Dual]. 25 4C]-ethanolamine 14 Figure 6. Whole tomato plants were fed 1 mM [1 1 specific activity 0.60 cpm pmol"1 (upper figure) and 1 mM [1,2 C]- choline 0.99 cpm nmol.1 (lower figure), in the nutrient solution. The plants were extracted and the extracts were chromatographed as described in the methods section. In the upper figure P-ethanolamine o----o; ethanolamine o——o; PE -" ' "; PC 3""‘3; and CDP-ethanolamine A----A. In the lower figure choline A—-—-A; P-choline o----o; PC o----o; CDP-cho line '3' ° ' 'U. 26 .H oustm A335 22.... N _ o 4'0 — — 2.50 25 a» .ho . _ fiEMQIAAQ \ ..1... l N ,-uom m use» 5) cm x was 2:20.18 $5.23-.. 1 ‘- b—u L (D 27 4 CDP- choline . a L- ?” ooooooooooo a ooooooooo no oooooooo o 00000000000000000 1 -—1 —".—‘”‘— O l ”fiholine , - [’0’ p/ I / /—”P/lws:hotidylcholine O l 1 l TWElhours) EDP-choline 2r a ........ b. ......... a... ..... o P‘?gL‘-p--—9———o”’P-choline I . Chofine cm 3: I03 (9 fresh m root)" cm 3: I04 (a fresh m root)" PhOSpholidylcholine --—i-f-—-9-- —9 TIME (minutes) Figure 2. Figure 3. CPM x I04 (9 fresh wt root)" .b 28 l l l V T ’/ EDP-ethanolamine Ethonolomme f—u". O .1.-I" P-ethonolomine Chofine '! ........... t ooooooooo In1 ........ L L ............. ! 2 4 6 TIME (hours) -1 29 CPM x I05 (9 fresh wf roof)" — N C) .5 CPM x I03 (9 fresh wt root)’| N Phosphotidylethonolomine Phosphotidylcholine ’,o a” 0’0" " 1 C) 8 I6 24 TIME (hours) Phosphotidylethonolomine q Phosphotidylcholinf?”o O 1—""" 1 I5 30 415 60 TIME (minutes) Figure 4. Figure 5. 30 N CPM x I03 (9 fresh wt leaf)"I T Phosphotidylcholine "‘.o ’Phosphafidylefhonolamine ° 4 ... TIME (hours) I“; I ” A A -o” . _ ”s". 4‘ ,H/ : ’0’ COP-choline 3 I .c / . 3 / t I, ...--"“ 3 2 - I, .......... P-cholune _. (0 .fi" 2 I ........ " fl. ....... E I" °°°°°° a; Phosphotidylcholine o M l 0 TIME (hours) 31 "L g 2 I- ; _______ _.°_________o ‘ ‘5 P-ethonolomine o I- ”. EthonoIomine 3 ' _ V ..... ,....---I Q 0'. oooooooo E E- oooooooo “... ........... .n.“ . ”.0 $2. - ; ”’"' COP-ethanolamine CL 1 4L__ 0 0 4 6 1 TIME (hours) § 3- _ h 3 5 E 2' d 3 I09 l’lP’ChOHDC x " _ 2 O. o sssssssssssssssssss ”000° °°°°°°°° CDP-cholirle l 0 2 4 6 TIME ( hours) F1Sure 6. 10 11 12 13 14 32 References Bieleski RL 1973 Phosphate pools, phosphate transport and phosphate availability. Ann Rev Plant Physiol 24: 225-252 Bieleski RL GC Laties 1963 Turnover rates of phosphate esters in fresh and aged slices of potato tuber tissue. Plant Physiol 38: 586-594 Bregoff HM CC Delwiche 1955 The formation of choline and betaine in leaf discs of Beta vulgaris. J Biol Chem 217: 819-828 Dykes CW J Kay JL Harwood 1976 Incorporation of choline and ethanolamine into phospholipids in germinating soya bean. Biochem J 158: 575-581 Johnson RD H Kende 1971 Hormonal control of lecithin synthesis in barley aleurone cells : regulation of the CDP-choline pathway by giberellin. Proc Natl Acad Sci 68: 2674-2677 Katayama M S Funahashi 1969 Metabolic pattern of phospholipids during germination g; mung bean, Phaseolus raditus var typicus I The incor- poration of P-orthophosphate into phospholipids. Biochemistry 66: 479-485 Rates M 1954 Lecithinase systems in sugar beet, spinach, cabbage and carrot. Can J Biochem Physiol 32: 571-583 Kates M 1955 Hydrolysis of lecithin by plant pla8tid enzymes. Can J Biochem Physiol 33: 575-589 Kennedy EP SB Weiss 1956 The function of cytidine coenzymes in the biosynthesis of phospholipides. J Biol Chem 222: 193-214 Macher BA JB Mudd 1976 Partial purification and properties of ethanolamine kinase from spinach leaf. Arch Biochem Biophys 177: 24-30 Maizel JV AA Benson NE Tolbert 1956 Identification of phosphory- choline as an important constituent of plant saps. Plant Physiol 31: 407-408 Morre JD S Nyquist E Rivera 1969 Lecithin biosynthetic enzymes of onion stem and the distribution of phosphorylcholine-cytidyl transferase among cell fractions. Plant Physiol 45: 800-804 Murushige T FS Skoog 1962 A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15: 473-497 Mudd JB RA Devor 1971 Biosynthesis of phosphatidylcholine by enzyme preparations from spinach leaves. J Lipid Res 12: 403-411 15 16 17 18 19 20 21 22 23 24 33 Quarles RH RMC Dawson 1969 The distribution of phospholipase C in developing and mature plants. Biochem J 112: 787-794 Ramasarma T LR Wetter 1957 Choline kinase of rapeseed (Brassica compestris L.). Can J Biochem Physiol 35: 853-985 Rowan WA RH Hammerstedt 1977 A.method for measurement of the specific radioactivity of the choline moiety of choline phospholipids. Anal Biochem 81: 175-185 Roughan PG RD Batt 1969 The glyerolipid composition of plants. Phytochem 8: 363-366 Setty PN PS Krishnan 1972 Choline kinase in Cuscuta reflexa. Biochem J 126: 313-324 Tanaka K NE Tolbert AF Gohlke 1966 Choline kinase and phosphoryl- choline phophatase in plants. Plant Physiol 41: 307-312 Tang WJ PA Castelfranco 1968 Phospholipid synthesis in aging potato tuber tissue. Plant Physiol 43: 1232-1238 Tolbert NE H Wiebe 1955 Phosphorus and sulfur compounds in plant xylem say. Plant Physiol 31: 407-408 Vandor SL KE Richardson 1968 Incorporation of ethanolamine -l,2 14C into plant microsomal phospholipids. Can J Biochem 46: 1309-1315 Willemot C WC B011 1967 Incorporation of serine-14C and ethanolamine- 140 into nitrogen containing phosphatides and effects of medium con- taining ethanolamine on phosphatide biosynthesis in excised tomato roots. Can J Bot 45: 1863-1876 CHAPTER 2 PHOSPHATE ESTERS AND INORGANIC PHOSPHATE IN XYLEM EXUDATE OF TOMATO I. LABELLING KINETICS ABSTRACT Phosphorus is present in the xylem of plants as inorganic phosphate and as 2 phosphate esters, phosphorylcholine and phosphorylethanolamine. Inorganic phosphate in xylem exudates is labelled by [32P]-orthophosphate from a rapidly exchanging "root pool" and a slowly exchanging "metabolic pool" in the root. Phosphoryl- choline and phosphorylethanolamine in xylem exudates of tomato plants are labelled with [32P]-orthophosphate from only the metabolic pool. Phosphorylcholine and phosphorylethanolamine in xylem exudates were also labelled by feeding the 14C-labelled bases to roots. Then there is about 10 times as much [14C1-phosphorylcholine as [14C]-choline, and 85-90% of the ester phosphate in xylem exudates was phosphoryl- choline. The remaining 10-15% of the ester phosphate was [1°C] phosphorylethanolamine. The amount of [14C]-phosphory1ethanolamine and ethanolamine in the xylem sap are about equal. Labelling experi- ments with whole plants indicate that phosphorylcholine and phosphorylethanolamine are accumulated in the leaves over the same interval of the time and in the same proportion as observed in the 34 35 xylem exudate. This indicates that phosphate ester transport in the xylem may be independent of transpiration rate. 36 Introduction In 1933 organic phosphorus was estimated to be approximately 32% of the total phosphorus exuded by field grown, corn plants (9). Subsequent investigations (11) revealed that two P-esters were labelled in xylem exudates of barley, bean, willow, and tomato plants after [32P]-Pi was fed to their roots. The major labelled phosphate ester was phosphorylcholine, which under their experimental conditions con- stituted 20% of the total 32P-phosphorus in tomato xylem exudate, and in barley 6% (7). The other phosphate ester which constituted 0.6-l.0% of the label remained unidentified. Some acid-soluble organic phos- phorus compounds have also been detected in xylem exudates from pumpkin roots (4). Since the original report of organic phosphorus in xylem exu- dates there have been reports that only Pi is present in xylen exudates (2). Xylem exudate collected from passively exuding willow roots, labelled with [32P]-Pi has been shown to contain phosphate esters, but xylem exudate collected by vacuum extraction either did not contain any phosphate esters or they represented a much smaller fraction of the total label (8). Similarly when xylem exudate was collected by mild suction from tea plants organic phosphorus compounds were not detected (10). In this investigation tomato plants were placed in nutrient solution containing [32P]-Pi, [1,2-14C]-choline or [1-14C]-ethanolamine to evaluate whether organic and inorganic phosphorus in xylem exudates are labelled from the same pool. The second unknown phosphate ester in xylem exudate has been identified as phosphorylethanolamine. Whether these phosphate esters are normally translocated from the root to the 37 shoot of intact tomato plants has been studied by comparing their labelling pattern in xylem exudate and leaves after short periods of time. Materials and Methods Reagents. [32P]-orthophosphate was from Amersham, [1,2-14CJ-choline and [1-14C]-ethanolamine were from Research Products. All other reagents were from Sigma. Plant Growth. Tomato (Lycopersicon esulentum) cv VF-36 plants were grown in aerated nutrient solution containing 8 mM KN03, 8 mM CaN03, 1 mM NH NO 6 uM MnCI, 42 uM 4 3’ ZnCl, 8 uM Fe- EDTA, .05 MM NaMoO 2 mM M3504, 1 mM anpoé, 24 11M NaB03, , and .16 pH CuSO in a growth 4 4 chamber. Daylength was 16 h (300 uE sec”1 M-2) and temperature was maintained at 25°C day and 20°C night. Two plants were grown per 200 m1 polystyrene beaker and the plants were supported by circles of high density polyuretane foam (2.5 cm X 6.5 cm). Plants were generally 20-40 days old when they were used for