ABSTRACT CONTROL OF NITRATE ASSIMILATION IN CULTURED TOBACCO CELLS By Yair M. Heimer The objectives of this study were to examine the nitrate uptake system of the XD strain of cultured tobacco cells, its regulation and the relationship between its regulation and that of the other steps of the nitrate assimilation pathway. The development of nitrate reductase and nitrite reductase activities in this cell line had been previously reported to be induced by nitrate and repressed by certain amino acids. Nitrate uptake was studied in cells lacking fully induced nitrate reductase, or possessing nonfunctional nitrate reductase formed in the presence of tungstate. In either case, the disappearance of nitrate due to reduction was minimized. The nitrate uptake system was found to be inOperative in nitrogen starved cells and in nitrogen rich cells lacking nitrate. Nitrate induced or possibly activated the nitrate uptake system. The rate of nitrate uptake increased linearly with time beginning immediately upon eXposure of the cells to nitrate. The rate increased ten times more rapidly in Yair M. Heimer nitrogen rich cells compared to nitrogen starved cells. However, the final nitrate uptake rate was only twice as high in nitrogen rich cells. The rate of nitrate uptake is dependent upon the concentration of nitrate in the medium. The apparent Km is 4 x 10‘“ M. The cells accumulate nitrate to an internal concentration which is at least 10 times the external con- centration. Accumulation is inhibited by dinitrophenol and cyanide, indicating energy dependence. Amino acids, either as a mixture or as individuals, inhibit the development of the nitrate uptake system. A variant cell line was selected in which the nitrate uptake system deve10ped in the presence of amino acids, but the development of nitrate reductase was still strongly inhibited. Thus the regulation of the uptake system can be altered without an identical alteration of the regula- tion of nitrate reductase. Apparently, amino acids regu- late the steps of the nitrate assimilation pathway similarly but independently. Nitrate does not have to accumulate within the cells to induce nitrate reductase. Accumulated nitrate cannot keep nitrate reductase induced when exogenous nitrate is removed, although accumulated nitrate can be reduced. Thus, most of the nitrate within the cells can act as sub- strate, but not as inducer of the nitrate assimilation pathway. CONTROL OF NITRATE ASSIMILATION IN CULTURED TOBACCO CELLS By Yair MfVHeimer A THEE IS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Biochemistry 1970 C9—- 6 Z; [/2 6 TO MY WIFE, ESTHER ii The ‘ gratitude t and encourc Pru‘ ‘ very much a to the p90? S‘lUDOI't GUT fission. ACKNOWLEDGMENT The author wishes to eXpress appreciation and gratitude to Dr. Philip Filner for guidance, interest and encouragement during the course of this study. Fruitful suggestions by Prof. J. E. Varner were very much appreciated. The author is also grateful to the members of the guidance committee, Drs. A. Lang, F. M. Rottman and J. A. Boezi. Last but not least the author is very grateful to the peOple of The United States for their financial support during this study, via the Atomic Energy Com- mission. 111 DEICATIOEI ACKEOKLELJIL LIST CF TAB 1131‘ 0? FIG DIBCDUCI‘IO ZITEAI‘URE Act: Tran Tran Ahic The 3816 Kit: M‘s: . -m, TABLE OF CONTENTS Page DEDICATION . . . . . . . . . . . . . . . . . . . . . ii ACKNOWLEDGMENT . . . . . . . . . . . . . . . . . . . iii LIST OF TABLES . . . . . . . . . . . . . . . . . . . vi LIST OF FIGURES O O O O O O O O O O O O O O O O O O V111 IDITRODUCTIOIJ O C O O O O O C O O O O O O O O O O O O 1 LITERATUREREVIEW................. 4 Active Uptake: General Consideration . . . . 4 TranSport Systems in Bacteria . . . . . . . . 5 TranSport Systems in Higher Plants . . . . . 9 Anion 36813118131011 0 o o o o o o o o o o o o o 11 The Carrier . . . . . . . . 12 Selection of Variant Plant Cell Lines . . . . 15 Nitrate Assimilation in Higher Plants . . . . 16 I‘LATEBIAIS AND METHODS o o o o o o o o o o o o o o o 19 The Tobacco Cell Lines . . . . . Growth Conditions . . . . . . . . O O O O O O O O O O O O H \0 Growth Media . . . 20 Selection of the xnnthr Cell Line . 21 Harvesting of Cells and Preparation of Extracts . . . . . . . . . . . . . . 22 Determination of Nitrate . . . . . . . . . . 22 Determination of Total Protein . . . . . . . 24 Assay of Nitrate Reductase Activity . . . . . 24 Analysis of Free Amino Acids . . . . . . . 25 Thin Layer Chromatography and Autoradio- graphy of Amino Acids . . . . . . . 26 Determination of Total Uptake of Radio- active Amino Acids . . . . . . . . . . . 26 Determination of Incorporation of Radio- active Amino Acids Into Protein . . . . . 26 Determination of 15N Atom Percent . . . . . . 27 iv RESULTS Part A: VI. VII. VIII. Part 3: II, III. IV RESULTS Part A: I. II. III. IV. V. VI. VII. VIII. Part B: II. III. IV. Part C: Nitrate Uptake System and Its Regulation in XD Cells of Tobacco . . . . . . . . . Nitrate Accumulation in XD Cells . . . . Effect of Tungstate on Nitrate Assimi- lation in XD Cells . . . . . . Growth of XD Cells on Urea as the Only Source of Nitrogen . . . . Nitrate Accumulation by XD Cells Grow- ing EXponentially on Urea and Shifted to Nitrate . . . . . . . . . . . . . Effect of Tungstate and the Concentra- tion of Nitrate in the Medium on the Accumulation of Nitrate in EXponential Phase Cells . . . . . Inhibition of Nitrate Accumulation by DinitrOphenol and Cyanide . . . . The Effect of Amino Acids on the Accu- mulation of Nitrate . . . The Effect of Different Metabolites of the Nitrate Assimilation.Pathway on the Accumulation of Nitrate . . . . . . summary I O O O O O O I O O O O O O O O The Relationship Between the Regulation of the Nitrate Uptake and the Regulation of the Rest of the Nitrate Assimilation P at hway O O I O O O O O O O O O O O O O The Effect of Casein Hydrolysate on the Accumulation of Nitrate and the DevelOp- ment of Nitrate Reductase Activity . . . A Variant Cell Line: Growth Character- istics O O O O O O O O O O O O O O O O O A Possible Mechanism of Resistance . . . Nitrate Assimilation in XDRthI' Cells . . Summary 0 O O O O O O O O I O O O I O 0 Do Individual Amino Acids Inhibit Growth by Specifically Inhibiting Nitrate Assim- ilation? C O I O O O C O O I I O O O O 0 DISCUSSION.................‘.. BIBLIOGRAPHY O O O O O O O O O O O O O O O O O O Page 28 28 28 31 #4 45 55 61 62 69 75 76 78 87 101 105 116 138 10. 11. Table 1. 10. 11. 12. LIST OF TABLES Effect of Tungstate on the Formation of Active Nitrate Reductase and on Nitrate Accumulation in XD Cells . . . . . . . . . . 34 Inhibition by Tungstate of the Incorpora- tion of[:15N] N05 into Protein . . . . . . . 35 Inhibition by Cycloheximide of the Total Upfiake and Incorporation Into Protein of C] L-Arginine in XDRthr Cells . . . . . . 4O The Rates of Deve10pment of the Nitrate Uptake System and the Nitrate Reductase Activity in Stationary Phase and EXponen- tia1.Phase XD Cells . . . . . . . . . . . . 51 Effect of Dinitrophenol and Cyanide on Nitrate Accumulation in XD Cells . . . . . . 61 Effect of Casein Hydrolysate on the Accu- mulation of Nitrate and the DevelOpment of Nitrate Reductase Activity in Cells Treated with Tungstate . . . . . . . . . . . 77 Effects of Amino Acids on Growth of XD and mRthr cells 0 O O O I I O O O O O O O O 82 Growth of XD and XDRthr Cells on Casein Hydrolysate as the Only Nitrogen Source . . 88 Endogenous Levels of Some Amino Acids Dur- ing Growth of XDRthr Cells Shifted From Inhibition of Growth of XD and XDRthr Cells by Canavanine . . . , , , , , , . . . 91 Effect of L_Threonine on Nitrate Accumu- lation and Development of Nitrate Reduc- tase Activity in XD and XDRthr Cells . . . . 95 Effect of Casein Hyd€olysate on Nitrate Uptake in XD and XDR hr Cells . . . . . . . 104 vi “\v t .s. at 3: 11 0 TA «1 0 «1 r... «I 0. gr. n Au .1. A E P... 0 9w 0 ”My «(10/ )IU, a“ Q]. l at. Table 13. 14. Page Growth of XD Cells on quminobutyric Acid in the Presence of L-Threonine and L- Arginlne O O I O O O O O I O O O O O O O 0 O 110 Effects of IpThreonine and L-Arginine on Growth of XD Cells Maintained Continuously on Nitrate or on Urea . . . . . . . . . . . 11h vii Figure 1. 7. 10. 11. LIST OF FIGURES Nitrate Accumulation by XD Cells of TObaCCO O O O O O O O I I I O O O O O O I The Molybdate Reversal of the Inhibition of Nitrate Reductase Activity by Tungstate, in vivo . . . . . . . . . . . Reactivation of a Non-Functional Nitrate Reductase by Molybdate in vivo, in the Absence of Protein Synthesis . . . . . . Growth of XD Cells on Urea as the Sole Nitrogen Source . . . . . . . . . . . . . Kinetics of Nitrate Accumulation and Development of Nitrate Reductase Activ- ity in XD Cells Shifted from Urea to Nitrate O O O O O O O O O O O O O O O O 0 Effect of Addition of Casein Hydrolysate or Removal of Nitrate on the Decay of Nitrate Reductase Activity and Nitrate content I O O O O O O O O 0 O O O O O O 0 Effects of Tungstate and the Concentra- tion of Nitrate in the Medium on the Nitrate Accumulation in Cells Shifted from Urea to Nitrate . . . . . . . . . . Dependence of the Rate of Nitrate Uptake on Its Concentration in the Medium . . . Kinetics of Inhibition of Nitrate Accumu- lation by DinitrOphenol or Cyanide . . . Effect of IPThreonine on the Accumulation of Nitrate . . . . . . . . . . . . . . . Effect of Casein Hydrolysate on the Accu- mulation of Nitrate and the DevelOpment of Nitrate Reductase Activity . . . . . . viii Page 30 38 43 47 49 54 58 6O 64 66 68 17. 13. 20. 21 a A, H o .43.! 129' IV M" {~1‘ ‘\ \. Figure 12. 13. 14. 15. 16. 17. 18. 19. 20. 21, Page Effect of Casein Hydrolysate or Ammonia on the Accumulation of Nitrate . . . . . . . . . 71 Effect of Nitrite on the Accumulation of Nitrate O O I O I O O O O O O O O O O O O O O 73 Inheritance in XDRthr Cells of the Resis- tance to Growth Inhibition by L-Threonine . . 81 Growth Characteristics of XDRthr Cells . . . 85 Nitrate Content and Nitrate Reductase Activ- ity in XDRthr Cells Under Different Growth conditions 0 o o o o o o o o o o o o o o o o 94 Effect of Casein Hydrolysate on Nitrate Accumulation in XD and XDBthr Cells . . . . . 98 Effect of Casein Hydrolysate on the Development of Nitrate Reductase Activity in XD and XDRthr Cells . . . . . . . . . . . 100 Effect of Various Concentrations of Casein Hydrolysate on Nitrate Accumulation and Nitrate Reductase Activity in XD and )CDRthr cells 0 O O O O O O O O I 0 O O O O O 103 Total Uptake and Incorporation Into Pro- tein (10% Trichfioroacetic Acid Precipitable Material) of [1 C] L-Threonine Under Various Growth Conditions . . . . . . . . . . 108 Effects of L-Threonine and L—Arginine on the Growth of XD Cells Using Nitrate or Urea as Nitrogen Source . . . . . . . . . . . 112 ix INTRODUCTION Nitrate is one of the forms in which nitrogen is available to higher plants. In order to incorporate the nitrate nitrogen into cell constituents, the plants con- vert the nitrate to ammonia. The pathway by which nitrate is assimilated by higher plants can be summarized as follows: 1) Nitrate 2) Nitrate 3) Nitrite uptake reductase reductase N03(outside) -—9 NOS-(inside) —> N05 —-> NH3 ——9 amino acids In XD cells of tobacco the nitrate reductase and the nitrite reductase activities deveIOp in reSponse to nitrate (Filner, 1966; Chroboczek-Kelker, 1969) and nitrite (Chroboczek-Kelker,i969). The two enzyme activities are subject to end product regulation. Casein hydrolysate, i.e., a mixture of amino acids which can substitute for nitrate as a nitrogen source, inhibits the development of nitrate reductase activity (Filner, 1966) and nitrite reductase activity (Chroboczek-Kelker, 1969) without inhibiting growth. Some individual amino acids also inhibit the development of nitrate reductase activity, while other amino acids do not inhibit this process. Furthermore, the non-inhibitory amino acids antagonize the 2 inhibitors (Filner, 1966). The amino acids which inhibit the development of nitrate reductase activity also inhibit growth. Again, the non-inhibitors antagonize. It seemed most likely that the inhibition of growth was due to inhi- bition of nitrate assimilation. However, the Specificity of the inhibitory effects of amino acids for nitrate assimilation was not clearly established. Also, the mechanism by which amino acids inhibit the develOpment of nitrate reductase and nitrite reductase activities was not characterized in the earlier work. If we assume that in order to induce the nitrate reductase and the nitrite reductase activities, nitrate must enter the cells, then inhibition of this entry would be a sufficient condition to inhibit the induction. It is possible therefore that the end product regulation of the nitrate reductase and the nitrite reductase activities is merely a result of the regulation of the entry of nitrate into the cells. Using the KB cell line of tobacco as experimental material, an attempt has been made to answer the following questions: 1) Is there an uptake system Specific for nitrate in tobacco cells? 2) If there is such an uptake system, is it consti- tutive or adaptive? Does it develop in reSponse to nitrate and exhibit end product regulation? 3 3) If the uptake system is subject to end product regulation by amino acids, can this account for the inhi- bition by amino acids of the development of nitrate reductase and nitrite reductase activities? 4) Is the inhibition of the develOpment of the nitrate assimilation pathway by individual amino acids Specific, or is it a consequence of a more general inhibi- tory effect? LITERATURE REVIEW Active Uptake: General Consideration Living cells absorb or secrete solutes by passive or active processes (Woodbury, 1960). Several criteria were proposed by which the active tranSport is determined (Woodbury, 1960; Rosenberg, i959; Collander, 1959; Kepes and Cohen, 1962). The dependence of the rate of uptake or the flow of the solute into the cells on its concentration in the ambient solution gives a saturation curve which can be analyzed by using Michaelis-Menten concepts of enZyme reaction. An apparent Km value and competition kinetics by analogs or related compounds can be obtained. In a case where the trans- ported compound is not readily metabolized, or if its utilization can be inhibited without impairing other processes, including the tranSport itself, the compound accumulates within the cells. If the accumulated compound is free in solution (which is assumed in many cases) then the accumulation takes place against a concentration gradient by consuming metabolic energy. The most sophisticated active uptake systems are those which are also selective and Specific. Nevertheless as was pointed out by Cohen and Monod (1957), selective permeation does not have to be active, and active tranSport h may not be Specific. Some of the criteria of active tranSport are common to other tranSport processes. Thus, complete characteriza- tion of an active tranSport can be achieved only when all the criteria are tested. TranSQort Systems in Bacteria The most characterized tranSport systems are the sugar and ion tranSport systems in bacteria. The B-galactoside uptake of Escherichia coli deve10ps in reSponse to B-galactosides (see reviews by Cohen and Monod, 1957; Kepes and Cohen, 1962) and the process involves inactivation of a repressor protein by the inducing B- galactoside, i.e., derepression (Jacob and Monod, 1961). The repressor protein was isolated (Gilbert and Mfiller-Hill, 1966). In the presence of glucose or other related carbon sources the develOpment of the system is inhibited as a result of a catabolite repression (Magasanik, 1961). The permeation of B-galactosides (one step or the overall process) is coded by the y locus of the lac operon (Jacob and Monod, 1961). The operon is regulated as a unit and the three structural genes are eXpressed coordinately. However it was shown that the lac Operon can be eXpressed sequentially also (Alpers and Tomkins, 1966). A membrane-bound protein component, the "M" protein binds B-galactosides very Specifically. It is believed to be involved in the tranSport of B-galactosides (Fox and 6 Kennedy, 1965). The protein was isolated and somewhat purified (Kolber and Stein, 1966; Fox et al., 1967). The presence of "M" protein in the membrane of the cells is clearly related to the eXpression of the y gene of the lac Operon (Fox et al., 1967). Mutants of the y gene do not have "M" protein, and temperature sensitive mutants in permeation of B-galactosides were shown to have tempera- ture-sensitive "M" protein. The "M" protein has been postulated to be the carrier of the B-galactosides across the cell membrane (Fox and Kennedy, 1965; Kennedy, 1966). Thus