ma Item, 338 1 3 :99! ' I" I ~ *4 .f,’ ABSTRACT SELECTIVE EFFECTS OF THE HOST-SPECIFIC TOXIN FROM HELMINTHOSPORIUM CARBONUM ON CELLULAR ORGANELLES AND ON SOLUTE ABSORPTION by Olen C. Yoder Helminthosporium carbonum, race 1, causes a leaf spot disease of certain corn (£e_a m) lines. The fungus produces a toxin (HC- toxin) in culture which specifically affects susceptible corn. Both fungus and toxin cause the same physiological changes in susceptible tissue. An attempt was made to determine those cellular functions most closely related to the site of toxin action. The first detectable effects of toxin on susceptible corn tissues were stimulatory; inhibition became evident later. Experiments were designed to determine whether or not toxin action is: a) hormone-like or related to that of hormones; b) involved in energy production by the cell; c) related to synthesis or degradation of enzymes; or d) associated with inter- or intracellular solute movement. Toxin promotes seedling growth under certain conditions, sug- gesting gibberellin-like activity. Therefore, toxin and gibberellic acid (GA) were compared in several standard assays. Toxin did not pro- mote dark germination of tobacco seeds, cucumber hypocotyl elongation, or q-amylase synthesis by embryoless barley half-seeds; GA was Olen C. Yoder active in all these tests. Thus, there was no evidence that toxin had a hormone-like action on tissues. In contrast, helrninthosporol, a bio- active metabolite produced by certain Helminthosporium spp. , has gibberellin-like activity. Toxin was tested for possible effects on isolated mitochondria and chloroplasts. Toxin did not uncouple phosphorylation, inhibit elec- tron transport, alter phosphorylation capacity, inhibit the Krebs cycle, or affect conformational changes whether or not electron transport was occurring. The effect of toxin on EM activity of nitrate reductase (NR), a substrate inducible enzyme, was determined. Treatment with toxin for 4 hr before induction (that is before exposure to N03), caused an in- crease in NR in )Li_v_o activity which was apparent from the beginning of the induction period. This difference between toxin-treated and control tissues persisted but did not change throughout the induction period, indicating that toxin did not enhance the rate of NR induction. Toxin had no effect on NR activity when the enzyme was extracted from tissue and assayed Exit—ml. This also supports the conclusion that toxin had no effect on NR induction. Toxin-treated tissue accumulated N03 up to 3 times faster than did control tissue. This observation could explain the increased NR _'1_I_}_v_i\_r2 activity. The absorption of other substances (Na+, Cl', 3-O- methylglucose and leucine) was also stimulated by toxin-treatment. There was no effect on uptake of N02, K+, Ca2+, Pi, SO42", or glu- tarnic acid. Olen C. Yoder Toxin-enhanced permeability was not caused by a general de- rangement of cell membranes. Cells had an increased capacity to absorb and retain certain solutes. Substances accumulated in 30 min by toxin-stimulated tissues could not be washed out of such tissues in 30 rnin desorption. Toxin-treatment enhanced active influx, but had no effect on active efflux of 3—o-methy1glucose. Toxin-stimulated ion absorption did not require the presence of other mineral ions. Thus, toxin enhanced the uptake rate of N05 from a tris-NOg solution; toxin enhanced the uptake rate of N05 but not Ca2+ from a Ca(NO3)2 solution. There was no apparent effect of toxin on H move ment. For the following reasons it is suggested that toxin causes spe- cific changes in the characteristics of the plasmalemma. 1) Toxin- stimulated uptake is highly temperature sensitive, which suggests an active uptake process. 2) Toxin-treated tissues accumulate ions a- gainst a concentration gradient, and deveIOp a steeper gradient with some ions than do control tissues. Mechanism 1, the mechanism Of ion uptake which Operates at low ion concentrations, is generally thought to be in the plasmalemma and is stimulated by toxin. 3) Nega- tive results from tests Of organelles support the hypothesis that in- creased uptake is caused by a change at the cell surface. 4) All other physiological effects of toxin can be explained on the basis of stimula- ted uptake. Toxin concentrations which enhanced the absorptive capacity of _}_‘I_. carbonum-susceptible tissue had no effect on E carbonum-resistant tissue. When the toxin concentration was increased 500 times, resis- tant toxin-treated tissue accumulated solutes at a faster rate than did resistant control tissue. SELECTIVE EFFECTS OF THE HOST-SPECIFIC TOXIN FROM HELMINTHOSPORIUM CARBONUM ON CELLULAR ORGANELLES AND ON SOLUTE ABSORPTION by Olen C. Yoder A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOC TOR OF PHILOSOPHY Department Of Botany and Plant Pathology 1971 A CKNOWL EDGMEN TS Dr. R. P. Scheffer, my major professor, has worked closely with me throughout the course of this study. He was particularly helpful in evaluating ideas and providing encouragement, and in pre- paration of this manuscript. Drs. N. E. Good, P. Filner, and A. H. Ellingboe, the other members of my guidance committee, made valuable contributions through discussions, loans of materials and equipment, and assis- tance in preparing this manuscript. The success of the experiments with organelles was due to many hours of close guidance by Dr. S. Izawa. Dr. T. E. Ferrari made important suggestions concerning the work with nitrate pools. My wife, Jacqueline, has provided moral support, assisted in some of the experiments, and typed rough drafts and the final copy of the manuscript. This work was supported in part by the National Science Founda— tion (Grants (SB-6560 and (SB—24962). A Graduate Tuition Scholarship from Michigan State University (1970-71) provided additional financial support. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS ............................ ii LIST OF TABLES ............................... iv LIST OF FIGURES ............................... vii LIST OF ABBREVIATIONS ......................... ix INTRODUCTION ................................. 1 LITERATURE REVIEW ............................ 3 MATERIALS AND METHODS ........................ 15 Plant material ............................... 15 Toxin preparation ............................. 16 Amylase induction ............................. l9 Nitrate reductase induction ....................... 20 N03 and N02 determinations ...................... Z3 Isolation of chloroplasts ......................... 24 Isolation of mitochondria ......................... 26 Absorption of exogenous solutes by roots .............. 28 RESULTS ...................................... 33 Effect of toxin on seed germination and seedling root growth . 33 Tests for hormone-like activity of toxin ............... 40 Experiments with toxin and isolated mitochondria ........ 45 Experiments with toxin and isolated chloroplasts ......... 50 Effect of toxin on nitrate reductase activity ............. 54 Effect of toxin on N03 accumulation and compartmentation . . 67 Characteristics of toxin-stimulated N03 accumulation by roots ................................ 75 Effect of toxin on uptake of selected cations ,,,,,,,,,,,, 87 Effect of toxin on uptake of selected anions ............. 98 Effect of toxin on uptake of selected organic molecules . . . . 107 DISCUSSION ................................... 1 1 5 LITERATURE CITED ............................ 138 APPENDIX .................................... 149 iii LIST OF TABLES Table Page 1. Effect of toxin exposure time on germination and root growth Of susceptible corn seeds . . . . . . . . . . ..... 35 2. Recovery of susceptible corn from effects of toxin on root length and dry weight .................... 36 3. Effect of toxin on growth of corn seedling roots ....... 39 4. Effects of toxin and gibberellic acid on germination of tobacco seeds in the dark .................... 41 5. Effects of toxin and gibberellic acid on cucumber hypo- cotyl elongation .......................... 43 6. Effect of toxin and gibberellic acid on d-amylase production by embryoless barley half-seeds ........ 44 7. Effect of toxin on gibberellic acid-induced amylase production by susceptible corn endosperm ......... 46 8. Respiration and phosphorylation by corn mitochondria pre- incubated in the presence and absence of toxin ...... 48 9. Induction of nitrate reductase in corn seed tissues ...... 56 10. Effect of nutrients on capacity for nitrate reductase induction in corn embryonic axes ............... 57 11. Protein content of susceptible corn embryonic axes after induction Of nitrate reductase in the presence or absence of toxin .......................... 63 12. Effect of temperature on toxin-stimulated N03 accu- mulation by corn root tips ....... _ ............. 7O 13. The metabolic N03 pool in corn root tips ............ 74 14. Effect of toxin on N03 accumulation by aged and fresh corn roots .............................. 79 iv LIST OF TABLES (Continued) Page 15. Effect of exogenous ion content and concentration on toxin-stimulated N03 accumulation by corn roots . . . . 81 16. Variation in rates of N03 accumulation by corn roots under standard experimental conditions ........... 83 17. Effect of experimental conditions on toxin-stimulation of N03 accumulation by corn roots .............. 84 18. Loss of NO3 from toxin-treated and control corn roots during desorption ...................... 86 19. Absorption of K by toxin-treated and control corn roots . . 90 20. Effect of toxin on absorption of Na by corn roots ....... 93 21. Effect Of toxin on simultaneous accumulation of Na, K, and N03 by corn roots ...................... 94 22. Absorption of Ca by toxin-treated and control corn roots. . 96 23. Effect of toxin on absorption of C1 by com roots ....... .99 24. Absorption of P04 by toxin-treated and control corn roots. 101 25. Uptake and incorporation of P04 by toxin-treated and control corn roots ......................... 103 26. Absorption of 804 by toxin-treated and control corn roots . 104 27. Accumulation of N02 and N03 by toxin-treated and control corn roots .................. ' ....... 106 28. Effect of toxin concentration on N03 or Na accumulation by susceptible and resistant corn roots ........... 108 29. Effect of toxin on absorption and desorption of 3-0- methylglucose (MeG) by corn roots .............. 110 30. Leucine and glutamic acid accumulation by toxin- treated and control corn roots ................. 111 31. Selective stimulation of solute accumulation in susceptible corn roots by toxin ................ 114 LIST OF TABLES (Continued) Page 32. Effect of toxin on leucine content in ethanol-soluble and insoluble fractions from corn roots ........... 150 33. Effect of toxin-treatment time on size of the leucine pool in corn roots ......................... 152 34. Age of seedlings in relation to size of the leucine pool in toxin-treated and control corn roots ........... 154 35. Effect of toxin on size of the leucine pool in aged and fresh corn roots .......................... 155 36. Effect of coleoptile or seed removal on toxin-stimulation of the leucine pool in roots of corn seedlings ....... 156 vi Figure 10. 11. 12. 13. 14. LIST OF FIGURES Extraction of toxin from chloroform residue ......... Respiration rates and phosphorylation of corn mito- chondria in the presence and absence of toxin ...... Volume changes in toxin-treated and control mitochondria ............................ Electron transport and phosphorylation by corn chloroplasts in the presence and absence of toxin . . . . Light-scattering by corn chloroplasts in the presence and absence of toxin ....................... Effect of toxin on induction of NR in corn embryonic axes as affected by imbibition time ............. Effect of toxin on induction of NR in corn axes ........ Effect of toxin concentration on induction of NR in corn axes .............................. Effect of N03 concentration on toxin-stimulated induction of NR in corn axes .................. Effect Of toxin on degradation of NR in vivo .......... Effect of pretreatment with toxin on induction of NR in corn root tips ....................... Effect Of pretreatment with toxin on induction of NR as measured by in vivo and in vitro assays .......... Effect of pretreatment with toxin on N03 accumulation by corn root tips ......................... Effect of pretreatment with toxin on N03 uptake and reduction in the absence of NR induction .......... vii Page 17 47 51 52 58 6O 62 65 66 68 71 LIST OF FIGURES (Continued) Page 15. Evidence for a metabolic pool of N03 in corn leaves . . . 73 16. The metabolic pool of NO3 in toxin—treated and control corn leaves ............................ 76 17. Accumulation of NO3 by corn roots ............... 78 18. Rates of N03 accumulation in toxin-treated and control corn roots as a function of N03 concentration ...... 88 19. Effect Of toxin on leucine uptake, incorporation and soluble pool size ......................... 113 viii BSA Ca Ck Cl DNP EDTA Fecy GA GD H HC-toxin HEPES HV-toxin K MeG MES Na NADH N02 N03 NR 0. D. PCA Pi P:O P04 Rb RCR 804 TCA Tox Tricine tris uc ueq LIST OF AB BR EVIA TIONS bovine serum albumin calcium ion control chloride ion 2 , 4-dinitrophenol disodium ethylenediaminetetraacetic acid potassium ferricyanide gibberellic acid gramicidin D hydrogen ion Helminthosporium carbonum-toxin N-Z -hydroxyethy1pipe razine -N{-2 - ethanesulfonic acid Helminthosporium victoriae-toxin potassium ion 3-o-methy1glucose 2 -(N-morpholino)ethanesulfonic acid sodium ion reduced nicotinamide adenine dinucleotide nitrite ion nitrate ion nitrate reductase optical density perchloric acid phosphate ions (H2P04' and HPO42') moles ATP formed:atoms oxygen consumed phosphate ions (HZPO4' and HPO42') rubidium ion respiratory. control ratio = respiratory rate + Pi/ respiratory rate - Pi sulfate ion trichloroacetic acid toxin-treated N-tris (hydroxymethyl)methylglycine tris (hydroxymethyl)aminomethane microcurie microequivalent ix INTRODUCTION Helminthosporium leaf spot is a fungus disease of certain inbred corn (Zea mays) lines and corn hybrids (119). The causal organism, Helminthosporium carbonum Ullstrup (119), has a sexual stage known as Cochliobolus carbonum Nelson (79). A host-specific toxin produced by E carbonum race 1 was first described in 1965 by Scheffer and U11- strup (104). This toxin (known as HC-toxin) selectively affects corn lines that are susceptible to E carbonum (57). The toxin is a low MW peptide whose structure is not fully known because of problems with lability (88, 90, 92). Several lines of evidence show that HC-toxin is required by the fungus for initial colonization of susceptible corn, and for development of disease symptoms (102, 103, 105). Furthermore, resistance of corn to E carbonum is the same as insensitivity to HC- toxin (102, 105). TherefOre, susceptibility and resistance can be stud- ied on the level of individual chemical reactions without the complica- tion of 2 interacting metabolic systems. The basic problem with such a model is to determine the characteristics of plant cells that convey sensitivity or resistance to toxin. The initial interaction between toxin and a sensitive Site in the susceptible cell can be considered the key to understanding disease development. 2 This study was undertaken to determine which physiological functions of susceptible cells are most likely to be associated with the initial site of toxin action. The earliest physiological effects of toxin described to date are stimulatory (54, 56, 57); toxin at low concentra- tions can also cause increased growth (57). Therefore, stimulatory effects are probably more closely associated with initial toxin action than are inhibitory effects (118). Several potential modes-of—action were considered: 1) Possible hormone-like effects of toxin were ex- amined by comparison with effects of gibberellic acid and of helmin- thosporol. 2) Possible effects on energy metabolism or intracellular membrane systems were observed with tests of isolated mitochondria and chloroplasts. 3) Possible effects on synthesis or degradation of proteins were tested with inducible enzymes, amylase and nitrate re- ductase. 4) Effects on inter- and intracellular solute movement were studied by observing absorption of exogenous solutes by excised and intact roots, by observing changes in intracellular nitrate pools, and by observing ion fluxes across membranes of isolated organelles. All experiments with toxin gave negative results except those de- signed to measure effects on solute absorption. The data indicate that toxin-treatment altered the characteristics of the plasmalemma of susceptible cells such that active uptake and retention of certain substances was enhanced. Uptake of several other substances was not affected by toxin-treatment. Toxin at concentrations which af- fected susceptible cells had no effect on resistant cells. LIT ERA T UR E REVIEW There is a large literature on metabolic changes which occur in plants after infection by microorganisms (129). The objective of most of this work has been to understand the mechanisms by which pathogens cause disease. Generally, the remarkable host-specificity of many plant pathogens has not been considered in this literature. Direct ef- fects of pathogens on host cells have been proposed at various levels. There are reports on changes in respiration (78), nucleic acid metab- olism (38), levels of regulatory hormones (108), protein synthesis (120), cell walls (1), photosynthesis (13), and cell permeability (124). Most of these disease-induced changes occur long after the pathogen has established a parasitic relationship with the host. Therefore, they cannot be considered as primary events, essential to the success of the pathogen. Even when a physiological aberration occurs relatively soon after infection, it is difficult to establish such a change as the initial event which leads to disease development. In spite of all the efforts, we have a poor understanding of dis- ease at the molecular level. However, the diseases known to involve host-specific toxins Offer exciting possibilities for studies of the chemical interactions between host and pathogen. To date, 8 4 substances which affect only hosts of the producing fungus have been discovered (105). Only 4 of these have been investigated in sufficient detail to give some understanding of disease relationships. The four are: l) HV-toxin produced by Helminthosporium victoriae, specific for certain varieties of oats; 2) HC-toxin produced by _I:I_. carbonum, race 1, specific for certain corn lines; 3) HM-toxin produced byli: maydis, race T, specific for corn with certain types of male sterile cytoplasm; and 4) PC-toxin produced by Periconia circinata, specific for certain lines of sorghum. Compound names derived from the in- itials of the producing fungus have been assigned to these toxins to signify their special biological roles as genotype-specific molecules (92). Trivial names become confusing in differentiating host-specific toxins from the large group of nonspecific toxic metabolites produced by microorganisms. HC-toxin is the subject of this investigation. Several lines of evidence are outlined below, which show that HC-toxin is necessary for pathogenicity offl. carbonum race 1 and is required for initial establishment of the fungus in host tissue. Similar data are available for HV and PC-toxins (102, 103, 105). l. The most obvious indication of a role in disease is host-specificity. Corn genotypes which are susceptible to H carbonum are also sensi- tive to HC-toxin. Plants which are resistant are insensitive to the toxin. Plants with intermediate susceptibility to E carbonum have a corresponding intermediate sensitivity to the toxin (57). 