labelling experiments. Radiochemical Labelligg. Long term [BZPJ-Pi labelling experiments were done by putting the plants in a 20 L glass tank which contained 100 uM Pi, and the standard nutrient solution. The specific activity of the 5 to 6.0 Ci mol-l. This size [32P]-Pi in the tank varied from 2 X 10- of culture allowed a number of plants to be labelled simultaneously with [32P]-Pi at the same concentration and specific activity without severe phosphate depletion. [1,2 14C]-choline and [1°C]-ethanolamine were fed at a concen- tration of 100 uM unless otherwise noted, with specific activities 38 varying from 0.2-0.4 Ci mol-l. Radioactive compounds were isolated and counted with a liquid scintillation counter or a G-M counter as described in Chapter 1. Labelled xylem exudate was directly trans- ferred onto the origin of 2.5 X 46 cm strips of washed Whatman #3 chromatography paper or collected with a Pasteur pipette and stored in graduated centrifuge tubes at 4°C. Separation of Labelled Compounds. P-choline and P-ethanolamine were separated from P1 by chromatography on paper or cellulose TLC plates developed with water-saturated glass-distilled phenol. Their Rf values were 0.9, 0.3 and 0.1 respectively. Pi was also separated from ester phosphate by introducing 50 p1 of labelled material onto a 0.5 X 1.0 cm column of Dow-l-Cl (400 mesh) and eluting P-choline and P-ethanolamine with 2 m1 of 0.01 N HCl. Pi was then eluted with 2 ml of l N HCl. P-choline and P-ethanolamine were separated from choline and ethanolamine on 0.25 mm Brinkman TLC plates developed with butanol: propionic acid:water (Chapter 1). Their Rf values were 0.42 for P-choline, 0.35 for P-ethanolamine, 0.63 for choline, and 0.57 for ethanolamine. Identification of P-ethanolamine in.Xylem Exudate. The P-ester originally described in barley and tomato xylem exudates as unknown 2 (7), is the only labelled P-ester, other than P-choline, in tomato xylem exudates after feeding [32P]-Pi. [32P]-labe11ed xylem exudate was introduced onto a Dow-l—COOH column (50 X 1.5 cm), which had been washed with 20 ml of 5 mM‘NaBO3 pH 8.5. The column was developed with a 0-0.4 M HCOONH gradient in the same buffer.. The [32P]-1abelled 4 material which was eluted by 0.3 M HCOONH4, co-chromatographed with 39 authentic o-phosphorylethanolamine on Brinkman cellulose TLC plate in 3 solvent systems; water-saturated phenol, butanol:propionic acid:water (92:1:l.3), and methanol:ammonia:water (6:1:3). The labelled compound also co-chromatographed with authentic o-phosphorylethanolamine in high voltage electrophoresis (260 mAmps for 10 min), with 0.1 M NaBO as an 3 electrolyte. In all of the systems labelled material was located by autoradiograms and authentic P-ethanolamine was located with a ninhydrin spray. In addition to this evidence, the unknown P-ester which co-chromatographs with authentic o-phosphorylethanolamine was labelled within 0.5 h after feeding [l 14C]-ethanolamine to tomato I'OOCS o anntitation of Pi and P-esters in Xylem Exudate. Phosphate ester con- tent of xylem exudate was determined by enzymatic hydrolysis (see Chapter 3) and Pi was determined by the acid molybdate method (1), described in Chapter 3. Extraction of Plant Material. Plant material was extracted sequentially in isopropanol at 50°C for 10 min, in CHCl :methanol (2:1) for 30 min 3 and then in 20 mM CaCl2 in water as described in Chapter 1. This pro- cedure extracted both the water-soluble and lipid-bound forms of choline and ethanolamine. Degradation products from P-lipids were not seen on subsequent chromatograms. Results and Discussion Rate of Labelling the Phosphate Esters. The specific activities (not the total activity) of Pi and P-choline plus P-ethanolamine in xylem exudate, collected between 1 to 96 hr after feeding [32P]-orthophosphate 40 to roots, are shown in Figure l. The rapid increase of the specific activity of Pi in the xylem exudate within 4 hours, to the specific activity of the added Pi in the nutrient solution, is consistent with the hypothesis that Pi loaded into the xylem comes from a small rapidly exchanging pool of Pi, which has been called the root pool (2). The Pi in this pool exchanges very slowly with the bulk of the Pi in the root, which is thought to be stored in the vacuole (3). The specific activity of [32P]-choline did not reach that of the nutrient solution until 72-96 hours after adding [32Pi]-Pi to the nutrient medium. The precise time depended upon the size of the plant and the phosphate status of the root. The data indicates that the P-esters in the xylem exudate are synthesized in a portion of the root that has access to or exchanges with the bulk of the phosphate. P-choline and P-ethanolamine were labelled at about the same rate and to the same specific activity (Table 1). Thus both are probably in equilibrium with the same source of phosphate. Pulse-Chase Experiments. The pulse-chase data in Figure 2 also sup- port the hypothesis of two phosphate pools. Pi in the xylem exudate was maximally labelled 20 min after adding [32P]-Pi. Label in Pi then rapidly declines upon adding unlabelled Pi although labelled Pi could still be detected in xylem exudate 24 h after the 10 min pulse. This long term labelling of Pi in the xylem exudate was probably coming from the slowly exchanging pool of Pi. P-choline in xylem exudate was maximally labelled 40 min after feeding [32Pl-Pi (Figure 2) which was slower than for labelling Pi. There was only 0.1% of the total 32P label in P-choline 30 min after feeding [32PJ-Pi, but the percentage 41 increased to l to 2% after 1.5 to 2 hours of chase. Thus both Pi and P-choline in xylem exudate, at the end of the chase period, probably came from the slowly exchanging pool of P1 in the root. Effect of Nutrient Phosphate Concentration. The phosphate concentra- tion in the nutrient greatly affected the ratio between phosphate and P-choline in the xylem exudate (Table 2). Plants were grown for 7 days in 50 liter containers which had 1, 10 and 100 uM Pi rather than 1 mM as is in the standard nutrient solution. They were transferred to 200 ml containers with the same Pi concentrations as that in which they had been grown, supplemented with [32P]-Pi to give each treatment a constant specific activity (1200 cpm uMol-l). Four hours later the xylem exudate was collected for 30 min. The plants with 1 uM Pi had removed all of the [32PJ-Pi from the medium after 4 hours, and the Pi and P-choline in the xylem exudate were probably coming from the slowly- exchanging metabolic pool. Thirty-four percent of the 32P-label in the exudate was in P-choline. The plants in 10 uM Pi had much more [32P]-Pi in the xylem exudate and the percent 32F in P-choline was 4.6% of the total label. The plants in 100 uM Pi had not exhausted their nutrient Pi and had most of the 32F in their xylem exudate in Pi, which may have been from the root pool. In other experiments, to be described in Chapter 3, plants that had been Pi starved for 7 days had approximately 30% of the phosphate in the xylem exudate as P-esters. Thus a much higher percentage of the phosphate in xylem exudate is present as P-choline when available Pi is low. Presumably this P-choline arises from the storage pool of phosphate. 42 Labelling with_11,2-1°Cl-choline and [l-IACj-ethanolamine. When plants were supplied with [1,2-14C]-choline and xylem exudate was collected 8 hours later, 90% of the label was in P-choline and the rest was in choline. When [1-14C]-ethanolamine was supplied for the same time interval less than 50% of the label was in P-ethanolamine and the rest in ethanolamine. The results from time course experiments in which the IAC-labelled compounds were fed to roots, and xylem exudates were col- lected, are shown in Figure 3. The specific activity of the labelled P-esters in the xylem exudate reached that of added compound in the nutrient after about 2 hr (data not shown). A further increase in total P—ethanolamine and P-choline in the xylem exudate can be attributed to the fact that exogenously applied choline or ethanolamine stimulated their flux into the xylem exudate. The distribution of 14C after adding 14C-labelled choline or ethanolamine to root nutrient medium is shown in Table 3. During the time interval between 2-4 hours after adding the labeled bases, the rate of accumulation into the roots was about linear. After 2 h of feeding the labelled compounds the flux rate into the xylem exudate was also a linear function with respect to time. The rate of P-choline and choline flux into the xylem exudate was 5.4 i 0.9 nmol min”1 (g fresh weight root)-1. The mean accumulation rate of choline and P-choline into the leaves during the time period 2-6 h after feeding 14C-labelled choline to the roots was 5.5 i 1.0 nmol min“1 (g fresh weight root)-l. The mean rates of ethanolamine and P-ethanolamine flux into the xylem exudate was 0.75 t 16 nmol min.l (g fresh weight root)-l. The rate of accumulation of label into the leaves during the time period 2-6 hours 1 after feeding 14C-labelled ethanolamine was 0.76 t 0.18 nmol min- (g 43 fresh weight root)-l. This data indicates that the flux rate of P-choline and P-ethanolamine into xylem exudate is approximately the same as the rate of accumulation of these P-esters into the leaves of whole plants. Summary Adding [32P]-Pi and [1,2-1°C]-choline and [1-140]- ethanolamine indicate that Pi in xylem exudates is labelled by exogenously supplied [32P]-Pi from both a small, rapidly-exchanging pool and a slowly exchanging pool of Pi in the root. P-choline and P-ethanolamine however are primarily labelled from the slowly exchanging pool which is probably associated with the metabolic pool of Pi which is associated with ATP synthesis. P-choline accounts for 85-90% of the P-ester content of xylem exudate, but the percentage of the total phosphate in the exudate in P-choline is determined by the phosphate status of the root. For tomato plants P-choline may be about 30% of the total phosphate for phosphate deficient plants on down to 1% of the total when excess Pi is available. From added choline and ethanolamine P-choline accounts for 90% of the choline in xylem exudate, while P-ethanolamine accounts for 50% of the ethanolamine. Judged by appearance of these compounds in leaves P-choline and P-ethanolamine seem to be translocated into xylem exudate at about the same rate as they are translocated in whole plants. 