the "M" protein would be the recognition site and the carrier as well. According to the model proposed by Kennedy and co- workers (Fox and Kennedy, 1965; Fox et al., 1967; Kennedy, 1966) the metabolic energy is involved in the process only when active accumulation takes place. Perhaps the energy requiring process is a change in the protein con- formation which would prevent the back flow of the trans- ported sugar. The site(s) in which metabolic energy is involved in the tranSport of B-galactosides in E. go}; and the sul- fate tranSport in §, typhimuriun (see below) is not yet known. There is however one tranSport system widely encountered among bacterial Species in which the site of energy involvement is much better understood. It is the 7 phoSphoenolpyruvate: sugar phOSphotransferase system. The system was first described as a "novel phOSphO- transferase system" by Kundig et al. (1964) and was later proposed to have a role in tranSport of sugars across the cell membrane (Kundig et al., 1966). Similar but not identical systems were reported in several bacterial Species (see review by Anderson and Wood, 1969). The phOSphotransferase system is composed of essen- tially three protein components, enzyme I, enzyme II and HPr, the latter being heat stable. They are located on the cell membrane (Kundig et a1..1966; Kaback, 1968). Enzyme II is strongly bound to the membrane, while enzyme I and HPr are readily solubilized. Enzyme I catalyzes the phOSphorylation of HPr using phosphoenolpyruvate as donor. The phOSphorylated HPr is the phOSphate donor for the phOSphorylation of the sugar, catalyzed by enzyme II (Kundig et a1” 1964; Kundig et al., 1966). A fourth component which is required for maximal affinity of enzyme II for fructose was described in Aerobacter aerogenes (Hanson and Anderson, 1968), and other bacterial Species (see review by Anderson and Wood, 1969). Enzyme II and the fourth component, the Specifier (Hanson and Anderson, 1968), are inducible and sugar specific (Kundig et al., 1966; Simoni et al., 1967). Enzyme II mutants are unable to tranSport a particular sugar or a 8 group of related sugars, while enzyme I or HPr mutants are pleiotropic mutants unable to tranSport a wide range of sugars (Simoni et a1" 1967; Tanaka et alw 1967). Thus enzyme I and HPr participate in several phOSphotransferase reactions using sugar Specific enzyme II. A proposed scheme for the tranSport mechanism was suggested by Pardee (1968 a), in which enzyme II is the recognition site and the translocator. Using cell-free vesicles of E. 2211 supplemented with phosphoenolpyruvate, Kaback (1968) showed accumulation of phOSphorylated glucose inside the membrane structure. The best studied ion tranSport system is the sulfate tranSport in Salmonella typhimurium (Dreyfuss, 1964). The system which is the first step of sulfate assimilation (Dreyfuss and Monty, 1963 a) is derepressed under low- sulfur conditions and repressed by the end product of the pathway, L-cysteine, even in the absence of subsequent utilization of sulfate (Dreyfuss, 1964) as is the rest of the pathway (Dreyfuss and Monty, 1963 b). The uptake of sulfate is believed to involve at least two steps (Pardee et al.,1966; Pardee, 1968 a,b). The first one is the reCOgnition and the binding of sulfate ions to a Specific binding protein, located at the external surface of the cells (Dreyfuss and Pardee, 1965; Pardee and Watanabe, 1968). The binding protein was isolated, purified and crystallized (Pardee and.Prestidge, 1966; Pardee, 1966, 1967). 9 The second one is the translocation of the sulfate ions into the cells (Pardee et al" 1966). The binding is related to tranSport but the two activities are not identical. This was determined by using tranSport nega- tive mutants of the cys A region which can bind sulfate but cannot translocate it, by the energy requirement for the translocation but not for the binding and by kinetic evidence (Pardee et a1” 1966). The cys A region is composed of three cistrons unlinked to the rest of the assimilation pathway. Mutants of the cys A region have normal sulfate binding capacity which is regulated similarly to the rest of the pathway. Presumably these mutants are defective in the transloca- tion step since they bind sulfate but do not accumulate it. Mutants of the cys SP region are defective in their bind- ing capacity and are tranSport negative (Pardee et al” 1966). These turned out to be regulatory and not structural mutants, however. TranSport_§y§tems in Higher Plants Plants are autotrOphic and depend mainly on the avail- ability of inorganic salts, C02, H20, 02 and light in the external environment. Needless to say, the ions which are essential for plant growth are taken up by the plant (Epstein, 1965) and some of them are also accumulated within the cells to concentrations much higher than that in the ambient medium (Steward and Sutcliffe, 1959; MacDonald 10 et al., 1960). The process of absorption and accumulation cannot be eXplained on a basis of free diffusion alone (Brouwer, 1965). The uptake process exhibits a high degree of Speci- ficity and selectivity (Epstein and Hagen, 1952; Epstein, 1961; Elzam and Epstein, 1965; Welch and Epstein, 1968; Pitman et al., 1968). The process of absorption and accumulation depends on metabolic energy as shown by eXperimental observation concerning the influence of oxygen tension (Hoagland, 1948; Epstein and Hagen, 1952), temperature (Epstein et al., 1962; MacDonald and Laties, 1963; Elzam and Epstein, 1965), metabolic poisons (Machlis, 1944; Ordin and Jacobson, 1955; Elzam and Epstein, 1965; Birt and Bird, 1956) and darkness (Van Lookern Campagne, 1957; MacRobbie, 1962; Chen and Ries, i969; Weigl, 1967). The energy requirement seems to be the only certain thing which can be stated about the process (Brouwer, 1965). Any interference with aerobic reSpiration which results in decrease in available energy, inhibits the absorption and accumulation of ions (Ordin and Jacobson, 1955; Higinbotham, 1959). Several mechanisms have been proposed to account for the process of ion tranSport and its characteristics (Brouwer, 1965; Epstein, 1965). None of these theories however accounts for all the features of this phenomenon (Brouwer, 1965). Two theories which are often discussed 11 are the anion-reSpiration theory originated by LundegArdh (i955. 1960 a,b) which coupled the ion transport to the reSpiratory electron flow, and the carrier theory deveIOped by Epstein and Hagen (1952) (see also Epstein 1961, 1962; Legget, 1968), which attempted to describe the tranSport processes in higher plants by concepts of classical enzyme kinetics. Anion RSSpiration EXposure of plant tissue to salt solution is accom- panied with increased rate of reSpiration, which can be inhibited by cyanide and carbon monoxide (inhibition by the latter is reversed by light) (LundegArdh, 1955; Ordin and Jacobson, 1955). The inhibition of reSpiration is corre- lated with inhibition of anion tranSport and accumulation. The concomitant inhibition of anion tranSport and reSpira- tion by cyanide and carbon monoxide indicate a close coupl- ing of the two phenomena. It was shown that eXposure of wheat roots to salt solution resulted in rapid oxidation of reduced electron carriers (cytochromes) (LundegArdh, 1960 a). The anion-reSpiration hypothesis implies that anions are advanced along the electron tranSport chain in reverse to the electron flow. The cytochromes are believed to be the anion carriers according to LungegArdh. At the time the hypothesis was develOped it was believed that the electron tranSport chain was located on 12 the cell surface (LundegArdh, 1955). Since then however, it was clearly demonstrated that the cytochromes are located in the mitochondria. The anion reSpiration hypothesis was revived by suggesting that the mitochondria serve as intermediate Sites for ion tranSport and accumu- lation (Robertson et al.,1955; Millard et a1",1964). The Carrier The relationship between the rate of uptake and the external concentration is a hyperbolic function of the external concentration. This suggests that some finite entity is being occupied. At increasing external concen- trations the maximal rate of absorption is approached asymp- totically. The maximal rate of absorption is achieved when the entity (carrier) is completely saturated. Based on such kinetic observations and by employing concepts of classical enzyme kinetics, the carrier theory was first used to describe the absorption of rubidium ion by excised barley roots (Epstein and Hagen, 1952). It was shown that the process could be described using Michaelis— fienten concepts for enzyme kinetics. ‘ It was later shown that the theory was applicable to other plant systems and ions (Kahn and Hanson, 1957; Smith and Epstein, 1964; Lyclama, 1963) and for solutes other than inorganic ions (Bieleski, 1960, i962; Birt and Hird,.1956; Nissen and Benson, 1964). The carriers which are postulated to be located in 13 the cell membrane are thought to combine with the ions on the external interface to form ion-carrier complexes. The complexes move across the membrane to its inside surface (Epstein and Hagen, 1952; Epstein, 1965; Brouwer, 1965; Legget, 1968) where the ions are released from the carriers. The leakage of the accumulated ions is prevented by the impermeability of the cell membranes to free ions (Collander, 1959; Arisz,1964; Brouwer, 1965). It is assumed that the release of the ion from the ion-carrier complex in the inside surface of the membrane is not a result of a Simple dissociation but rather a result of a chemical change of the carrier molecule. The alternate modification of the carrier molecule is believed to be the process where metabolic energy gets involved (Goldacre, 1952; Epstein, 1965). Prior to the formation of ion-carrier complex, the carrier molecule may be phoSphorylated using ATP. In the inside surface, the carrier molecule is dephOSphorylated thereby altering its configuration and the ion is released. When the generation of ATP is interrupted by using poisons (Machlis, 1944; Ordin and Jacobson, 1955; Elzam and Epstein, 1965; Birt and Bird, 1956; Higinbotham, 1959), the activation of the carrier molecule, i.e., phoSphoryla- tion, is therefore inhibited and the process of accumulation steps. According to Epstein (1965) this also accounts for 14 the increase in rate of reSpiration upon exposure of tissue to salt solutions. He suggests that if reSpiration is controlled by the level of phoSphate acceptor (ADP), then liberation of ADP upon phoSphorylating of the carrier molecules will increase the rate of reSpiration. The theory assumes that the selectivity of the tranSport system is based upon different affinities of the absorption sites or carrier molecules for various ions or groups of ions (Epstein, 1962). In general, a group of ions which share a transport system consists of ions of an essential element and its analogs (Epstein, 1965). At present the carrier theory seems to be the most satisfactory one, and provides a reasonable model for tranSport and accumulation of ions which take place in Spite of the impermeability of the cell membrane, and for the Specificity and selectivity of the tranSport system (Brouwer, 1965). Most of the work on ion absorption and accumulation in higher plants was done on either intact plants or excised roots (see review by Legget, 1968). The observed phenomena in higher plants are the summations of interactions between the different tissues and organs of which they are composed. In order to eliminate interactions between roots and shoots (translocation) excised roots were employed. Nevertheless, the root system is composed of several tissues. It is not known whether all of the tissues and cells are active in 15 ion absorption and accumulation or only some. The most complete studies on tranSport systems have been done on lower organisms, mostly unicellular, i.e., bacteria and fungi, which lack the complications due to the interactions between tissues and organs. In order to achieve that degree of freedom of inter- actions in a higher plant system, one is led to cell sus- pensions. Under these conditions the highly organized plant is reduced to a state which somewhat resembles that of cultures of microorganisms. The system can be studied by using many of the methods which were originally developed to study microorganisms. The selection of mutant or variant cell lines might make it possible to study interactions and regulation of metabolic pathways. Selection of Variant Plant Cell Lines Selections of variant cell lines of plant systems were reported in several cases. Tulecke (1960) isolated a cell line derived from GinkgoIaollen which required arginine for growth. Arya et a1 (1962) and Sievert and Hildebrandt (1965) described variations in the ability of secondary clones derived from Single cells of common clones to use various sugars as carbon source. Blakely and Steward (1962, 1964) reported the isolation of cell lines of Haplopappus gracilis which varied in their anthocyanin contents from a common cell line. They also reported the 16 isolation of a variant cell line of Daucus carota resistant to inhibition by acriflavin from a sensitive one. Fox (1963) reported the selection of tobacco cell lines which had lost their requirement for an exogenous supply of plant growth substances. Miura and Miller (1969) reported the selection of a cytokinin-independent cell line from a culture of soybean cells which were cytokinin-dependent. Nitrate Assimilation in Higher Plants The steps in Which the nitrate is reduced to nitrite and then to ammonia are very well documented (Kessler, 1964; Beevers and Hageman, 1969; Filner et a1" 1969). The two reductases develop in reSponse to nitrate (see review by Beevers and Hageman, 1969), and nitrite (Ingle et al, 1966). The induction of nitrate reductase activity depends on genetic factors (Warner et a1" 1969), hormones (Borriss, 1967; Lips and Roth-Bejerano, 1969) and some environmental factors including light, temperature and COZ (see reviews by Kessler, i964 and Beevers and Hageman, 1969). Molybdenum was shown to be required for the induction (Afridi and Hewitt, 1962, 1964; Hewitt and Afridi, 1959). Inhibitors of nucleic acids and protein synthesis inhibit the develop- ment of the two reductases (see Filner et a1” 1969). End product control of nitrate reductase and nitrite reductase activities has been demonstrated in cultured tobacco cells (Filner, 1966; Chroboczek-Kelker, 1969) and Lgmna (Sims et al. 1968; Joy, 1969), but not in other systems (Ingle et al, 1966). 17 In contrast with the two reduction steps, the uptake, or as it is commonly referred to in the literature, the absorption, of nitrate and its regulation are much less understood and documented. The dependence of the rate of nitrate absorption on its concentration in the nutrient solution and nitrate absorbed as a function of time have been studied in intact plants or excised roots of tomato (Jensen, 1962), nitrogen rich wheat plants (Kihlma-Falk, 1961), low nitrogen excised wheat roots (Stenlid, 1957), intact plants or excised roots of perennial rye—grass (Lyclama, 1963). The results of these kinetic studies were interpreted to mean that nitrate absorption is an active process. The process of absorption depends on the presence of calcium ions in the ambient medium (Nightingale et a1” 1931; Skok, 1941; Minotti et a1” 1969 a,b). It was shown however, that calcium is required for the maximal rate of absorption of other ions and was proposed to have a role in the integrity of the membranes or the tranSport system (Epstein, 1965). Arnon et a1.(1942) found a small effect of the pH of the nutrient solutions on the absorption of nitrate. Van den Honert and Hooymans (1955), Fried et al.(1965), and Lyclama (1963) showed a strong pH effect on the absorption of nitrate. The process was shown to be affected by temperature (Van den Honert and Hooymans, 1955; Lyclama, 1963; Chen and Ries, 1969), light (Hageman et a1” 1961; 18 Knipmeyer et a1” 1962; Kannangara and Woolhouse, 1967; Beevers et a1” 1965; Chen and Ries, 1969) and C02 (Kannangara and Woolhouse, 1967). Nitrate absorption was Shown to be inhibited by ammonia (Weisman, 1950, i951; Lyclama, 1963; Fried et al. 1965; Minotti et a1" 1968, 1969 a,b). It was interpreted as the inhibition of nitrate reduction (Lyclama, 1963). Minotti et al.(1969) however, demonstrated that the uptake system rather than the reduction was inhibited by ammonia. They suggested that derivatives of ammonia assimilation rather than the ammonium ions inhibited the uptake. Ferguson and Bollard (1969) concluded that ammonia inhi- bited both the uptake and the reduction of nitrate. Minotti et al.