2. All isolates of_l-_l_. carbonum race 1 which produce toxin are also pathogenic to susceptible corn. All isolates which do not produce HC- toxin are nonpathogenic to corn. This is true for a large group of wild type isolates collected from all over the world (100), for mutants which have lost ability to produce HC—toxin (unpublished), and for as- cospore progeny of crosses of a pathogen with a nonpathogen (100). _I:I_. carbonum is sexually compatible with H: victoriae. Progeny of such crosses segregate in a ratio of 1:1:l:l for ability to produce HC-toxin, HV-toxin, both toxins, or neither toxin. One gene pair controls pro- duction of HC-toxin by _1:I__. carbonum, ' whereas another gene pair con- trols production of HV-toxin by H: victoriae. Again, toxin producing ability is correlated with ability to cause disease in specific host plants. Progeny which produce both toxins are pathogenic to both oats and corn, whereas progeny which produce neither toxin are not pathogenic to either oats or corn (100). 3. HC-toxin is required by E carbonum for successful colonization of susceptible host tissue. When exogenous HC-toxin is added to spores of_H_. victoriae (nonpathogenic to corn) it colonizes H. carbonum-sus- ceptible corn tissue just as does E carbonum (105). 4. HC-toxin causes the same physiological changes that are found in Ii. carbonum-infected tissues. These include increases in respiration, dark fixation of COZ (56), and electrolyte leakage (54). There are several reviews which discuss the biological signifi- cance of host-specific toxins in more detail (102, 103, 105). The 6 literature on physiological changes in _I:I_. carbonum- infected tissue has also been reviewed (54). Therefore, I am summarizing the known ef- fects of HC-toxin on susceptible corn. These effects are compared with effects of HV-toxin on susceptible oats. Uptake and/or activity of HC-toxin on susceptible corn is depen- dent on time, temperature and cellular metabolism (55). Roots of growing seedlings require at least 4 hr exposure to toxin to accumulate enough toxin to inhibit later growth. As toxin exposure time is in— creased, the degree of later root growth inhibition increases. If toxin exposure time is held constant, later inhibition becomes progressively greater as temperature is increased from 5 C to 37 C. To test the need of oxygen for toxin uptake and/or activity, susceptible seedlings were placed in toxin solution under nitrogen for 20 hr, then were rinsed and transferred to solutions without toxin in air for 4 days. Toxin had no effect on seedling root growth when seedlings were exposed to toxin under anaerobic conditions (55). In similar experiments susceptible seedlings were exposed to toxin in the presence of metabolic inhibitors (DNP, KCN, or NaN3) for 20 hr, then transferred to solutions without toxin or inhibitors. There was no inhibition of growth when tissues were exposed to toxin under such conditions (55), which suggests that the inhibitors interferred with uptake of toxin. To date, there is no direct evidence that HC-toxin is actually taken up by corn tissue (54, 55). This statement is based on data which show that toxin cannot be recovered from either susceptible or resistant tissue. Cuttings were exposed to a high concentration of toxin (500 pg/ ml) for 20 hr, then homogenized and extracted with water. Water ex- tracts were assayed for host-specific toxicity. None was found. In another experiment, 3 large masses of roots were each exposed in turn for 12 hr to the same small volume of toxin solution. Bioassays of the residual solution showed no loss in host-specific toxicity, even though roots had accumulated toxic doses (54, 55). A similar experi- ment with 14C-labelled toxin showed that susceptible and resistant plants removed equal amounts of radioactivity from solution (54). There have been attempts to identify the site of toxin action by use of 14C-labelled toxin (54). Susceptible tissues were exposed to HC-toxin, then tissues were homogenized and subcellular particles were separated by differential centrifugation. Most of the radioactivity (80%) remained in the 105, 000 g supernatant. Small amounts of radio- activity were in ribosomes (11%), mitochondria (8%), and nuclei and chloroplasts (1%). The results were inconclusive as to the site of toxin action (54). There are inherent problems in such an experiment. It is difficult to obtain a homogeneous toxin preparation which is free of nonhost-specific peptides and breakdown products produced by spon- taneous toxin inactivation (88, 92). In addition, both resistant and sus- ceptible tissues completely inactivate the toxin. Thus, even if a pure toxin preparation is used, labelling of cellular sites could be caused by toxin breakdown products. 8 The ability Of HC-toxin to induce electrolyte leakage from corn tissue was tested (54). Leaves infiltrated with toxin and incubated for 2 hr were then rinsed and leached in distilled water. No toxin-induced electrolyte leakage was evident before 8 hr after initial exposure to toxin. Toxin exposure of 8-10 hr resulted in 30-70% increase in leak- age of electrolytes from toxin-treated leaves, as compared to controls. HC-toxin was tested for its ability to stimulate respiration of susceptible leaves (56), as HV—toxin is known to do. Cuttings were allowed to take up toxin for varying periods, then oxygen uptake by leaf sections was measured. Toxin exposures up to 4 hr had no effect on respiration; 8 hr exposure caused 30% increase in oxygen uptake, as compared to nontreated controls. The potential of corn tissues to fix C02 in the dark is known to be increased after infection with Ii. caibonum (54). The ability of HC- toxin to duplicate this effect was tested. Cuttings were allowed to take up toxin for 4 hr, then leaf sections were exposed to 14C02 for an ad- ditional 4 hr. Toxin-treated tissues fixed as much as 200% more C02 than did control tissues. In a second experiment, toxin-treated and control tissues were homogenized, centrifuged, and the supernatants were used as sources of CO2 fixing enzymes. When ribose-S-phos- phate was the substrate, enzymes from toxin-treated tissues fixed up to 99% more COZ than did control enzymes. Toxin—treatment had no effect on cell-free activity when other substrates involved in dark C02 fixation were used. When toxin was added directly to enzymes extracted 9 from nontreated tissues, there was no effect on COZ fixation by the ribose-S-phosphate system. Therefore, stimulation of C02 fixation in the dark is a secondary effect of toxin (56). The effects of toxin on the ability of corn tissues to fix 14tC- labelled amino acids and uridine into TCA—insoluble cellular compo- nents (54) were examined. Incorporation was not affected by toxin ex- posures up to 6 hr. When tissues were treated with toxin for 8 hr, then exposed to l4C-amino acids for 4 hr, there was a 23% increase in incorporation by susceptible tissues and a 43% increase by resistant tissues. Similar results were obtained with uridine. Longer toxin ex- posures (22 hr) inhibited incorporation by susceptible tissues 45% but stimulated incorporation by resistant tissues 20% (54). HV-toxin, produced by I_-I_. victoriae and specific to certain vari- eities of oats, has been used as a model with which to compare other host-specific toxins (102, 103, 105). HV and HC-toxins are in the same chemical family but there are differences. HV-toxin is a ses- quiterpenoid complex (C17H29NO) (89) combined with a peptide consis- ting of aspartic acid, glutamic acid, glycine, valine, and leucine (105). The MW is between 800 and 2000. HC-toxin is a cyclic peptide (C32 H50N6010) containing 2 molecules of alanine, 1 Of proline (88), an un- saturated analog of leucine (2-amino-2, 3-dehydro-3-methy1pentanoic acid) which has an unstable double bond, and an unknown hydroxyamino acid (90). It has a MW of approximately 679 (88). HV-toxin causes complete inhibition of root growth of susceptible oats at 0. 0002 pg/ml 10 (105). Resistant oats tolerate >400, 000 times higher concentrations with no effect (105). HC-toxin causes 50% inhibition of root growth of susceptible corn at 0. 2 pg /ml (57); resistant corn is affected to the same degree at approximately 100 times this concentration (92). Nei- ther toxin was recovered from susceptible or resistant plants (55, 102) which took up known amounts of toxin. Any effective concentration of HV-toxin invariably inhibits root growth; HC-toxin, at low concentra- tions, stimulates root growth (57, 105). The physiological effects of HC-toxin on corn, described above, differ from those of HV-toxin on oats in several categories. In con- trast to HC-toxin, uptake and/or aetivity of HV-toxin is not dependent on time, temperature, or cellular metabolism. HV-toxin has an im- mediate effect on susceptible cells (98, 105), is not affected by temper- ature during the exposure time, and acts in the presence of metabolic inhibitors (101). Thus, HV-toxin appears to act by a simple physical process. HV-toxin causes rapid leakage of electrolytes from suscep- tible cells immediately after exposure (103, 105). In contrast, HC- toxin causes only slight leakage after 8-10 hr exposure. When suscep- tible tissues are exposed to HV-toxin, there is a rapid rise in the res- piration rate (102), whereas the respiratory response to HC-toxin is small and is delayed. HV—toxin has no effect on isolated mitochondria (102). Under certain conditions, HC-toxin stimulates incorporation of 14C-labelled amino acids and uridine into TCA insoluble material. HV- toxin invariably causes a rapid loss of ability to incorporate amino acids and uridine (102). 11 The two toxins also have physiological effects in common. When tissues were exposed to l4C-labelled HV-toxin, the same type of in- conclusive results were obtained as described above for HC-toxin (Samaddar, unpublished). Both HV and HC-toxins stimulate dark fixa- tion of C02 by their respective susceptible tissues (56). Most of the physiological effects of these toxins are probably secondary (105). The site-of—action of HV-toxin is thought to be in the plasmalemma because it quickly alters many characteristics of the plasmalemma (103, 105). There is some evidence that a toxin receptor site is associated with a membrane protein (25). There are no clues to date to the site-of—action of HC-toxin. Data in this thesis suggest that HC-toxin alters characteristics of the plasmalemma, but in a dif- ferent way than does HV-toxin. Part of my research included a study of the effects of toxin on pro- tein synthesis, using indirect methods with an inducible enzyme, ni- trate reductase (NR). A brief summary of NR literature for background purposes is sufficient, because there are detailed reviews (3, 50). Much of the nitrogen absorbed by plants is in the form of nitrate (N03), which must be reduced before it is incorporated into organic materials. This requires at least 3 steps: reduction of N03 to N02 is catalyzed by NR; reduction of N02 is catalyzed by nitrite reductase (a complex of enzymes); and incorporation of NH3 into OL-ketoglutarate to form glu- tamic acid is catalyzed by glutamic acid dehydrogenase (24). NR ap- pears to regulate this pathway because it is: a) the first enzyme in the 12 sequence; b) the rate limiting step; c) substrate inducible; and d) relatively unstable with a high turnover rate (3). Although NR has not been completely purified, some of its characteristics are known. It has a MW of 500, 000-600, 000; a requirement for Mo, Fe, and FAD as cofactors and NADH or NADPH as electron donors; is unstable both 12 ill? and 13m and is induced by N03 and by Mo (3). Regulation of NR levels 32m is not solely dependent on levels of N03; concentra- tions of N03 can be high in tissues with low NR activity (3). This could be attributed to effects of light (4) or regulation by endogenous metabo- lites (23, 106). Results of many studies indicate that NR is found in all living plant cells. The main evidence for this is that tissues can be grown in media which contain N03 as the sole nitrogen source (3). Induction of NR has been reported in green leaves (3, 4), stems and petioles (51), roots (51, 99), germinating embryos Of wheat (113) and rice (110), bar- ley aleurone cells (20, 21), corn scutella (3), and tobacco pith cultures (23). Green leaves usually contain the highest levels of NR. In corn, roots contain only 20% of the activity found in leaves (3). The amount of NR in corn leaves increases in light and decreases in darkness (4), suggesting an association of NR with chloroplasts. However, several workers have reported that NR is not a chloroplastic enzyme but prob- ably is located either free in the cytoplasm or in cytoplasmic particles (74. 96, 115). 13 Development of aninm NR assay (21) has made possible great- er experimental rnanipulation. The technique is based on the fact that under anaerobic conditions N03 is converted to NOZ but N02 is not fur- ther reduced (53) and can be recovered from tissue (21, 22). In green leaf tissue, darkness is required in addition to anaerobiosis for maxi— mum N02 production (94). 19.3222 NR activity is directly related to the rate of N02 production. A major portion of my work involved studies of ion uptake by corn roots. There is a vast literature including a number of reviews (17, 59, 61, 72) on salt absorption by plants. In brief, ion accumulation is generally considered to be dependent upon metabolic energy (61). This conclusion is based on observations that ion uptake is affected by tem- perature, anaerobiosis, and metabolic inhibitors (67, 68, 117). In ad- dition, ions are transported by roots against concentration (70) and electrochemical gradients (39). Active transport of many ions is de- pendent on the presence of Ca (16). The role of Ca is unknown, but it probably stabilizes the configuration of the plasmalemma and thus keeps ion carriers in an active form (93). Uptake Of most ions is mediated by at least 2 mechanisms; one operates at low ion concentrations and is called *mechanism 1, the other Operates at high ion concentrations and is called mechanism 2 (17, 59, 61, 72). Uptake rates by both mechan- isms lend themselves to kinetic analysis (18). With increasing ion con- centrations, maximum rates are attained. The dual mechanisms of Uptake have been described for several ions, including K, Rb, Cs, NH4, 14 Na, Mg, Sr, Cl, Br, 504, and choline sulfate. At least 11 plant spe- cies including both monocots (corn among them) and dicots are known to have dual uptake mechanisms (17). MAT ERIALS AND METHODS Plant material. -- Ii carbonum-resistant (PR 1 x K61) and sus- ceptible (Pr x K61) corn hybrids were used in all experiments involving corn. Seedlings were grown from seeds planted in vermiculite and in- cubated at 24 C under Sylvania Gro-Lux lamps (18 hr light/day). They were watered with White's inorganic nutrient solution (125) or nitrate- less modified White's solution containing the following in mg/l: CaClz' ZHZO, 176; NH4C1, 74. 2; KCl, 80; NaHzPO4' H20, 18. 7; MnSO4°HZO, 5; ZnSO4' 7H20, 2. 6; H3BO3, 1.5; K1, 0. 75; MgSO4, 360; Fe (EDTA chelate), 5; NazMoO4- ZHZO, 0. 025. Green leaf tissue was obtained from tertiary leaves of lS-day-old plants. Mitochondria were isolated from 7 to 8-day-old etiolated shoots. Roots used in ion uptake experiments were grown as follows: 250 seeds were soaked 8-12 hr in water, washed 10 times in water and placed embryo down on cheesecloth stretched over a ring of stiff poly- ethylene tubing. Seeds were covered with a second layer of cheese- cloth, and suspended over 4000 ml 0. 2 mM CaClz or CaSO4 in a 4000 ml beaker. The beaker was covered with Saran Wrap and the solution was aerated with filtered air which passed through a gas dispersion tube suspended in the solution. After 3 days, primary roots had grown into the solution and Saran Wrap and top cheesecloth covering were 15 l6 removed. Primary roots were used after 4 days when they were 8-12 cm long; 90-95% of seeds produced usable roots. Roots for other types of experiments were grown as follows. Seeds were placed embryo down in 30 ml White's solution (40-50 seeds/ 15 cm petri dish) at 24 C for 60-84 hr, when roots were 4-8 cm long. Each day the seedlings were placed in clean petri dishes containing fresh solution. In later experiments, agar (0. 