44 Table l.--The Specific Activities of Pi, P-choline and P-ethanolamine in Xylem Exudate After Labelling Roots in Nutrient Solution Containing [3 P]-Pi. The nutrient solution was 1 mM Pi with 2000 cpm nMole-1 [32P]-Pi. The P-esters were separated from Pi by TLC developed with 80% phenol-water. The quantities of P-choline and P-ethanolamine in the xylem exudate were determined by multiplying the total P-ester content, by the fraction of total P-ester that each represents at 96 hours. Time Pi P-ethanolamine P-choline hr cpm nmol-1 24 2173 : 70 625 t 110 856 t 48 51 2060 1 40 682 2 163 1006 1 46 72 1985 1 100 1330 t 151 1490 t 51 96 2025 i 5 1950 t 100 2150 i 50 45 Table 2. The Effect of Phosphate Concentration on the Labelling Rate of Pi and P-choline in Xylem Exudate. 32P]-Pi was fed at a constant specific activity of 1.2 cpm I uMol-l. P-choline was separated from Pi by paper chromatograms devel- oped with 80% phenol-water. Further experimental details are described in the text. P-choline a a Nutrient Exudate Exudate % of total SZP Pi (uM) cpm cpm % l 111 58 34.0 10 4,598 220 4.6 100 1.198.636 12,175 1.0 46 Table 3.-—The Distribution of Radioactivity in Tomatoes After Feeding [1,2-14C]-choline and [l 14C]-ethanolamine. The bases were fed at concentration of 0.1 mM in standard nutrient solution and the plants were decapitated every 2 hours. The specific activity of [1,2 14C]-choline was 598 cpm nmol.1 and [l 14C]- ethanolamine was 991 cpm nmol-1. The bases were separated from their P-esters by TLC developed in butanol:propionic acid:water (l:2:l.3). The 1 fresh weights were 0.111 t .015 g (roots), 0.324 t .022 (leaves) and .097 i .022 (stems). Xylem Exudate Time cpm min.1 (g fresh weight root).-1 hr [1,2 14C]-choline [1 14C]-ethanolamine P-choline choline P-ethanolamine ethanolamine 2 5811 585 246 273 4 3805 801 187 158 6 4300 729 237 260 Total Incorporation into Plant Tissue Time cpm min (g fresh weight root)-1 hr [1,2 14C]-choline [l 14C]-ethanolamine leaves stems roots leaves stems roots 2 240 1,792 8,848 186 150 295 4 2,321 2,414 12,624 188 151 330 6 3,895 2,258 12,215 143 362 524 47 .Figpre Legends Figure 1. The specific activities of Pi and P-choline plus P- ethanolamine in xylem exudate of tomato plants. The nutrient solution had 100 uM Pi with 47 cpm nmol-1 of 32Pi. The labelled P-esters were separated from 32Pi by Dow-l-Cl columns, and P-ester quantities were determined by hydrolysis with alkaline phosphatase and phosphate quan- titation by the molybdate method. Error bars indicate SD. Figure 2. The amount of [32F] in phosphate and P-choline after a 10 min pulse with [32Pi]-phosphate and a 80 min chase with [32P]-phosphate. The 32P-labelling was run with 2 plants per 200 m1 of 100 uM Pi in nutrient. The chase was done in a 50 L tank to dilute the label which was carried with the roots into the chase nutrient. P-choline was separated from P1 by paper chromatograms developed with 80% phenol-water. Figure 3. The amount of radioactivity in P-choline, choline P- ethanolamine and ethanolamine after adding 100 uM [1,2 14C]-choline or 14C]-ethanolam.ine to the nutrient solution at specific activities [1 of 991 and 598 cpm mol-1 respectively. The bases were separated from their P-esters by TLC developed with butanol:propionic acid:water (2:1:1.3). 48 .H ouame $.35 m2: 0.0 0.0 0.. 0.0 0 \\\ \s \w 00 o \x\ .0 \\ W \\ u \\ mw oEESocoéorn +oc__ofinn\W\ mun \\ ll \\ cs \ 1T _ . _ _ 00 49 IOOO 13f 3 Pi E3: Unlobelled O +- .Jl I 500 i | l '0 Q " l 2 O I 0.. o I c—L— . I’ ‘\~ I / . . ‘\P'CI'IOIIIIO I . \. 'r | / ' ‘541 | I arts: | I ’J/ o 1 1 l l 1 IO 30 60 90 Figure 2. TIME (mlnufes ) 50 0 ””0 2~ ,,/”°’ 5 x”P-choline / 0 0 1 |- ’ d 2 o o 1; Chohne 1__.__, t“‘ 3' o - I I l —l_rl EEC, 2 4 6 8 lfifii c. 2- TIME (hours) a) o .9. X g Ethanolamine ° I _ ‘fl—flfiu“ -—--°"""- P-efhonolomine 1 1 1 44. I—L Figure 3. TIME (hours) 10 11 51 References Chen PS TY Toribara 1956 Microdetermination of phosphorus. Anal Chem 28: 1756-1758 Crossett RN BC Loughman 1966 The absorption and translocation of phosphorus by seedlings of Hordeum Vulgare (L). New Phytol 65: 459-468. Greenway HB Klepper 1968 Phosphorus transport to the xylem and its regulation by water flow. Plata 83: 119-136 Kazuto, 0H 0F Tueva 1977 Effects on the roots on phosphorus metab- olism of the leaves during aftereffect of phosphorus starvation. Fiziol Rast 24: 351-356 Loughman BC RS Russell 1957 The absorption and utilization of phos- phate by young barley plants IV The initial stages of phosphate metabolism in roots. J Exp Bot 23: 280-293 Lundegarde H 1945 Absorption, transport and exudation of inorganic ions by plant roots. Arkiv Bot 32A no 12: 1-139 Maizel JV AA Benson NE Tolbert 1945 Identification of phosphoryl choline as an important constituent of plant saps. Plant Physiol 30: 407-408 Mbrrison TM 1965 Xylem sap composition in woody plants. Nature 207: 1027 Pierre WH CG Pohlman 1933 Preliminary studies of the exuded plant sap and the relation between the composition of the sap and the soil solution. Agron J 25: 144-160 Selvendran RR 1969 Changes in the composition of the xylem exudate of ten plants (Camellia sinensis L.) during recovery from pruning. Ann Bot 34: 825-833 Tolbert NE H Wiebe 1955 Phosphorus and sulfur compounds in plant xylem sap. Plant Physiol 30: 407-408 CHAPTER 3 PHOSPHATE ESTERS AND INORGANIC PHOSPHATE IN XYLEM EXUDATE II. FACTORS INFLUENCING THE FLUX AND CONCENTRATIONS OF PHOSPHATE ESTERS AND INORGANIC PHOSPHATE IN XYLEM EXUDATE ABSTRACT Xylem exudate of tomato plants contains inorganic phosphate and two phosphate esters, phosphorylcholine and phosphorylethanolamine. The concentration of inorganic phosphate in xylem exudate was dependent on phosphate concentration in the nutrient solution, decreasing from 1700 to 170 uM when phosphate in the nutrient solution was decreased from 50 to 2 nM. The concentrations of phosphate esters in the xylem exudate was not affected by the inorganic phosphate concentration in the nutrient solution unless it was below 1 nM. After 7 days of phos- phate starvation, the concentration of inorganic phosphorus in the xylem exudate decreased from 1400 pH to 130 uM while the concentrations of phosphate esters remained unchanged. The concentration of phosphate esters in the xylem exudate was increased by addition of choline, ethanolamine and (2—chloroethyl) trimethylammonium chloride to the nutrient solution. These compounds had no effect on the concentration of inorganic phosphorus in the xylem 52 53 exudate. Thus no relationship between the transport of phosphoryl- choline and phosphorylethanolamine to the transport of inorganic phos- phate in the xylem was established. The amount of P-esters in xylem exudate may be more closely related to the distribution of choline and ethanolamine in plants. 54 Introduction In 1933 organic phosphate was reported to represent 30 i 14% of the phosphate translocated in the xylem exudate of field-grown corn 3 plants (13). [ 2P]-orthophosphate fed to the roots of barley and tomato plants was subsequently shown to label two phosphate esters in the exuded xylem sap (18). The ester containing 6-20% of the total 32P- label was identified as P-choline (12). The ester which contained 0.2—0.6% of the total label has been identified as P-ethanolamine (Chapter 2). P-choline has a t during acid hydrolysis of 30-38 h (9), a 1/2 property which has led to erratic results (7) when investigators have attempted to estimate the Pi and ester-P content of xylem exudate by conventional acid hydrolysis techniques (19, 15). This resistance to hydrolysis or detection by Pi spray tests may be why some early investi- gators (11) found only P1 in the exuded xylem sap of plants. When plants are Pi starved for 7-14 days and placed in nutrient solution containing Pi, the transport of phosphate from roots to leaves increases 2-4 fold (7, 4). In one of the investigations only total 32P-label in xylem exudate was measured (4), due to the supposition that only Pi was present, and in the other study (7) organic phosphate determinations were erratic. Therefore, little is known about the role ester-P transport may play in plants grown in low Pi, during Pi starva- tion, or during recovery from Pi starvation. Phosphate distribution