(1968) observed a lag period in the absorp- tion of nitrate when low nitrogen plants were eXposed to nitrate. They prOposed that a synthesis of components of the nitrate uptake system took place during the lag period. MATERIALS AND METHODS The Tobacco Cell Lines Two cell lines were used as eXperimental material in this study. The XD cell line which was isolated from tobacco stems by P. Filner (1965). This cell line can grow in liquid suSpension on a chemically defined medium. The second cell line was isolated from the XD cell line in the course of this study. It was selected for its ability to grow on nitrate containing medium in the presence of the inhibitory amino acid, L-threonine. The selection procedure of this variant cell line, which is designated XDRthr, is described below. Growth Conditions Stock cultures were grown in 1000 ml flasks con- taining 500 ml medium, on an horizontal shaker with a dis- placement of 4 inches at 80 cycles per minute at 28°C. Two week old cultures were used for routine subcul- tures and eXperimental material. Subcultures were carried out by diluting the cell suSpensions twenty fold into fresh media using wide-tip pipets. When large size inoculations were required, graduated cylinders were used. The following procedure was used for eXperiments in which cells were shifted at mid-exponential growth from 19 20 urea containing medium to new media. Stationary phase XD cells were diluted 12.5 fold into 1000 ml flasks contain- ing 500 ml nitrate-less M-ID supplemented with 3.0 mM urea. The cells were allowed to grow for 5 days. They were then aseptically harvested by filtration on Miracloth, rinsed with nitrate-less M-ID and resuSpended in new medium. Two or three flasks of cells growing on urea were pooled together to give 10-15 g/l cell suSpension in the new medium. Growth Media The basic medium used was the N-ID described by P. Filner (1965). It is composed of the following in moles x10'5/l: Ca(N03)2°4 H20, 84.8; KNO3, 79.1; NaHZPOh‘HZO, 11.9; Nasou, 141.0; Mg804°7 H20, 146.0; KCl, 87.1; F906H507°3 H20, 0.67; MnSOu'4 H20, 2.2; Zn804’7 H20, 0.52; H3B03, 2.4; KI, 0.45; nicotinic acid, 0.41; pyridoxine°HCl, 0.049; thiamine'HCl, 0.03; 2,4-di- chlorOphenoxyacetic acid, 0.23; sucrose, 5,840. The pH of the medium was adjusted to 6.2-6.5 prior to autoclaving. Nitrate-less M-ID was made by replacing the nitrates of calcium and potassium with their reSpective chlorides. When supplemented media were required, sterile solutions of the supplements were diluted into the sterile media. The pH of the supplement solutions was adjusted to 6.2-6.5 prior to sterilization which was carried out either 21 by autoclaving or filtration. Urea was purified by the following procedure. Bio-Rad AG2-X8 ion exchange resin was equilibrated with 2 N KOH. After equilibration it was rinsed well with deionized distilled water to remove the excess of potas- sium hydroxide. One g of the resin (OH’ form) was mixed with 100 ml of 1 M urea solution and stirred for 30 min. The process was repeated twice. The urea solution was then concentrated under vacuum and excess water removed by acetone. The acetone was removed by vacuum. Selection of the ththr Cell Line About 0.7 g fresh weight (2 x 106 cells) of two weeks old XD culture was subcultured into each of three 1000 ml flasks containing 500 ml M-ID plus 0.1 uM N-methyl- N'~nitro-N-nitrosoguanidine. This level of the mutagen has almost no effect on the growth of XD cells. (After five days, the cells were harvested aseptically by fil- tration on Miracloth and resuSpended in 1000 ml flasks containing 500 ml M-ID plus 50 OM L-threonine. This was a sublethal concentration of the inhibitory amino acid so that the cells could grow. After seven days, the cells were subcultured into 1000 ml flasks containing 500 ml M-ID plus 100 CM L-threonine. Five subcultures were made from each of the three replicates, about 0.7 g fresh weight per subculture. The cells were grown until the density of the cell suSpension was comparable to that of XD cells 22 after 14 days of growth on M-ID. Of the 15 subcultures, only three obtained that density in less than five weeks. These three cultures were serially subcultured on M-ID plus 100 0M L-threonine. After the third subculture, the threonine-resistant cell lines were also transferred to M-ID, so that threonine-resistant cells were cultured in parallel on M-ID with and without threonine. Harvesting of Cells and Preparation of Extracts Cells were harvested by vacuum filtration on Whatman No. 1 filter paper, rinsed with deionized distilled water and weighed to determine the fresh weight. The cells were suSpended in 0.1 M Tris (tris(hydroxymethyl)- aminomethane)0 HCl pH 7.5 buffer containing 1 mM L- cysteine. Five ml of the buffer were used per gram of cells. In eXperimentS in which urea was used, the con- centration of L-cysteine was 5 mM. The cells were homo- genized at 4°C with thirty strokes of motor driven Teflon- glass homogenizer. Determination of Nitrate The method of Lowe and Hamilton (1967), which depends upon the ability of soybean nodule bacteroids to reduce nitrate to nitrite, was used with slight modifica- tions. Prior to planting, soybeans were inoculated with soybean inoculant kindly provided by the Nitragin Co. of Milwaukee. The plants were grown on vermiculite in the 23 green house, with the nitrogen-free nutrient solution pro- vided via the bottom of the pots. Nodules were harvested from 6-8 week old soybean plants. The nodules were col- lected in ice-cold deionized distilled water, then rinsed several times with ice-cold deionized distilled water, wiped dry and weighed. The nodules were ground in a cold mortar in 0.1 M potassium succinate buffer pH 6.8, 5 ml of buffer/gram of nodules . The homogenate was filtered through four layers of cheesecloth and the filtrate was centrifuged for five min at 6000 x g in Sorvall RC2-B centrifuge. The supernatant was discarded and the pellet was resuSpended in the same buffer and centrifuged again at 6000 x g for 5 min. The supernatant was discarded and the pellet was resuSpended in the same buffer. The bacteroid suSpension was kept frozen at -20°C. The assay mixture was composed of the following: 0.5 ml of 0.1 M potassium succinate buffer pH 6.8, 0.4 ml of the extract and 0.1 ml of undiluted bacteroid suSpension. The assay tubes were incubated for 30 min at 45°C. The amount of nitrite in the assay tube was determined by the colori- metric method of Snell and Snell (1949). Before reading the absorbancy at 540 mu the assay mixtures were cleared by centrifugation at 15,000 x g for 10 min. The assay, which is linear in the range of 0-100 mumoles of nitrate per assay tube, was carried on the total homogenate after apprOpriate dilution. 24 Determination of Total.Protein The method of Lowry et a1.(i951) was used. An 0.5 ml aliquot of the total homogenate was precipitated with 10% trichloroacetic acid. After 24 hours at room tempera- ture, the precipitate was collected by centrifugation and rinsed twice with 95% ethanol. After air drying, the pro- tein in the precipitate was dissolved in 1 N NaOH and the undissolved material was removed by centrifugation. The assay was carried out on the supernatant fraction. Bovine serum albumin dissolved in 1 N NaOH was used as a standard. Assay of Nitrate Reductase Activity A modification of the method of Paneque et al.(1965) using FMNH as electron donor was used. The following are mixed and equilibrated at 25°C: 0.5 ml of 0.1 M potassium phoSphate buffer pH 7.5, 0.1 ml of 0.1 M potassium nitrate, 0.1 ml of 2 mM FMN, 0.25 ml of enzyme plus water. The reaction is started by adding 0.05 ml freshly made 0.046 M sodium hydrosulfite in 0.095 M sodium bicarbonate followed by slightly agitating the tube. Care is taken not to excessively agitate the tube, which results in rapid air oxidation of the hydrosulfite. After 20 min incubation at 25°C, the reaction is stopped by vigorously shaking the assay tube until the FMNH is reoxidized, as indicated by the return of the bright yellow color. The product of the reaction, nitrite,was determined colorimetrically by the method of Snell and Snell (1949), as in the nitrate assay. 25 Reaction mixture stopped at zero time was used as the blank. The 10,000 x g supernatant of the total homogenate which is obtained after 15 min centrifugation in Sorvall RC2-B centrifuge was used as enzyme source when the XDRthr cells were examined alone. However, the protein of the 10,000 x g supernatant, precipitable by 50% saturated ammonium sulfate was used in comparisons of the XD and the XDRthr cells, because of the presence of an inhibitor in the XD homogenate. For the xnathr cells, the activity measured in the 10,000 x g supernatant was equal to that in the protein fraction pre- cipitable by 50% saturated ammonium sulfate. Analysis of Free Amino Acids Cells were harvested as described above, and homo- genized in 70% ethanol (final concentration) in the ratio of five ml per gram fresh weight. The homogenate was stored at 3°C overnight and then clarified by centrifuga- tion at 10,000 x g for 10 min. The supernatant was saved and the pellet was rinsed twice with 70% ethanol. The ethanol fractions were pooled and vacuum dried. The residue was redissolved in sodium citrate buffer pH 2.0. The solution was cleared by centrifugation. After approp- riate dilution with the same buffer, 0.5 ml was applied to a Technicon amino acid analyzer. The amount of each amino acid was calculated by cutting out and weighing each peak of the chart. The results were compared to a standard analysis made on a solution containing 100 mumoles of each amino acid. 26 Thin Layer Chromatography and Autoradiography of Amino Acids Aliquots of the 70% ethanol extracts (2-10 ul) were applied to cellulose powder thin layer plates. The plates were develOped in two solvent systems and in two dimen- sions: n-butanol:formic acid:H20 (15:3:2) and pyridine: H20 (4:1). The plates were dried for 30 min at 72°C between the two runs. Reproducibility was improved by scraping the cellulose powder from the plates at the region of the front of the first solvent system. Alanine and proline dissolved in 70% ethanol were used as internal standards . Autoradiograms were prepared by eXposing the plates after the two runs to Kodak X-ray film for eight days. Between 500 and 1500 counts/min 140 were used per plate. Determination of Total Uptake of Radioactive Amino Acid Aliquots (0.1-0.2 ml) of total homogenates were added to scintillation vials containing 10 ml of Bray's (1960) scintillation liquid supplemented with 35 g/l thixotropic gel (Cab-O-Sil), to keep the homogenate suSpended during scintillation counting. Determination of Incorporation of Radioactive Amino Acids into Protein The initial procedure is identical to that described in the section on protein determination. An aliquot (0.5-0.7 ml) of the 1 N NaOH redissolved 27 precipitate was neutralized by 1 N HCl. Aliquots of the neutralized solution were added into scintillation vials containing 10 ml of Bray's (1960) scintillation liquid. Determination of 15N Atom.Percent The 10% trichloroacetic acid insoluble fraction of the total homogenate was collected and rinsed twice with 95% ethanol. The precipitate was digested with 2-4 ml of concentrated H2304 in the presence of 1 ml of 10% CuSOu and 0.5 g K2304. The digestion was carried out for about 7 hrs and was completed by adding 0.2 ml of 30% H202 to the digestion tube. The ammonia which was released during the digestion was distilled using micro-Kjeldahl distilla- tor and collected in 2% boric acid. The ammonium borate solution was concentrated under vacuum. The ammonia was oxidized to nitrogen gas using alkaline hypobromite (Burris and Wilson, 1957). Five ml of bromine were slowly added into 15 ml of 40% NaOH with slow stirring at 0°C. After the bromine was dissolved another 15 ml of 40% NaOH were added. A white precipitate deve10ped and was dis- carded. The supernatant was used as the source for the hypobromite. The 15N atom percent was determined using the MAT Berschreibung Mass Spectrometer CD 150. RESULTS 22.22.11: Nitrate Uptake System and Its Regulation in XD Cells of Tobacco I. Nitrate Accumulation in XD Cells In order to be reduced, nitrate must enter the cells. One of the objectives of this study was to explore this process of entry. Stationary phase cells were subcultured into M-ID (2.5 mM nitrate). The time-dependent nitrate accumulation was then examined. The results are summarized in Figure 1. The points in the accumulation curve were eXperimentally determined. The curve was drawn to best fit the points. The plot of the rate of uptake was calculated from the curve rather than from the eXperimental values. Station- ary phase cells have little or no nitrate. Following subculture, nitrate accumulates and the rate of accumula- tion increases linearly until it reaches a constant value. The rate of uptake extrapolates to zero at zero time. The constant rate of uptake (1.8 umoles of nitrate taken up/hr/g fresh weight) is obtained at about 10 hours after the subculture. The rate of increase in the rate of uptake taken from this plot is 0.16 umoles of nitrate taken up/hr/g fresh weight/hr. The values of nitrate 28 29 A Figure 1. Nitrate Accumulation by XD Cells of Tobacco Stationary phase cells maintained on M-ID were subcultured into the same medium. At various times the nitrate content (0—0) and the rate of nitrate accumulation in each time interval (———) were determined. 30 stores; momma ext: \oS Exes mg 6.36: i 4O _ _ 4 _ _ _ O 0 O 3 2 I: atoms momma o\ moz mm .5: 1 24 I6 TIME(HR) 31 content at various times after subculture calculated from the equation: nitrate content = ktZ’ where k is the con- stant of increase in the rate of uptake and t the time period after subculture, are very similar to those obtained experimentally (Figure 1). It should be noted that although nitrate reductase activity develOpS during the first 24 hours after subcul- ture, its influence on the nitrate level in the cells is small. The accumulation therefore reflects the uptake but it is not identical with the uptake. This conclusion is based on the following observations: 1) there is a lag period of about 10 hours before nitrate reductase activity starts to develop, and at this time the uptake system has reached its constant value already, 2) after 24 hours the amount of nitrate reduced per unit time, is at the most one third of the amount taken up in the same time period. Thus, the nitrate uptake system is inoperative in stationary phase cells. It deve10ps immediately upon eXposure of the cells to nitrate and reaches its maximal rate of operation in 10 hours. II. Effect of Tungstate on Nitrate Assimilation in XD Cells Nitrate ions, like those of sulfate and phOSphate, are not only taken by plant cells but are also metabolized. The accumulation of nitrate is therefore different than the accumulation of ions which are taken up but not 32 metabolized, e.g., chloride, sodium and potassium (Hoagland, 1948; Epstein, 1962). The level of nitrate in the cells is determined by the rate of uptake and by the rate of utiliza- tion. During the first 24 hours after subculturing XD cells into fresh medium, nitrate reductase activity develOpS and reaches a level of about 700 mumoles nitrate reduced/hr/g fresh weight. After 24 hours the nitrate accumulation rate is about 2000 mumoleS/hr/g fresh weight. Is it possible to inhibit nitrate reduction without impairing nitrate uptake, thereby making possible studies of nitrate uptake per se? Since nitrate reductase negative mutants of higher plants were not available, a different means of blocking nitrate reduction was developed. Tungstate is a competitive inhibitor of molybdate uptake and utilization in Azotobacter vinelandii (Keeler and Varner, 1957. 