9%) was added to the nu- trient solution, and 100 ml/lS cm petri dish was used; this made the daily change of solutions unnecessary. Toxinpreparation. -- Toxin was isolated from filtrates (8 l) of cultures grown for 23 days at 24 C in Fries medium plus 0.1% yeast extract (91). Toxicity of each fraction obtained in the purification pro- cedure was assayed against susceptible and resistant seedlings as pre- viously described (100). The isolation procedure was a modification of that described previously (54, 55). Pringle's (92) procedures were fol- lowed through chloroform extraction. The chloroform extract was evaporated .12. M to a thick yellow syrup. The flow diagram in Fig- ure 1 describes separation of yellow pigment from the toxin. There were about equal amounts of toxin in ethanol—ether soluble and insoluble fractions. Extraction of the yellow syrup was repeated until most of the toxin was removed. Toxic Bio-Gel fractions were pooled and evaporated to dryness, leaving a tan residue of l. 25 g. This material was dried EEC—l1? over CaClz at 4 C for 24 hr. It was stored under nitrogen in the presence of CaClz at -20 C, and used for all 17 Figure l. Extraction of toxin from chloroform residue. Yellow syrup from chloroform extract. Dissolve in 5 ml absolute ethanol, add 100 ml diethyl ether, and store at 5 C 24-72 hr. Yellow residue. Ethanol-ether solution. Dissolve in water. Decant and evaporate to dryness. Filter Oily residue. Wash 5 times with 2 ml water. Yellow syrup. Oil (discard). Aqueous (filter). Evaporate to dryness. If solution is yellow, repeat extraction, Repeat ethanol-ether beginning with ethanol- extraction. ether. If solution is clear or slightly 0- paque, concentrate and use gel filtration (54, 55). 18 experiments described here. The preparation caused 50% inhibition of root growth of susceptible corn at 0. 2 ng/ml. The probable 'molecular weight of this toxin is 679 (88). Thus, activity was evident at >2. 95 x 10’7 M, which is as good as the best crystalline preparations (88, 92). Under the storage conditions used, this dry preparation did not lose ac- tivity or host-specificity in 2 yr. Lyophilized toxin is reported to be more stable than crystalline preparations (90). For experiments, 20- 30 mg was dissolved in water (1 mg/ml), stored at -20 C in 1 and 2 m1 ali- quots and used as needed. One such aliquot was thawed and refrozen at least 6 times without losing activity or host-specificity. A small amount of crystalline toxin was prepared following Pringle's procedure (92). One toxic Bio-Gel fraction produced a clear residue when evap- orated to dryness. This residue was dissolved in 5 ml absolute ethanol, 100 ml diethyl ether was added and the solution was placed at 5 C for several days. A small amount of fluffy, colorless, needle-shaped particles precipitated; this preparation caused 50% inhibition of suscep- tible corn root growth at 0. 2 pg/rnl. In most cases, toxin-containing solutions were assayed against susceptible and resistant seedlings (100) before and after completion of the experiment. This was done for all experiments which showed little or no effect of toxin. In all cases, there was 50% inhibition of suscep- tible and no effect on resistant seedling root growth at 0. 2 jig/ml, indi- cating no loss of toxin activity during the experiment. Inactivated toxin was prepared as described for HV-toxin (98) or the toxin solution was 19 sealed in a glass tube and autoclaved 1 hr on each of 10 consecutive days. This destroyed essentially all toxic activity. Amylase induction. -- (gt-amylase induction in embryoless barley half-seeds was determined by a procedure 'modified after that of Varner ital (7, 46). Barley seeds were placed in 50% H2504, stirred vigor- ously for 1 hr to remove husks (9), and washed 10 or more times with distilled water. Seeds were cut transversely with a razor blade and embryoless halves were surface sterilized 20 min in 1% sodium hypo- chlorite, then washed 3 times in sterile distilled water and incubated in a beaker of sterile water at 24 C for 24 hr. Half-seeds were placed aseptically in sterile 50 m1 flasks (IO/flask) containing 2 pmoles sodium acetate (pH 4. 8), 40 pmoles CaClz, 20 pg chloramphenicol with or with- out 2 n'moles gibberellic acid (GA) or varying amounts of toxin in a total volume Of 2 ml. Acetate and CaClZ were sterilized by autoclaving; other components were sterilized by filtration (0. 22 p. Millipore filters) and added to the medium. Flasks were placed on a reciprocal shaker (100 oscillations /rnin) at 24 C for 24 hr. The medium from each flask was decanted into a 15 ml glass centrifuge tube, and half-seeds were rinsed 3 times with 1 m1 volumes of water. The water from each rinse was added to the medium. Half-seeds were ground in a mortar with a pinch of sand and 0. 8 ml grinding medium (44 mM KHzPO4, 2 mM Ca C12). One ml grinding medium was added and the slurry was poured into glass centrifuge tubes. The mortar was rinsed twice with a total of 3 m1 grinding medium and rinsings were added to homogenate. Tubes 20 containing incubation medium or seed ho-mogenate were centrifuged 10 min at 2000 g; supernatants were assayed for (pk-amylase. Aliquots (0. 02-0. 2 ml) were diluted to 1 ml with water and incubated with 1 ml starch solution (46) for 5 rnin. One ml iodine solution (46) was added, then colored solutions were diluted with water and the color intensity was measured using a Klett-Summerson colorimeter with a #62 filter. Enzyme activity was quantified by defining 1 unit d-amylase as a change of 10 Klett units in 1 min. Amylase activity in corn endosperm was determined by a method modified from that used for barley half-seeds. Corn seeds were incu- bated in water 3 hr, then embryos were dissected and discarded. En- dosperms were rinsed in distilled water, surface sterilized in 1% so- dium hypochlorite for 20 min, rinsed 4 times in sterile water, and in- cubated 24 hr in sterile water at 24 C. Endosperms were then trans- ferred aseptically to 4 ml incubation medium (described for barley half-seeds) in 50 ml flasks and incubated on a shaker (120 oscillations/ min) for 48 hr. Amylolytic activity was determined in endosperms and medium as described for barley. The type of amylolytic activity was not ascertained; Dure (12) has shown that corn endosperm contains pri- marily fi-amylase. Nitrate reductase induction. -- Nitrate reductase (NR) was induced in aseptic corn embryonic axes (axis = embryo minus scutellum) dis- sected with a sterile scalpel from corn seeds which were surface steri- lized 15 min in 1% sodium hypochlorite, and washed 4 times in sterile 21 water. Axes were rinsed in sterile water, then allowed to imbibe in petri dishes on filter paper wetted with sterile water or nutrient solu- tion containing filter-sterilized chloramphenicol (20 jig/ml) or a com- bination of penicillin, mycostatin, and streptomycin (10 pg of each/nil). The nutrient solution was modified after that of Dure (11) and contained the following in mg/l: CaClz- 2HZO, 190; KCl, 65; NaHzPO4' HZO, 16.5; MgSO4' 7H20, 36; iron citrate, 30; MnSO4‘HZO, 0.3; and glucose, 20 g/l. Induction of NR was initiated by transferring axes to similar solutions containing nitrate (N03) and shaking at 200 oscillations/min. Experimental results were not affected when antibiotics were omitted from sterile media. Solutions from all experiments were plated on potato-dextrose agar to detect possible microbial contamination. None was found. Procedures for induction under nonsterile conditions in corn roots and leaves are described in a later section. The Em method of Ferrari and Varner (21) and the EM method of Filner (23) were modified to determine NR activity in corn tissues. For the Exile assay, axes were placed in 1. 7 ml assay medium in 15 ml glass centrifuge tubes. The assay medium contained 0.1 M phosphate buffer (pH 7. 5), 0. 01 M KNO3, and 5% (v/v) ethanol. Nitrogen was bubbled through the solution for 1 min and the vial con- taining axes was stoppered. Axes were then incubated 15-30 rnin at 24 C on a wrist action shaker at high Speed. After incubation, the reaction was stopped by adding an equal volume of each color reagent (1% sul- fanilarnide in 3 N HCl and 0. 02% N-l -naphthy1ethy1enediamine 22 dihydrochloride). Color was allowed to develop for at least 15 min. Turbid solutions were centrifuged 5 min at 5000 g. The color intensity was measured with a Klett-Summerson colorimeter using a #54 filter. The procedure for root tips was similar to that for axes except the volume of the assay medium was 0. 5 ml, anaerobic incubation was in 3 ml glass vials, and after incubation contents of vials were boiled briefly before color reagents were added. For the i2_v_i£_r_o_assay, 5 mm root tips were excised, rinsed in ice cold water, and homogenized in 1 ml ice cold medium in a 2 -ml glass homogenizer (10 tips/replicate). The homogenizing medium con- tained 25 rnM KZHPO4, 5 mM disodium ethylenediaminetetraacetic acid (EDTA), and 10 mM cysteine-HCl, pH 8. 8 (4). The homogenizer was rinsed twice with 0. 5 ml aliquots of homogenizing medium; homogen- ate and rinsings were centrifuged in a 15 m1 glass centrifuge tube at 2 C for 10 min at 15, 000 g. Enzyme activity was determined in a reac- tion mixture which contained 0. 5 m1 0. l M phosphate buffer (pH 7. 5), 0.1 ml 0. 1 M KNO3, 0.1 ml 0. 001 M NADH, and 0. 3 m1 supernatant. The reaction was started by transferring tubes from ice to a 28 C water bath. The reaction was stopped after 0 and 30 min by adding 1 ml of each color reagent; color was developed for at least 15 min and Optical density (O. D.) at 540 nm was determined with a Coleman spectrophoto- meter. NR activity was determined from a standard nitrite (NOZ) curve, and was expressed as nmoles NOZ produced/unit of tissue /hr. 23 N03 and N02 determinations. -- The quantitative bacterial assay of Lowe and Hamilton (65) was used to determine N03 content of boiled tissue extracts. Boiling released soluble intracellular materials into the suspending medium. Reaction mixtures contained 0. 02 M sodium succinate (pH 6. 8), 0.1-0. 2 m1 soybean root nodule bacteroids (65), and NO3-containing extract in a total volume of 1. 0-1. 7 ml. Reaction was allowed to go to completion (30-60 min), then an equal volume Of each color reagent was added. Color reagents were added directly to boiled and nonboiled tissue extracts to determine NOZ content. Color intensity Of solutions was measured with a Klett-Summerson colori- meter. Concentrations of N02 were determined from N02 standards. The metabolic N03 pool (22) was demonstrated in tertiary leaves of 17-day-Old corn seedlings grown in vermiculite watered with White's solution containing 10 lei KNO3 + Fe and MO. Leaf sections (5 m2) were placed in 2 ml 0. l M phosphate buffer (pH 7. 5) in 50 ml flasks (100 mg/flask). Nitrogen was bubbled through the solution for l min in dim light; flasks were then stoppered and incubated in total darkness at 30 C (94). To demonstrate the capacity of the metabolic N03 pool, flasks were opened at intervals, 2 m1 phosphate buffer was added (to keep final volumes constant), contents were boiled, and N02 in 1 ml of boiled extract was determined by adding 1 m1 of each color reagent. To demonstrate refilling of the metabolic pool, flasks were Opened after metabolic pool N03 was exhausted. Buffer (2 ml) with and without 0. l M KNO3 was added, flasks were aerated 1 min, incubated aerobically 24 15 min, flushed with nitrogen 1 min, and returned to darkness at 30 C. Periodically, contents of flasks were boiled and N02 determinations were made. In experiments with toxin, metabolic pool N03 was ex- hausted, and 2 ml buffer with or without toxin (40 pg /ml) was added to the flasks. Contents were aerated 1 min and incubated aerobically 4 hr. Flasks were flushed with nitrogen l min and returned to darkness at 30 C. After 2 hr contents of flasks were boiled and N02 determinations were made. The size of the metabolic N03 pool was estimated in roots. Tis- sues (grown in N03 solution) were placed in 0. 4 m1 0. l M phosphate buffer (pH 7. 5) in a 3 ml glass vial. The vial was flushed with nitrogen 1 Inin and stoppered. At intervals contents were boiled, cooled, and amount of N02 produced was measured. Isolation of chloroplasts. -- Corn chloroplasts were isolated at 4 C by a method modified after that of Miflin and Hageman (75). Two- week-old seedlings were held in darkness 24 hr prior to use to elimin- ate starch from leaves. Tertiary leaves (10 g) were cut in 1 cm pieces, chilled and homogenized 15-20 sec in a Waring blender in 80 m1 homo- genizing medium containing 0. 01 M NaCl, 0. 05 M Tricine-NaOH (pH 7. 8), 0. 002 M MgClz, 0. 4 M sucrose, 0. 01 M glutathione and 12 mg polyethylene glycol (Carbowax 4000)/‘rnl. Glutathione was added to the medium just prior to blending. Homogenate was squeezed through 8 layers of cheesecloth and centrifuged 4 min in 50 ml centrifuge tubes at 2500 g. The supernatant was discarded; each pellet was gently 25 dislodged with a camel's hair brush and suspended in 40 m1 washing medium which contained 0. 01 M NaCl, 0. 01 M Tricine-NaOH (pH 7. 5), 0. 002 M MgClz, and 0. 4 M sucrose. Tubes were centrifuged slowly 30 sec in a clinical centrifuge to remove large debris. Supernatants were decanted into clean tubes and centrifuged 3 rnin at 2500 g. Superna- tants were discarded, pellets were suspended in <10 ml washing 'medium and chlorophyll content was adjusted by a method modified from that of Mackinney (71). An aliquot of the unadjusted stock sus- pension was diluted 1:100 with 80% acetone and centrifuged 5 Inin at 5000 g. The O. D. of the supernatant was determined at 663 nm (chlorophyll a), 645 nm (chlorophyll b) and 652 nm (the wavelength at which absorption spectra of chlorophylls a and b intersect). Chloro- phyll content was determined from Mackinney's extinction coefficients: 0. D. 663 (12. 7) - O. D. 645 (2. 7) = pg chlorophyll a/ml acetone 0. D. 645 (23) - O. D. 663 (4. 7) = pg chlorophyll b/rnl acetone The combined equations, O. D. 663 (8) + O. D-645 (20. 3), give the total pg chlorophyll/ml acetone. TO check accuracy of the determination, O. D. 652 (28. 5) should also equal total pg chlorophyll/ml acetone. The ratio of chlorophyll a:b was about 3:1 for corn. The stock chloroplast suspension, which contained 0. 4 mg chlorophyll/ml, was stored on ice. The reaction medium for chloroplasts contained 0.1 M sucrose, 0. 01 M KCl, 0. 05 M Tricine-NaOH (pH 7. 8), 0. 001 M MgClz, 20 pg chlorophyll/m1, and other components as indicated in Results in a total volume of 2 m1. Changes in light-scattering were estimated by measuring 26 changes in the apparent O. D. (small angle light-scattering) of a chloro- plast suspension at 540 nm in a 1 cm cuvette. Electron transport was induced with a beam of actinic light (>600 nm) at right angles to the de- tecting beam. The rate of electron transport was measured by follow- ing the decrease in O. D. of potassium ferricyanide at 420 nm. The method is Similar to that of Izawa and Good (45). Isolation of mitochondria. -- Mitochondria were isolated and tested following the general procedures of Miller e_t£ (77). All man- ipulations were done in the cold room (4C) and mitochondria were stored on ice. Seven-day-old etiolated Shoots (75 g) were cut in 1 cm seg- ments and chilled. They were ground 30 sec in a mortar on ice with 150 ml grinding medium which contained 0. 4 M sucrose, 0. 03 M HEPES, 0. 005 M EDTA, 0.1% bovine serum albumin (BSA), 0. 05% cysteine. The pH was adjusted to 7. 5 with NaOH. Cysteine was added just prior to grinding. Homogenate was squeezed through 8 layers of cheesecloth and centrifuged in 50 ml tubes for 5 min at 28, 000 g. The supernatants were removed with an aspirator; each pellet was gently dislodged with a camel's hair brush, suspended in 40 m1 grinding medium minus cys- teine, and centrifuged 3-4 min at 2500 g. The pellets were discarded and supernatants were centrifuged 5 rnin at 28, 000 g. In some experi- ments the pellets were washed once more in grinding medium minus cysteine to remove all endogenous substrates. Supernatants were as- pirated; pellets were dislodged with a brush (leaving behind as much starch as possible) and suspended in 1 ml 0.4 M sucrose. An aliquot 27 of the mitochondrial suspension was diluted 1:40 with 0. 4 M sucrose. The O. D. was measured at 520 nm, and protein content was determined from a standard curve plotting O. D. 520 vs. protein. The stock mito- chondrial suspension was adjusted to 5 mg protein/ml with 0. 4 M su- crose. The standard curve was plotted from protein and O. D. 520 deter- minations made on a series of mitochondrial dilutions. After 0. D. 520 measurements were taken, 1 ml 20% (w/v) trichloroacetic acid (TCA) was added to 1 ml 'mitochondrial suspension in 15 ml centrifuge tubes. After 24 hr, 8 ml 10% TCA was added and suspensions were centrifuged 10 -min at 20, 000 g. The pellets were washed in 10 ml 95% ethanol, air dried, and dissolved in 1 m1 1 N NaOH ()1 hr required). Protein con- tent was determined by the method Of Lowry _e_t_a_._l_ (66) using BSA in 1 N NaOH as the standard. The reaction medium used in experiments with mitochondria con- tained 0. 2 M KCl, 0. 02 M HEPES (pH 7. 5 with NaOH), 0. 002 M MgClZ, 0. 1% BSA, 250 pg mitochondrial protein/ml, and other components as indicated in Results in a total volume of 2 ml. Volume changes in rnito- chondria suspended in a 1 cm cuvette were determined by changes in O. D. at 520 nm using a Bausch and Lomb Spectronic 505 spectropho- tometer. Respiration rates were measured by inserting a Clark oxygen electrode into a special temperature-controlled cuvette (28 C) contain- ing 2 m1 reaction medium and monitoring oxygen loss from the solution with a recorder. Phosphorylation was determined by including ADP and 28 32Pi in the reaction mixture. After 5-10 min reaction time, 1 m1 of the radioactive reaction mixture was quickly frozen in ethylene glycol at -20 C. Incorporated Pi was determined by the method of Saba and Good (97) as described later in this section. Corrections were made for volume changes in reaction mixtures caused by additions during the course of the experiment. Absorption of exogenous solutes by roots. —- A modified standard procedure (19) was used to determine absorption of N03, N02, K, Na, Cl, Ca, P04, 504, leucine, glutamic acid, and 3-o-methy1g1ucose (MeG) by roots. Four-day-Old seedlings were placed in 15 cm petri dishes containing 50 ml 0. 5 mM CaClz or CaSO4 with or without toxin (10 pg/ml), and incubated for 4 hr. Toxin-treated and control seed- lings were rinsed in separate 4 1 beakers containing 3000 ml 0.5 mM CaClZ, unless otherwise indicated. Root tips 6 cm long (0. 3-0. 5 g/ 6-10 roots) were placed in a cheesecloth bag, rinsed again in 0. 5 mM CaClz, unless otherwise indicated, and twirled rapidly in the air to spin out excess solution. Bags were incubated for 30-45 min in vigor- ously aerated solutions containing the appropriate solute under test. Absorption periods were terminated by rinsing roots 6 times in 1500 m1 0. 