between the roots and leaves of plants is known to be altered during Pi starvation (4, 10, 14). As plants are grown in decreasing concentrations of Pi, or are starved for Pi, the roots accumulate Pi to a greater extent than the shoots. These 55 observations have led to a hypothesis that phosphate transport may be modulated by the phosphate status of the root. There is nothing known about how ester-P transport may be related to phosphate allocation in plants. The purpose of this investigation was to determine how the flux of P-esters into xylem exudate is affected by Pi stress, and to examine the relationship between transport of Pi and ester-P in plants. The effects of choline, ethanolamine and CCC on Pi and ester-P concentra- tions in xylem exudate was also examined. Materials and Methods Reagents. Alkaline phosphatase (E.C. no. 3.1.3.1) type VII, P-choline and P-ethanolamine obtained were from Sigma, CCC was from Eastman and all other reagents were from Mallinckrodt. Plant Growth. Tomato (Lycopersicon esculentum) cv VF-36 plants were grown in nutrient solution as described in Chapter 3. The plants were grown in controlled environment chambers with a 16 h daylength (300 uE sec-l M72) and 25°C day, 20°C night. Nutrient solutions were changed daily except when plants were grown in very low concentrations of P1. In these experiments plants were grown in 50 and 100 L containers which were stirred by vigorous aeration. Pi concentration was monitored daily in these containers and KHZPO4 was added to maintain desired con- centrations of Pi. Collection of Exudate. The beakers containing the plants were placed in a water bath at 25°C during the time of collection. If the root temperature was reduced below 20°C, exudation was slowed or stopped 56 entirely. Plants were out below the cotyledonary node with a razor blade and allowed to exude for 3 min. The stems were blotted dry and collection was started with a syringe to collect the exudate, which was put into acid washed graduated centrifuge tubes, which were standing in an ice bath. The collection period was 30 min, unless otherwise noted. Determination of Pi and ester-P. Ester-P content of xylem exudate was determined by measuring the difference in phosphate content between samples of xylem exudate before and after treatment with alkaline phos- phatase. Hydrolysis of ester-P was achieved by mixing 25 ul aliquots of xylem exudate with 0.225 ml of 50 mM NaHCO pH 10.4, 1 mM MgCl 3 2’ 0.1 mM ZnSOA and 0.5 pg of alkaline phosphatase (1 unit), in acid washed tubes and incubating the samples for 16-18 h. Then 0.75 ml of 6% trichloroacetic acid was added to acidify the mixtures and bring the total volume to 1 ml. Inorganic phosphorus was determined by mixing 25 ul of xylem exudate with 0.225 ml of 6% trichloroacetic acid. These samples were incubated in the water bath, along with aliquots containing the alkaline phosphatase. After the hydrolysis period, 0.75 ml of 6% trichloroacetic acid was added to all of the tubes to bring the volume to 1 ml. Phos- phorus determinations (3) were made by adding an equal volume of a freshly made mixture of 015% (NH Mo 0 , 1.2 N H 4)6 4 24 2 acid to the tubes and standards, which contained 0-100 nmoles of phos- 804’ and 2% ascorbic phate. The color was developed for 90 min at 37°C. Absorbance was read at 820 nM on a Gilford 2400-8 spectrophotometer. This method of enzymatic hydrolysis quantitatively hydrolyzed authentic P-choline and 57 P-ethanolamine added to tomato xylem exudate at concentrations from 0-200 uM. These phosphate esters were not hydrolyzed either during the trichloroacetic acid incubation or during the phosphate deter- minations. Flux of Pi and P-esters into xylem exudate were calculated from their concentrations using the formula (nmoles ° mlul) (m1 exudate collected min-1) (g fresh weight root)-1. Since volume cancels out values are expressed as nmoles min-1 (g fresh weight root)-1. Results and Discussion Effect of Nutrient Phosphate Concentration. In Figure 1 the concen- trations of Pi and P-esters in xylem exudate are plotted as a function of the concentration of Pi in the nutrient solution. In tomato plants, the flux of Pi into the xylem was not saturated until the concentration of Pi in the nutrient solution reached 40 nM. which is comparable to phosphate transport in barley (5). Since the optimum Pi concentration in the nutrient solution culture for growth and develOpment of tomato plants was 5-16 uM P1 (l6, l7), tomato plants have the potential to transport Pi at a far faster rate than is needed for current metabol- ism. It has been suggested that excess Pi acquired during periods of high supply may be stored in the vacuole for reallocation during periods of inadequate supply (2). The flux of the P-esters, P-choline and P- ethanolamine, into xylem exudate, was saturated by a P1 concentration in the nutrient solution of 1-2 uM. Plants grown at this concentration of Pi were as large as plants grown at 40 pH Pi, but have foliage which was darker green. Plants grown in Pi concentrations below 1 uM Pi were small and had very dark purple-green foliage. At 2 pM Pi in the 58 nutrient, phosphate esters represent approximately 30% of the phosphorus in the xylem exudate of tomato. This value is similar to the percentage of total phosphate that was reported to be in the organic form in field grown corn (13) and 2 uM Pi is in the range expected in most soils. In nutrient solution (1 mM Pi) P-esters only represent 1-2% of the total phosphate in the xylem exudate. Appendix A shows the percentage of phosphorus, that is translocated as P-esters in the xylem exudate of field grown tomatoes in Michigan. In the experiments shown in Table 1, plants were grown in 2 uM Pi, 1 mM Pi and in nutrient solution without Pi for 10 days. Two hours before cutting off the top of the plants, 1 mM Pi was added to the plants which had been Pi starved. After Pi starvation and then addition of Pi, the amount of Pi in the xylem exudate greatly increased over plants grown with Pi as has been previously reported (4, 7). However the amount of P-ester flux into the xylem sap remained unchanged by the treatments. These experiments suggest that the modulating influence the Pi status of the root has on Pi transport does not affect P-ester transport. Effect of Phosphate Starvation. In Figure 2 data are shown on the rate of exudation and the flux rates of Pi and P-esters into xylem exudate as a function of the number of days of Pi starvation. The concentration and the flux rate of Pi in the xylem exudate decreased nearly 2-fold after the first day of Pi starvation, but the concentrations of P-esters did not change during the course of the experiment. The flux rates of the P-esters decline in proportion to the decline in exudation volume. After 7 days of Pi starvation the concentration of P1 in the xylem 59 exudate was reduced from 1400 1 60 pM to 130 t 9 HM and the concentra- tions of P-esters remained unchanged at 35 t 9 uM. When plants were Pi starved for longer times such as 14, 28 and 35 days they failed to exude when the shoots were cut off. The ability that plants have to increase Pi transport when Pi becomes available to roots after a period of Pi starvation, may be a reflection of how plants absorb Pi from the soil during their lifetime. Soil in the U.S.A. has a modal Pi concentration of 1.5 0M (1), with the concentration rarely exceeding 8 pH. In addition to this low concen- tration, Pi becomes rapidly depleted in zones 1-2 mm from roots due to its slow diffusion rate through the soil (2). During periods of Pi availability (such as after a rain) roots may rapidly take up Pi and transport it to maintain a high diffusion gradient in the direction of the root. When plants have depleted the root zone and the soil dries out, Pi transport slows, which may allow further root growth to search for P1 in undepleted soils. P-ester flux does not respond rapidly to Pi stress, which indicates that their transport or P-esters is con- trolled by factors other than Pi availability. Effect of Ethanolamine and Choline in the Nutrient. In Figure 3 data is shown on flux rates of the P-esters in xylem exudate asva function of the concentrations of choline or ethanolamine in the nutrient solu- tion. Addition of exogenous choline or ethanolamine at concentrations as low as 25 uM significantly stimulated the flux of P-esters into xylem exudate. After supplying ethanolamine, this increase leveled off or declined at concentrations higher than 100 nM. but the stimulation from added choline increased up to a concentration of 10 mM choline (data 60 not shown). Since tomato roots can form approximately 10 nmoles min.