1958). Tungstate inhibits growth of this organism when nitrate or nitrogen gas is the nitrogen source (Takahashi and Nason, 1957). Tungstate competi- tively inhibits molybdate function in Aspergillus niger when nitrate is the nitrogen source (Higgins et a1, 1956). Molybdenum (as molybdate) is believed to be an essential micronutrient for higher plants. Molybdenum- deficient plants will not develOp nitrate reductase activ- ity when growing on nitrate as nitrogen source (Afridi and Hewitt, 1964). Molybdenum-deficient plants grow better on 33 ammonia or nitrite as nitrogen source than on nitrate (Agarwala, 1952). Molybdenum was reported to be associ- ated with soybean nitrate reductase through several puri- fication steps (Evans and Hall, 1955). The effect of tungstate on nitrate accumulation and the development of nitrate reductase activity in XD cells was examined (Table 1). AS the concentration of tungstate is increased, the activity of nitrate reductase detected after 24 hours of exposure to nitrate decreased. Nitrate reductase activity cannot be detected when the concentra- tion of tungstate is 50 uM or higher. At the same time, the level of nitrate increased 2-3 fold over the untreated control. Tungstate at a concentration of 1.0 mM does not inhibit nitrate reductase activity in 21253. The effect of tungstate on the incorporation of [15NJ N03 into protein was studied. Tungstate inhibits the detectable nitrate reductase activity by 82% while at the same time bringing about a 2 fold increase in the level of nitrate (Table 2). The inhibition of the incorporation of 15M into protein is very similar to that of the nitrate reductase activity (Table 2). De Renzo (1954) reported that tungstate inhibited the stimulation of intestinal xanthine oxidase in rats. Mitidieri and Affonso (1965) showed that tungstate inhi- bited the develOpment of xanthine dehydrogenase in Eseudomonas aeroginosa.in;zlzg. The inhibition could be overcome in_vivo by molybdate. 34 S.H: o ooa rm «.3: 0 on em mos ms om SN 5.5: sma oa em 3.3 mmm m rm m.wfi own 0 SN :o.o o o o pswdos Smohm pswaoz mosh w\H£ Aznv Ahnv w\moz moaoad \ooosomh :oz moHosda soapmhpsoosoo pampsoo opmhpaz ommposoom opmhpdz opwpmwzse doahmm SPSOHQ .oosHaaopoo cams mpabapom ommposoos mpmeHs map use p:op£oo mpwhpas on» use oopm0>9m£ ohms mHHoo 02p manos :N songs .opmpmwSSp adapow mo mamboa unohowmao wsHsHmpSoo QH:2 opSa ooHSpHSCQdm one: maaoo ax omega mammoapmpm maaoo ax Ga soapmasa500< opmhpaz no one ommposoom opmapaz obapo< mo :opraHom on» no mumpmwssa ho poommm .a magma 5 3 .HoHSSoo oopmospss 05p mo unoohog on» unmmohaon mamoszCHSQ Ea mohswamt *Amm V sma.o *Amsmv os.oe ears v omm opennessa z: oo« + QHIZ *Aooav osm.m *Aooav oo.mm *Aooav 6mm.s oHuz p: Hos showy unwaoz cream pamsnoahsm R m\ioz moHoan w\h£\ooosooh moz moHoaja sodpwsoanoosH zmH unopsoo opmspaz owoposoom opmapaz Haddoz I! ll .oosdahopoo ohms Sampoam SH ZmH mmooxo & Hope on» use mpabdpom ommposomn opmhpd: on» .psopsoo opdhpas 02p use oopmm>sm£ ones mHHoo on» npzohm go thon :N songs .opmpmwQSp 21 ooH unonpas use Spas mozflmZmHu mmooxo R aopw oa wsasawpsoo QHIS SH popcodemoa one topmobhm: maamoapaomm owes mHHoo CSp mmwo m Hopw< .omhs 25 o.m Spas posses ICHQQSm QHIS mmoauopmhpas op:« ooHSpHSODSm ohms mHHoo ax omega mammodpmpm saopoam ousH moz mamau Go soaaoaoaaoosH esp so ocopnmssa an soapanassH .N canoe 36 If the effect of tungstate on XD cells is due to interference with one or more molybdenum-dependent processes, then this inhibition must be overcome by molybdate. It can be seen (Figure 2), that molybdate does overcome the tungstate inhibition ig‘zizg. Cells which are treated with tungstate deveIOp very little nitrate reductase activity after 24 hours. However, molybdate when given to the cells at the 24 hour point, overcomes the tungstate inhibition and the nitrate reductase activ- ity increases with time. An attempt was made to understand the mechanism by which tungstate inhibits the deve10pment of nitrate reductase activity. Wray reported (1969) that NADH:cytochrome c reduc- tase activity is induced by nitrate in barley seedlings. The activity is associated with NADH and FMNH: nitrate reductase activities in a sucrose gradient. In seedlings treated with tungstate only the NADH:cytochrome 0 reductase activity is induced by nitrate. The activity has the same S value as the activity which develOpS in seedlings not treated with tungstate. Wray concluded that the nitrate reductase apoenzyme is synthesized in the presence of tungstate. If this is correct, it might be possible to activate the apoenzyme by molybdate in 2132 in the absence of protein synthesis. Attempts to activate the apoenzyme with molybdate lg vitro were unsuccessful. Based on Wray's 37 Figure 2. The Molybdate Reversal of the Inhibition of Nitrate Reductase Activity by Tungstate, 121122 Stationary phase XDRthr cells maintained on M-ID were subcultured into two, 6000 ml flasks containing 4000 ml M-ID plus 100 uM tungstate. After 24 hours of growth the nitrate reductase activity in the cells was determined in 500 ml samples taken from the flasks. At that time one of the flasks was given molybdate to a final concentration of 100 CM. At various times thereafter the nitrate reductase activity in 500 ml samples was determined. Molybdate added (c)), No molybdate ( O ). 38 _ _ 0 0 m 0 4. 2 a toms tough s\tm\om§qmt moz mm .5: is LO TIME(HR) 39 observation of the development of the NADH:cytochrome 0 reductase activity in the presence of tungstate, the following assumption was made. Non-functional nitrate reductase is formed in cells which are exposed to tungstate. Provided that the apoenzyme can be activated, if then after exposure to tungstate protein synthesis is, inhibited and molybdate is added, one may expect to see an increase in nitrate reductase activity. A two week old culture of XDRthr cells maintained on M-ID was subcultured into the same medium. After 11 hours of growth (zero time) the nitrate reductase activ- ity was determined, and the flasks were treated as described in the legend of Figure 3. Table 3 summarizes the effect of 4 ug/ml cycloheximide on total uptake and incorporation into protein of uniformly labeledl:1uC] L-arginine. The two processes are greatly inhibited by the inhibitor. The incorporation into protein is stopped within two hours after the application of the inhibitor, and the total uptake is inhibited within four hours. It is very unlikely that the incorporation into protein may be due to the inhibition of the total uptake. In the untreated cells the incorporation into protein is prOpor- tional to the total uptake. If the incorporation into protein depends only on the total uptake, one would expect to see an additional increase in the absolute amount of counts in the protein of the treated cells from the 2nd to 40 .mmmohosa psoonma psmmoaamh mamCSonth SH mmhswams *Aaosv omm.a *ABSNV cos.aa *Asmav ooo.:m whammy oom.smm 0 *hmoav omm.a *Asmmv oom.oa *Asmav ooo.mm *ASHNV oos.sma s *Aooav 0NN.H *AO0Hv 000.: *Aooav 000.H0 A *AO0Hv 000.H0 m oudaaxosoaomo + ovaadxosoaoho I odaaaxonoaomo + ovaaaxosoaomo I pawaos Smohm w\amo unwaos Swenm w\amo AHSV Samsonm opQH noHpmathoosH campus Hmpoe made .vosdasopou cams Samsonm opsa Anabapomoadoh ho Soapmaoahoosa 0cm zpabapomodpma mo manna: Houop cap hopwmmho£p mHSos 0 use 3 .N 94 .Ha\w1 : no SoHpSApsCCSoo Honda S on ovaaaHCSoHomo Sobam ohms moHSSHsC 0:» mo meow .msasawhmlq modau Uoflopma maahomdss 01 N mafia osasawhmlq 28 no.0 Sobaw ohms mHHoo on» .Aoaap osouv mason :m hopu< .opmpmwQSp 21 00a mafia QHIS opsd UCHSpHSCQSm Cams man so dosaopSamB mHHoo hapmmx omonm mammoapmpm naaoo enemas as osasamasrq mosmu uo adopoam opsH soaaoaoaaoosH one crowds Hence one so ooaaawosoaoao a soapaaassH .m canoe 41 the 4th hours. This is not so. Although the inhibition of total uptake complicates the interpretation of the results, they tend to indicate that protein synthesis is inhibited within two hours after the application of cyclo- heximide. Figure 3 summarizes the activation of non-functional nitrate reductase by molybdate in 111g in the absence of protein synthesis. In the untreated cells the nitrate reductase activity increased linearly for the first 4 hours. The net increase was 100 mumoles of nitrate reduced/hr/g fresh weight. The activity in the cells which were treated at zero time with tungstate and cycloheximide, and with molybdate at the 6 hour point did not increase above the initial (zero time) value. However, the cells which were treated only with tungstate at zero time and were exposed to it for 4 hours before protein synthesis was inhibited by cycloheximide, developed nitrate reductase activity after the addition of molybdate at the 6 hour point. Six hours after the addition of molybdate the net increase in the nitrate reductase activity was similar to that of the untreated cells during the first 4 hours. The results show that cells treated with tungstate do make the nitrate reductase apoenzyme. The apoenzyme can be activated lg 1119 by molybdate under conditions of inhibition of protein synthesis (Table 3). For the apo- enzyme to be synthesized, protein synthesis must not be inhibited. 42 Figure 3. Reactivation of a Non-Functional Nitrate Reductase by Molybdate in vivo, in the Absence of Protein SynthesIs Rthr cells were subcultured Stationary phase XD into three, 6000 ml flasks containing 4000 ml M-ID. After 11 hours (zero time), the nitrate reductase activity was determined on 500 ml samples, and the flasks were treated as follows: one flask was given tungstate (W) to a final concentration of 100 CM at zero time, cycloheximide (CX) to a final concentration of 4 ug/ml at the 4 hour point and molybdate (Mo) to a final concentration of 100 uM at the 6 hour point (I); one flask was given tungstate and cycloheximide to final concentrations of 100 uM and 4 ug/ml reSpectively, at zero time and molybdate to a final concentration of 100 CM at 6 hour point (A); one flask was kept untreated (0). At 2 hours intervals the nitrate reductase activ- ity was determined on 500 ml samples. 43 Ho... 18) R H F. w 14.! m w 10 _ _ _ O O O O O O .0 6 4 2 RIQNE Imumtk o\mt\em§qmm moz 632.115 44 It can be concluded that tungstate inhibits the formation of nitrate reductase activity when applied to the cells. This inhibition is reflected by a low level of incorporation of [15le N03 into protein. The inhibi— tion can be overcome in 1112 by molybdate and the inhibi- tion is due to the fact that the nitrate reductase is non-functional. The nitrate level in the cells treated with tungstate is always higher than in the untreated cells. Tungstate was employed in some of the experiments in which nitrate accumulation was studied. III. Growth of XD Cells on Urea as the Only Source of Nitrogen Stationary phase cells develOp the nitrate uptake system immediately upon exposure to nitrate (Figure 1). The nitrate reductase activity starts to develop only after a 10 hour lag period (Filner, 1966 and Figure 11). An attempt was made to study the develOpment of the two processes in eXponentially growing cells. A search was undertaken for a reduced nitrogen compound which would support the growth of XD cells, but which was neutral in the regulation of nitrate assimilation. Once the compoundwas found the cells could be grown on it as a sole nitrogen source. During exponential phase, the cells could be shifted to nitrate containing medium and the accumulation of nitrate and the develOpment of nitrate reductase activity could be followed. 45 It was reported that urea can support the growth of higher plants (Pharis et a1” 1964; Bollard, 1966), and will induce urease activity (Bollard et a1” 1968; Cook, 1968; Bollard and Cook, 1968). The ability of urea to support the growth of XD cells was examined. Figure 4 summarizes the growth kinetics of XD cells using 3.0 mM urea as the sole nitrogen source. It can be seen that urea can support the growth of XD cells. The fresh weight and the total protein increase linearly and in parallel during the first two weeks. However, compared to growth on nitrate, that on urea is Slow and yields less fresh weight at the end of the growth period. The increase in protein content in cells growing on nitrate is much more rapid than the increase in fresh weight. The doubling time is 30 hours and 48 hours for the protein content and the fresh weight, reSpectively (Filner, 1965). In cells growing on urea the increase in protein content is parallel to that of the fresh weight. The doubling time is three days. Thus it can be concluded that urea can support the growth of XD cells. IV. Nitrate Accumulation by XD Cells Growing EXponentially on Urea and Shifted to Nitrate The kinetics of nitrate accumulation and the development of nitrate reductase activity in cells shifted at mid-exponential phase from urea medium to nitrate medium were examined (Figure 5). The nitrate level and the 46 Figure 4. Growth of XD Cells on Urea as the Sole Nitrogen Source Stationary phase XD cells maintained on M-ID were subcultured into nitrate-less M-ID supplemented with 3.0 mM urea. After 5 subcultures into the same medium, 2-3 weeks each, the growth kinetics were determined. Fresh weight/l (0); total protein/l (0). Fresh weight/l of X0 cells growing on M-ID (———) (taken from P. Filner, Ph.D. thesis, 1965). Results were plotted on logarithmic scales. 47 38:: 2E: SE 0 nlu IO d 38:165.: 1%Mtk 24 IS TIME(DAYS) 48 .moflaamm Ha 00m So vocassopop cams Anny mumps“: mo SoapwHSESoom mo ones oSp use Anuv hpabapom ommpodooa mpmapas map .AHUV pamp2oo mpmnpas map mead» msoaamb p4 .Anv Sons as 0.m mafia QHIS 0am Adv mHiz Ga 000:09m5moh use umpmobhoa mHHmoHonmm Soap 0mm mmwd m pom wsHSOHm ohms maaoo 0:9 .mohs as 0.m Spas umpSCSCHQQSm QHIS whoaiopmnpas opsa deSpHSCQSm macs 0H1: so cosHSpSams mHHoo ax omega haosodpmpm ouoanaz op moan Sosa oouwasm waaoo as ea assesses ommposvom mpmhpaz mo psoaaoambom use modpmHSSSCC< opmhpaz ho moaposam .m ohswam 49 1H9/3M HSBHJ 5/UH/E0N 337017 77’ 9 IO r 1 ' I a 9 77’ .U-CIDQIHM HSSHJ /_0N $370W 0 (D 2000 8 1149/3»: H3383 fi/aH/aaan 03H §o~ $37011! fiw TIME(HR) 5O nitrate reductase activity were eXperimentally determined. The curves of the nitrate accumulation and the develOpment of the nitrate reductase activity were drawn through the experimental values. The rate of nitrate accumulation was calculated from the experimental values of the nitrate content. In this eXperiment tungstate was not used to inhibit the develOpment of active nitrate reductase. Thus the results reflect the uptake but are not identical with the uptake. Cells growing on urea contain little or no nitrate and have no nitrate reductase activity. Following the shift from urea to nitrate, nitrate accumulates and nitrate reductase activity deveIOpS, during at least 6 hours after the shift. Urea, if present in the nitrate medium, does not have any strong effect on the development of the two processes. If urea has any effect at all, it enhances them somewhat (Figure 5b). The nitrate uptake system and the nitrate reductaSe activity increase linearly following the shift, without any detectable lag. Thus, SXponential phase (nitrogen rich) cells are able to deve10p the two processes immediately after exposure to nitrate. EXponential phase cells not only lack the lag in the develOpment of the nitrate reductase activity (Figure 5) but they develop the two processes much faster than do stationary phase cells (Table 4). 51 .m esswam Bosh uepeHSCHeotrs .HH mHSmHm Bosh uepeHSOHeU** .H esswam soak uepeHsCfieoa sweeps reeo.a where swapsosoawm atom *0H.0 emess usesoapepm as\asma s seeps .as\nsmams nose w\ss\ueosues :oz moHosms w\ss\ss sexes :oz meaosn emeposuem opespaz exepso epespaz mHHeo ax emesm Heausesossm use emesm asesoapepm sa mpabapos emeposuem epespaz esp use ampmmm esepsa muespaz esp so mpsessoaebem so mepem ese .: eases 52 Upon exposure of nitrogen rich cells to nitrate, nitrate reductase begins to deve10p immediately. The rate of development of the nitrate reductase activity is con- stant for at least 6 hours. The rate of nitrate uptake also increases at a constant rate, so the level of nitrate in the cells during the same period of time increases greatly. If the rate of development of nitrate reductase activity depended upon the absolute amount of nitrate in the cells, one would eXpect to see an increasing rate of development of the enzyme activity. This is not the case. The cells are able to deVSIOp the nitrate reductase activ- ity at the maximum rate before any substantial accumulation of nitrate occurs. It can be concluded that the deve10p- ment of nitrate reductase activity does not depend on the absolute amount of nitrate in the cells. Additional support for this hypothesis comes from the results shown in Figure 6. In this eXperiment XD cells were grown on nitrate for 48 hours. At that time (zero time) some of the cells were given casein hydrolysate to a final concentration of 1.5 g/l, some were aseptically harvested and resuSpended in nitrate-less M-ID and some were left untreated. The nitrate content and the nitrate reductase activity were determined as a function of time. Soon after Shifting the cells to nitrate—less M-ID the nitrate reductase activity and the nitrate content in the cells declined. In Spite of the initial high level of 53 Figure 6. Effect of Addition of Casein Hydrolysate or Removal of Nitrate on the Decay of Nitrate Reductase Activity and Nitrate Content Stationary phase XD cells were subcultured into three, 4000 ml flasks containing 2000 ml M-ID. After 48 hours (zero time) one of the three cultures was harvested and resuSpended in nitrate-less M-ID ((3), one was given casein hydrolysate to a final concentration of 1.5 8/1 (I) and the third was left untreated (O ). At zero time and at various times thereafter the nitrate reductase activity (---) and the nitrate content (———) were determined on 200 ml samples. The nitrate content and the nitrate reductase activity are plotted on a logarthmic scale. 