5 mM CaClz, unless otherwise indicated. The amounts of each test solute in tissue were then determined by methods described below. Possible effects Of toxin on efflux were tested by desorption (described in Results) in aerated solutions at 24 C. Removal of ions from free space by desorption is usually done at 5 C (22). Variations in the usual procedure are indicated where appropriate. 29 Absorption of all solutes except N03 and N02 was detected by use of isotopes. These were: 86RbCI, Na2H32PO4, Na235804, ZZNaCI, 45CaClz, and Na36Cl, all of which were carrier free. Organic com- pounds had the following specific activities: 3-O-methy1-14C-D-glucose, 10 mc/mrnole; 1"LC-l-L-leucine, 12.2 mc/rnrnole; and l4C-l-L-glu- tamic acid, 20 mc/mmole. None of the isotopes made a significant contribution to the solute concentration of the experimental solution. To extract N03, N02 and C1, roots were cut in 3 cm sections, placed in 5 ml water in a 50 m1 flask, boiled and frozen. To determine Cl content, 2 ml aliquots were placed in aluminum planchets, dried and counted in a gas-flow detector. Amounts of N03 and N02 in the ex- tracts were determined as described in the previous section. Quanti- ties of N03 (or N02) in tissue are expressed as "accumulated" rather than ”absorbed. ” This is because some of the N03 taken up during the absorption period was reduced, and only the "accumulated" N03 was detected in the boiled extracts. For all solutes other than N03 and N02, "absorption" indicates total solute taken up during the absorption period. Total N03 uptake could be determined by observing N03 loss from the external solution, or by suppressing NR activity with tungstate, which would eliminate N03 metabolism (37). To determine content of other ions in roots, 1-2 cm root sections were placed on aluminum planchets, dried, and ashed 2-3 hr at 500 C. Ash was spread with 0. 5 ml 1% Triton X-100, dried, and counted. Cor- rection was made for self-absorption, which occurred only with 450a and 14C. 30 Labelled organic *materials were extracted from roots cut into 1 cm sections. Sections were placed in 3 ml vials containing 1. 5 ml of the appropriate solvent. Roots containing MeG were placed in 95% ethanol and held 24 hr at -20 C (31). Leucine was extracted in 95% ethanol at 24 C or in boiling water. Glutamic acid was extracted in boiling water. Aliquots were dried in aluminum planchets and counted. To determine efficiency of amino acid extraction, roots were homogenized in ethanol or water. The homogenate was dried in plan- chets, counted, and counts were corrected for self-absorption of the root residue. Boiling water extracted 64% of the glutamic acid and 42% of the leucine from roots. Ethanol extracted 66% of the leucine from roots. Since a significant portion of the labelled amino acids remained in the roots, total uptake was calculated as the sum of the counts in the soluble and insoluble fractions. Phosphorous absorbed by roots was separated into organic and inorganic fractions. Roots (0. 5 g) were homogenized in 2 ml 10% per- chloric acid (PCA) at 5 C. Homogenate and 1 ml 10% PCA rinse were placed in a 15 ml glass centrifuge tube and centrifuged at 5 C for 10 min at 20, 000 g. The pellet was washed in 7 ml 10% PCA, suspended in 1 ml water, placed in an aluminum planchet with 1 drop of 1% Triton X- 100 and counted without drying to prevent explosion of residual PCA. Organic and inorganic phosphate in the combined supernatants was separated and quantified by a procedure modified from that of Saha and Good (97). One ml 10% ammonium molybdate was added to the super- natant to complex Pi. Acetone (2. 2 ml) was added and phosphomolybdate 31 was extracted with 2 volumes (6. 5 ml each) of 10% PCA-saturated bu- tanol-benzene (1:1, v/v). The aqueous phase (containing organic phos- phate) and the organic phase (containing phosphomolybdate) were counted in a Geiger-Muller immersion tube (97). Starting times were adjusted so that all toxin-treated seedlings were exposed to toxin 4 hr 1’ 15 min. After toxin-treatment there was a 15-30 min handling period and 30-45 min exposure to experimental solutions. Elapsed time from beginning of toxin-treatment to termina- tion of experiment was approximately 5 hr. Short absorption periods have several advantages (19) including a brief post-toxin-treatment period. The volume of experimental solutions varied, depending on the solute under examination and solute concentration (19). There was often approximately 1 g tissue/500 ml solution. Loss of solute from experimental solutions never exceeded 5-10%. Some workers report little change in pH of experimental solutions during short term experiments (15, 93), although others (70, 85) have observed alterations in pH. My experimental solutions were prepared at pH 5. 8. In some experiments the pH changed from 5. 8 to a final value of 4. 8-5. 3, regardless of the solute used or solute concentration. There was no pH change when the volume of experimental solution was large compared to the amount of tissue used. The pH change was not caused by aeration, presence of cheesecloth, string, or solutes, or cut ends of excised roots. Extensive washing Of roots during preparation 32 did not prevent pH change. Ion uptake is known to be affected by pH, but not in the range of pH 5-7 (68). Furthermore, similar results were obtained in nonbuffered solutions and in solutions buffered at pH 5. 8 with 2 mM MES. There was no difference in pH change caused by toxin-treated and control roots. There is a report that H release in small volumes Of solution (100-500 ml/g roots) is related to total salt uptake (85). The efflux Of H was proportional to salt concentration (above 0. 5 HM) and to the rate of salt uptake. Thus, the pH change observed in the present study could be related to salt uptake but cannot be explained on the basis of charge balance or effect of toxin (see ex- periments with Ca(NO3)z). All glassware used in uptake experiments was washed with deter- gent and rinsed in tap water, distilled water, 0. 5 N HCl, and then 6-8 times in distilled water. Cheesecloth and string were boiled in 0. 05 N HCl and rinsed many times in distilled water before use. Similar re- sults were obtained with tap distilled (>1, 000, 000 ohms) or glass dis- tilled (>900, 000 ohms) water. All experiments were repeated, with or without variations, one or more times except where indicated. Ranges, indicated by '1' in tables, are the result of 2 replicates. RESULTS Effect of toxin on seed germination and seedlingroot growth. -- Samaddar found that oat seeds exposed to HV-toxin for 1 hr will not germinate (98), which indicates that metabolically inactive tissues are sensitive to toxin. Thus, it seemed important to determine the effect of HC-toxin on the germination process in corn seeds. In the first ex- periment, susceptible and resistant seeds were exposed to White's solution with or without toxin (25 seeds/25 nil/15 cm petri dish), for 24, 48, and 72 hr, washed 1 hr in running tap water, and placed in fresh White's solution without toxin in clean petri dishes. After 4 days incu- bation, % germination was determined and roots were measured. Ex- posure of seeds to toxin for 24 and 48 hr had little or no effect on germ- ination or root growth of susceptible and resistant seedlings at any con- centration. Exposure for 72 hr did not affect % germination, but root growth of susceptible seeds was inhibited 95, 82, and 35% at 20, 2, and 0. 2 pg toxin/Inl, respectively. There was no effect on resistant seed- lings at any concentration used. In a similar experiment, toxin at 20 pg/ml had no effect on sus- ceptible root growth after 24-40 hr exposure times but caused 54% in- hibition after a 48 hr exposure. Results Of these 2 experiments were uncertain because germination and root growth were reduced in the 33 34 controls by the washing procedure. Even brief washing in tap or dis- tilled water was found to cause a decrease in germination and growth. Washing was omitted in later experiments; instead, seeds were blotted on paper towels before transfer to fresh solutions. Results of a third experiment show that toxin had no effect on seed germination (Table 1). Exposure of seeds to toxin for 40 hr caused slight inhibition of the root growth which occurred after remov- al of seeds from toxin. Exposure times longer than 40 hr caused greater inhibition of later root growth. Inhibition was more obvious at 3 days than at 1 day after treatment. A fourth experiment was designed to determine whether or not seedlings can recover from a toxin exposure capable of causing about 50% inhibition of later root growth. Seeds were exposed to White's so- lution with or without toxin for 32, 44, or 56 hr. These exposures gave no inhibition, partial, and complete inhibition, respectively, of later root growth (Table 2). Immediately after treatment, seeds exposed to toxin for 32 hr had not germinated, whereas seeds exposed for 44 hr had germinated normally, and seeds exposed for 56 hr had germinated but further growth was inhibited. Roots of seedlings exposed to toxin for 32 hr grew normally throughout the experimental period, indicating that seeds did not accumulate a toxic dose in that time. When 44 hr toxin exposure was used, root lengths and dry wts l and 2 days after the treatment were less than for controls. Four days after treatment, the roots of toxin-treated and control plants were equal in length; dry 35 Table 1. Effect of Toxin Exposure Time on Germination and Root Growth of Susceptible Corn Seeds Seeds were placed in White's solution with or without toxin (20 pg/ml) for times indicated, then removed, blotted, and placed in fresh White's solution. There were 25 seeds/treatment, with 5 seeds/ 10 ml in each of 5 petri dishes (10 cm). Roots were measured 1 and 3 days later. A seed was considered germinated if it had a 5 mm root. , Interval Control Toxin Inhibi- Toxm- ° T After tion reTatment Removal Germ- Avg Root Germ- Avg Root of Root Ime from Toxin ination Length ination Length Growth hr days % mm % mm % 40 l 100 34 96 27 19 44 100 40 92 20 51 48 96 40 88 13 68 52 100 43 92 9 98 56 96 33 96 9 ' 97 40 3 100 63 37 44 114 50 57 48 115 34 71 52 110 20 82 56 111 10 91 36 Table 2. Recovery of Susceptible Corn From Effects of Toxin on Root Length and Dry Weight Seeds were placed in toxin solutions (20 pg/ml) or in control solutions for 32, 44, or 56 hr. Seeds were then placed on cheesecloth suspended over 150 ml White's solution in glass staining jars. There was a 12 hr difference in age Of seedlings of the 3 treatments. Thus, each treatment can be compared only with its own control. There were 10 roots/treatment. Between 4 and 10 days after treatment, primary and secondary roots formed a tangled mat and seedlings could no longer be removed individually. Therefore, the entire mat was excised, dried and weighed. Avg wt is total wt divided by the number of seeds contributing roots to the mat. This number varied from 13-20. Inte rval Toxin After Treat- Avg Length Toxin- Avg Wt Toxin- T . Induced Induced ox1n ment _ , E . Control Tox1n Change Control Toxin Change xposure Time days hr mm mm % mg mg % 0 32 0 0 . . . 0 0 ... 44 3.4 3.9 +15 0.70 0.81 +16 56 23.0 5.1 -78 2.86 1.04 -64 1 32 13.3 13.7 +3 1.66 1.57 -5 44 29.1 17.0 -42 3.20 1.90 -41 56 46.7 7.0 -85 4.57 1.11 -76 2 32 28.5 31. 7 +11 3.08 3.27 +6 44 45.4 33.5 -26 4.74 2.64 -44 56 60.6 7.4 -88 5.19 0.86 -83 4 32 75.4 65.1 -13 5.87 4.09 -30 44 101.8 96.5 -5 8.73 5.78 -34 56 115.9 17.4 -85 8.86 1.77 -80 10 32 22.20 24.70 +11 44 24.10 23.00 -4 56 24.10 18.20 -24 37 wt of roots indicated partial recovery by toxin-treated seedlings. By 10 days there was no difference between dry wts of the control root mat and the root mat from seeds exposed to toxin 44 hr, which suggests complete recovery. However, a small effect on dry wt of primary roots could have been masked by the large mass of secondary roots in the mat. Exposure of seeds to toxin for 56 hr resulted in essentially complete inhibition of primary root growth throughout the experimen- tal period, indicating that roots cannot recover from the effect of toxin if a large dose is accumulated. The apparent increase in growth of 56 hr primary roots at 4 days is due to 2 roots which grew abnormally long (73 and 89 mm). The average length of the other 8 roots was 9 mm, indicating complete inhibition. Primary roots from seeds ex- posed to toxin 56 hr had a brown discoloration throughout the experi- mental period. Exposure to toxin for 56 hr had little or no effect on growth of secondary roots, as shown by the large root mat at 10 days. The mat from toxin-treated seedlings weighed less than that of the control, probably because all primary roots in the mat were dead and shriveled by that time. It was concluded that intact, resting corn seeds do not accumu- late a toxic dose of HC-toxin in the same way that intact, resting oat seeds accumulate a toxic dose of HV-toxin. In contrast to its inhibitory effect on root growth, toxin can also stimulate root growth if used at the proper concentration (57). My data on this subject were published previously (57). The experiments were 38 with a toxin preparation which caused 50% inhibition of growth of sus- ceptible and resistant roots at 0. 2 and 20 pg toxin/ml, respectively. This same toxin preparation stimulated growth of susceptible and re- sistant roots at 0. 025 and l. 0 pg/ml, respectively. Further results (Table 3) show that there is a corresponding increase in dry wt of re- sistant roots, which was statistically significant at the 10% level. There was no significant increase in dry wt of susceptible roots, per- haps because of the small sample size used. There were 25 observa- tions /treatment for root length but only 5 observations /treatment for dry wt (roots were handled in groups of 5 to facilitate the dry wt pro- cedure). As with root length analysis (57), a large number of Obser- vations would be required to Show that toxin-stimulation of dry wt is highly significant. Increased dry wt could mean that toxin enhanced movement of materials from seed to root. The toxin preparation which inhibited root growth of ii. carbonum- susceptible corn at 0. 2 pg /ml was found to inhibit root growth of radish, oats, and tomato at 3pg/ml; barley, cucumber, sorghum, wheat, and fl. carbonum- resistant corn were inhibited at 45 )ug/rnl. Tomato root growth was stimulated 15% by toxin at 0. 2 ,ug/ml, barley roots were stimulated 15% by 3 pg /ml, and sorghum roots were stimulated 34 and 40% by 0. 2 and 3pg toxin/ml, respectively (57). Stimulation of sor- ghum root growth was much greater than stimulation of either suscep- tible or resistant corn. 39 Table 3. Effect of Toxin on Growth of Corn Seedling Roots Roots were measured after 87 hr exposure to White's solution with or without toxin, then placed in groups of 5, dried and weighed. Avg length is an average for 25 roots; avg wt was determined from 5 observations of 5 roots each. Susceptible and resistant seedlings were exposed to 0. 025 and l. 0 pg toxin/ml, respectively. Avg Length /Root Av; Wt/5 Roots Corn Hybrid Control Toxin Control Toxin mm mm mg mg Susceptible 115 133a 42. 5 45. 5 (Pr x K61) Resistant 123 134a 37. 5 40. 5b (Prl x K61) 3‘Significantly different (P< . 01) from control. bSignificantly different (. 05 < P < . 10) from control. 40 The stimulatory effects of toxin evidently precede the inhibitory effects (54); thus, they may be related to the Site of toxin action. Fur- ther experiments were built upon stimulatory effects in an attempt to understand the earliest effects of toxin on plant cells. Several possi- bilities were considered: 1) Toxin may act as a growth hormone; 2) toxin may interfere in energy metabolism of the cell; 3) synthesis or degradation of regulatory substances such as proteins may be affected by toxin; 4) toxin may alter inter— or intracellular solute movement. Experiments were designed to determine whether any of these possi- bilities is related to toxin action. Tests for hormone-like activity of toxin. -- Toxin-stimulation of growth of host and nonhost plants, as well as stimulation of certain physiological processes (54, 56, 57), suggested that toxin may have hormone-like activity for plants in general. Such is the case with hel- minthosporol, a metabolite produced by certain Helminthosporium spp. which has gibberellin-like activity. To test for hormone action, toxin was assayed in three biological systems known to be stimulated by both gibberellic acid (GA) and helminthosporol (36, 48, 81). Maryland Mammoth tobacco seeds ordinarily require light to germinate, but will germinate in darkness in the presence of GA or helminthosporol (36). My data (Table 4) show that GA, but not toxin, stimulated dark germination Of tobacco seeds. After the experimental period, all nongerminated seeds were incubated in the light for 1 week. Seeds in all categories listed in Table 4, except those exposed to >1. 0 pg toxin/m1, germinated and grew. 41 Table 4. Effects of Toxin and Gibberellic Acid on Germination of Tobacco Seeds in the Dark Maryland Mammoth tobacco seeds (SO/treatment) were placed on 3 filter paper discs saturated with 2 ml White's solution with or without GA or toxin in 5 cm petri dishes (25 seeds/dish). They were incubated in total darkness or in diffuse light on the laboratory bench for 6 days. Treatment Concentration Germination % Control - Dark . . . 0 Control ~ Light . . . 100 GA - Dark 10’3 M 88 10'4 M 86 10'5 M 4 Toxin - Dark 1000. 0 pg/ml 0 100. 0 pg /ml 0 10. 0 pg /m1 0 l. 0 pg/ml 0 0. 1 pg/ml O 0.01 pg/rnl 0 42 The rate of cucumber hypocotyl elongation is stimulated by GA or by helminthosporol (48). For comparison, GA or toxin was applied to apices of cucumber seedlings. Data in Table 5 Show that GA, but not toxin, stimulated cucumber hypocotyl elongation. Production of d—amylase in embryoless barley half-seeds is in- duced by GA and by helminthosporol (81). An experiment was designed to determine whether or not toxin can induce ot-amylase production, or affect GA-induced o(-amylase production in barley half-seeds. Two cultivars of barley (cv. Betzes and cv. Hudson) were used. Toxin alone did not induce oC—amylase production and had little or no effect on GA-induced o(-amy1ase production by half-seeds of cv. Betzes (Table 6). However, toxin at 1 and 0. 