-l (gram fresh weight).1 of P-choline and P-ethanolamine by kinase activity (Chapter 1), and it is unlikely that ATP concentration limited these low phosphorylation rates, the increase shown in Figure 3 would appear to be the result of increased availability of ethanolamine and choline. The lower amount of P-esters formed with exogenous ethanolamine may be the result of slow conversion of ethanolamine to choline in plants, and the lower concentration of P-ethanolamine in the xylem exudate (Chapters 1 and 2). The stimulatory effect of choline on P-ester flux in xylem exu- date was shown to saturate within 2 hours after adding [1,2 14 C]- choline to the nutrient solution (Chapter 2). Since specific activity of P-choline was the same as the added choline at this time, P-choline in xylem exudate may be formed by a kinase. When the labelled P-choline arrives in the leaves it is rapidly incorporated into phosphatidyl- choline with only a small amount of hydrolysis to choline and Pi (Chapter 1). These data suggest that the transport of P-esters in the xylem may be a way to insure a steady supply of these phosphorylated bases for phospholipid synthesis in the leaves. Effect of CCC. Table 2 contains data on the effect from the continuous application in the nutrient culture of the growth regulator, CCC, on tomato plants during 10 days of treatment. The fresh weight of leaves and roots decreased, particularly from 10"3 M CCC, when some yellowing of the leaves also occurred. The weight of the stems decreased at all concentrations of CCC and the plants were more compact. The rate of exudation was significantly decreased at all concentrations of CCC 61 tested. The concentration of P1 in the xylem exudate was not signifi- cantly affected by CCC, but the concentration of P-esters was increased at 10-4 and 10.3 M CCC. It is possible that this effect could be the result of the conversion of CCC to choline over the 7 days of feeding (6, 14). However [14C]-CCC when fed to wheat leaves, was not con- verted to any products (Tolbert, unpublished). The flux rates of Pi and P-esters were decreased by treatment with CCC, but this may be a secondary effect caused by the decrease in exudation rate. The CCC mediated decrease in exudation rate could be a result of a decline in salt transport into the xylem elements, if the phenomenon of root pres- sure is a consequence of carrier mediated secretion of ions into con— ducting vessels as has been suggested (8). Summary (1) Pi flux into the xylem exudate was stimulated by Pi concentrations in the nutrient solution above 2 uM, while P-ester fluxes were not. (2) During Pi starvation the concentrations of P-esters in xylem exu- date did not decrease for at least 7 days, while the concentration of Pi in the xylem exudate was decreased 2-fold after 1 day. (3) Pi flux into the xylem exudate of Pi starved plants was greatly stimulated by the addition of Pi to the nutrient, but the flux of P-esters into xylem exudate was not affected by this treatment. (4) The flux of P-esters into the xylem exudate was stimulated by add- ing choline and ethanolamine to the nutrient solution. This sug- gests that the amount of choline and ethanolamine in the roots were limiting P-ester transport. 62 (5) CCC, a choline analogue, slightly increased the concentration of P-esters in xylem exudate but caused a 3-fold decrease in exuda- tion rate. 63 asses N How He as H mH.o H mm~.o m H H.o~ m H H.HH mHH H ma~.m + msee oH Hem Hm oe mo.o H emH.o ~.H H H.HH NH H o.me HNH H ooH.H He as H ao.o H omH.o ~.o H ms.o oH H H.He om H HHH He 2: o.~ umumolm Hm umummlm Hm uflmn—UNUHH H Huoou ua.amoum my H :Ha Hex: oumvflxu Ha Hoz: .mmOSuoa mom mHmHumumE cH vonHHummo mm ommmmmm mma acoucou Houmolm new Hm msu mam :HE on new vouooHHou mos mumoaxm ENme .mucmHa mm>umum Hm ecu wcHaHmuooo cOHuaHom uaoHuuac onu on women was Hm za H waHuuau enemas meson 039 .mhmv 0H you anonm Hm mo m=OHumuuamucoo onu :H caouw mums muamHm .mumummfm mam Hm mo xaHm mam mGOHumuucmucoo onu so Hm wchHmucoo ucmHuusz ou apnoea mam :OHum>umum Hm mo ammo oH mam Hm :5 H .Hm :2 N no muuomwm one .H oHan 64 eH H m.em mH H H.HOH HH H m.ee OH H H.oe H-Hmusesxe Hey HHHHHHIH seHeze oo~ H Han omH H mmeH osH H mseH emH H HHHH H-Hsumeexo Hav Hm HHHoze mo.o H H~m.o mo.o H smm.o Ho.o H mee.o HH.o H Hue.o H-HeoeH assessed we eHa Hesaere Home o.H H m.e H.H H H.HH H.H H H.H o.~ H e.mH H-HHooH Ha Hesse we HreHa He Hose m.o H H.H s.o H m.H e.o H N.» m.o H H.HH H-Huooe Ha geese we HreHe «assess HeHeze N.o H mm.o m.o H ¢.H m.o H ¢.N o.o H ¢.N Hwy muoou us nmoum m.o H H.H H.H H o.e m.o H H.H e.o H H.H va eases Ha Hesse s.e H e.s H.H H H.H o.H H H.H o.H H m.e Hwy me>ssH Ha Hesse coo z mIOH one 2 eroH coo z mroH wawemw .ucoaHuoaxo you some mucmHm N mo waHumHmcou m:0HumUHHaou m .mucoaHummxm m Scum aOHumH>mv vumvcmum mam coma wnu ucomoummu moaHm> may .uao coca mHo ammo OH whoa mucmHa .mamo 0H How mucoaumouu coo o:u :H :Bouw who? muaqu any .muamHm cumEOH mo mummsxm EmHhx any :H mumummlm mo m:0Humuuamuaoo msu mam Hm mo coHumHucmoaoo may .oumm :oHummaxm msu do 000 we muumwwm was .N oHan 65 Figure Legends Figure l. The concentrations of Pi and P-esters in xylem exudate as a function of Pi concentration in the nutrient solution. o————o represents Pi and o----o represents P-esters in the exudate. The plants were grown at the Pi concentrations for 20 d prior to the experiment. Figure 2. The effect of Pi starvation on the flux of Pi and P-esters into xylem exudate. o-——-o represents Pi, o----o represents P-esters and A————A represents pl of exudate min-1 (3 fresh wt root)-1. The plants were 41 d old at the time they were decapitated. The Pi starvation was done in 200 ml beakers in minus Pi nutrient that was changed daily. Figure 3. The effect of exogenous choline and ethanolamine on the flux of P-esters into xylem exudate. o—-—-o represents the flux when choline was added to the nutrient and o----o represents the flux when ethanolamine was used. The compounds were supplied for 16 h before the plants were decapitated. 66 nxxz . AXE on ,-Iwalow11 31vonx3 WB'IAX NI sealsa—d a :23 a 55.52 H) _ H oc.Eo.oco£ord N .H ouame l-luJ Glow“ 31VOOX3 WBWAX NI Id 67 7:02 ts :3: 3 56 BE: Humanoid 0. R~ nu _ _ _ J U 4 H P-chofine+ --0\ P-efhonolomine \ 6 4 Pi STARVATION (days) 1 2 fly 5 AU A\ o H {1. 5 Av wee East 3 _...._e E390. .1 Lee E as. 3 was .95. a Figure 2. 68 N I + Choline P-ESTERS nmoles min' (9 fresh wt roof)" 1 OJ I.O I0.0 CHOLINE OR ETHANOLAMINE (mM) Figure 3. 10 11 12 13 14 69 References Barber SA JM Walker EH Vasey 1962 A survey of 135 U.S. soils. New Zealand Inter Conf Proc 3 Bieleski RL 1973 Phosphate pools, phosphate transport and phosphate availability. Ann Rev Plant Physiol 24: 225-252 Chen PS TY Toribara 1956 Microdetermination of phosphorus. Anal Chem 28: 1756-1758 Clarkson DT J Sanderson CB Scattergood 1978 Influence of phosphate stress on phosphate absorption and translocation by various parts of the root system of Hordeum Vulgare L. (barley). Planta 139: 47-53 Edwards DG 1970 Phosphate absorption and long distance transport in wheat seedlings. Aust J Biol Sci 23: 255-264 Intieri C K Ryugo 1974 Uptake, transport and metabolism of (2-chloroethyl) trimethyl ammonium chloride (CCC) in vegetables 2. communication: biochemical studies in the metabolism of CCC in kohlrabi, cauliflower and tomatoes. Qual Plant 2: 179-186 Kazuto 0N 0F Tueva 1977 Effect of the roots on phosphorus metabol- ism of the leaves during the aftereffect of phosphorus starvation. Fiziol Rast 24: 351-356 Lauchli A AR Spurr A Epstein 1971 Lateral transport of ions into the xylem of corn roots. 11 Evaluation of a stelar pump. Plant Physiol 48: 118-124 Leloir LF CE Cardini 1957 Characterization of phosphorus compounds by acid lability. Methods Enz III: 840-850 Loughman BC RS Russell 1957 The absorption and utilization of phos- phate by young barley plants IV. The initial stages of phosphate metabolism in roots. J Exp Bot 8: 280-293 Lundegarde H 1945 Absorption, transport and exudation of inorganic ions by roots. Arkiv Bot 32A no 12: 1-139 Maizel JV AA Benson NE Tolbert 1956 Identification of phosphoryl choline as an important constituent of xylem saps. Plant Physiol 31: 407-408 Pierre WH CG Pohlman 1933 Preliminary studies of the exuded plant sap and the relation between the composition of the sap and the soil solution. Agron J 25: 144-160 Russell RS RP Martin 1953 A study of the absorption and utiliza- tion of phosphate by young barley plants I. The effect of external concentration on the distribution of absorbed P between roots and shoots. J Exp Bot 4: 108-127 15 16 17 18 19 70 Selvendran RR 1969 Changes in the composition of the xylem exudate of tea plants (Camellia sinesis L.) during recovery from pruning. Ann Bot 34: 825-833 Sommer AL 1936 The relationship of the phosphate concentration of solution cultures to the type and size of root systems and the time of maturity of certain plants. J Agr Research 52: 133—148 Tidmore JW 1930 Phosphate studies in solution cultures. Soil Sci 30: 13-31 Tolbert NE H Wiebe 1955 Phosphorus and sulfur compounds in plant xylem sap. Plant Physiol 30: 499-504 Truog E AH Meyer 1929 Improvements in the deniges colormetric method for phosphorus and arsenic. Ind and Eng Chem Anal Ed 1: 136-139 CHAPTER 4 P-CHOLINE LABELLING IN TOMATO XYLEM EXUDATE AS AFFECTED BY N03, DEFOLIATION 0R TRIACONTANOL TREATMENTS Introduction Although P-choline has long been recognized as a component of the xylem exudate of plants (3), the effect of various treatments on its concentration in the xylem is unknown. The experiments described in this chapter test whether some physiological factors other than exo- genous phosphate and choline might affect the concentration of P-choline in xylem exudate. Triacontanol has been reported to stimulate the growth of rice seedlings (l) and tobacco, bean and tomato callus (2), but its mode of action remains unknown. Because triacontanol was proposed to affect membrane transport and because at least one membrane must be