54 erodes teeth s\ mg were: 1 24 IOO- store: teeth s\tt\qmoSQMt moz 636: is 'TIMEmR) 55 nitrate in the cells (25 umoleS/g fresh weight), they can- not maintain a constant level of nitrate reductase activity. If all of the nitrate in the cells is capable of inducing the nitrate reductase activity, one would eXpeCt to see maintainance of an induced level of activity until the nitrate content drops below the inducing threshold. Thereafter the nitrate reductase activity would decline. The previous experiment indicated that the threshold is low. This eXperiment indicates that a high level of endogenous nitrate cannot induce nitrate reductase activity. One is inclined to conclude that most of the nitrate in the cells is neither necessary for, nor capable of inducing the nitrate reductase activity. If casein hydrolysate is applied to the nitrate- containing medium, a similar phenomenon is observed. In Spite of an initial high level of nitrate in the medium and in the cells, nitrate reductase activity declines. However, nitrate persists for a longer time in cells treated with casein hydrolysate. This aSpect of the effect of casein hydrolysate will be discussed in detail later. V. Effect of Tungstate and the Concentration of Nitrate in the Medium on the Accumulation of Nitrate in EXponential Phase Cells Cells growing on urea can be used to study nitrate accumulation. However, Since nitrate reductase activity develop immediately after the Shift from urea to nitrate and reaches a relatively high level shortly thereafter, 56 (Figure 5), it is necessary to inhibit this activity in order to obtain reliable information about the uptake process. The effect of tungstate and of nitrate concentra- tion in the medium on the accumulation of nitrate was examined (Figure 7). In control cells shifted from urea to 3.0 mM nitrate (Figure 7b) the kinetics of nitrate accumulation and the development of nitrate reductase activity (inset in Figure 7a) are very similar to those in Figure 5. In cells shifted to 3.0 mM nitrate plus 100 uM tungstate, the develOpment of nitrate reductase activity is inhibited (inset in Figure 7a). The kinetics of nitrate accumulation however, are very similar to that of the control cells although the absolute amount of nitrate accumulated is higher. This establishes the validity of the interpretation of the nitrate accumulation data in Figure 5. Cells shifted from urea to tungstate- containing media plus different concentrations of nitrate Show similar kinetics. There is initial period, of 2-3 hours, during which the rate of uptake increases, followed by a second period in which the rate of uptake is constant. The length of the first period is not affected much by the concentration of nitrate in the medium. However, the rate of uptake in the second period does depend on the concen- tration of nitrate. A typical saturation curve is Shown in Figure 8a. The double reciprocal plot gives an apparent KIn value of about 4 x 10'” M (Figure 8b). 57 dampness» as 03 A00 Hosp? use A: spas 383s as o.m soeam assoc 5. mpabapoe emeposues onespas “pemsH .A1DV 00p sss mes epepmmsSp pdospds epespas ss o.m as osspsso Hoassoo s A o0 333: as 0.0m £00 333s as 0.6 .A n: 3833 as o.m .5: Beards as To .3: 383s as To . 1: Basis as 8.0 . ADV 382s ass 96 £32 oossosesassh. opspnwsss as ooa mass as whoauepwsfls sa ueusesmSees esez mHHeo ese .m esswam sa we maaepeu Hessesasessm muespaz op ems: Boss uepwasm mHHeo sa soapeas85004 onespaz esp so ssauez esp sd muespaz mo soapespseosoo esp use epepmwsse mo mpoewsm .m essmam 58 ¢ N 189/3»: 85383 6/88/ U a39na38 EON s3 70w 11w .0 a: I DJ 2 _. Am}- I — w _m E I )— q-g ; M l l o 8 O IO 0 E 1H9l3M H3383 5/ -0/v S370w 77’ 59 .AO . O V mpsesasemcne 23 80.3 sexes oses epeu esa .Aev sH mpHSmes esp mo poas Heoossaoes eassou s As .ssHues esp sH A280 epespas so scapespseosoo esp pmsaewe mHSos sum esp use sud esp semapes as sexes Apswaez smesm w\meflosnv epespas mo ps5ose esp so poas s Ae sasocs esp sH sofipespseosoo mpH so exepsb onespaz so epem esp so eoseusesea .m mssmfim I l )- o‘o -3 _ -0 N _. Lg o o 1 O l .0 Q' m 0 (588 mm N3)! VJ. few 93 70w r/M S(mM) 60 40 20 '/8 mm" 61 VI. Inhibition of Nitrate Accumulation by Dinitrophenol and Cyanide XD cells can accumulate nitrate to a concentration much higher than that in the external medium (Figure 1, 5, 7, and Table 1). A common approach for demonstrating the energy dependence of a tranSport system is the appli- cation of energy uncouplers and poisons (Cohen and Monod, 1957; Ordin and Jacobson, 1955). The effect of 2,4—dinitrophenol and cyanide on the accumulation of nitrate in XD cells was examined (Table 5). Table 5. Effect of Dinitrophenol and Cyanide on Nitrate Accumulation in XD Cells Stationary phase XD cells were subcultured into the indicated media. After 17 hours of growth the nitrate content of the cells was determined. Medium Nitrate Content tumoles NOE/g fresh weight M-ID 15.9 (100)* M-ID + 1 mM dinitrophenol 0.4 ( 3)* M-ID + 1 mM KCN 0.6 ( 4)* *Figures in parenthesis represent percent of control. The level of nitrate in cells treated for 17 hours with dinitrOphenol or cyanide is very low compared to untreated cells. However, one can argue that the effect of these compounds over a 17 hour period may be non-Specific. 62 Kinetics are Shown in Figure 9. The inhibition by dinitro- phenol is observed much earlier and is more pronounced than that by cyanide. Dinitrophenol not only stops further accumulation, but seems to bring about a leakage of the nitrate already in the cells. VII. The Effect of Amino Acids on the Accumulation of Nitrate The effect of L—threonine on the accumulation of nitrate was examined (Figure 10). It can be seen that 100 uM L-threonine inhibits nitrate accumulation. The level of nitrate in the cells treated with L-threonine is about 0.5 umoles/g fresh weight after 24 hours of growth. This level is a 1/6 of the external concentration and 1/70 of that in the untreated cells. Thus, L-threonine not only inhibits the formation of nitrate reductase activity (Filner, 1966), but it also Shuts off the accumulation of nitrate, the inducer and the substrate for the enzyme. The effect of casein hydrolysate on the accumulation of nitrate was also examined (Figure 11). The results are similar to those of Figure 10. At 0.1 g/l, casein hydro- lysate inhibits nitrate accumulation. After 27 hours of growth the treated cells contain 10% of the nitrate in the untreated ones. Casein hydrolysate at 0.1 g/l also inhibits the formation of nitrate reductase activity by about 60%. Thus under growing and non-growing conditions amino acids inhibit nitrate accumulation. 63 Figure 9. Kinetics of Inhibition of the Accumulation of Nitrate by DinitrOphenol or Cyanide Stationary phase cells were aseptically harvested and resuSpended in M-ID. After 6 hours the cells were treated as follows: control (C)), 1 mM dinitrophenol (final concentration) ([3), 1 mM KCN (final concentra- tion) ([3). The nitrate content of the cells was determined. 64 DNP KCN l5 _ O 5 atoms 1:me 3 mg 6.30: 1 l2 TIME (HR) I“ C)" ‘-—-»v--— 65 Figure 10. Effect of L-Threonine on the Accumulation of Nitrate Stationary phase XD cells were subcultured into M—ID (o) and M—ID plus 100 ,uM L-threonine (O ). At various times as indicated the nitrate content of the cells was determined. 66 32- _ 4. 6 8 O 2 .I , kisses teeth s\.moz ESSA TIME (HR) 67 Figure 11. Effect of Casein Hydrolysate on the Accumu- lation of Nitrate and the DevelOpment of Nitrate Reductase Activity Stationary phase cells maintained on M-ID were subcultured into fresh M-ID (O , O) and M-ID plus 0.1 g/l - casein hydrolysate (D,,l). The nitrate con- tent (open symbols) and the nitrate reductase activity (filled symbols) were determined. 68 m. sheik tomes 3:02 6.30:1 m 8 4 m _ _ _ _ _ 0 O 8 klwxmss _ I _ _ 0 0 O O O 0 0 6 4 2 South 3t: \ 363mm Inez 936: is TIME (HR) 69 VIII. The Effect of Different Metabolites of the Nitrate Assimilation Pathway on the Accumulation of Nitrate The effect of amino acids, ammonia and nitrite on the accumulation of nitrate in exponential phase cells were examined (Figures 12 and 13). In these eXperiments the reduction of nitrate was inhibited by 100 uM tungstate. Each of the nitrogenous metabolites inhibited the accumu- lation of nitrate. Casein hydrolysate at 4.0 g/l (a much higher concentration of casein hydrolysate was used in this experiment than in those reported above because of the high inoculation of cells used), markedly lowers the steady state level of nitrate. It also lowers the rate of uptake to some extent. Ammonia at 0.5 mM lowers only the steady state level. Nitrite at 0.5 mM appears to inhibit the rate of uptake. In cells treated with nitrite the steady state level of nitrate was not reached after 10 hours. The effect of nitrite on the steady state level was not therefore determined. Another piece of information can be obtained from the experiments presented above. The metabolites tested can affect the nitrate accumulation even in the absence of functional nitrate reductase. The significance of this observation is discussed later. Summary The nitrate uptake system is inoperative and nitrate reductase activity is undetectable in either nitrogen- 70 Figure 12. Effect of Casein Hydrolysate or Ammonia on the Accumulation of Nitrate Stationary phase cells were subcultured into nitrate-less M-ID supplemented with 3.0 mM urea. After 5 days of growth the cells were aseptically harvested and resuSpended in nitrate-less M-ID supplemented with 100 uM tungstate. The following were then added to the cultures: 0.9 mM nitrate (I): 3.0 mM nitrate (o); 0.9 mM nitrate plus 0.5 mM ammonia (A); 3.0 mM nitrate plus 0.5 mM ammonia (A5); 0.9 mM nitrate plus 4.0 g/l casein hydrolysate (I): 3.0 mM nitrate plus 4.0 g/l casein hydrolysate (E1). At various times the nitrate content in the cells was determined. 71 4O _ P 0 0 3 2 stoic: teeth s\ moz 8.84633. TIME (HR) 72 Figure 13. Effect of Nitrite on the Accumulation of Nitrate EXperimental details as in Figure 12. The cells were resuSpended in nitrate-less M-ID supplemented with 100 CM tungstate. The following were then added to the cultures: 0.3 mM nitrate ([3); 0.3 mM nitrate plus 0.5 mM nitrite (A); 0.9 mM nitrate (o); 0.9 mM nitrate plus 0.5 mM nitrite (o): 3.0 mM nitrate (D): 3.0 mM nitrate plus 0.5 mM nitrite (I). At various times the nitrate content in the cells was determined The level of nitrate in the cells was determined by using soybean nodule bacteroids which convert nitrate to nitrite quantitatively (see Materials and Methods). The actual amount of nitrate was determined by subtract- ing the amount of nitrite in the extract before the bacteroid assay from the amount of nitrite in the extract after the assay. The nitrate present in the extract before the assay did not exceed 300 mumoles/g fresh weight. In most instances, no nitrite was detectable. 73 _ _ 0 0 O 4. 2 armies teeth s\.moz 6.30:1 TIME (HR) 74 deficient (stationary phase) or nitrogen-rich (eXponential phase) cells. Upon eXposure to nitrate the two systems develop. The nitrate uptake system starts to develop immediately without any lag period. In exponential phase cells it develOpS faster and reaches a higher rate of uptake than in stationary phase cells. This is also true for the nitrate reductase activity. In addition, the lag period which is observed in stationary phase cells before the nitrate reductase activity starts to develop is elimin- ated in exponential phase cells. Tungstate can be used to inhibit the formation of active nitrate reductase and thereby allow studies of nitrate uptake in the absence of nitrate reduction. The inhibitory effect of tungstate which can be overcome by molybdate, seems to be very Specific for nitrate reductase function. At least part of this inhibition is due to the formation of non-functional nitrate reductase. By the use of tungstate it was clearly shown that the uptake of nitrate does not depend on the presence of functional nitrate reductase, nor on nitrate reduction. The develOpment of nitrate reductase activity does not depend on the absolute amount of nitrate in the cells. This is based on the observation that there is no lag before the development of nitrate reductase activity begins in exponential phase cells exposed to nitrate while there is a lag before nitrate accumulates. 75 The accumulation of nitrate is inhibited by individ- ual amino acids which also inhibit growth, and by a mixture of amino acids which does not inhibit growth. Thus the effect of amino acids is not limited to inhibition of the development of nitrate and nitrite reductases, but can be observed on the process of nitrate accumulation too. This inhibitory effect by amino acids does not depend on the presence of functional nitrate reductase. Nitrite and ammonia also inhibit nitrate accumula- tion. XD cells accumulate nitrate to a concentration much higher than that in the medium, and the process is energy dependent. Part B: The Relationship Between the Regulation of the Nitrate Uptake and the Regulation of the Rest of the Nitrate Assimilation.Pathway In the preceding section the nitrate uptake system and its regulation were described. An uptake system for nitrate develops in stationary phase and exponential phase cells in reSponse to nitrate. The uptake system is subject to end product regulation by amino acids very similar to that described for nitrate reductase (Filner, i966) and nitrite reductase (Chroboczek-Kelker, 1969) activities. In other words, the three steps of the nitrate assimilation pathway deve10p in tobacco cells in reSponse to nitrate and 76 in each case this develOpment is inhibited by amino acids. Thus the question about the relationship between the regulation of the nitrate uptake system and the rest of the assimilation pathway arises. I. The Effect of Casein Hydrolysate on the Accumulation of Nitrate and the Development of Nitrate Reductase Activity In.Part A (Figure 12) it was shown that casein hydrolysate lowered the level of nitrate in cells treated with tungstate. Similar results are shown in Table 6. Casein hydrolysate at a concentration of 0.3 g/l lowers the level of nitrate and the amount of detectable nitrate reductase activity in the cells. Tungstate similarly inhibits the deveIOpment of nitrate reductase activity. However, tungstate increases the level of nitrate two fold over that in the cells not treated with tungstate. If casein hydrolysate is applied together with tungstate, there is a further reduction in the amount of detectable nitrate reductase activity. There is also a very pro- nounced reduction in the level of nitrate, compared to that in cells treated with tungstate alone. The results presented in Figure 7 and Table 6 indi- cate that neither the development of the nitrate uptake system, nor the inhibition of the development by amino acids depends on the presence of functional nitrate reduc- tase. 77 os m.m opensaososs saoneo me m.o + caepwwssp :1 or + sans 0H N.: epemhaosums sHemeo H\w m.0 + openness» sa om + ssus Os o.mm muesnmsss a: or + asus mm o.:m epepwmssp as om + man: as m.s epshasososs saonwo H\m m.o + asus mmm o.sa sHus pswaez smess as Hes seess w\ss\ueosues moz meaosus w\ioz meaosn onwsosoom maessaz psopsoo muessaz snaoos .uesdssepeu mam: mpabdpoe emeposues epespfis esp use psepsoo epespas esp suSosm so endos SN sepss .eaues uepeoausa esp cpsa uoHSPHsCDSm oses mHHeo Chess ahesoapepm epepmwssa suds uepeese mHHeo sa hpabdpos emeposuem epespaz so usessoambem esp use epespaz so sodpeasasoos esp so epemmaosuhm sdeeeo so poessm .0 eHQeB 78 II. A Variant Cell Line: Growth Characteristics In the following section some characteristics of a variant cell line which seems to have an altered nitrate uptake system, will be described. Based on these charac- teristics, it will be concluded that the effect of amino acids on nitrate assimilation is not brought about by keeping the nitrate out of the cells. The cell line is one of three isolates, originally selected for their ability to grow on nitrate in the presence of the inhibitory amino acid L-threonine. The selection was conduCted in order to obtain a cell line with a lesion in the regulatory mechanism by which the amino acids regulate the assimilation pathway. The selection procedure is described in the section of Materials and Methods. Only one of the three cell lines, the XDRthr , was studied extensively