1 pg/ml caused striking stimu- lation Of GA-induced ot-amylase production by half-seeds of cv. Hud- son. Control seeds of CV. Hudson had much less amylase production than did control seeds of cv. Betzes. A germination test showed that all Betzes seeds were viable, but (20% of Hudson seeds were viable. Toxin (0.5 pg/rnl), GA (Io-6 M), or GA (lo-6 M) + toxin (0.5 pg/ml) had no effect on germination of Hudson seeds, compared with control seeds in water. Another batch of Hudson seeds which gave 99% germination was used to test the effect of toxin on oL-amylase production. Results were similar to those obtained with barley cv. Betzes (Table 6). Thus, toxin has no effect on o<-amy1ase production in normal barley seeds, but it can enhance the effect Of GA in certain seeds with low viability. 43 Table 5. Effects of Toxin and Gibberellic Acid on Cucumber Hypocotyl Elongation The procedure of Katsurni e_t_ai (48) was followed. National Pickling cucumber seedlings (lo/treatment) were treated with 0. 01 ml 95% ethanol with or without GA or toxin, and incubated 3 days in light. m Avg Hypoc otyl Treatment Dosage Elongation ng /plant mm Control (nontreated) . . . 2 Control (ethanol) . . . 2 GA 10. 0 11 l. 0 7 Toxin 1000. 0 0 (dead) 100. 0 0 (alive) 10. 0 2 1. 0 2 0. 1 2 0. 01 2 0. 001 2 44 Table 6. Effect of Toxin and Gibberellic Acid on 0< -Amy1ase Production by Embryoless Barley Half-Seeds One unit °<-amylase is defined as a change of 10 Klett units/ min. GA concentration was 10'9 M. u-Amyla se /Half-Seed Treat- Toxin Barley cv. ment Concn. _ Medium Seeds Total pg /ml units units units Betzes Control . . . 0 70 70 GA . . . 117 105 222 Toxin 0.1 0 67 67 GA & Toxin 10. 0 101 103 205 1. 0 140 108 248 0.1 109 95 205 0. 01 113 99 212 0. 001 99 98 198 Hudson Control . . . 1. 5 4. 5 6. 0 GA . . . 9. 0 7. 5 16.5 GA 8: Toxin 100.0 7.0 6.5 13.5 10. 0 5. 0 6. 5 11. 5 1 0 16 5 13 5 30 0 45 The production of soluble sugar by embryoless corn endosperms can be stimulated by exposure to GA (43). This implies an increase in amylase activity, which according to Dure (12) is primarily fl -amy1ase. Toxin at 10 and 1 pg/ml was found to inhibit GA-induced amylase ac- tivity in susceptible corn endosperms (Table 7). Toxin at low concen- trations had little or no effect on amylase activity. Experiments with toxin and isolated mitochondria. -- Toxin- enhanced growth and metabolism should require increased expenditure of cellular energy, Both toxin and infection are known to increase the respiration rate of intact tissues (56). The possibility of an effect of toxin on energy metabolism or on other intracellular sites was tested with isolated organelles. Respiration rates and phosphorylation by corn mitochondria in the presence of exogenous NADH were measured in a series of experi- ments. Results (Figure 2) show that toxin did not affect electron trans- port rates of either phosphorylating or nonphosphorylating mitochondria, nor did it affect the respiratory control ratio (RCR) or the P:0 ratio. A known uncoupler, 2, 4-dinitrophenol (DNP), caused an increase in the rate of electron transport. These data agree well with published re- sults using corn mitochondria (76). Oxidation and phosphorylation after preincubation of mitochondria in the presence and absence of toxin were determined in another series of experiments using 2 different substrates (Table 8). With NADH as the substrate, mitochondria which were not preincubated had normal 46 Table 7. Effect of Toxin on Gibberellic Acid-Induced Amylase Production by Susceptible Corn Endosperm One unit amylase is defined as a change of 10 Klett units/min. GA concentration was 10'5 M. Amylase/Endosperm Treatment Toxin Concn. Medium Endosperm Total pg /m1 units units units Control . . . 80 392 472 GA . . . 164 652 816 GA & Toxin 10. 0 40 220 260 1. 0 95 528 623 0. 1 180 600 780 0. 01 158 712 870 0. 001 192 632 824 47 HC-Toxin: Respiration rates of corn mitochondria NADH 1 O 7< ._g O X 100 nmoles 02 I-—l min——1 Rate - nmoles Ozlmg protein/min Figure 2. Respiration rates and phosphorylation of corn mito- chondria in the presence and absence of'toxin. The reaction medium contained 0. 2 M KCl, 0. 02 M HEPES (pH 7. 5), 2 mM MgC12, 1 mg BSA/m1 and 250 pg mitochondrial protein/m1 in a final volume of 2ml. The following additions were made as indicated: 1 pmole NADH, 2 pmoles ADP, 9 pmoles NazHPO4 labelled with Na2H32PO4, 0. 08 pmole DNP, and 40 pg toxin. 48 Table 8. Respiration and Phosphorylation by Corn Mitochondria Preincubated in the Presence and Absence of Toxin Reaction conditions and additions are given in Figure 2. ADP was present in all reaction mixtures; Pi was added as indicated. Malate-pyruvate was 20 pmoles each/2 ml reaction mixture. Prein- cubated mitochondria were held 1 hr at 24 C in 0. 3 ml 0. 38 M sucrose containing 750 pg mitochondrial protein with or without 6 pg toxin or inactive toxin. To measure respiration and phosphorylation, 0. 2 ml Of this mixture was added to reaction mixture (See Figure 2). RCR = respiratory control ratio. 02 Uptake Substrate Tregirreent RCR P:O at -Pi +Pi nmole 5 /mg protein/min NADH None 127 280 2.21 1.63 1 hr Control 143 170 1.19 0.96 1 hr Toxin 204 214 1. 05 1.46 1 hr Inactive Toxin 164 218 _ 1.33 1.50 Malate- None 45 69 1. 53 2. 33 Pyruvate 1 hr Control 48 39 0. 81 2. 24 1 hr Toxin 40 36 0. 90 2. 24 1 hr Inactive Toxin 57 36 0. 63 2. 06 49 respiratory rates, RCR, and P:O ratios. Mitochondria incubated for 1 hr at 24 C had slightly higher nonphosphorylating respiratory rates than did mitochondria stored on ice, but the phosphorylating rates, RCR, and P:O ratios were below the values for mitochondria stored on ice. Preincubation of mitochondria for 1 hr in the presence of toxin or inactive toxin at 24 C caused slight stimulation of respiratory rates, but had essentially no effect on RCR and P:O. The P:0 of the preincu- bated control was unusually low in this experiment, for unknown rea- sons. With malate-pyruvate as the substrate, respiratory rates of fresh mitochondria were relatively low but P:O ratios were comparable to those reported for similar experiments (76). Preincubation of rnito- chondria for 1 hr in the presence of toxin or inactive toxin at 24 C had no effect on mitochondrial functions. The mitochondrial functions just described are dependent on the integrity of the mitochondrial membrane for normal operation (76, 126). No change in these functions after toxin treatment suggests that the mitochondrial membrane is immune to toxin. However, negative evi- dence is not conclusive and another parameter of membrane integrity was used. Light-scattering is a direct measure of volume changes in mitochondria (64); volume changes reflect water flow caused by solute fluxes across the membrane (82, 114). If toxin affects the rnitochon- drial membrane, it should affect the light-scattering properties of mitochondria . 50 Corn mitochondria swell spontaneously when placed in a buffered salt solution (76, 114). Toxin had no effect on this spontaneous swel- ling (Figure 3) but gramicidin D increased the swelling rate, as others have found (76). Addition of NADH caused swelling to stop and mito- chondria contracted slightly, as they normally do (76). Toxin had no effect on stabilized mitochondria, but gramicidin D reversed the con- tracted state. Exgeriments with toxin and isolated chloroplasts. -- Chloroplasts were used in experiments similar to those with ‘mitochondria. Results (Figure 4) show that toxin had no effect on electron transport by phos- phorylating or by nonphosphorylating chloroplasts. Ammonium sulfate completely uncoupled electron transport, indicating a normal response of chloroplasts to an uncoupling agent (52). Toxin had no effect on light-scattering by chloroplasts in the pre- sence or absence of actinic light (Figure 5, left). This indicates that toxin did not induce active or passive swelling of chloroplasts. Ammo- nium sulfate was included as a control to confirm that chloroplasts can be induced to swell in light and shrink in darkness (45). Toxin had no effect on light-scattering when added during the experiment in the ab- sence of actinic light (Figure 5, right). This eli'rninates the possibility of an instantaneous effect of toxin on passive swelling by chloroplasts. Triton X-100, which affects chloroplast membranes in the dark (44), was included as a control to Show induced passive swelling. Chloro- plast functions and membrane integrity were not affected by toxin. At 51 HC-Toxin; Volume changes in corn mitochondria NADH Toxin Gram D i t l A0D520-0.1 J— r—l min—t _ Figure 3. Volume changes in toxin-treated and control mito- chondria. The reaction medium contained 0. 2 M KCl, 0. 02 M HEPES (pH 7. 5), 2 mM MgC12, 1 mg BSA/ml, and 250 pg mito- chondrial protein /ml in a total volume of 2 ml. Additions of l pmole NADH, 40 pg toxin, or 5 nmoles gramicidin D were made as indicated. 52 HC-Toxin: Ferricyanide--Hill Reaction in Corn Chloroplasts i thton *~\ - \ -ADP-PI T J I -ADP-Pi /‘ 4: 0° + ' ooom-o. 1 ‘5‘ mm 54 +ADP+Pi J. . . . + Toxin Degre'ilse +ADP+P' ./ J R -ADP-Pi I? NH4+ 30'? Rate: pmoles Fecy/hrlmg Chl. 1—30 Sec-—l Figure 4. Electron transport and phosphorylation by corn chloroplasts in the presence and absence of toxin. Chloroplasts were suspended in a reaction mixture containing 0. l M sucrose, 0. 01 M KCl, 0. 05 M Tricine-NaOH (pH 7. 8), 0. 001 M MgClz, 0. 4 mM potassium ferricyanide, 20 pg chlorophyll/m1 and the following components where indicated: 4 mM (NH4)ZSO4, 1 mM ADP, 4. 5 mM NaZHPO4, or 20 pg toxin/ml. 53 HC-Toxin: Light Scattering by Corn Chloroplasts Control W T 1 ' t t Inactive W Water Toxun 1' NH4+ M T t Toxin A00540'0. l 1‘ Triton -L DARK Figure 5. Light-scattering by corn chloroplasts in the presence and absence of toxin. Conditions for experiment on left were as des- cribed in Figure 4, except KCl was 0. 1 M. Conditions for experiment on right were as those on the left except ferricyanide was omitted and 0. 00025% Triton X-100 was added where indicated. 54 the end of each experiment with chloroplasts and mitochondria, toxicity of the toxin preparation was bioassayed. Full toxicity and host-speci- ficity was retained in all cases. Preliminary experiments by Samaddar (unpublished) indicated that susceptible oat chloroplasts were not affected by HV-toxin. I have used HV-toxin with chloroplasts from susceptible oats (cv. Park) in electron transport and light-scattering experiments. The procedure was the same as described for corn chloroplasts. HV-toxin, from a preparation which completely inhibited susceptible roots at 0. 0016 pg/ ml, was used at 1. 6 and 0.16 pg/ml. Results showed no effect of HV- toxin on oat chloroplasts. Effect of toxin on nitrate reductase activity. -- Enhanced meta- bolic activity could result from increased enzyme synthesis. Toxin is known to enhance incorporation of 14C-amino acids into TCA insoluble materials of susceptible corn tissues (54). However, toxin had little or no stimulatory effect on GA-induced amylase activity in barley (a nonhost) or in susceptible corn. Another inducible enzyme was selec— ted to further test the possibility that toxin affects protein synthesis £232. Nitrate reductase (NR) is induced by its substrate, is easily manipulated, and has been studied extensively in corn (3). Recent de- velopment of an EM assay (21) has greatly facilitated NR investiga- tions. Seed tissues were used for the first series of experiments, be- cause they are easy to handle under aseptic conditions. Microbial con- tamination can be an important source of error in enzyme studies. 55 Induction of NR in various seed tissues was tested in preliminary experiments. Tissues were allowed to imbibe 24, 48, or 72 hr; NR was then induced by transferring tissues to NO3—containing solution. The results show that inducibility of intact seeds was low but increased with age whereas inducibility of embryos, scutella, and axes was high and decreased with age (Table 9). There was negligible NR induction in endosperms. Axes were selected for further studies because they are more active than the other tissues on a dry wt basis. More NR was induced in axes which were allowed to imbibe in nutrient solution than in those allowed to imbibe in water (Table 10). Induction of NR in axes was not affected by light, darkness, (NH4)ZSO4 (500 mg/l), or substitution of White's solution for modified Dure's solution. NR induction was not affected in axes that imbibed toxin solution for 12 hr. Toxin exposures of 24-72 hr inhibited NR induction but did not eliminate it (Figure 6). In fact, NR activity in toxin-treated axes was higher at 72 hr than at 24 and 48 hr. The same pattern was evi- dent when the data were plotted on a dry wt basis. Other experiments showed that there was less NR induction in embryonic shoots than in embryonic roots. Induction Of NR in shoots was inhibited more by toxin than was NR induction in roots. The rate of NR induction was about the same whether embryonic roots were separated from embry- onic shoots before or after imbibition. The toxin exposure time required to affect NR induction was tested by including toxin in the induction medium. Axes were allowed 56 Table 9. Induction of Nitrate Reductase in Corn Seed Tissues Seeds were surface sterilized and dissected aseptically. Five tissues/treatment were allowed to imbibe in 12 ml water containing 20 pg chloramphenicol/m1 in 10 cm petri dishes. Enzyme was induced aseptically in 4 ml 0. 05 M KNO3 containing 20 pg chloramphenicol/ml in 50 m1 flasks shaking at 160 oscillations/min for 7 hr. Imbibition Time Tissue N02 Production hr nmoles/g dry wt/hr 24 Whole Seed 20 Endosperm 2 Embryo 1103 Scutellum 957 Axis 6559 48 Whole Seed 146 Endosperm 4 Embryo 485 Scutellum 222 Axis 2468 72 Whole Seed 261 Endosperm 4 Embryo 75 Scutellum 201 Axis 1043 57 Table 10. Effect of Nutrients on Capacity for Nitrate Reductase Induction in Corn Embryonic Axes Axes (4 /treatment) were allowed to imbibe aseptically in 5 cm petri dishes in 2 ml Of water or modified Dure's nutrient solution. The induction medium corresponded to the imbibition ‘medium and con- tained 0. 05 M KN03. Induction time was 6 hr. Imbibition Time Nutrients N02 Production hr pmoles/g dry wt/hr - 4. 9 24 + 6. 0 - 1. 7 48 + 10. 2 _ 2. O 72 + 13.9 58 30 ’ Control t- 1 E 3 20 - ; N S E g . : Toxm 10 ' i "1 l 1 l l 0 24 48 72 Imbibition time (hr) Figure 6. Effect Of toxin on induction of NR in corn embryonic axes as affected by imbibition time. Axes imbibed aseptically in 2 ml modified Dure's solution with or without toxin (2 pg/ml) in 5 cm petri dishes (4 axes/dish). Enzyme was induced aseptically in 2 m1 imbibi- tion solution containing 0. 05 M KNO3 in 50 m1 flasks incubated 8 hr on a shaker (200 oscillations/min). 59 to imbibe. for 36 hr; NR was then induced by placing axes in NO3-solu- tion with or without toxin. At intervals NR activity in the axes was determined. The data (Figure 7) show no effect Of toxin during the first 2 hr Of induction. Between 2 and 8 hr of induction NR activity de- creased in the control but continued to increase in toxin-treated axes. Thus, the earliest observable effect of toxin was stimulatory rather than inhibitory; it was evident 4 hr after exposure to toxin. The effect Of toxin or of N03 on NR induction in axes was exam- ined by varying toxin and N03 concentrations in the induction medium. Results in Figure 8 show the effect of toxin concentration on NR induc- tion. Toxin concentrations of 2 pg or more/rnl caused maximum stim- ulation of NR induction. Considering variability between replicates, there was probably no real difference between 2, 20, and 200 pg toxin/ m1. A N03 concentration of 0. 05 M was optimum for both induction and toxin-stimulation of NR activity (Figure 9). Toxin-stimulated NR induction suggested that toxin may enhance total protein synthesis. To test this possibility, the experiment just described was repeated and total protein in the axes was estimated. Toxin apparently had no significant effect on total protein content (Table 11). Since induced NR is less than 0. 1% of the total protein (42), its contribution was negligible. A similar experiment with green corn leaf tissue gave Similar results. The effect of toxin on NR degradation in corn axes was tested. Axes imbibed for 36 hr; NR was induced for 2 hr in the presence or 60 8 nmoles Nozlaxislrr N O Induction time (hr) Figure 7. Effect of toxin on induction of NR in corn axes. Axes imbibed aseptically for 36 hr in 15 m1 modified Dure's solution on filter paper in a 15 cm petri dish. Enzyme was induced aseptically in 10 m1 imbibition solution which contained 0. 05 M KNO3 with or without toxin (20 pg/ml) in 300 m1 flasks (3 axes/flask) on a shaker (200 oscil- lations / min) . 61 (,0. e 40- g N o z .3 . O E C 20- 0 4 3 Induction time (hr) Figure 8. Effect of toxin concentrate on induction of NR in corn axes. Axes imbibed aseptically for 36 hr on filter paper wetted with 15 m1 modified Dure's solution in a 15 cm petri dish. Enzyme was induced aseptically in 4 m1 imbibition solution containing 0. 05 M KNO3 with or without toxin in 50 ml flasks (3 axes/flask) on a shaker (220 oscillations/min). Toxin concentrations (pg/m1) are indicated. 62 4o .. h Toxin 5 g N o O z In 0 2 O E 20 )- contrOI o 1 1 J u 0 0.5 5.0 50.0 500.0 log N03 concentration (mM) Figure 9. Effect of N03 concentration on toxin-stimulated induction of NR in corn axes. Axes imbibed aseptically for 36 hr on filter paper wetted with 15 m1 modified Dure's solution in a 15 cm petri dish. Enzyme was induced aseptically in 6 m1 imbibition solution containing 0. 05 M KNO3 with or without toxin (2 pg/ml) in 50 m1 flasks. Induction was for 4 hr on a shaker (220 oscillations/ min). 63 Table 11. Protein Content of Susceptible Corn Embryonic Axes After Induction of Nitrate Reductase in the Presence or Absence of Toxin Axes (5 /treatment) imbibed aseptically in 15 ml modified Dure's solution on filter paper in 15 cm petri dishes for 36 hr. For induction, axes were incubated in 10 ml imbibition solution + 0. 