crossed by P-choline en route from the site of synthesis into the xylem, the effect of triacontanol on the concentration of P-choline in the xylem exudate was measured. Materials and Methods Tomato species (Lycopersicon esculentum L. and L. pimpinellifolium Mill) were grown in nutrient solution with 1 or 16 mM 71 72 P1 as described in Chapter 2. The plants were 28 to 40 days old when they were used for these experiments. The plants were labelled by placing them in 200 m1 beakers which contained Hoagland's nutrient and l‘mM [32P]-Pi for 4 hours. The plants were cut at their cotyledonary node and xylem exudate was collected onto the origin of a 5 X 42 cm strip of Whatman number 3 chromotography paper for 30 min. The chromatograms were developed in 80% phenol-water. The amount of radioactivity in Pi and P-choline was measured with a Geiger-Muller counter. Results and Discussion In Table 1 data are presented on the effect of defoliation upon P-choline labelling relative to the amount of [32P]-Pi in the xylem exudate of tomatoes. At the time the exudate was collected in these experiments, Pi in the exudate had the same specific activity as the P1 in the nutrient solution (Chapter 2). In this experiment P-choline was rapidly labelled because the plants were Pi starved for 48 hours before the experiment, and the metabolic pool of P1 was depleted. There was no significant difference between the plants grown in l and 16 uM Pi but the defoliation treatment did decrease the labelling of P-choline by 50%. It also slightly decreased the amount of P1 in the exudate (data not shown). The decrease in P-choline after defoliation may reflect sink demand, a decrease in the amount of pre- cursor available for P-choline synthesis, or a decrease in the ATP/ADP ratio which might slow the phosphorylation of choline. The effect of N03 concentration in the nutrient culture of tomato plants for 30 days before the experiment on P-choline labelling 73 with [32P]-Pi is shown in Table 2. While the total labelled Pi in the exudate increased with decreasing N03 concentration, the percentage of phosphorus labelled as P-choline remained unchanged. The results suggest that P-choline synthesis and transport are not very sensitive to N0 deficiency. The plants grown in 0 mM N03 were very light green 3 and much smaller than the plants in the other N03 treatments. Experiments were designed to test the effect of triacontanol on P-choline labelling in the xylem exudate of tomatoes (Table 3). There was an increase in the amount of P-choline labelled relative to P1, and the Pi concentration in the nutrient was decreased, probably due to a smaller metabolic pool of Pi in the roots of these plants, as discussed in Chapter 2. Triacontanol treatment had no significant effect, at any of the Pi concentrations tested, on P-choline labelling. In summary, defoliation decreased either the rate of labelling or the total quantity of P-choline in xylem exudate, while N03 and triacontanol treatments had no effect. 74 Table 1. The Effect of Defoliation on P-choline Labelling in Tomato Xylem Exudate. Tomato plants (Lycppersicon pimpinellifolium) were grown for 28 days in normal Hoagland's nutrient. The plants were starved for Pi for 48 hours before the experiment. Half of them were defoli- ated 24 hours before cutting at the cotyledonary node. The exudate was collected onto the origin of a paper chromatogram and developed with 80% phenol-water. 32P-Label in P-choline Treatment % of total 32P 1 uM P1 16 uM Pi Control 15.7 i 1.7 13.3 t 0.4 Defoliated 7.8 t 2.3 6.9 t 1.3 75 Table 2. The Effect of NO- Concentration on P—choline Labelling in Xylem Exudate. Tomato plants var. VF-36, 47 days old, had been grown in 3 plants were fed 1 mM [32P]-Pi for 4 hours and then shoots were cut Hoagland's nutrient with 0, 4, 8, and 16 mM N0 for 30 days. The off at the cotyledonary node. Xylem exudate was collected onto the origin of a paper chromatogram for 30 min. Total 32P in exudate Phosphate as [32P]-choline Treatment 3 cpm X 10 % 16 mM N03 9.1 s 3.0 0.63 s 0.03 8 mM 1901'3 15.5 1 9.3 0.60 s 0.50 4 mM no?3 23.5 s 15.3 0.99 z 0.29 0 mM 190?3 38.1 t 17.2 0.72 s 0.18 76 Table 3. The Effect of Triacontanol on P-choline Labelling in the Xylem Exudate of Tomatoes. Tomato plants var. VF-36 were grown for 30 days in 0.1, 1.0, 32P]-Pi was added to the nutrient and the plants 10 and 100 uM Pi. [ were cut at the cotyledonary node 4 hours later. The triacontanol was dissolved in the nutrient solution by putting enough on a filter paper disc to give a final concentration of 10 ppm and allowing the disc to sit in the nutrient solution for 24 hours before the experiment. Xylem exudate was collected onto the origin of paper chromatograms. Labelled as P-choline Z of total 32P Pi in the nutrient uM Control 10 ppm triacontanol 0.1 11.5 1 7.0 11.9 1 9.0 1.0 12.5 1 7.7 8.1 1 3.8 10 2.3 1 0.9 1.4 1 0.3 100 1.4 l+ 0.8 1.6 l+ 0.9 77 References l Bittenbender HC DR Dilley V Wert SK Ries 1978 Environmental param- eters affecting dark response of rice seedlings (Oryza sativa L.) to triacontanol. Plant Physiol 61: 851-854 2 Hangarter R SK Ries 1978 Effect of triacontanol on plant cell cul- tures in vitro. Plant Physiol 61: 855—857 3 Maizel JV AA Benson NE Tolbert 1956 Identification of phosphoryl choline as an important constituent of plant saps. Plant Physiol 31: 407-408 CHAPTER 5 THE DISTRIBUTION OF P-CHOLINE IN TOMATO PLANTS Introduction Nutrient movement into leaves from xylem elements in plants necessarily follows the xylem stream, but nutrients may move laterally in the xylem, or be redistributed in the plant by the phloem after arriving in the leaves. It has been shown that phosphorus fed to bean roots circulates throughout the plant (1), but is redistributed pref- erentially to young leaves. Amino compounds which have been identified in xylem sap of legumes are distributed differentially, to the fruit phloem sap and leaflets. Valine and asparagine accumulate in the fruit, while aspartate and arginine tend to remain in the leaves (3). While it has been known for many years that P-choline is a component of xylem exudates there are no published reports on its localization in the plant. The purpose of the experiments presented in this chapter was to examine the distribution of P-choline in tomato plants from the xylem stream and to compare its distribution to that of Pi. Materials and Methods Tomato plants var. VF-36 were grown in nutrient culture as described in Chapter 2. The plants were 38 days old and had 8 to 9 78 79 leaves per plant at the time of sampling. The plants were cut at the cotyledonary node and cut shoots were placed in test tubes containing a 1:5 dilution of xylem exudate which had been collected from VF-36 plants. They were illuminated with a photoflood lamp at a light inten- sity of 300 uE mfz sec-1. The air surrounding the plants was kept at about 25°C by blowing a stream of water-saturated air over the leaves. A 20 min pulse of either [32Pl-orthophosphate (Amersham) or [32P]-P- choline, synthesized by the method described in Chapter 1, was added to the exudate. This was followed by a 20 min chase with the unlabelled compounds. Both compounds were fed at a concentration of 1 mM and a specific activity of 1.9 X 10.2 Ci mol-l. The leaves were cut from the petioles and stems and placed in 5 X 0.5 cm petri plates. The plates were placed under the thin end window of a Geiger-Muller tube for counting. The leaves were air dried at 100°C for 6 weeks to obtain the dry weights. For studies on distribution of [32P]-Pi in whole plants, they were labelled in a 20 liter tank described in Chapter 2, with 100 uM 3 [32P]-Pi (specific activity 3.3 X 10- Ci mol-l). Results and Discussion In Table 1 data are presented on distribution of radioactivity in excised tomato plants, after a 20 min pulse of [32P]-Pi or [32P]-P- choline followed by a 20 min chase with the unlabelled compounds. These times were used as they were shown to be optimal for amino acid distribution studies (3). There was no significant difference in the distribution of the 32F from feeding Pi or P-choline. 80 The data in Table 2 are from a similar experiment, but they are presented on a dry weight basis. The dry weights indicate that leaves number 3 and 4 were fully expanded and that leaves numbered 5-8 were expanding. The results suggest that the young expanding leaves do not accumulate either Pi or P-choline to a greater extent than the older leaves, since there was no highly significant difference between the cpm (mg dry weight)-1 in the leaves. The results in Table 2 are expressed on a percentage basis because there was a difference in the specific activity of the [32PJ-1abelled Pi and P-choline when they were fed to the plants. In Table 3 data are presented which indicate that the distribu- tion of radioactivity in whole tomato plants, whose roots were placed in [32P]-Pi for 50 hours, was nearly identical to that observed in the short pulse-chase experiments with excised tomato shoots. This experi- ment also indicates that tomato plants which are grown in adequate Pi, do not preferentially accumulate phosphate in the young leaves. Although 32F from labelled P-choline and Pi, which were trans- ported in the xylem, had the same distribution in whole plants, the 32P products into which Pi and P-choline accumulate were not measured. It was possible that much of the [32P]—P—choline was hydrolyzed to Pi and distributed as Pi among the leaves, but this possibility was ruled out in Chapter 1. The experiments did not measure redistribution by phloem transport. Since P-choline has been reported to contribute up to 4% of the carbon in phloem tissue (2) redistribution probably occurs by these tissues. 