and the results of this study are given below. The question whether the resistance to the inhibi- tion of growth by L-threonine was a stable inherited char- acteristic of the cell line arose. It was answered in the following way. The resistant cell line XDRthr, maintained on M-ID plus 100 uM L—threonine was subcultured into M-ID and maintained on it for 10 consecutive subcultures. After each serial subculture on M-ID the cells were subcultured into M-ID plus 100 CM L-threonine and the growth was deter- mined after 3 weeks. XD cells not previously exposed to 79 L-threonine were used as control. The results are summar- ized in Figure 14. The ability of the XDRthr cells to grow in the presence of 100 uM L-threonine was not lost during 10 serial subcultures (about 40 generations) in the absence of L-threonine. In the course of the experiment a tran- sient effect was observed. The average amount of growth for the 50 replicates dropped after the second consecutive subculture in the absence of L-threonine, and then rose to a constant high level following subsequent subcultures. thr cells This phenomenon could not be reproduced with XDR which were kept on M-ID plus 100 CM L-threonine for many more subcultures before beginning the series of subcultures in the absence of L-threonine. The results indicate that the resistance to the inhibition of growth by L-threonine is a stable inherited characteristic of the cell line. The inhibitory amino acid, L-threonine, represents a group of amino acids which bring about the same effect when applied to XD cells (Filner, 1966). These amino acids may act via one common mechanism, or different ones. If there is one common mechanism, it is eXpected that the L-threonine-resistant cells might grow in the presence of the other inhibitory amino acids. The ability of the XDRthr cells to grow in the presence of other inhibitory amino acids was therefore examined (Table 7”. XDRthr cells are resistant to some 80 Figure 14. Inheritance in XDRthr Cells of the Resistance to Growth Inhibition by L-Threonine Stationary phase XDRthr cells maintained on M-ID plus 100 uM L-threonine were serially subcultured into M—ID. After each consecutive subculture on M-ID the cells were subcultured into fifty, 500 ml flasks contain- ing 200 ml M—ID plus 100 uM L-threonine (()). A control group of 50 subcultures into M-ID plus 100 uM L-threonine of XD cells maintained on M-ID was run each time (o ). After 3 weeks of growth on the medium supplemented with Lathreonine the average fresh weight in a liter culture was determined. 81 0 Oh— 2 ll 3st Seas sweat IO SERIAL SUBCULTURES 82 Table 7. Effects of Amino Acids on Growth of XD and XDRthr Cells Stationary phase XD cells maintained on M-ID, or XDBthr cells maintained on M-ID + 100 uM L—threonine were subcultured into the indicated media. Three replicate cultures were harvested after 16 days. Medium g Fresh Weight/l XD XDRthr Nitrate-less M-ID <0.25 0.30 M-ID 38.6 27.6 M—ID + 100 uM L-arginine 45.4 36.6 M-ID + 100 uM glycine <0.25 4.50 M-ID + 100 CM L-histidine 2.80 20.8 M-ID + 100 uM L-leucine 1.50 16.2 M-ID + 100 uM L-lysine 41.7 35.1 M-ID + 100 uM L-methionine 0.60 3.80 M-ID + 100 0M L-threonine <0.25 23.9 M-ID + 100 uM L-valine 1.25 20.1 83 other inhibitory amino acids but are not equally resistant to all of them. It may indicate that there is more than one mechanism by which the inhibitory amino acids act. Selection of other variant cell lines resistant to the inhibitory effect of other amino acids can shed more light on this aSpect. The amino acid L—threonine does not inhibit the growth of XDRthr cells. However, it might have an effect on the growth kinetics of the cells, as compared with that of XD cells on M-ID. The kinetics of growth of XDRthr cells on M-ID, M-ID plus 100 uM L-threonine and M-ID plus thr cells which were maintained 100 uM L-valine and of XDR continuously on M-ID and subcultured into M-ID plus 100 MM L-threonine, was determined (Figure 15). The XDRthr cells exhibit virtually the same growth kinetics on M-ID as do the KB cells. The growth of XDRthr cells on M-ID plus 100 CM L-threonine or M-ID plus 100 uM L-valine on the other hand is not quite as rapid, nor to as high a final yield as on M-ID. Nevertheless, the XD cells cannot grow on either of the media with an inhibitory amino acid (Filner, 1966). Stationary phase XDRthr cells maintained on M-ID plus 100 uM L-threonine grow with little or no lag, following subculture into the same medium. When Rthr cells maintained on M-ID are sub- stationary phase XD cultured into M-ID plus 100 CM L-threonine, there is a lag period of eight days before the cell mass starts to increase (Figure 15a). 84 .esasocsspus :1 2: + Bus op Biz sows assoc Essen 33 “858% case of op essaeblq s: 00H + QHiz Boss mHHeo s max Anvv ”ssHuos esem es» op esasoessplq as s1 2: + as sows mflmo sea A: 56.2 on as sows nfloo mes A3 as» .83 “seasons dim .33 38:33 3.2 cu Bus sows nice as All .385 :HSC epeCAHses eessp so 03p so ewesebe esp ma psaos seem .meHeom ossspssewoa so ueppoas use uesassepeu one: saeposs Hepop ADV use pswaez seems Aev mesap mSOHHeb p4 .ssHues as com ops“ ueHSpHSCDSm who: mHHeo emess maesoapepm mHHmO HSmeN so moapmahmpoeheso £93090 .ma ehsmam 85 I UM) N131 0w 0. N. Am>lq 21 ooH + QHiz on use esfisoesspuq as ooa + asuz Asa .eHus see so wsssosw wHHmo assess omega ssesoasepm wsospsosoo sssoso psmaosssm sous: mHHco assess ss assessos oweposoom opsspaz use psopsoo osespdz .oa ossmse O to l 94 1H9/3M H5381 6/ 501v ssnorv r/ 8 9 o l l 1H913M HS‘HHJ 5/HH /030003a 501v 53 70w n’w l6 l2 TIME(DAYS) 5 Q/ omma oooH m.mm m.:a esasoessplq :1 ooH + 0H1: aspmmx osmm ommm m.os m.sa mHus gnomes an m: oa.a mmm.o osasoesspuq :1 ooa + Qle ax ossm ONOH m.o: H.mm QHIZ ex .33 anew as? .53 psmaes seeps m\s\ueosuos ssHuez mHHeo mpwhfldfl mOHOEjE emeposuem epespdz psmaez seems m\meHosj usepsoo epehpaz emeposues epespas use epespaz use psepsoo epespas .mssos w: QHuz so sees ues mHHeo sspmmx esa QHus so uesaepssea seep ues sodss mHHeo HSmeN use so useasoaebem use soapeHsESoos .esdp ones pe eapawaawos one maebea .uesaasepeu who: mpdbapoe emeposues epehpas use em sense .messpHSopsm Heasom mess» hos .eauoa uepeoausd esp ops“ ueHSpHsoDSm who: mHHeo Hspmmx use ax emess mHesOapepm as as spatssos emeposoom oneness oposaaz so osasooasaiq so soouum .Ha oases 96 and XDRthr cells. The inhibition however is much stronger in XD than in XDBthr cells as compared with the two cell lines growing in the absence of the amino acid. Moreover, in XDRthr cells the effect of L-threonine on the deve10p- ment of nitrate reductase activity is much stronger than on the accumulation of nitrate. More information was obtained to support this observation (Figure 17). The kinetics of nitrate accumulation and the reSponse of the accumulation to casein hydrolysate are similar to that which was described in.Part A. However there is a marked difference between the two cell lines in the magnitude of the reSponse. In XD cells, casein hydrolysate inhibits the accumulation of nitrate very strongly. As a result the steady state level of nitrate is low (10% of that in the untreated cells). Nitrate accumulation in XDRthr cells is less affected by casein hydrolysate. This is reflected in a much higher steady state level of nitrate (40% of untreated cells). Casein hydrolysate also brings about inhibition of the development of nitrate reductase activity. Although the absolute amount of nitrate reductase activity in XDRthr cells is higher than in the XD cells, the percent decrease brought aabout by casein hydrolysate is the same (Figure 18). 97 Figure 17. Effect of Casein Hydrolysate on Nitrate Accumu- lation in XD and XDRthr Cells Stationary phase )CD (El , I ) and )CDBthr (O . 0) cells were subcultured into M-ID (filled symbols) and M-ID plus 0.1 g/l casein hydrolysate (open symbols). At various times the nitrate content was determined. 98 _ O O 2 .l klmrmss 19$: .u\ inc? 9933‘ 3‘ TIME (HR) 99 Figure 18. Effect of Casein Hydrolysate on the Development of Nitrate Reductase Activity in XD and XDR hr Cells EXperimental details as in Figure 17. 100 / I O; m; [8 ,- 10 O _ O 0 8 200— 400 - s toms tomes self owes owe moz we so: is TIME (HR) 101 A similar effect of casein hydrolysate is observed when a wide range of its concentration is used (Figure 19). The absolute level of nitrate reductase activity is much higher in XDBthr cells than in XD cells, but the percent decrease caused by casein hydrolysate at any given concen- tration is essentially the same. This is however not so in the case of the nitrate content. The absolute levels of nitrate (except for the untreated cells) are not only 3-4 times higher in XDRthr cells than in XD cells, but the percent decrease is 5-7 times smaller. The unequal effect of casein hydrolysate on the nitrate content in the two cell lines can also be observed when the develOpment of active nitrate reductase is inhibited by tungstate. XDRthr cells accumulate 3-5 times as much nitrate as do XD cells when both lines are treated with tungstate and casein hydrolysate (Table 12). From the results presented above it is concluded that the difference between the two cell lines may reside in the nitrate uptake system or in the control of this system. Summary In Part B information was presented concerning the relationship between the regulation of the nitrate uptake system and the regulation of the rest of the nitrate assimilation pathway. Two lines of evidence were presented. 102 Figure 19. Effect of Various Concentrations of Casein Hydrolysate on Nitrate Accumulation and Nitrate Reductase Activity in XD and XDRthr Cells Stationary phase XD (0 , I) and XDRthr (O , D) cells maintained on M-ID were subcultured into M-ID supplemented with various concentrationscfl‘casein hydrolysate. After 24 hours the nitrate content (I , D) and the nitrate reduc- tase activity (0 ,7 0) were determined. The 100% nitrate content is 20.500 and 28,000 mumoles nitrate/g fresh weight for XDRthr and XD cells, reSpectively. The 100% nitrate reductase activity is 3,300 and 715 mumoles nitrate reduced/hr/g fresh weight for XDRthr and XD cells, reSpectively. 103 l.0 0.8 0.6 0.4 0.2 CASEIN HYDROLYSATE (g/ L) _ 0 O 3bu$k$< LZMUQMQ 9.53522 bee NMQRUDQNQ MKSW‘R‘Z 104 fr... s.. 4).-H.!ii-..I..h .:. adv“... a. rat‘s m.mm N.ma ms.m mo.a opemmaosums saemeo axe m.o + osus m.ms N.mm 3.0H wo.m epemhaosums sdemeo H\m H.o + asus m.sm s.sm m.©s s.sm oHuz epepmwsSB + epepmwsse I epepmwsse + epepmwsse I sspmmx ax ssauez pswaez seeps m\meH081 psepsoo epespaz .s :N sense uesassepeu me: usepsoo euespaz .Rom sesp esos as emeposues opespas HesoaposSs so scapeasos evanassa evenness» so Hebea ease .epepmwsSp :1 om pSOspas use spas .eauos uepeoausd 0:» opsa uoHSpHSonsm one: QHIZ so uesaepsaea mHmSoaboss mHHeo Hspmmx use QN emess maesoapepm mHHoo sssmmx use ex ea camps: ossspsz so oponsaosuam ssowso so poossm .ms cases 105 The first one dealt mainly with the effect of casein hydrolysate on nitrate accumulation in cells treated with tungstate. The results showed that nitrate accumulation could take place and still be inhibited by casein hydrolysate in the absence of functional nitrate reductase. The second one dealt with the characteristics of the variant cell line XDRthr, resistant to the growth inhibition by L-threonine. The develOpment of nitrate reductase activity in the variant cell line, XDRthr, was inhibited by casein hydrolysate in a similar manner as that of the XD cells. The nitrate uptake system on the other hand was less sensitive to inhibition by casein hydrolysate than that of XD cells. That is, the XDRthr cells accumulate nitrate in the presence of casein hydrolysate, but the develOpment of the nitrate reductase activity is inhibited nevertheless. 222.22: Do Individuaermino Acids Inhibit Growth by Specifically Inhibiting Nitrate Assimilation? Casein hydrolysate which is a mixture of amino acids inhibits the development of the nitrate assimilation path- way, but does not inhibit growth. However, individual amino acids which inhibit develOpment of the assimilation pathway, inhibit growth as well. Inhibition of nitrate assimilation would be a sufficient condition to inhibit growth. On the other hand it is possible that individual amino acids inhibit growth first, with the inhibition of 106 nitrate assimilation being an indirect and non-Specific consequence. The effect of individual amino acids on nitrate assimilation would be a Specific regulatory phenom- enon only if it is Specific to the assimilation pathway and not an indirect consequence of a more general effect, i.e., inhibition of growth. Two approaches were applied to the study of this phenomenon. The first one was based on the assumption that if an inhibitory amino acid, L-threonine, has a general toxic effect it should be observed in the absence of any nitrogen source, and might reasonably be eXpected to influence energy requiring processes, such as total uptake of radioactive amino acid and its incorporation into protein. If the amino acid affected nitrate assimi- lation Specifically these processes should not be impaired. Stationary phase XD cells were subcultured into various media as indicated in the legend to Figure 20. At various times the total uptake of uniformly labeled [:1ch L-threonine and its incorporation into protein were deter- mined. In 32 hours the cells can concentrate the amino acids about 3h0 times over the concentration in the medium. The rate of total uptake of the amino acid was the same in all media tested. In other words, L-threonine which inhibits growth and deveIOpment of the nitrate assimilation pathway did not affect the uptake process. Incidentally, it is 107 .uesHShepeu ohms psmdez Smehs m_sesfinv saeposm ops“ sodp lesossoosa esp use Aev esepss Hepoa .osasoessplq meHoan ooa\esasoohsplq mpsau ueHeQeH massosflss on N mean esasoosspiq 21 00H uosaessoo edues esp HH4 An: osssswsuuq 21 cos mafia Biz use A: 3-2 .A 0V QHIZ needlepespas cps“ ueHSpHsoDSm eyes mHHeo ax emesa usesoapepm msoapausoo spaces mSoHHe> sous: esHsoessaiq mo auwso Aaeasopez eHerasHerm uaos oapeoeosoasoasa ROHV saopoam o sH soapesossoosH use exepsb Hence .om esswam 108 l l )3 _ . _ <1- N F— \ ' - fi' I <\ ‘9 1 I o 8 8 o o o an e .L H9/3M H5384! 6 /NlW/S‘.L Am 09) I I 8 l O 0 q- 9 _ 01 x (1 H9/3M HSSHJ 6/N/rv/ 51 mo 0) l O O N 24 l2 TIME (HR) 109 . clear from this experiment that Learginine does not antagon- ize the effect of L-threonine by preventing its uptake. A similar picture can be observed in the incorporation of the radioactivity into protein. During the first 12 hours after subculture, the rate is very similar under all condi- tions. Except for the medium with nitrate and arginine, the incorporation continued linearly up to 32 hours. In the medium containing L-arginine, the rate of incorporation of the radioactive amino acid increased after the 12 hour point. It coincided with the first detection of nitrate reductase activity (Filner, 1966) and may reflect an increase in protein synthesis. The experiment was also done with radioactive arginine, with and without nitrate, with and without L-threonine. The same results were obtained. It is clear that if there was any general toxic effect it certainly was not manifested within the first 32 hours, in the total uptake of amino acids and their incorporation into protein. Thus the effect of L-threonine, and presumably other inhibitory amino acids, on the development of the nitrate assimilation pathway cannot be attributed to a general toxic effect. The second approach was based on the assumption that if the effect of individual amino acids on nitrate assimi- lation is a Specific regulatory phenomenon, then individual amino acids should inhibit growth only when nitrate is the 110 nitrogen source. It was shown in Part A that urea as the only nitro- gen source can support the growth of XD cells. y-Amino- butyric acid was reported to support the growth of §pirodela oligorrhiza (Bollard, 1966). As can be seen from.Table 13, y-aminobutyric acid can support the growth of XD cells as well. Table 13. Growth of XD Cells on y-Aminobutyric Acid in the Presence of L-Threonine and L-Arginine Stationary phase XD cells maintained on nitrate-less M-ID plus 3 mm y-aminobutyric acid were subcultured into the same medium supplemented with the indicated amino acids. Duplicate 0.5 l cultures were harvested after two weeks. Amino Acid.Added g Fresh Weight/l Percent of Control None 23.4 100 100 uM L-threonine 22.9 97 100 UN L-threonine + 100 uM L-arginine 24.1 103 100 uM L-arginine 23.9 102 The effect of L-threonine, one of the most effective inhibitory amino acids (Filner, 1966) and that of L-arginine, one of the antagonistic amino acids (Filner, 1966) on XD cells growing on either urea, y-aminobutyric acid or nitrate was examined (Figure 21, Table 13). Inhibition of growth of XD cells by L-threonine only occurred when nitrate was 111 .meaeom oHssuHHewoa so ueppoas use uesdasepeu who: sfieposs Hepop use pswaez seeps ese .A By esdsawseuq 21 ooa .Ho Alv esasawselq 21 cos + oessoesssis es ooa . A 0v esssoosspis 2:. cos .Aev csos ”messes Isoo .maebapoesmes eaues eaem esp ops“ uesspHSoDSm esez .Apswasv eehs as o.m + QHuz meeanepespas so “useav QHIE so uesaepsaes mHHeo ax emess anesoapepw eonsom sewosuaz me ems: so epespaz asses nines as so spsoso one so esssseseus use esssoessaus so soossm .am 33.3 112 l00 1 O I 7/ 5) lH9/3M HSHUJ 0.l I2 I2 TIME (DAYS) 113 the nitrogen source. L-threonine did not inhibit the growth of XD cells when urea or y-aminobutyric acid were used as sources of nitrogen. This result seems to indi- cate that the effect of individual amino acids is Specific for the nitrate assimilation pathway. However, the XD cultures used in the eXperiments reported in Figure 21 and Table 13 had grown for several subcultures on urea or y-aminobutyric acid reSpectively. It is possible therefore, that as a result of the growth on nitrogen sources other than nitrate the cells have changed and lost their susceptibility to the inhibition of growth by L-threonine. This possibility was examined by comparing the susceptibility to L-threonine of XD cells maintained continuously on nitrate or urea and transferred to nitrate, nitrate plus urea or urea. The results (Table 14) seem to support the hypothesis that the cells were changed. Cells maintained continuously on nitrate were sensitive to L—threonine even when trans- ferred to urea. Cells maintained for only one subculture on urea on the other hand were not sensitive at all. Since the growth period on urea (one transfer) was insufficient for selection, this means that the cells were physiologically modified by the growth on urea so that they lost the sensitivity to L-threonine. 