05 M KNO3 with or without toxin (20 pg/ml) in 300 m1 flasks shaking at 200 oscillations/ min. Axes were homogenized and protein was extracted and estimated by the methods of Filner (23). Ranges between 2 samples are indi- cated. Pr ote in C ontent Induction Time Control Toxin hr pg/axis pg /axis 0 203 f54 4 233 T17 251 I1 8 238 i12 265 is 64 absence of toxin. Then axes were placed in NO3-less solution with or without toxin and loss of NR activity was observed. Results (Figure 10) show that about half the i_nm NR activity disappeared in both toxin-treated and control axes in 4 hr. NR is reported to have a half- 1ife of 4 hr in corn tissues (107). Root tips and green leaves were used in experiments similar to those with axes, except that nonsterile conditions were employed. Ad- dition of toxin to NR induction medium of root tips gave results similar to those shown in Figure 7 for axes. Toxin had no effect on NR activity after 2 hr induction. Between 2 and 4 hr, NR activity decreased slight- ly in the control but continued to increase in toxin-treated tips. This resulted in 88% stimulation by toxin after 4 hr induction. The effect of toxin on initial kinetics of NR induction in corn root tips was studied. Seedlings were first exposed to toxin 4 hr, then root tips were excised and placed in NR induction medium. Results (Figure 11) show an increase in NR activity of toxin-treated axes which was apparent from the beginning of the induction period. This difference between toxin-treated and control tissues persisted but did not change throughout the experiment. Similar parallel lines were obtained with axes after 4 hr toxin pretreatment. Stimulation of NR activity by toxin at 0 induction time and the failure of toxin-treatment to increase the slope of the induction curve suggested that the effect of toxin was not on induction of NR. To fur- ther check this point, NR activity in root tips was estimated 65 Toxin nmoles Nozlaxis/hr Control 20‘; Degradation time (hr) D Figure 10. Effect of toxin on degradation of NR .131 X139; Axes imbibed aseptically for 36 hr on filter paper wetted with 15 m1 modi- fied Dure's solution in a 15 cm petri dish. Enzyme was induced asep- tically in 6 m1 imbibition solution containing 0. 05 M KNO3 with or without toxin (2 pg/ml) in 50 ml flasks on a shaker (200 oscillations/ min). After 2 hr induction, axes were rinsed in sterile water and transferred to sterile NO3-less modified Dure's solution with or without toxin (2 pg/ml) in 50 m1 flasks on a shaker (200 oscillations/ min). At intervals (0, 2, and 4 hr), i_nvi_vo NR activity was deter- mined. 66 nmoles Nozlroot tip/hr Induction time (hr) Figure 11. Effect of pretreatment with toxin on induction of NR in corn root tips. Seedlings were grown in petri dishes in N03- less modified White's solution. Seedlings were placed in 30 ml of the same solution with or without toxin (20 pg/ml) in 15 cm petri dishes for 4 hr, then NR was induced by placing seedlings in 30 m1 White's solution containing 10 mM KNO3 and 0. 104 pM Na2M004. At intervals 5 mm root tips were excised and assayed for NR acti- vity in 0. 5 ml assay medium in 3 m1 vials (5 tips/vial). Contents Of vials were then boiled and N02 production was determined by adding color reagents. Fresh wt of a 5 mm root tip is about 3. 5 mg. 67 simultaneously with both i_n_v_iv_o and i_nv_itr_c_>_ assays. Results of the i_r_1_vi_vg assay (Figure 12) showed the usual stimulatory effect of toxin. However, when NR was extracted from the tissue and an 323.1152 assay was performed, toxin-treatment appeared to have had no effect on, or perhaps inhibited, NR inducibility. Thus, toxin-enhanced NR activity i_n_vi_vq did not appear to result from an effect of toxin on induction of NR. Effect of toxin on Nolaccumulation and compartmentation. ~- The tissue extracts which were used for the i_n__vi_tI£ assay of NR ac- tivity (Figure 12), were tested for N03 content. Toxin-treated root tips accumulated N03 at a faster rate over the 3 hr induction period than did control root tips (Figure 13). Results of another experiment showed that toxin-induced N03 accumulation was affected by tempera- ture (Table 12). The ing/319 assay for NR activity was used in a further test of the effect Of toxin on N03 uptake. This experiment was based on the fact that N03 must enter the cell to be reduced. If NR content is held constant, any increase in N03 reduction ‘must be caused by increased N03 uptake. Noninduced toxin-treated and control leaf sections were placed directly into i_1_i__v_i_v_9 assay medium rather than into induction medium. Within 10 min, more N03 was reduced in toxin-treated than in control leaves, and the slope of the curve for toxin-treated leaves was steeper than that for the control (Figure 14). Thus, N03 entered toxin-treated cells at a faster rate than it entered control cells. 68 HC-‘I’oxin Pretreatment In Vivo & In Vitro Assn O N I a I In Vivo — nmoIes N02 /root tip/hr «h —I In Vi'fo r-- 0 1.5 3 Induction Time (hr) Figure 12. Effect of pretreatment with toxin on induction of NR as measured by i_riziyg and in vitro assays. Seedlings were grown in NO3-1ess modified White's solution, then placed in 30 m1 of the same solution with or without toxin (20 pg/rnl) in 15 cm petri dishes for 4 hr. Enzyme was induced by placing seedlings in 30 m1 White's solution con- taining 10 rnM KNO3 and 0. 104 pM NaZMoO4, with or without toxin (20 pg/ml). At intervals, 5 mm root tips (10 tips/replicate) were excised and NR activity was determined by i_n_ £1.12 and E vitro methods. 69 HC-‘l’axin Pretreatment Effect on N01 Uptake nmoles NO3/raat tip 1.5 3 Induction Time (hr) Figure 13. Effect of pretreatment with toxin on N03 accum- ulation by corn root tips. Supernatants from the i_n_vitro assay described in Figure 12 were assayed for N03 content. 70 Table 12. Effect of Temperature on Toxin-Stimulated N03 Accumulation by Corn Root Tips Seedlings, grown in 15 cm petri dishes in NO3—1ess modified White's solution, were transferred to the same solution with or with— out toxin (20 ug/ml) in 15 cm petri dishes for 4 hr. They were then placed in White's solution containing 10 rnM KNO3 at 3 or 24 C. At intervals 5 mm root tips were excised, rinsed and boiled in O. 8 ml water (5 tips/replicate). The N03 content of the water was deter- mined. NO3 Accumulation Time of 010 Exposure 3C 24 C 130 N03 Control Toxin Control Toxin Control Toxin hr nmoles/root tip nmoles/root tip 1 l. l 1. 5 2 2.1 3.0 15.8 32.1 2.61 3.09 4 4.4 6.2 71 HC-Toxin Pretreatment N03 Reduction by Leaves .8 n O \ N 0 Tax 2 . 0 0 '3 ’1’ E .4 - I c I / / ,’Ck . .II / [I --'40’ J 10 3'0 Incubation in N03 (min) Figure 14. Effect of pretreatment with toxin on N03 uptake and reduction in the absence of NR induction. Seedlings were grown for 12 days in NO3-1ess modified White's solution. Cut- tings were placed in 15 ml water with or without toxin ( 20 pg/ml ) in a 50 ml beaker under Gro—Lux lamps for 4 hr. Tertiary leaves were cut into 5 mm2 sections, 100 mg was placed in 3 ml 0.1 M phosphate buffer (pH 7. 5) + 0. 1 M KNO3 in a 50 ml flask. Nitrogen was flushed through the solution 1 min, flasks were stoppered and incubated in the dark at 30 C. At intervals contents of flasks were boiled and assayed for N02. 72 A recent report (22) indicates that N03 is compartmented in plant cells. A small portion of the cellular N03 is available for re- duction by NR and is known as the metabolic pool; the rest of the cel- lular N03 is not available to NR and is called the storage pool. This compartmentation was demonstrated in corn leaves (Figure 15). Seed- lings were grown in the presence of N03 so that tissues contained both N03 and NR. Leaf sections were placed under anaerobic conditions in the dark. This allows reduction of N03 to N02, but N02 accumulates and is excreted from the cells. Accumulation of N02 continues as long as N03 is available to NR. Production of N02 was linear for 2 hr (Figure 15). Production of N02 then stopped, which resulted in a pla- teau at approximately 2 pmoles NO3/g tissue. The plateau indicated that all N03 available to NR was exhausted, although only 8% of the total cellular N03 (24 pmoles/g in this experiment) had been reduced. Reduction of N03 was resumed when exogenous N03 was added to the incubation medium. This shows that N03, not some other factor, was limiting N02 production. The capacity of the metabolic pool in corn leaves is about 2 pmoles N03 /g leaf tissue (22 and Figure 15). An attempt was made to determine the capacity of the 'metabolic pool in corn root tips. Results (Table 13) show that the pool in root tips contains about 0. 03 pmole NO3/ g and is depleted so rapidly that it is almost nondetectable. Ethanol re- leased NO3 from the storage pool of root tips, as it does in corn leaves (unpublished), tobacco cells (22), and barley aleurone layers (21). 73 Metabolic N03 Pool in Corn Leaves 4k——N2———9 (——N2———)l .... II, I "2 N03 ’p. nmoles NOz/g \T‘ Z .9. I l 2 4 Incubation Time (hr) Figure 15. Evidence for a metabolic pool of N03 in corn leaves. Total N03 in tissue was 24 nmoles/g; the metabolic pool contained approximately 2 pmoles/g. 74 Table 13. The Metabolic N03 Pool in Corn Root Tips Seedlings were grown in White's solution containing 20 rnM KNO3 + Fe and M0 for 60 hr. Root tips 5 mm long (10 tips/replicate) were placed under anaerobic conditions within 1-2 min after excision. Ethanol (5%) was included in the incubation medium where indicated. Anaerobic Incubation Time N02 Production min nmoles/g 10 27 20 36 40 2'7 60 27 30 + ethanol 1057 75 Similar results were obtained with 1 cm root sections taken 5 mm be- hind the root tip (unpublished). Since toxin affected movement of N03 into cells, it seemed ap- propriate to determine if toxin affected 'movement of N03 within the cell, i. e. , from storage to metabolic pool. Results of an experiment designed to test this possibility are shown in Figure 16. Leaf tissue containing N03 and NR was incubated anaerobically for 2 hr to exhaust the metabolic N03 pool. Buffer with or without toxin was added and leaves were incubated aerobically 4 hr to give toxin time to act. One group of leaves received exogenous N03 15 min before the end of the aerobic period. This was a control to determine if NR was still active 4 hr after metabolic pool N03 was exhausted. Leaves were then re- turned to anaerobic conditions and N02 production was monitored. The treatment which received exogenous N03 produced N02, indicating the presence of active NR. Control and toxin-treated leaves did not pro- duce additional N02, indicating that toxin did not release N03 from the storage N03 pool. Characteristics of toxin-stimulated N03 accumulation by roots. -- A toxin—induced change in the plasmalemma could lead to enhanced N03 uptake. This is the most plausible explanation considering that mito- chondria, chloroplasts, and storage pools of N03 are not affected by toxin. The nature of the stimulatory effect was explored in further ex- periments with N03 accumulation. One experiment showed that N03 accumulation from a 10 mM solution was linear with time for at least 76 HC-Taxin Intracellular localization of N03 fl +N 2 ~- Ck . T T Tox ' Tox N03 Nz'fie 02 flé—Nz—Q i l a Incubation Time (hr) Figure 16. The metabolic pool of N03 in toxin-treated and control corn leaves. Ck = control without addition of exogenous N03; Tox = toxin-treated without addition of exogenous N03. 77 4 hr (Figure 17). The results of the experiment with corn root tips pretreated with toxin (Figure 13) suggest that toxin-stimulated NO3 accumulation is also linear with time. The condition of roots during the toxin—treatment period affected the rate of N03 accumulation and toxin-stimulated N03 accumulation (Table 14). Excised roots and roots of intact seedlings were exposed to solutions with or without toxin for 4 hr. This resulted in a 4 hr aging period for excised roots. Excised roots were aerated as they aged. Roots of intact seedlings were excised after the toxin-treatment period and can be considered fresh tissue. All roots were placed in N03 solution and the rates of N03 accumulation were determined. Aged control roots accumulated N03 twice as fast as fresh control roots (Table 14). Toxin-treatment caused a large stimulation of N03 accu- mulation by fresh roots but had only a small stimulatory effect on aged roots. An additional treatment included excised roots which were not aerated, but floated on the surface of a solution during the toxin- treatment period. Such roots had a reduced rate of N03 accumulation, perhaps because of poor aeration. Toxin-treatment caused a marked increase in the rate of NO3 accumulation by aged, nonaerated roots. There was evidence that NO3 accumulation is an active process. One such indication was the effect of temperature on N03 accumulation and toxin-stimulated NO3 accumulation (Table 12). Accumulation of N03 against a concentration gradient by both toxin-treated and control 78 8 6 2’ M 0 2 § ‘5’ 4 q 2 o Absorption time (hr) Figure 17. Accumulation of NO by corn roots. Seedlings were grown over 4000 ml 0. 2 mM CaClz. They were then placed in 50 ml 0. 5 mM CaCl2 + 10 rnM KNO3 in 15 cm petri dishes for the times indicated, and rinsed 3 times in 0. 5 mM CaClz. Roots were excised, blotted, weighed (0. 5 g/replicate), and N03 content was determined. 79 Table 14. Effect of Toxin on N03 Accumulation by Aged and ° Fresh Corn Roots Fresh roots were prepared as follows. Intact seedlings were incubated 4 hr in 50 ml 0. 5 mM CaClz with or without toxin (10 pg/ml) in 15 cm petri dishes. Roots were excised after incubation. For aged tissues, roots were excised and incubated 4 hr in 50 ml 0. 5 mM CaClz with or without toxin (10 ug/rnl) under one of the following conditions: a) aerated in a 100 ml beaker; b) shaken at 150 rpm in a 300 ml flask; or c) floated in a 15 cm petri dish. Fresh and aged roots were al- lowed to absorb NO3 from solutions containing 0. 5 mM CaClz and 0. 4 mM KNO3. Condition Condition NO3 Accumulation , Toxin-Induced of for St' 1 . Tissue Aging Control Toxin 1mu ation nmoles/g/hr % Fresh .. . 265 383 45 Aged Aerated 510 623 22 Shaken 575 642 12 Floated 172 401 133 80 roots is another indication of an active process. Toxin-treated roots exposed to a 0.4 rnM N03 solution for 50 min acquired an internal NO3 concentration of 1.12 mM. Control roots did not establish a concentra- tion gradient under these conditions. However, control roots exposed to a 0. 04 mM NO3 solution for 45 min accumulated an internal concen- tration of 0. 068 mM. The rate of N03 accumulation by control roots was less than that by toxin-treated roots. This explains the failure of control roots to establish a concentration gradient in 0, 4 HM NO3 during a brief exposure. The rate of N03 accumulation by control roots (ca 300 nmoles/g/hr) in 0. 4 HM N03 indicates that they begin to accu- mulate N03 against a concentration gradient within l-2 hr. Accumula- tion of N03 is linear for at least 4 hr (Figure 17). There were several experiments to test the effect of other ions (K, Ca, C1, Na, $04) in the experimental solution on N03 accumulation. Seedlings were exposed to solutions with or without toxin for 4 hr. Roots were excised and placed in NO3-containing solutions in the pre- sence of varying concentrations of other ions. The data (Table 15) suggest the following. 1) There was considerable variation in the rates of N03 accumulation by toxin-treated and control roots in different ex- periments and also between similar treatments in the same experiment. 2) These experiments, and experiments to be described later, indi- cated that Ca is required for the maximum rate of N03 accumulation but not for toxin-stimulation of N03 accumulation (See Table 17). 3) Presence or absence of other ions or combinations of ions had no 81 Table 15. Effect of Exogenous Ion Content and Concentration on Toxin-Stimulated N03 Accumulation by Corn Roots Seedlings were exposed to 0. 5 leI CaClz with and without toxin (10 ug/ml) for 4 hr. Roots were excised and placed in experimental solutions for 30-45 rnin. Ion Concentrations NO3 Accumulation Expt. Stimulation No. , by Toxin K Ca N03 Cl Control Toxm mM mM mM mM nmole s /g /hr % l 0.4 0.5 0 4 1.0 218 470 115 0.5 1.0 . .. 338 600 78 0 4 0.5a 0.4 . .. 232 495 114 . . 0. 7 0 4 l. 0 286 423 48 0. 4 0. 05 0 4 0.1 148 240 62 2 0 04 0.4 0.04 0.8 90 110 22 0.4 0 4 0.4 0. 8 316 437 39 4 0 4 4. 0 930 1160 25 0 . . 0. . 181 288 59 0.4 0.4 0.4 320 455 42 0.4” 171 238 39 3 0.4 0. 5 0.4 1. 0 249 550 120 10.0 .. . 10.0 1488 1780 20 9. 9 0. 05 10.0 1760 2000 14 9.0 0.5 10.0 1660 2040 23 5. 10.0 . . 1520 1975 30 0. 5 0.4 0. 6 276 404 46 aCa was added as 804 salt. bNO3 was added as Na salt. 82 consistent effect on the rate of N03 accumulation or on toxin-stimula— tion of N03 accumulation. 4) The percent stimulation by toxin may have been less at high than at low N03 concentrations. Variability in N03 accumulation between identical experiments run simultaneously and between replicates of treatments within experi- ments was tested. Maximum variability between replicates and be- tween experiments was 14 and 13%, respectively (Table 16). Root tips 3 cm long accumulated NO3 faster, on a fresh wt basis, than did whole roots (Table 16). In subsequent experiments, 5-6 cm root tips were used as a compromise between efficient use of tissue and use of the most active portion of the root. Several experimental conditions were evaluated for their effect on N03 accumulation and toxin-stimulated N03 accumulation. Seedlings were exposed to solutions with or without toxin for 4 hr. Conditions for the NO3-absorption period were varied as follows. 1) Toxin- treated and control roots were excised and placed in identical experi- mental solutions in separate flasks. 2) Toxin-treated and control in- tact seedlings were placed in identical experimental solutions in separ- ate flasks. 3) Toxin-treated and control roots were excised and placed in the same experimental solution, buffered at pH 5. 8 with MES. 4) Toxin-treated and control roots were excised and placed in the same experimental solution containing tris as the counterion for N03. Re- sults (Table 17) indicate the following. 1) The same results were ob- tained whether toxin-treated and control roots were in the same or 83 11 m3... Sm N a. 93 m if” amt“ 4S m ... m; 3.... mmm a 3:. boom cod N w cmm m?“ voN N am». may 300% mm mm ..T wow H u. wvm H 305$ om. u£\w\moHoEa u£\w\moHoE: mxmmfim mom—moflmom mxmmfim moumofimom 5N0 .H. fixo .H. Ho .Sao O . 02 >3 oucmm owcmm Mmmgh 2638 83135828 Gowuafisaao o< mOZ .muoou mo ammo. Houuaoo N was woumouuuaflxou N pafifidoo Mom: comm .mOZM 25 v .O was NaOmO 28 m .o wadfimuaoo mxmmm 5 @6039 can @33on who? mm? uoou So m no muoou 3055 ..E v HOW Sufi m1 c3 5x3 305?? no £3, NHOmO 28 m .o E cocoa one? mwafipoom 2.532500 Hmuaoefinomxm peopafim nopcb Boom auoO >3 cofluafiagaoo< mOZ .«o moumm E aofimwum> A: 3an 84 Table 17. Effect of Experimental Conditions on Toxin-Stimulation of N03 Accumulation by Corn Roots Seedlings were placed in 0. 5 mM CaClz with or without toxin (10 pg/rnl) for 4 hr. Roots were exposed to N03 solutions under the following conditions. A) Toxin-treated and control roots were ex- cised and placed in separate flasks. B) Toxin-treated and control intact seedlings were placed in separate flasks. C) Toxin-treated and control roots were excised and placed in the same flasks, which contained solution buffered at pH 5. 