81 Summary (1) There was no significant difference between the distribution of Pi and P-choline to the leaves of excised tomato plants by xylem transport. (2) The distribution of P1 in whole plants was nearly identical to that measured in excised tomato shoots in experiments of short duration. 82 32 32 Table l. The Distribution of [ P]-Pi and [ P]-P-choline to the Leaves of Excised Tomato Shoots. The values represent mean and SD obtained from 2 experiments, which each had 2 replications per treatment. The leaves were numbered from the bottom. Added Compound Leaf No. [32PJ-Pi [32P]-P-choline percentage of radioactivity + SD 1 3.2 1 1.0 3.5 1 1.9 2 10.9 1 4.6 9.0 1 4.4 3 13.0 1 3.5 12.5 1 7.7 4 23.9 1 11 16.9 1 1.5 5 21.8 1 5.3 25.2 1 7.4 6 12.0 1 3.4 13.7 1 6.3 7 5.6 1 3.3 4.9 1 3.0 8 1.1 1 0.4 2.2 1 0.5 meristem 0.4 1 0.1 0.9 1 0.4 stems 8.3 1 0.8 11.2 1 3.8 83 32 32 Table 2. The Distribution of [ P]-Pi and [ P]-P-choline in the Leaves of Excised Tomato Plants. The values represent the mean and SD obtained from 2 experi- ments each of which had 2 replications of each treatment. The leaves were numbered from the bottom of the plant, were air dried at 100°C for 6 weeks before they were weighed. Percent of total cpm (mg dry wt)’1 1 SD Leaf No. X dry wt Pi P-choline 1 76.5 6.8 1 1.4 11.3 1 6.0 2 229.0 9.6 1 3.6 5.4 1 8.9 3 295.0 5.1 1 1.1 8.9 1 2.0 4 364.0 9.3 1 3.3 10.5 1 0.3 5 250.0 13.5 1 3.8 11.9 1 5.5 6 166.0 13.2 1 5.3 14.3 1 2.4 7 78.0 26.5 1 15 12.2 1 7.5 8 49.0 5.6 1 4.2 11.5 1 5.0 meristems 21.0 6.0 1 2.7 9.4 1 1.7 stems 353.0 4.0 |+ 0.3 4.8 l+ 4.0 84 Table 3. Labelling of Tomato Leaves by [32P]-Pi Fed to the Roots. Whole tomato plants were placed in a 20 liter tank containing Hoagland's nutrient solution and [32P]-orthophosphate, 7.3 cpm nmol-1. The leaves were excised after 50 hours of labelling and radioactivity was counted. Leaf No. cpm X 10 Z of cpm uMoles 1 5.6 3.6 0.78 2 13 l 8.4 1 8 3 21.9 13.97 3.0 4 29.4 18.78 4.1 5 22.8 14.53 3.1 6 17.3 11.06 2.4 7 ll 6 7.4 1.6 stems 34.2 21.8 4.7 85 References l Biddulf O S Biddulf R Cory H Koontz 1958 Circulation patterns for phosphorus, sulfur and calcium in the bean plant. Plant Physiol 33: 293-300 2 Bieleski RL 1966 Accumulation of phosphate, sulfate and sucrose by excised phloem tissues. Plant Physiol 49: 447-454 3 McNeil DL CA Atkins JS Pate 1979 Uptake and utilization of xylem borne amino compounds by shoot organs of a legume. Plant Physiol 63: 1076-1081 CHAPTER 6 EXPERIMENTS ON THE pds/pds (PHOSPHATE DEFICIENCY SYNDROME) MUTANT OF TOMATO (LYCOPERSICON ESCULENTUM L.) Introduction Tomato plants which contained a single recessive allele (pds), when homozygous, exhibited what appeared to be severe phosphate deficiency symptoms, i.e., small stature, purple coloration and chlorosis of older leaves, when grown on supraOptimal concentrations of Pi (1). In addition when pds/pds scions were grafted onto wild type root-stocks the plants appeared phosphate deficient. When the reciprocal graft was made, wild type scion onto pds/pds root-stock, the plants grew normally (C. Rick and J. Fobes personal communication). This evidence suggested that pds/pds shoots were unable to produce a metabolite necessary for normal growth. The roots were either able to produce the metabolite or it was imported from normal leaves. In this chapter experiments are presented which were designed to determine whether the pds mutation was expressed in callus made from pds plants, and to determine whether pds contained a lesion in either P-choline transport or metabolism. Further grafting experi- ments and eXperiments with whole plants were also performed to confirm previous reports and to attempt to achieve phenotypic reversion of the pds phenotype. 86 87 Materials and Methods Tomato (Lycopersicon esculentum L.) var VF-36, VF-l45 and pds/ pds in var VF-36, as well as_Lycopersicon_pimpinellifolium seeds used in these experiments were obtained from Dr. Rick, University of California, Davis, through Dr. Jon Fobes. The seeds were germinated by placing them on the basic MS (Murashige and Skoog) medium described in Chapter 1. The plants were allowed to grow for 10 days on MS medium. The hypocotyls were excised and placed, 4 per plate, onto MS medium with 8.9 uM benzyladenine and 10.8 uM napthaleneacetic acid, in 100 X 15 mm plastic petri plates (33 ml volume). Callus was grown for 30 days in the light with a 16 hour day, 70 uE sec"1 m-Z, and at 25°C. Callus was removed from the outside edge of the developing mass and sub- cultured onto fresh medium. After another 30 days of growth, pieces were taken from these calli for the experiments. The growth of callus was monitored during the experiments by individually weighing each of 4 pieces of callus which were used in each replicate plate, then weigh- ing each of them at the end of the experiment. All of the experiments were done with 10 replicate plates per treatment unless otherwise noted. The plates were sealed with parafilm for the duration of the experi- ments, which was usually 30 days. Whole plants of VF-36 and pds/pds in homozygous VF-36 background were grown heterotrophically by placing germinated seeds into 2 liter glass jars which contained in a 100 m1 volume Hoagland's salts, 88 mM sucrose and 0.9% Difco bacto agar. The plants were grown in a growth chamber on a 16 hour day, 100 uE sec.1 mfz, at 30°C day and 25°C night temperature. 88 The grafting experiments were performed on plants grown in the greenhouse at 30°C day and 25°C night temperatures in the summer. The pds/pds genotypes used as scions were 15-20 days old. The pds/pds plants which were fed P-choline through the nutrient solution, were grown as described in Chapter 2. The plants were grown in 1 liter jars, and the nutrient solution was changed daily. Results and Discussion Effect of P-choline and Choline on Callus Growth. To compare the growth of pds/pds callus to that of VF-36 callus, the variety with which pds/ pds is isogenic, the strains were grown at 10.6 to 10-3 in Pi (Table 1). They both had the same response to the concentration of Pi in the medium. This experiment indicated that the pds/pds mutant phenotype was probably not expressed in cultured cells obtained from the hypo- cotyls of this mutant. Since the mutation was expressed in the shoots of whole plants, the effect of P-choline on the induction of primary callus was tested. There was no significant effect of P-choline treatment on induction or growth of either VF-36 or pds/pds callus (Table 2). The only significant effect was an inhibition by P-choline of callus growth from L.4pimpinellifo1ium hypocotyls. The Effect of P-choline on pds/pds Plants. Figure 1 shows a 70 day old pds/pds plant which was grown in the greenhouse. A VF—36 plant of this age would have been about 1.5 m tall and have fruit on it. This mutation (pds/pds) is not expressed until the plants are 15 to 20 days 89 old. At that time the leaves began to turn yellow. The purple colora- tion occurred when the plants were grown in soil. Treatment of similar plants with a foliar spray of P-choline of choline (1 mM) resulted in necrosis or death. Pds/pds shoots which were grafted onto a VF—l45 root stock did not grow (Figure 2) and never attained a size any larger than shown in this figure. The reciprocal graft of a VF-l45 shoot onto a pds/pds root grew normally. These experiments indicated that only the shoot was affected by the mutation. In Figure 3 the growth of a pds/pds shoot, grafted onto a VF-l45 shoot, is shown. The pds/pds shoot grew normally for about 40 days after which it started turning yellow. (This grafting method was used to generate pds/pds seed, from plants which had 10-15 leaves.) The addition of 10'”7 to 10.3 M P-choline to whole plants in the nutrient solution stimulated the growth of the pds/pds plants (Figure 4). The pds/pds mutant plants grew better in solution culture than in soil. This can be seen by comparing the control plant in Figure 4 with the plant in Figure 1, both were about 70 days old. The P-choline treatment resulted in pds/pds plants which were taller and greener than the control plant but their growth was not as fast as VF-36. Normal tomato plants would be 4 times as large as those shown in Figure 4. Bacterial contamination in the nutrient was a problem in these experiments, and not enough plants of each treatment could be obtained for statistical analysis. When pds/pds plants were labelled with [32P]-orthophosphate as described in Chapter 2 and xylem exudate was collected, there was as much P-choline in the exudate as was in that obtained from VF—36 plants (data not shown). This result is consistent with the results 90 of the grafting experiments which showed that pds/pds roots were apparently similar to normal roots. From data with choline feeding to roots, which is described in Chapter 3, 1 mM choline resulted in a P-choline flux in the xylem exudate which was 4 times larger than normal. The stimulation of pds/pds growth by P-choline (Figure 4) may have been due to a high P-choline flux into the leaves. However there is nothing in the data to suggest that this effect was specific for the mutant. The effect of high nutrient nitrogen on growth of pds/pds plants, grown on a 30% sucrose medium in the light, was tested to determine how the growth of the pds/pds mutant would compare to that of VF-36 when photosynthesis was not a limiting factor (Table 3). This experiment was performed at 2 levels of nitrogen, the normal level of - + 16 mM N03 and 1 mM NH4 in Hoagland's nutrient and a high level of 40 mM N03 and 20 mM NHZ. The pds/pds plants in the high nitrogen medium grew 3-4 times larger than those grown in normal Hoagland's nutrient. The VF—36 plants exhibited the opposite response, they grew twice as well in the Hoagland's solution than in the high nitrogen medium. While it was clear that the high nitrogen treatment did not reverse the mutation, this medium stimulated their growth far more than P-choline treatment. The significance of this observation to my studies was that the pds/pds mutant may not be a phosphorus metabolism mutant. It could be a lesion in the metabolism of nitrate. In speculation the P-choline enhancement of growth may have been due to the catabolism of choline and use of the nitrogen and carbon as well as the phosphorus. 