114 0.0HH m.Hm no.0 no.0 osasoessplq + eons + onespss 00H 0.0m 00H 0.mm eess + epespas o.moa e.mm o.uoa o.em essssmseuq + onessss 0.0HH m.om 0.0HH 0.0N esasawhelq + esasoessplq + epespas m.mm m.sm no.0 No.0 esssoesspnq + epespss ooH u.sm ooa m.sm epespss a H\sswso3 guess w s H\pswsc3 snoss w assume ems: so uosaepsaez mHHeo epespaz so uesaepsaez eHHeo .maebapoesmes .21 00H use 21 00H .28 o.m .28 o.m ”eyes esasamselq use esfisoessuiq .eoss .epespas so msodpespseosoo .uesassepeu mes pswaez seeps esp use uepmebses esez mHHeo esp mseu HH Hesse .eauea uepeoausa esp opsd ueHSp lasossm eses eess so so epespas so uesaepsaes mHHeo 0x emess muesoapepm ems: so so epespaz so mamsossapsoo uesaepsdez mHHeo QR ho SQBOHO So esdsdwhslq use esHSOeHSBIA so mpoesmm .da eHDeB 115 m.moa o.mm N.mo oos 0.nm o.NoH o.es e.es s.es o.ea use o.mms m.os ss.o ooa m.so n.00 m.ss s.oa No.0 m.ms m.mm m.mm esdsamseiq + eehs esdsawselq + osasoessplq + ems: esasoessvlq + eons eess esasawselq + eeHs + evespfis esasawseiq + esasoesspwq + eess + epespas DISCUSSION Nitrate reductase negative mutants of higher plants would be the best system for studying nitrate uptake in the absence of its reduction, as was done in the study of sulfate uptake in bacteria (Dreyfuss, 1964). Such mutants, however, were not available. Therefore a different approach was used. Tungstate was shown to be an analog of molybdate function and incorporation in lower organisms (Keeler and Varner, 1957, 1958; Takahashi and Nason, 1957; Higgins et al., 1956; De Renzo, 1954). The nitrate reductase of higher plants was reported to be molybdenum dependent (Afridi and Hewitt, 1964; .Agarwala, 1952; Evans and Hall, 1955). Tungstate was used therefore to inhibit the forma- tion of active nitrate reductase in XD cells of tobacco. Tungstate indeed inhibited the development of nitrate reductase activity in the cells without impairing the deveIOpment of the nitrate uptake system. The nitrate level in cells treated with the Optimal concentration of tungstate increases at least two fold over the control. The tungstate inhibition of the development of the nitrate reductase activity can be overcome by molybdate 22:2332. A similar molybdate reversal of tungstate inhibi- tion was shown for the xanthine dehydrogenase of 116 117 Pseudomonas aeroginosa (Nitidieri and Affonso, 1965). In addition to nitrate uptake, the develOpment of nitrite reductase activity was not inhibited by tungstate (Chroboczek-Kelker, 1969). It is tempting to conclude there- fore, that the effect of tungstate, although not necessarily limited to inhibiting the develOpment of nitrate reductase activity, is Specific for molybdate-dependent processes, and others are not affected. Some attempt was made toward better understanding the mechanism of tungstate inhibition. Its effect can be explained in one of three ways: 1) tungstate has a non- Specific toxic effect, one of its consequences being the inhibition of the deveIOpment of nitrate reductase activ- ity, 2) tungstate Specifically inhibits the formation of the nitrate reductase apoenzyme, 3) tungstate inhibits the incorporation of molybdate into the apoenzyme and thereby renders the enzyme non-functional. The first possibility was already excluded above. It was shown in this study that cells which were exposed to tungstate did make the nitrate reductase apoenzyme. The apoenzyme became functional in ZiXB» in the absence of protein synthesis, following the addition of molybdate. These results agree with J. L. Wray's conclusion (1969) that NADH:cytochrome c reductase is a partial activ- ity of non-functional nitrate reductase which deve10ps in the presence of tungstate. 118 It is therefore most likely that the inhibition of the development of nitrate reductase activity by tungstate was the result of the inhibition of molybdate incorporation into the apoenzyme. This inhibition could take place at the site of molybdate uptake or at the Site of assembly of the enzyme molecule. In cells treated with tungstate the nitrate level is at least two fold higher than that in the untreated cells. This could be due to one of two reasons. The first is that the increased level of nitrate is simply due to the inhibition of its reduction. In other words, the level of nitrate in the untreated cells is a steady state level determined by the rate of uptake and by the rate of reduc- tion and does not represent the true capacity of the cells to accumulate nitrate. The second one is that the increased level of nitrate is due to increased capacity of the cells to accumulate nitrate. Amino acids, the end products of nitrate assimilation are involved in the regu- lation of the pathway, as shown for nitrate reductase (Filner, 1966) and nitrite reductase, (Chroboczek-Kelker, 1969). It is possible that inhibition of production of amino acids by tungstate, under conditions which otherwise favor their production, would result in increased activity of the entire pathway, including the capacity to accumulate nitrate. Indeed, there is evidence that the steps of the pathway are more active in the presence of tungstate. It 119 was shown that tobacco cells treated with tungstate had higher activity of nitrite reductase (Chroboczek-Kelker, 1969) and barley seedlings treated with tungstate had higher activity of NADH:cytochrome c reductase (Wray, 1969) than the untreated controls. Regardless of the correct explanation for the two fold increase in nitrate accumula- tion, one can conclude that tungstate can be used to inhibit the develOpment of active nitrate reductase and thereby make possible the study of nitrate uptake in the absence of its reduction. Since the nitrate uptake system develOpS upon expo- sure of cells to nitrate and the cells can accumulate nitrate to a concentration higher than that in the medium, several questions were asked. Is the deve10pment of the nitrate uptake system the result of derepression (the removal of a compound(s) from the medium), induction (the addition of inducer to the medium), or activation (the addition of a compound(s) to the medium which causes the activation of a preexisting non-functional system)? Is the accumulation of nitrate energy dependent? Is the rate of uptake affected by the concentration of nitrate in the medium? These questions are discussed separately. The nitrate uptake system is inOperative and the nitrate reductase activity is undetectable in nitrogen starved stationary phase cells. It can be concluded that 120 nitrogen deficiency does not derepress the nitrate assimi- lation pathway. The two systems deve10p upon exposure of the cells to nitrate. The nitrate uptake system begins to deve10p immediately and its activity increases linearly from the time of subculture until it reaches a final value. Thereafter the nitrate is taken up at a constant rate. The steady state level of nitrate is at least 10 times higher than that in the medium. In stationary phase cells the development of nitrate reductase activity begins after a lag period of about 10 hours. After the lag the activity increases linearly. The metabolism of nitrogen starved cells (stationary phase) changes when given nitrate. This change does not occur instantaneously but requires a certain period of time. Thus, phenomena which are observed during this period of time are sometimes difficult to interpret. It was desirable therefore, to use nitrogen-rich exponential phase cells instead of nitrogen-poor stationary phase ones. A similar approach was used in the study of sulfate uptake and assimilation in Salmonella typhimurium (Dreyfuss and Monty, 1963 a,b; Dreyfuss, 1964). In order to avoid the use of resting cells, djenkolic acid (a reduced sulfur source) was used to support the growth of the cells. The sulfate uptake and assimilation were therefore studied on exponential phase cells. The same reasoning was adopted 121 in this study and a search was undertaken for a neutral reduced nitrogen compound which would support the growth of XD cells. The results obtained indicate that urea can support the growth of the cells. However, compared with cells growing on nitrate those on urea have a longer generation time (2 days and 3 days reSpectively). and a longer time for doubling the protein content (1.25 days and 3 days reSpectively). This might be due to slow entry of urea into the cells, slow assimilationcu'both. Urea is there- fore somewhat Similar to djenkolic acid, a sulfur source which supportsa slow growth of §. typhimurium. Moyed and Umbarger (1962) suggested that a compound which is only Slowly converted to repressing metabolites often supports slow growth but brings about derepression of a pathway. It was therefore possible that growth on urea would derepress the nitrate assimilation pathway. The results obtained with exponential phase XD cells growing on urea and shifted to nitrate or nitrate plus urea, clearly Show that this is not the case. Since nitrate reductase activity was not detectable and the nitrate uptake system was ineperative in cells growing on urea, growth on urea did not derepress the nitrate assimi- lation pathway. Following the shift from urea medium to nitrate or nitrate plus urea, nitrate accumulated and nitrate reduc- tase activity develOped. Urea, when present in the medium 122 together with nitrate, slightly enhanced the two processes. The rate of nitrate uptake and the nitrate reductase activ- ity increased linearly without any detectable lag. Thus exponential phase cells (nitrogen rich) are able to begin to deve10p the two systems immediately upon eXposure to nitrate. Nitrogen rich cells not only lack the lag period in the develOpment of nitrate reductase activity but they develop the two processes much faster than nitrogen defi- cient, stationary phase cells. The develOpment of the nitrate uptake and nitrate reductase activities in stationary phase cells is either induction or activation but not derepression since the two activities deve10p in reSponse to nitrate. Nitrogen star- vation, however, affects the kinetics of development. Using nitrogen-starved wheat seedlings for studying nitrate uptake, Minotti et al. (1968) also observed a lag period before a constant rate of accumulation of nitrate occurred. They suggested that a synthesis of components of the nitrate tranSport system took place during the lag period, but provided no evidence to support this idea. It might be correct to assume that components of the tranSport system are synthesized following the expo- sure of a plant to nitrate. However, this does not necessarily account for the lag period. The lag period may merely be a period necessary for recovery from nitrogen starvation. Examining high nitrogen plants (after growing 123 them on ammonia or urea) would shed more light on this aSpect. As in stationary phase cells, the development of the nitrate uptake system and the nitrate reductase activ- ity in eXponential phase cells Shifted from urea to nitrate could reflect derepression, induction or activa- tion. Urea may repress the nitrate assimilation pathway. When urea is removed from the medium and nitrate is given to the cells, the pathway may be derepressed. If this is correct one would expect that urea, if present in the nitrate containing medium too, will keep the pathway repressed. This, however, is not the case. Urea, if present together with nitrate, does not slow down the deve10pment of the pathway. In fact, it seems to enhance the deveIOpment somewhat. Although growth on urea is slower than growth on nitrate, urea does not derepress the nitrate assimilation pathway. Urea as the sole nitrogen source in the medium is not a sufficient condition for the cells to deve10p the nitrate assimilation pathway. On the other hand it does not repress the pathway either. The pathway deve10ps very similarly, with or without urea, upon exposure of the eXponential cells to nitrate. Thus urea can be considered a nitrogen source which is neutral insofar as the regulation of the nitrate assimi- lation pathway is concerned. 124 The combination of urea as a nitrogen source and tungstate as an inhibitor of nitrate reduction made it possible to study nitrate uptake and its regulation in nitrogen-rich eXponential phase cells. Nitrate accumulates against a concentration gradient and at the steady state, the level of nitrate in the cells (per gram of cells) is at least 10 times higher than that in the medium (per ml medium). Some results were obtained which Show that the accumulation of nitrate is energy dependent. The accumulation of nitrate was inhibited by dinitrOphenol and cyanide. DinitrOphenol also brought about leakage of nitrate already in the cells. However, unless it is shown that the effects of dinitrOphenol and cyanide are reversible, and the inhibition of nitrate accumulation is not due to permanent damage to the cells, the results can be considered indicative and not a final proof. The length of the period during which the rate of nitrate accumulation increases does not depend on the con- centration of nitrate in the medium (up to 9.0 mN). Apparently, during this period a fixed quantity of compo- nents of the transport system becomes available. Once they are fully available the rate of transport depends on the degree of their occupation (which depends on the concentra- tion of nitrate in the medium), being maximal at their saturation (Epstein and Hagen, 1952; Epstein, 1965). 125 The dependence of the rate of uptake on the concen- tration of nitrate in the medium gives a saturation curve which can be analyzed to give an apparent Michaelis-Menten constant of about 4 x 10'” N. This value is similar to other reported Km constants for nitrate uptake: 6 x 10'“ M for rice seedlings (Fried et al., 1965). 