8 with 0. 2 mM MES. D) Toxin- treated and control roots were excised and placed in the same flask, containing 0. 4 mM HNO3 adjusted to pH 6. 0 with tris. Tissues for flasks A, B, and C were rinsed in 0. 5 mM CaClz and exposed to ex- perimental solutions containing 0. 5 HM CaClz and 0.4 le KNO3. Roots for flask D were rinsed well with water before exposure to the experimental solution. Conditions N03 Accumulation . . Expt. Stimulation for N03 N b T , Absorption 0' Control Toxin Y ox1n nmole s /g /hr ‘70 A. Toxin-treated and 1 286 785 175 control excised roots in separate 2 398 815 105 flasks. B. Toxin-treated and l 559 1570 181 control intact seed- lings in separate 2 504 1083 116 flasks. C. Toxin-treated and l 382 570 50 control excised roots in the same 2 270 567 110 flask, solution was buffered. 3 282 645 128 D. Toxin-treated and 1 160 392 146 control excised roots in the same flask containing tris-N03. 85 different flasks. 2) Roots of intact seedlings accumulated N03 at a faster rate than did freshly excised roots. Toxin-treatment caused the same percentage stimulation of N03 accumulation rates in excised roots and intact seedling roots. Entry of N03 through cut ends of toxin-treated and control roots were the same in buffered and non- buffered solutions. 4) Accumulation of NO3 occurred in the absence of other mineral ions, i. e. , when tris was the counterion. The accu- mulation rate was low, probably because Ca was absent. Toxin-treat- ment caused the same percentage stimulation of accumulation that would be expected from a conventional N03 solution. A change in membrane characteristics resulting in either in- creased influx or decreased efflux could result in an increase in N03 accumulation. To test these possibilities, the rate of N03 loss from roots was determined. When a 30 min absorption period was followed by a 30 rnin desorption period there was little or no detectable loss of N03 from roots (Table 18). This indicates that efflux of N03 is Inini- mal in short term experiments with roots. In all 3 experiments (Table 18) toxin-treated roots lost about the same proportion of NO3 during desorption as did control roots. The absorption rates of many ions from a range of ion concen- trations provide evidence for dual mechanisms of uptake (17, 59). The possibility that corn roots absorb N03 by more than one mechanism was investigated. After the series of N03 experiments was concluded, N03 accumulation rates were collected from each different NO3 86 Table 18. Loss of N03 From Toxin—Treated and Control Corn Roots During Desorption Seedlings were placed in 0. 5 rnM CaClz with or without toxin (10 pg/ml) for 4 hr. Roots were excised and exposed to solutions con- taining 0. 5 mM CaClz and 0. 4 mM KNO3. Roots were desorbed in 3 l aerated 0. 5 mM CaClz. One rnin desorption is the time required to remove N03 from free space with 6 rinses of 0. 5 mM CaClz. N03 Content Toxin- Expt. De sorption Stimula - T... Gets 322:. 922.339 min nmole s / g % nmole s / g % "/0 1 1 119i2 144‘f21 21 30 112 i 5 -6 156 f24 +8 39 2 1 128'f‘4 ... 249 115 ... 95 3o 90 1‘13 -29 198 i 9 -21 120 3 1 179'114 ... 407‘f 7 ... 127 30 212 f 6 +18 398 izs -2 88 60 211 ":11 +17 343 i41 -15 62 87 concentration in each NO3 experiment. These data were plotted on a linear scale and are shown in Figure 18. Surprisingly, data from a diverse group of experiments, done over a period of several months, show evidence of dual mechanisms of NO3 uptake. The pattern is qualitatively similar to that described for other ions (93). Mechanism 1 has a high affinity for N03 and operates'at concentrations up to about 1 mM, with logarithmic increases in accumulation rates with increased NO3 concentration. Mechanism 2 has a low affinity for N03 and oper- ates at concentrations above 1 mM, with linear increases in accumula- tion rates with increased N03 concentration. Toxin-treatment appears to increase the maximum velocity (Vm) and decrease the Km of mech- anism 1. Little can be said about mechanism 2 except that toxin ap- pears to increase the slope of the curve, which suggests greater affin- ity of a carrier for N03. The data in Figure 18 were analyzed with double reciprocal and Woolf-Hofstee (10) plots. Results supported the suggestion that N03 is absorbed by 2 mechanisms. These plots also supported the possibility, mentioned above, that toxin-treatment in- creased Vm and decreased Km. However, points were too few and too scattered for firm conclusions. There is evidence that mechanism 2 is sensitive to temperature before and after toxin-treatment (Table 12). The data given in Figure 18 must be confirmed by experiments designed specifically to test for dual uptake mechanisms. Effect of toxin on uptake of selected cations. -- The implications from experiments with N03 were that toxin causes changes in the 88 4 o 3 l- é 2" 5‘ Z % 2 1- . E : Toxin Control 1 .. o 0 4 8 12 16 20 N03 concentration mm Figure 18. Rates of N03 accumulation in toxin—treated and con- trol corn roots as a function of N03 concentration. Rates shown are for 7 N03 concentrations collected from 5 different experiments over a period of several months. The stimulation percentages are: 0. 04 mM, 22%; 0. 2 mM, 125%; 0. 4 mM, 120%; 1. 0 mM, 78%; 4. 0 mM, 25%; 10.0 mM, 23%; 20.0 mM, 35%. 89 plasma membrane which increase the efficiency of N03 uptake. A sur- vey of selected solutes was made to determine if stimulation of uptake is a general phenomenon. Potassium (K) or rubidium (Rb) is actively accumulated in corn roots by at least 2 mechanisms (117). Mechanism 1 has a high affinity for K and operates at K concentrations (0. 5 mM. Mechanism 2 has a low affinity for K and operates when K concentration is between 1 and 50 mM. The effect of toxin on both mechanisms was tested using 86Rb as a label to estimate K absorption. Some workers have questioned the validity of this technique (69), but recent results with corn roots show that when proper conditions are used, 86Rb gives only a slight over- estimate of actual K absorption (60). I used conditions required for a valid estimate, and based my experiments on those of other workers (15, 60). Seedlings were exposed to solutions with or without toxin for 4 hr. Roots were excised and placed in solutions containing K. Re- sults (Table 19, experiment 1) show that toxin-treatment did not in- crease the rate of K absorption at any K concentration used. Exposure to toxin for 0. 5 to 8. 0 hr did not stimulate the K absorption rate (Table 1‘), experiment 2). The decrease in uptake rate of control roots after 0. 5 hr could be caused by lack of aeration of roots during the toxin- treatment period. In experiment 3 (Table 19), K solutions were pre- pared with the NO3 rather than the Cl salt and both K and N03 accumu- lation rates were measured simultaneously. Toxin-treatment enhanced 90 Table 19. Absorption of K by Toxin-Treated and Control Corn Roots Seedlings were placed in 0. 5 HM CaClZ with or without toxin (10 ng/ml) for 4 hr. Roots were excised and exposed to the following experimental solutions: experiment 1, KCl and 0. 5 mM CaClz; ex- periment 2, KCl and 0. 2 mM CaClZ; experiment 3, KNO3 and 0. 5 mM CaClZ. Uptake of both K and N03 was determined in experiment 3. Solutions were labelled with 0. 01 11c 86Rb/p.mole K. E Toxin- K or NO3 Accumulation Toxin— ;pt' Treatment KConcn. Induced 0' Time Control Toxin Change hr mM nmoles/g/hr % 1 4.0 0.02 556 1‘122 384 1‘ 34 - 31 4.0 0.2 816 1' 36 648 ”£103 - 21 4. 0 20.0 4760 1‘ 95 4832 ”£260 + 1 2 0.5 0.02 428 1‘ 41 416 if 27 - 3 2.0 0.02 270 1‘ 42 276 "f 76 - 2 4.0 0.02 310 ”I 3 310 ”I 27 0 8.0 0.02 305 1' 31 231‘! 61 - 24 3 4.0 0.2 1700 1' 1 1140 i185 - 33 4. 0 0. 2a 1220 1‘270 905 f145 - 26 4.0 0.2b 131 1’ 14 149 ”I 1 + 14 4.0 0.2C 192 1‘ 19 433 1' 73 +125 aRoots were desorbed 30 min in 1500 ml aerated solution con- taining 0. 5 lei CaClZ and 5 mM RbC1. bExperimental solution and rinsing solution were held at 5 C. CData from this treatment are for N03 uptake. 91 the accumulation of N03 but not that of K. Desorption caused 28% and 21% loss of K from treated and control roots, respectively, indicating that toxin did not affect K efflux. Sensitivity of K absorption to low temperature (experiment 3, Table 19) is characteristic of active accumulation (61). Further evi— dence for active uptake is movement against a concentration gradient (70). After 30 min absorption in experiment 1 (Table 19), control roots in 0. 02, 0. 2, and 20 mM K solutions had internal K concentrations of 0. 278, 0.408, and 2. 38 mM, respectively. Ion movement against a gradient occurred in 0. 02 and 0. 2 mM solutions but not in the 20 mM solution. More than 30 min may be required for roots to reach an in- ternal K concentration of 20 mM. Sodium (Na) is not known to be an essential element in higher plants, but is thought to be actively absorbed (32, 34) by both K uptake mechanisms (93). However, Na uptake by mechanism 1 is almost eliminated in the presence of K and Ca. Na is preferred over K by mechanism 2 (93). This information was interpreted to mean that a Ca-stabilized plasma membrane in the presence of physiological con- centrations of Na and K will discriminate against the nonessential ele— ment Na. Mechanism 2, which operates at abnormally high concentra- tions, transports Na more efficiently than K. The effect of toxin on Na uptake by both mechanisms was tested using modifications of previously described procedures (32, 34). Seed- lings were exposed to solutions with or without toxin for 4 hr, then 92 placed in solutions containing Na. The results of 3 experiments (Table 20) show that toxin-treatment caused an increase in the rate of Na ab- sorption by both absorption mechanisms in the presence and absence of Ca. In each experiment, some of the experimental solutions con- tained N03. Toxin-treatment stimulated N03 accumulation to a greater degree than Na absorption- A 30 min desorption period indicated that toxin—treatment did not cause leakage of Na from roots. Experiments with plant roots have shown that Na uptake is inhibi- ted by the presence of Ca (32, 93). My data show the same thing; mechanism 1 operating alone was more sensitive (about 80% inhibition) than both mechanisms operating together (about 40% inhibition). Corn roots absorbed Na equally well from N03 and chloride (Cl) solutions (experiments 2 and 3, Table 20). Results of individual experiments with Na and K indicated that toxin-treatment distinguished between these 2 ions. To re-evaluate this, accumulation rates of Na, K, and N03 from the same solution under identical conditions were -measured. Toxin-treated roots selec- tively accumulated Na and N03 at increased rates with no change in the rate of K absorption (Table 21). This phenomenon was observed for both uptake mechanisms. Plant cells require Ca to maintain membrane integrity and ab- sorption mechanisms (16, 60, 93). Actual uptake of Ca occurs slowly in most plants and uptake is mostly nonmetabolic (68). However, corn roots absorb Ca at rates similar to those of actively absorbed ions. 93 Table 20. Effect of Toxin on Absorption of Na by Corn Roots Seedlings were exposed to 0. 5 mM CaClz with or without toxin (10 pg/rnl) for 4 hr. Roots were excised and placed in experimental solutions. All rinsing was with water. Solutions were labelled with 0. 03-0.1).1c Na/ml. Ion Concentrations Na or N03 Accumulation Toxin- Expt. Test Induced No. Ion Na Ca Cl N03 C ontrol Toxin Stimula " tion rnM mM mM mM nmoles/g/hr % 1 Na 20. 0 20. 0 2920 1‘220 4360 ”£210 49 Na 0.2 .... 0.2 371 i“ 26 390 1' 4 5 Na 0.2 o. 1.2 .. 55f 6 941 20 71 Na 0.2 o. 0.8 .4 66 1‘ 3 87 f 17 32 N03 0.2 0. 0.8 .4 368 1' 12 720 ’5105 96 2 Na 20. 0 .. 20. 0 3020 i380 3480 f120 15 Na 20.0 0.5 21.0 1422 i“ 62 2320 ’5 30 63 Na 0.2 0.2 335 f 22 449 i' 42 34 Na 0.2 0. 1.2 .. 68 1‘ 4 102 i 13 50 N03 0.2 0. 0.8 0.4 225 i 8 520i 10 131 3 Na 20.0 20.0 3200 “i140 4290 $270 34 Na 20. 0 0. 1.020. 0 2520 1’130 2910 i140 15 Na 20.0 0 5 21.0 .. 1900 ”$330 2800 1L 40 42 Na 0.2 0.2 316 f 2 662 i“ 40 97 Na 0.2 0. 1. 0. 9o 1‘ 7 137 1‘ 31 52 Na 0.2 1. 113 f 35 143 i’ 2 27 N03 0.2 1.0 o. 388 1' 38 655 i 27 69 N03 0.2 o 158 265 1‘ 5 68 94 Table 21. Effect of Toxin on Simultaneous Accumulation of Na, K, and N03 by Corn Roots Seedlings were exposed to 0. 5 mM CaClz with or without toxin (10 ug/nrll) for 4 hr. Roots were excised and placed in experimental solutions which contained 0. 3 mM CaClz, 0. 2 mM Ca(NO3)2, and chlorides of Na and K at indicated concentrations. Solutions were labelled with ”Na or 86Rb at o. 1 pc/m1. Ion Accumulation Toxin- Test Ion Ion Induced Concn. Control Toxin Change rnM nmoles/g/hr % Na 0.2 74 it 4 116 1'16 + 57 K 0.2 420 i“ 20 390 1536 - 7 N03 0.4 368 ’5 16 748 ‘5 8 +104 Na 20. 0 1954 ”5114 2430 ”590 + 24 K 20.0 3020 15160 2880 1' 1 - 5 N03 0.4 254 ‘3 9 668 1‘63 +163 95 There are indications that absorption is dependent on cell metabolism (33, 68). In addition, Ca uptake by corn roots is ‘mediated by dual mechanisms which operate in the concentration ranges found for other ions (68). The effect of toxin on Ca uptake was studied because of this peculiarity of corn, and because it provides an opportunity to observe simultaneous absorption, by a Ca-stabilized membrane, of both ions from a simple one-salt solution, Ca(NO3)2. Experiments with Ca were patterned after those described else- where (33, 68). Seedlings were exposed to solutions with or without toxin. Roots were excised and placed in Ca-containing solutions. Toxin-treatment had no effect on the rate of Ca absorption by either of the dual mechanisms (Table 22). Desorption in water caused about 15% loss of absorbed Ca from both toxin-treated and control roots. Similar results were obtained in another experiment when roots were desorbed in 0. 5 rnlvi CaClz, except Ca losses ranged from 30-50%. This indi- cate s that 45 Ca influx was due in part to exchange. The following additional observations were made. Roots exposed to 0. 2 mM Ca for 40 min had accumulated Ca to a concentration of 0. 59 mM, indicating movement of external Ca against a concentration grad- ient. Absorption of Ca by mechanism 1 was slightly inhibited by K (Table 22). The accumulation rate of the counterion, N03, was stimu- lated )100% by toxin-treatment (Table 22). High or low Ca concentra- tions did not affect N03 uptake or toxin-stimulated N03 uptake (Table 22). In another experiment, N03 was accumulated from a 10 mM 96 Table 22. Absorption of Ca by Toxin-Treated and Control Corn Roots Seedlings were exposed to 0. 5 HM CaClZ with or without toxin (10 ug/ml) for 4 hr. Roots were excised and placed in experimental solutions containing Ca(NO3)2 with or with 45Ca (0. 05 pc/ml). All rinsing was with water. Ca or N03 Accumulation Toxin- Expt. Test Ion Special Induced No. Ion Concn. Conditions Control Toxin Change mM nmoles/g/hr % 1 Ca 10. o ... 1618 ”£53 1490 i150 - 8 Ca 0.2 82812 782’.r 39 — 6 Ca 0.2 KCla 592 662 T 80 + 12 . b + + N03 0.4 ngh Ca 382 -18 850 -114 +122 N03 0. 4 Low c4C 394 1'48 790 i“ 25 +101 2 Ca 0.2 1110 1'20 1030 1' 22 - 7 Ca 0.2 Desorbedd 887 ":35 855 I“ 35 - 4 Ca 0.2 KCle 959 ”529 927 t 59 - 3 + + No3 0.4 282 - 4 645 - 15 +128 3‘KCl (0. 1 mM) was included in experimental solution. bExperimental solution contained 0. 2 mM Ca(NO3)Z + 9. 8 HM CaClz. cExperimental solution contained 0. 2 mM Ca(NO3)2. dRoots were desorbed for 35 min in 1000 ml aerated water. 6KCl (0. 2 ranl) was included in experimental solution. 97 Ca(NO3)2 solution (20 mM N03) at 2900 nmoles/g/hr and toxin caused , 35% stimulation. Differential absorption from a solution containing only Ca and . N03 provided an opportunity to determine if the pH change of the ex- perimental solution, observed occasionally (see Methods), was related to unequal absorption of charged particles. During one 40 min absorp- tion period control roots removed 0. 708 uequivalents (ueq) of Ca and 0. 326 ueq of NO3 from the experimental solution. The pH changed from 5. 8 to 4. 8 indicating that 15. 9 ueq of H were added to the solu- tion. Excess Ca uptake required H loss of 0. 382 ueq to balance the charges. Thus, H loss from tissue was much greater than that re— quired to compensate for excess Ca uptake. Toxin-treated roots under similar conditions removed 0. 662 ueq of Ca (no stimulation) and 0. 672 ueq of N03 (101% stimulation) while the pH changed from 5. 8 to 4. 8. In this case there was slightly more N03 than Ca accumulation, which would require an increase in pH (0. 01 ueq of H uptake) to balance charges. Instead the pH dropped the same as with control roots. Values for NO3 accumulation are conserv- ative estimates of total N03 uptake, because some of the N03 taken up was reduced during the absorption period. Thorough washing of roots for 1 hr before the experiment in 4 l aerated 0. 5 mM CaClz did not affect the pH. The pH of a salt solution containing cheesecloth and string was not changed after aeration. It was concluded that the pH change, which occurred only when the volume 98 of the experimental solution was small compared to the amount of tis- sue in it, was caused by the tissue, as reported by others (85), and was not related to toxin-stimulation of ion uptake. Effect of toxin on uptake of selected anions. -- Absorption of Cl by corn roots is dependent on cell metabolism and is mediated by dual uptake mechanisms similar to those described for other ions (67, 70). The effect of toxin on both mechanisms was tested. Seedlings were ex- posed to solutions containing toxin for 4 hr. Roots were excised and placed in solutions containing C1. Toxin-treatment caused a small stimulation of C1 absorption rates by both mechanisms (Table 23). Progressively faster rates occurred when solutions contained 0. 1 and 0. 5 lei Ca. Toxin-stimulated Cl absorption occurred in the presence and absence of Ca. Roots in 0. 2 mM Cl solution contained 0. 95 mM Cl after a 40 min absorption period, indicating Cl ‘movement against a concentration gradient. Another experiment showed that desorption for 30 min in 4 1 of 0. 