91 Summary Callus derived from the pds/pds mutant, which appeared to be extremely phosphate deficient when grown in the soil, grew as well as callus derived from VF—36, the normal Lycopersicon esculentum line with which it was isogenic. Addition of choline of P-choline to the culture medium did not stimulate the growth of callus derived from either VF-36 or pds/pds plants. Grafting experiments with pds/pds and VF-l45, a normal line of tomato, demonstrated that pds/pds shoots grafted onto VF-145 roots exhibited a mutant phenotype, while VF-l45 shoots grafted onto pds/pds roots grew normally. In addition it was shown that a pds/pds shoot grafted onto the top of a VF-145 plant grew normally. Thus the muta- tion would seem to affect only the growth of the top of the plant. P-choline stimulated the growth of whole pds/pds plants grown in nutrient culture but did not reverse the mutation. Treatment of pds/pds plants with 40 mM N03 and 20 mM NH: stimulated the growth of pds/pds plants but inhibited the growth of VF-36 plants. This treatment appeared to stimulate growth to a greater extent than P-choline treatment but it also did not completely reverse the mutation. The bulk of the evidence suggests that the pds/pds mutation is a developmental lesion in the shoots which may be involved with nitrogen metabolism. 92 Table 1. The Effect of Pi Concentration on the Growth of VF-36 and pds/pds Callus. The callus was initiated and grown as described in the methods section. The initial inoculum was 4 pieces of callus per plate, each weighing approximately 400 mg. The callus was allowed to grow for 30 d in the light. Treatment Callus Growth (g) [Pi] pds/pds VF-36 10'3 M Pi 4.86 5.28 10'“ M Pi 1.30 0.94 10"5 M 91 1.06 0.73 10'6 M Pi 0.88 0.60 Sfi - 0.367. 93 Table 2. The Effect of P-choline on Callus Induction and Growth. The VF-36, pds/pds and L. pimpinellifolium seeds were germinated and the seedlings were grown on MS medium. The hypocotyls were excised after 10 d of growth and 4-1 cm segments, each obtained from 1 seedling were placed on the various media. The callus was weighed after 30 d. X Callus wt (8) Treatment L. pimpinellifolium VF-36 pds/pds Control 79.9 20.6 21.0 10"6 M P-choline 69.6 16.6 28.2 10’5 M P-choline 66.9 17.4 33.1 10" M P-choline 51.9** 21.1 17.1 10‘3 M P-choline 55.4* 12.6 19.8 *LSD .05 8 18.75 **LSD .01 - 24.64 SD ' 9.6 94 0.8 w n.HH o.w w q.a~ mm H o.o~H cm H o.MH~ omnm> H.H M m.m s.o w m.H s H «.mm m M n.6H mea\mea w w w w 6 m m e m m +22 28 ON + 102 28 cc Ioz :8 ca +22 28 ON + I02 28 co Ioz :8 ca huowum> spasms mun sauce newsms swans Hmuoa .umn won madman q mums muons .mp6“ 6 a“ mucmaa way mo unwama some msu uowmmuomu mm=Hm> was muamaa «so was someone nuaouw m :« vmoman mums much may .mhmc on How 3ouw Ou wokoHHm mums .umwm Ouumn Osman Nm.o cam omouosm 26 mm .usmfiuusc m.vcmawmom w:H:«Mucoo mum“ uumsu N 6% voomaa mums mvomm vmumcaahou .asfivoz :owouufiz swam was Hmahoz so aaouu mucmam omlm> cam mvo\mva mo auaouo 058 .m magma 95 Figure Legends Figure l. Pds/pds plants grown for 70 days in the greenhouse in the summer. Plants were watered with Peters 20—20-20 fertilizer (200 ppm) about once a week. Figure 2. A pds/pds shoot grafted onto the top of a VF—l45 rootstock, the graft was done 40 days before the photograph. The shoot never grew any larger than is shown in this photo. Figure 3. A pds/pds shoot grafted onto the top of a VF-145 plant at the same time as the plant in Figure 2. The shoot grew normally for 40 days, and then started to turn yellow. The plant in this photo had 4-5 VF—l45 leaves. Figure 4. Pds/pds plants grown in nutrient culture. The plants were 74 days old in this photo, and had received the P-choline treatments indicated for 30 days. 96 Figure l. 97 Figure 2. 98 Figure 3. 99 PHOSPHORYL CHOLINE, MOLAR Figure 4. 100 References 1 Rick CM RW Zobel R0 Pena 1970 Tomato Genetics Cooperative Report 20: 53 CONCLUDING DISCUSSION In this thesis evidence is presented for P-choline synthesis in the root by a soluble kinase, P-choline transport in the xylem exudate, and in whole plants, P-choline incorporation into phosphatidylcholine. The magnitude of this P-choline transport may contribute significantly to the total synthesis of phosphatidylcholine in leaves. This possi- bility is evaluated below. Why P-choline and P-ethanolamine are the only phosphate esters in xylem exudate is unknown. In Appendix A data on the Pi and P-ester concentrations in xylem exudate of field grown tomato plants are added. This prelimi- nary experiment indicates that P—choline is present in the exudate of field grown plants at about the same level as in plants grown in nutrient culture. The Pi and P-ester concentrations in the xylem exu- date were high, perhaps due to the slow exudation rate of field grown plants, in comparison to those grown in nutrient culture. Appendix B has the calculations which attempt to compare P- choline flux with phosphatidylcholine synthesis in the leaves of VF-36 tomato plants. In these calculations the following assumptions were made. P-choline moves into xylem exudate at the same rate as in whole plants (Chapter 2). P-choline flux into xylem exudate has a mean value of 0.4 nmolmin"1 (g fresh weight root).1 (Chapter 3). Tomato leaves have about 1 umol (g fresh weight leaf).1 of phosphatidylcholine (20). 101 102 The roots do provide a significant amount of the P-choline needed to synthesize the quantity of phosphatidylcholine needed for growth. In plants 0 to 25 days old, P-choline import seems to be adequate for phosphatidylcholine synthesis in the leaves. In older plants the P-choline, which arrives from the xylem, could only provide 30 to 402 of the quantity of P-choline needed for phosphatidylcholine synthesis. A similar observation was made with young barley plants which transport more P-choline than do older plants (25). The reason why P-choline and P-ethanolamine are the only P- esters in xylem exudate of tomatoes was not answered by our investiga- tions. Both esters exist as intramolecular salts with a low net charge and their membrane transport may be different from the transport of other phosphate esters, which are highly ionized at physiological pH values. Phosphatidylcholine contains 20 to 30% of the ester phosphate in tomato leaves and phosphatidylethanolamine contains another 10 to 15% (la). Tomato plants, as well as other species, may have evolved P-choline transport as a mechanism to assure adequate phosphate and choline availability in leaves for phospholipid synthesis. APPENDICES APPENDIX A THE PI AND P-ESTER CONCENTRATIONS IN XYLEM EXUDATE OF FIELD-GROWN TOMATO PLANTS APPENDIX A THE PI AND P-ESTER CONCENTRATIONS IN XYLEM EXUDATE OF FIELD-GROWN TOMATO PLANTS The plants were 90 days old and had a X leaf weight of 94 g at the time of the experiment. There were 2 plants per replicate. nmoles ml—1 2 Replicate Pi P-esters P-esters as Z of total phosphate 1 3212 442 12-1 2 2996 255 7.8 3 2640 1144 30-0 4 1493 316 17.4 i 2595 539 16.9 103 APPENDIX B THE GROWTH RATE OF TOMATO PLANTS (LYCOPERSICON ESCULENTUM) VAR VF-36 IN STANDARD NUTRIENT CULTURE AND THE REQUIRED RATE OF PHOSPHATIDYLCHOLINE SYNTHESIS (PC) NECESSARY TO MAINTAIN GROWTH .AN wouamnov H Auoou ua £mmum mv mCHHozoIm mace: «.0 so vmmmm Awmma us smopw my on mace: o.H so vmmmmm HI m.wo oo~.o comm.o oo.m~ oooe.HH comm.n on 0.0: NHH.o omq~.c mm.qH comm.» oooe.~ me ~.mm oqa.o camc.o om.m~ oomo.n ooec.~ cc s.mm «0H.o oNom.o nn.HN ooqo.q ocmo.H mm m.cw moa.o oomH.o N¢.HH coma.H ommm.o Hm o.ma m~H.o coha.o ~o.~ omom.o Nmmo.o mm o.- o-.o oeao.o oo.H meH.o memo.o ON II II mmoc.o No.o oeoo.o omoo.o NH ocHHonolm Hlaua nmoum wv mo unmouom use Hos: HIAWMMMEMVmeMWMUHMEC ovouwsvmu mum“ mmswmw x va uB m va u3_m Amhmvv HIAmfimosuahm omv A mwmonuahm um I IIIIIIIII mw< vouuoaaa oswnonolm uo mama muoom uamHm WUOOfiw Ufim m0>001~ .aaonm omam ma mammcuahm om ou mafiaonuamuonomona ma coausnauuooo manammoo one 155% E554: 8. wimmmomz 6.: mHmmmHZWm mzHAomUAVQHH m<> AZDHZMADUmm zouHmmmmooqu mHz