1 x 10'” M for maize (Van den Honert and Hooymans, 1955). However, a dis- similar value was also reported: 3.3 x 10‘5 M for perennial rye-grass (Lyclama, 1963). Only nitrate can induce the deveIOpment of the assimilation pathway, including the uptake system. The compositions of the urea-containing medium and the nitrate- containing medium are the same except for the replacement of one nitrogen source with the other (see Materials and Methods). The nitrate assimilation pathway develops only when nitrate is introduced to the medium. The lactose tranSport system in E, ggli is inducible too, in the sense that it deve10ps in reSponse to lactose (Cohen and Monod, 1957: Kepes and Cohen, 1962). The sulfate tranSport in §, typhimurium, on the other hand is not induc- ible. It develOpS under conditions of sulfur deficiency (Dreyfuss, 1964), rather than in reSponse to sulfate, i.e., it is derepressible. In cells shifted from urea to nitrate, the nitrate reductase activity deve10ps before any substantial accumu- lation of nitrate in the cells. When fully induced cells 126 are shifted from nitrate to nitrate-less medium the nitrate reductase activity decays in Spite of the high level of nitrate initially in the cells. It can be concluded that most of the nitrate which is accumulated in the cells is neither necessary for, nor capable of inducing the nitrate reductase activity. This might be due to the compartmenta- tion of nitrate, probably in the vacuole. Casein hydrolysate when applied to fully induced cells causes a decay of nitrate reductase activity in Spite of the presence of nitrate in the medium and in the cells (Figure 6). It is possible that the induction of the assimilation pathway is brought about by nitrate flowing into the cells. Once it is in the cells it cannot induce the activity. Shutting off the flow of nitrate is a suffi- cient condition to inhibit the development of the pathway. Some evidence was indeed obtained which shows that the uptake of nitrate is subject to end product regulation Similar to that reported for the nitrate reductase (Filner, 1966) and nitrite reductase (Chroboczek-Kelker, 1969) activities. The develOpment of the nitrate uptake system is inhibited when an inhibitory amino acid is applied to the medium (non-growing conditions), or when a mixture of amino acids, casein hydrolysate, is applied (growing con- ditions). Thus, amino acids Shut off the flow of nitrate which is the inducer of the entire pathway and the substrate for the first two steps. 127 Using nitrate reductase negative mutants of A. nidulans, Pateman et al. (1967) showed that nitrate can induce nitrite reductase activity. A similar conclusion can be drawn from the work of Chroboczek-Kelker (1969) who showed that nitrate can induce nitrite reductase activity in XD cells of tobacco in which nitrate reduction was greatly inhibited by tungstate. Inhibition of nitrate uptake by amino acids raises the possibility that the effect of amino acids on nitrate reductase and nitrite reductase might be a direct conse- quence of their effect on the uptake system. The regulation of nitrate uptake by amino acids in tobacco cells is not an exceptional situation. It is an example of a wideSpread phenomenon of regulation of a tranSport system by the end product of the assimilation or dissimilation of the tranSported compound. Sims et al. (1968) and Joy (1969) demonstrated a regulatory effect of ammonia on nitrate assimilation in higher plant systems. .Ammonia was also shown to inhibit nitrate absorption by higher plants. El-Shishiny (1955) using potato tubers and Wiessman (1951) using wheat seed- lings, observed that nitrate nitrogen was absorbed less from media which also contained ammonia then from media containing nitrate alone. Lyclama (1963) using perennial rye-grass observed a similar phenomenon and concluded that the reduction process was inhibited by ammonia. Minotti et al. (1969) on the other hand, using wheat plants 128 concluded that the uptake process was inhibited but not the reduction. Ferguson and Bollard (1969) concluded that ammonia affects the uptake and the reduction of nitrate in Spirodela oligorrhiza. Using the same XD cell line of tobacco, Hart and Filner (1969) Showed that sulfate uptake is regulated by sulfur containing amino acids. The sulfate uptake system in.§. typhimurium is repressed by cysteine (Dreyfuss, 1964). Methionine trans- port in.Penicillium chgysogenum, which develops upon sulfur starvation is controlled by feedback inhibition and not by feedback repression (Benko et al., 1967). Sulfate uptake in the same organism also develOpS upon sulfur starvation (Yamamoto and Segel, 1966). The system is repressed by L-cysteine and L-methionine. The sulfate assimilation pathway of §, typhimurium is coded by several unlinked clusters of cistrons. Using mutants lacking various activities of the pathway it was shown that all the steps are regulated similarly but the repression of one activity does not depend upon the func- tioning of others (Dreyfuss and Monty, 1963 a,b: Dreyfuss, 1964). The lac operon in E. ggli which codes for the trans- port and dissimilation of lactose is regulated as a unit (Jacob and Monod, 1961). Genetic factors controlling the level and physical properties of nitrate reductase activity in maize were reported (Warner et al., 1969). Nevertheless, the overall 129 information about genetic control of nitrate assimilation in higher plants is much less detailed than that from lower eukaryotic organisms. Pateman et al. (1967) and Pateman and Cove (1967) concluded that there are structural genes for nitrate reductase and nitrite reductase in ASpergillus nidulans which are not linked (Cove and Pateman, 1963), and regu- lated by a regulatory gene. In Neurospora crassa, Sorger and Giles (1965) reported the existence of at least 4 unlinked genes which regulate nitrate reductase activity. The absence of such information about the genetic factors governing the steps of the nitrate assimilation in XD cells of tobacco makes it difficult or even impossible to study the regulation of the pathway at the genetic level. It is still possible however to study the regulatory rela- tionships of the steps of the pathway at the physiological level. Two approaches were applied. The first one dealt mainly with the deveIOpment of the uptake system and the nitrate reductase activity in reSponse to nitrate, and their regulation by casein hydrolysate in cells treated with tungstate. It was shown that the deveIOpment of the nitrate uptake system and its regulation by amino acids could take place in XD cells treated with tungstate. Similar results were obtained for the deveIOpment of nitrite reductase activity (Chroboczek-Kelker, 1969). Thus, the nitrate 130 uptake system can develop and be regulated by amino acids in the absence of functional nitrate reductase. It can be concluded that the regulation of the nitrate uptake does not depend on nitrate reduction, nor does it depend on a functional nitrate reductase. Amino acids do not regulate nitrate uptake by simply stOpping its reduction, since anything which will stop the reduction, such as tungstate, Should have the same regula- tory effect. In addition, the regulatory effect of amino acids is observed when reduction is already stopped by tungstate. Thus, the nitrate uptake system does not exhibit product (N03(in)) inhibition. Independence of the tranSport system and its regula- tion on subsequent metabolism of the tranSported compound was Shown for the sulfate uptake in §, typhimurium (Dreyfuss, 1964). Using cys CD-519 mutant, a deletion mutant lacking the first two enzymes in the sulfate reduction pathway, it was Shown that the tranSport system was regulated indepen- dently of further utilization of the tranSported compound. Although it is similar to the use of tungstate in tobacco cells, there is a major difference. Tungstate does not inhibit the formation of the nitrate reductase apoenzyme but rather renders it non-functional. Furthermore, the develOpment of nitrite reductase activity is not at all affected by tungstate (Chroboczek-Kelker, 1969). It is possible therefore that the regulation of the uptake system 131 is mediated via the regulation of nitrate reductase and/or nitrite reductase on the translational level rather than the functional level. Cove and Pateman (1969) indeed pro- posed that the nitrate reductase molecule is involved directly in the regulation of its own synthesis and pos- sibly in the regulation of subsequent steps in.A. nidulans. The condition for this regulation to take place is a func- tional enzyme. The second eXperimental approach to the relationship between the regulation and function of the steps of the nitrate assimilation pathway dealt with the characteristics of a variant cell line, XDRthr. It was selected for its ability to grow in the presence of the inhibitory amino acid, L-threonine, from the sensitive XD cell line. The resistance was shown to be a permanent characteristic of the line. The resistance to one inhibitory amino acid is associated with resistance to other inhibitory amino acids. The resistance to other amino acids varies however, which suggests that there might be more than one mechanism of inhibition. Selection of other variant cell lines resis- tant to other inhibitory amino acids may Shed more light on this aSpect. Although the XDRthr cell line is resistant to the growth inhibition by L-threonine, its presence in the medium significantly changes the growth behavior of the cells. This is reflected in slower rates of mass and pro- tein increase and different time profiles of nitrate 132 accumulation and deveIOpment of nitrate reductase activity than in cells growing in the absence of the amino acid. The sensitive XD cell line could have given rise to the resistant XDBthr cell line by any one of several changes including: 1) general exclusion of amino acids from inside the cells, 2) exclusion of the Specific inhibi- tory amino acid from inside the cells, 3) exclusion of the inhibitory amino acid from the Site of action (compartmen- tation), 4) rapid degradation of the inhibitory amino acid (detoxification), 5) elevated endogenous level of an antagonistic amino acid, 6) greater inherent stability or catalytic activity of the nitrate assimilation pathway, 7) lower endogenous level of the system which inactivates the nitrate assimilation enzymes, 8) desensitized primary site of action of the inhibitory amino acid, 9) a lesion in the subsequent inhibitory chain of events. The results obtained in this study rule out Bthr cells 1) general exclusion of amino acids, since XD could utilize casein hydrolysate as the only nitrogen source; 2) exclusion of the inhibitory amino acid, since XDRthr and XD cells took up radioactive L-threonine from the medium similarly; 3) degradation of the inhibitory amino acid, Since radioactivity fed in the form of L-threonine was found in the cells almost entirely as L-threonine. Also the high level of free L-threonine accumulated in XDRthr cells adapted to M-ID and shifted 133 to M-ID plus 100 uM L-threonine persisted beyond the lag period into the rapid growth phase. Rthr cells to canavanine may The resistance of XD indicate that these cells have a higher level of arginine. It is difficult to see how an elevated arginine level would account for the difference in the sensitivity of nitrate uptake and nitrate reductase to amino acids. Based on the characteristics of the nitrate uptake system, and the develOpment of nitrate reductase activity of the XDRthr cell line, an inherited change occurred that apparently altered the regulation of the uptake system. This fits best with either resistance mechanism 8 or 9 (above). It remains to be determined if this change can completely account for resistance of XDRthr cells to the growth inhibition by L-threonine. The formation of nitrate reductase is equally sensi- tive to casein hydrolysate in XDBthr and XD cells. The nitrate uptake system on the other hand is less sensitive in XDBthr cells than in XD cells. Thus, under conditions which equally inhibit the formation of nitrate reductase activity, XDBthr cells accumulate much more nitrate than do XD cells. This indicates that amino acids do not regulate the nitrate reductase activity by keeping the inducer, nitrate, out of the cells. Additional support for this conclusion can be obtained from the kinetics of nitrate accumulation compared to kinetics of the develOpment 134 of nitrate reductase activity in eXponential cells shifted from urea to nitrate. A high level of nitrate need not first accumulate in the cells before the develOpment of nitrate reductase activity can take place. Also, a high level of nitrate in the cells is not a sufficient condi- tion to maintain the nitrate reductase activity. The characteristics of the XDRthr cell line indi- cate that the three steps of the nitrate assimilation pathway are probably not regulated as a unit. It was shown that the regulation of nitrate uptake was altered without a similar change in the regulation of the other two steps. As was mentioned above, the results obtained with tungstate treated cells indicate that amino acids do not regulate the nitrate assimilation pathway by either keep- ing the nitrate out of the cells or by inhibiting its reduction. The actual mechanism by which the amino acids regulate the nitrate assimilation is still unknown. Nevertheless, it is clear that they similarly affect the three steps of the pathway in a manner which does not depend upon the sequential functioning of the steps of the pathway. It is not at all clear how the insensitivity of the nitrate uptake system to regulation by amino acids can enable the XDRthr cells to grow in the presence of inhibi- tory amino acids in Spite of the fact that the development 135 of nitrate reductase is still greatly inhibited. Possibly the nitrate dependence of the nitrate reductase reaction in XlZQ is reSponsible for resistance. The absolute nitrate level in XDRthr cells may increase the rate in 2132. of nitrate reduction sufficiently to compensate for small formation of the enzyme. In any event, the amount of nitrate reductase activity which is still present in cells treated with threonine is sufficient for growth. Several lines of evidence were obtained which sup- port the idea that the inhibitory effect of individual amino acids is Specific for the nitrate pathway, and the inhibition of growth is a direct consequence of inhibition of nitrate assimilation. Cells which could not grow because of nitrogen deficiency (nitrate-less M-ID), cells which were given nitrate but could not grow because of the presence of L-threonine, and cells that could grow when given nitrate, L-threonine and L—arginine, absorbed radio- active amino acid, accumulated it and incorporated it into protein at a very similar rate. If threonine did have a non-Specific toxic effect, it certainly did not Show up judging by the three tested parameters, namely, uptake rate, final accumulation and incorporation into protein of radio- active amino acids. The second line of evidence was based on the assump- tion that if the effect of individual amino acids is Specific for nitrate assimilation, then growth of cells on nitrogen 136 sources other than nitrate Should not be inhibited. L-threonine inhibited growth only when nitrate was the nitrogen source but not when urea or y-aminobutyric acid were the nitrogen source. Assuming that the only differ- ence between cells growing on nitrate and cells growing on other nitrogen sources is the dependence of the former on a functional nitrate pathway, it would appear that the amino acid inhibited growth by preventing nitrate assimi- lation. However, cells growing on urea were insensitive to L-threonine only after being adapted to growth on urea. This strongly indicates that a change had to occur in these cells which made them insensitive. Such a change could be an elevation of the endo- genous level of one or more of the antagonistic amino acids which would render the inhibitory mechanism inOperative. The relative levels of free amino acids in plants are known to change depending upon the nitrogen source used (Pharis et al., 1964: Ferguson and Bollard, 1969). There still can be a second kind of change. Sta- tionary phase cells maintained on nitrate, are sensitive to inhibition by L-threonine in Spite of the fact that the nitrate in the medium has long been assimilated and the activity of the assimilation pathway has decayed. One transfer, i.e., 4-5 doublings, on urea was enough for these cells to lose the sensitivity. One can imagine the 137 involvement of a stable component, which is made during growth on nitrate, in the inhibitory mechanism of L- threonine. This compound which does not decay or dis- appear very fast, and is present in stationary phase cells makes the cells sensitive perhaps by blocking the deve10p- ment of the urea assimilating enzymes. A brief period of growth on urea however, is enough to cause either dilution or decay of this compound. The third line of evidence is the variant cell line. The selection for a cell line resistant to the inhibition of growth by L-threonine also selected for a cell line in which the regulation of a step in the nitrate assimilation pathway had been altered. This selection is in accordance with the hypothesis that a single amino acid inhibits growth by inhibiting nitrate assimilation Specifically. It is also consistent with the hypothesis that amino acids inhibit growth by one mechanism and nitrate assimilation by a second independent one. Then, in order for cells to grow in the presence of the inhibitory amino acids, two indepen- dent lesions had to occur which seems highly improbable. 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