2 HM NaCl caused 4-7% loss of Cl from toxin- treated roots and 13-20% loss from control roots. Variation between duplicate samples was great enough to indicate that the difference in losses was not significant. Some of the experimental solutions also contained N03 (Table 23). Uptake of C1 by control roots was faster than accumulation of N03, although toxin stimulated N03 accumulation more than Cl uptake. The N03 accumulation rate was low when ex- perimental solutions contained no Ca. 99 Table 23. Effect of Toxin on Absorption of C1 by Corn Roots Seedlings were grown in 0. 2 rnM CaSO4 solution. They were ex- posed 4 hr to 0. 5 mM CaSO4 with or without toxin (10 ug/ml). Roots were excised and placed in experimental solutions. All rinsing was with water. Solutions were labelled with 0. 01 pc 3(’Cl/rnl. Test Ion Concentrations C1 or N03 Accumulation 1:33:22; 1°” Na K Ca c1 N03 Control Toxin $22: mM mM mM mM rnM nmoles/g/hr % c1 20.0 20.0 3300 1'130 4430 1' 85 34 C1 0.2 0.2 7111525 1010‘.r 25 42 c1 0.1 0.2 970i 70 1185 f155 23 c1 0.2 0.5a 0.2 1035 1' 35 1488 1'108 43 Cl 0.2 0.4 0.2 0.4 796 i“ 76 970 f145 22 N03 0.2 0.4 0.2 0.4 187 4741' 4 153 a‘Ca was added as 804 salt. 100 Of the ions discussed so far, K, Na, and C1 are not known to be metabolized by the cell. Metabolism does not appear to be a signifi- cant factor in toxin-stimulated uptake of N03 under my experimental conditions (see Discussion) and Ca is required primarily for stabiliza- tion of cell membranes (16, 60, 93). Therefore, it was advisable to test the effect of toxin on uptake of an essential ion such as phosphate (P04) which is rapidly metabolized by the cell. Corn roots absorb P04 by 2 mechanisms, both of which require Ca for maximum P04 ab- sorption rates (6). The high affinity mechanism operates at P04 con- centrations up to 10 pM and the low affinity mechanism operates at concentrations from 20-200 pM (6). Experiments on P04 uptake were similar to those described for previously discussed ions, except that P04 was included in experimen- tal solutions. Toxin-treatment did not stimulate P04 absorption at any P04 concentration (Table 24). Absorption of N03 from a P04 solution was enhanced by toxin-treatment. Roots in 2 and 20 1.1M P04 contained 41 and 91 11M internal P04 concentrations, respectively, after a 30 rnin absorption period. Thus, P04 moved against a concentration gradient. In one experiment, excised roots and roots of intact seedlings were ex- posed to solutions with or without toxin for 4 hr, which resulted in 4 hr aging of excised roots. All roots were then placed in solutions contain- ing P04. Aged control roots absorbed P04 about twice as fast as did fresh control roots. Toxin-treatment had no effect on P04 uptake by aged or fresh roots (Table 24). 101 Table 24. Absorption of P04 by Toxin-Treated and Control Corn Roots Fresh roots were prepared as follows. Intact seedlings were in- cubated 4 hr in 0. 5 lei CaClz with or without toxin (10 pg /rnl). After incubation roots were excised. For aging, roots were excised and aerated 4 hr in 0. 5 mM CaClz with or without toxin (10 pg/ml). Fresh and aged roots were placed in solutions containing 0. 5 mM CaClz and NazHPO4 (pH 4. 8) labelled with Na232PO4 such that the 200 11M solu- tion contained 12, 000 cpm/ml. One solution in experiment 2 also con- tained 0. 4 HM KN03. Roots placed in this solution were analyzed for N03 content. Eb’l‘St- 1:53;?“ Type P04 or N03 Accumulation 1:33:25 ' ' Ti 3 sue C ontrol Toxin Change 11M nmoles/g /hr % 1 200 Aged 230 i“ 2 256 T 6 +11 20 Aged 181 1' 1 138 “:12 -24 2 Aged 81 i 1 70 ”f 5 -13 20 Fresh 93 9:10 96 1' 9 + 3 2 400 Fresh 208 ”:13 200 “:12 - 4 400a Fresh 248 f40 438 ":33 +72 aData from this treatment are for N03 uptake. 102 Failure of toxin to affect total P04 uptake did not rule out a pos- sible effect on a specific phase of P04 metabolism. Such an effect could be masked by the large amount of total P04 absorbed. However, data show that toxin-treatment did not affect the P04 content of PCA- soluble organic or inorganic fractions or the PCA-insoluble residue (Table 25). Under identical conditions N03 accumulation was enhanced by toxin-treatment. Accumulation of N03 by control roots was faster than usual and toxin-stimulation was small, because aged roots were used in this experiment (see Table 14). The effect of toxin on sulfate (S04) uptake by corn roots was determined by modification of described procedures (35, 62). Toxin- treatment either had no effect on, or was slightly inhibitory to 804 absorption at all concentrations used (Table 26). Accumulation of N03 from the same solutions was stimulated by toxin-treatment. The usual amount of N03 stimulation was observed in experiment 3 (Table 26), but stimulation was abnormally small in experiments 1 and 2, for un- known reasons. Desorption removed about the same amount (10-25%) of 504 from both toxin-treated and control roots. Roots in 0. 002 and 0. 02 ran 504 solutions contained 0. 023 and 0. 066 rnM internal $04 concentrations, respectively, after a 40 min absorption period. Thus, SO4 was accumulated against a concentration gradient. The effect of toxin on N02 accumulation was compared with the effect on N03 accumulation. Seedlings were placed in solutions with or without toxin for 4 hr. Roots were excised and placed in solutions 103 Table 25. Uptake and Incorporation of P04 by Toxin-Treated and Control Corn Roots Aged roots were used for all treatments. Roots were excised, aerated 4 hr in 0. 5 HM CaClz with or without toxin (10 pg/ml), and placed in solutions containing 0. 5 mM CaCl , 0.4 mM KN03 and 0. 4 rnM NaZHPO4 (pH 4. 9) labelled with Na2H3 P04 to give 53, 000 cpm/ ml. Organic and inorganic P was separated. P04 or N03 Accumulation Toxin- Te st Ion Fraction Induced Control Toxin Change nmoles/g/hr ‘70 P04 Orthophosphate 126 128 + 2 Organic Phosphate 224 197 -12 Residue __6 __5_ -16 Total 356 330 - 7 N03 Total 554 820 +48 104 Table 26. Absorption of $04 by Toxin-Treated and Control Corn Roots Seedlings were exposed 4 hr to 0. 5 mM CaClz with or without toxin (10 11g /ml). Roots were excised and placed in experimental solu- tions. In experiment 1, experimental solutions contained 0. 2 mM Ca(No3)z and NaZSO4 labelled with NaZ3SSO4 such that 0. 2 mM solu- tion contained 2200 cpm/ml. Roots were desorbed 30 rnin in 3000 ml aerated 0. 5 mM CaClz and 0. 2 mM NaZSO4. In experiments 2 and 3, experimental solutions contained 0. 5 mM CaClZ, 0. 4 mM KNO3, and NaZSO4 labelled as in experiment 1. Roots were desorbed as in ex- periment 1, except NaZSO4 concentration was 0. 02 m.M. $04 or N03 Accumulation Toxin- Expt. Test Test Ion Desorp- Induced No. Ion Concn. tlon Control Toxin Change m.M nmoles/g/hr % 1 so4 0.2 ' + 97 1‘11 96 i“ 3 - 1 so4 0.2 - 109 1L 1 121 ”f 3 + 11 N03 0.4 - 334 “f 5 386 1“ 6 + 15 2 so4 002 + 491‘4 40f2 -18 so4 0.02 - 66 i“ 4 42 i 1 - 36 so4 0.002 + 34 1‘ 3 27 IL 1 — 19 + + No3 0.4 - 199 - 4 240 -16 + 21 3 so 0 02 + 57 +10 43 + 5 24 4 _ - _ No3 0.4 - 204 1‘ 4 500 1'25 +145 105 containing N02 or N03 or both N02 and N03. After absorption, roots were analyzed for N02 and N03 content. The usual stimulatory effect on N03 accumulation was observed, but toxin had no effect on N02 ac- cumulation (Table 27). This differential stimulation was observed when roots were exposed to N03 and N02 in separate solutions and when exposed to both ions simultaneously in the same solution. De- sorption caused 35% N02 loss from control roots and 22% N02 loss from toxin-treated roots. This differential loss of N02 was also ob- served after roots absorbed N02 and N03 simultaneously. Loss of N02 during a 30 min desorption period could be caused by leakage through cell membranes damaged by N02 poisoning, or the loss could indicate N02 metabolism within the cells. The latter possibility seems feasible because N02 is very reactive with many substances, including amines. In one experiment N02 and N03 were absorbed simultaneous- ly. During desorption some N02 was lost but N03 was not, indicating that membranes were not damaged by N02 poisoning. Roots accumulated N02 about 10 times faster than N03. At the pH used for this experiment (5. 8), a significant amount of N02 is un- dissociated and could passively diffuse into cells at a fast rate as HNOZ. A high intracellular pH would prevent rapid HNOZ leakage during de- sorption. The effect of pH was tested in solutions buffered at pH 7. 5 with 2 rnM HEPES. The rate of N02 accumulation was reduced 95% at pH 7. 5, as compared with pH 5. 8. The rate of N03 accumulation was also reduced (about 80%) at pH 7. 5, for unknown reasons. Although Table 27. 106 Accumulation of N02 and N03 by Toxin—Treated and Control Corn Roots Seedlings were placed 4 hr in 0. 5 mM CaClz with or without toxin (10 ug/ml). Roots were excised and placed in experimental solu- tions containing 0. 5 mM CaClz and 0. 4 mM KNOZ or KNO3 or both KNOZ and KN03 in the same solution. N02 or N03 Accumulation Toxin- Te st Ion Ion Content N03 N03 N02 N02 N023 N02 N03 N03 + N02 N02 N03 + NO2 Induced Control Toxin Change nmoles/g/hr ‘70 222 f 3 642 1‘ 77 +190 2400 ”£90 2050 i245 - 14 1565 1‘45 1595 1' 65 + 2 394 1L 5 617 “f 10 + 57 1950 i30 1765 1‘ 20 _ 10 aRoots were desorbed 30 min in 1000 ml aerated 0. 5 mM CaClz. 107 accumulation rates for both ions were slower at pH 7. 5 than at pH 5. 8, toxin-treatment still had no effect on N02 accumulation but in- creased the N03 accumulation rate by 270%. Estimation of N03 con- tent in the presence of N02 was inaccurate and quite variable because there was much more accumulation of N02 than of N03. Selective stimulation of uptake by toxin-treatment in resistant and susceptible corn was tested in several experiments. In one ex- periment, accumulation of N03 and Na by resistant corn roots was determined because uptake of these ions by susceptible corn is rela- tively sensitive to toxin. Results (Table 28) show that the rate of N03 accumulation by susceptible roots was enhanced by as little as 0. 2 pg toxin/m1. The rate of N03 accumulation by resistant roots was not affected by 2 and 10 pg toxin/n11 but was stimulated 138% by 100 pg/rnl. Uptake of Na by resistant roots was not stimulated by toxin at any con- centration used. The experiment comparing the effects of toxin on N03 and Na accumulation by resistant corn was not repeated; it should be confirmed before concluding that toxin-treatment has a differential effect on N03 and Na accumulation by resistant roots. Effect of toxin on uptake of selected organic molecules. -- Se- lective toxin-stimulation of ion uptake raised the possibility that this effect is specific to ions. This possibility was tested by studying up- take of a noncharged organic molecule, 3-o-methy1glucose (MeG). Use of MeG (an analog of glucose) simplifies study of sugar uptake because it is not metabolized by higher plant cells (31, 95), although it is 108 Table 28. Effect of Toxin Concentration on N03 or Na Accumulation by Susceptible and Resistant Corn Roots Seedlings were exposed 4 hr to 0. 5 mM CaClz with or without toxin. Roots were excised and placed in experimental solutions which contained 0. 5 mM CaClz and 0. 4 'mM KNO3 or 0. 2 mM NaN03 labelled with 22Na (0. 1 11C /ml). N03 or Na Accumulation Corn Toxin Hybrid Concn. NO Toxin-Induced Na Toxin-Induced 3 Change Change 11g /ml nmoles/g/hr % nmoles/g /hr % Resistant 0 442 102 2. 0 351 - 20 74 -27 10. 0 550 + 24 62 -39 100. 0 1050 +138 98 - 4 Susceptible 0 260 0. 2 802 +208 2. 0 1235 +395 10. 0 745 +186 109 actively accumulated by the glucose transport mechanism (95). In ad- dition, efflux of MeG in the presence of glucose is reported to require cellular energy (95). This provides a system for studying the effect of toxin on active efflux. Experiments with MeG were similar to those with ions; toxin- treated and control roots were placed in solutions containing MeG. After absorption, MeG was extracted from roots and quantified. Toxin- treatment caused 25-35% stimulation of the MeG absorption rate and 105% stimulation of N03 accumulation rate from identical solutions (Table 29). Desorption for 30-60 min in 0. 5 mM CaClz caused no loss of MeG from toxin-treated or control roots. Both toxin-treated and control roots lost 25% of their MeG when desorbed 60 min in the pre- sence of glucose. Thus, toxin-treatment enhanced active influx but had no effect on active efflux of MeG. Glucose competitively inhibits MeG uptake (95). My data showed that presence of 10 mM glucose in the experimental solution inhibited the rate of MeG uptake by 30% in both toxin-treated and control roots. Amino acids were used in further experiments to determine ef- fects of toxin on uptake of organic molecules. Toxin-treated and con- trol roots were exposed to amino acid-containing solution for 30-45 min. The amounts of amino acids in roots were then determined. Re- sults (Table 30) show that toxin had a selective effect on amino acid uptake. Leucine uptake was stimulated about 25% by toxin-treatment, glutamic acid uptake was not affected, and the N03 control showed 110 Table 29. Effect of Toxin on Absorption and Desorption of 3-o-Methy1glucose (MeG) by Corn Roots Seedlings were exposed 4 hr to 0. 5 'mM CaClz with or without toxin (10 pg/ml). Roots were excised and placed in solutions con- taining 0. 5 mM CaClz, 0. 4 mM KN03 and 10 mM MeG labelled with 1‘l’C-MeG (0. 01 uc/umole MeG). Roots were desorbed in 3000 ml aerated 0. 5 mM CaClz with 10 mM glucose where indicated. One min desorption was time required to remove MeG from free space with 6 rinses of 0. 5 mM CaClz. MeG or N03 Content Toxin-Stimulation edifice Desi???" . °f “80 °r N03 Control Toxin Content min nmoles/g/hr % MeG 1 1832 1' 82 2285 ":60 25 MeG 30 1805 ”$135 2440 1’85 35 MeG 60 1850 f 45 2400 ”£35 30 MeG 60 1400 1800 28 (in glucose) No 1 398 i“ 2 815 i45 105 Table 30. 111 Leucine and Glutamic Acid Accumulation by Toxin-Treated and Control Corn Roots Seedlings were exposed 4 hr to 0. 5 mM CaClz with or without toxin (10 pg/ml). Roots were excised and placed in experimental solu- tions containing 0. 5 mM CaClz and 0. 4 m.M KN03 with or without 0. 1 rnM leucine or glutamic acid or both. The corresponding 14C-l- labelled amino acids were included at 0. 01 11c /ml. All solutions were adjusted to pH 5. 8. Specific activities of l""C-l-leucine and glutamic acid were 12. 2 and 20. 0 mc/rnmole, respectively. Total Amino Acid or Test Supplements to Expt'l. Substance , Solutlon Leucine Leucine Glutamic Glutamic Acid Acid Leucine Leucine and Glutamic Acid Glutamic Leucine and Acid Glutamic Acid NO3 Leucine and Glutamic Acid N03 Accumulation 1:311:21 Control Toxin Change nmoles /g /hr ‘70 823 “535 1030 177 + 25 199 J! 5 200 i 4 0 879 1'10 1091 ":31 + 24 65 0 64 i” 1 0 288 1’40 745 1'32 +159 112 159% stimulation. Glutamic acid did not affect leucine uptake, but leucine inhibited glutamic acid uptake by 67%. In another series of experiments using different experimental conditions (see Appendix), leucine uptake and incorporation by corn root sections were studied. Toxin-treatment caused an increase in the rates of uptake and incorporation, and an increase in the size of the soluble leucine pool (Figure 19). Thus, there is agreement be- tween the 2 sets of data on leucine uptake. A summary of results of experiments on solute absorption by susceptible corn roots is presented in Table 31. 113 HC-Toxin Effect on leucine Uptake pmoles/root N l l 5 20 40 |.eu-Cl4 Exposure (min) Figure 19. Effect of toxin on leucine uptake, incorporation, and soluble pool size. Seedlings were grown in 15 cm petri dishes in White's solution. They were placed in 30 m1 White's solution with or without toxin (2 pg/ml) for 4 hr. Roots were then rinsed, 1 cm sections were excised 2 mm behind the root tip and placed in 10'6 M 14C-l- leucine in White's solution. At intervals, sections were removed, rinsed in 12C-leucine in White's solution, blotted and placed in 0. 5 ml 95% ethanol (10 sections/replicate) for 12 hr. Activity in aliquots was counted to determine the size of the soluble pool. Sections were then rinsed 3 times in 95% ethanol and hydrolyzed in 0. 5 m1 6 N HCl at 95 C 1 hr. Activity in aliquots was counted to determine leucine incor- poration. Tox = toxin, Ck = control, Sol = ethanol-soluble leucine, Insol = ethanol-insoluble residue. 114 Table 31. Selective Stimulation of Solute Accumulation in Susceptible Corn Roots by Toxin Solute Toxin-Induc ed Stimulationa % N03 100 N02 0 Na 50 K 0 C1 30 Ca 0 P04 0 504 0 3-o-methy1glucose 25 leucine 25 glutamic acid 0 a . . . . Values are estimates of the average stimulation observed in several experiments. DISC USSION The effect of HC-toxin on corn seed germination was compared with the immediate lethal effect which HV-toxin has on resting suscep- tible oat seeds (98). The 2 systems are strikingly different. Corn seed germination was not affected by massive doses of HC-toxin (Table 1). Intact seeds required 36-40 hr exposure to toxin before an effect on later root growth was observed. Kuo (55) found that growing corn seedlings required at least 4 hr toxin exposure to accumulate a toxicdose. The long toxin exposure necessary for toxin uptake by seeds is due in part to inability of toxin to penetrate the seedcoat. Thus, only 24 hr imbibition in the presence of toxin was sufficient to reduce the capacity for NR induction in excised embryonic axes. Inhibition of NR induction is a late effect of toxin-treatment and an insensitive parameter. Addi- tional studies are necessary to determine the sequence for activation of metabolic systems during germination, and the point in this sequence at which tissues can absorb effective doses of toxin. It is possible that toxin does not prevent germination of intact seeds because the initial stages of germination are stimulated rather than inhibited. 115 116 The earliest physiological effects of toxin observed to date are stimulatory and only later does inhibition become evident (54). It is generally agreed that the earliest observable effect of a biologically active molecule is closer to the site of action than effects observed later (118). Therefore, it seemed logical that mode-of-action studies should involve the stimulatory activity of toxin. Experiments were de- signed to determine whether stimulatory effects could be related to a particular aspect of cell metabolism. Toxin-stimulated growth of susceptible and resistant corn and nonhost plants suggested that toxin may have nonhost-specific hor- mone action. An analogy could be drawn with the gibberellin-like ac- tivity of helminthosporol (36, 48, 81). Possible hormone-like activity may or may not be related to host-specific activity. Toxin, when ap- plied over a wide concentration range, did not promote hypocotyl growth, seed germination, or enzyme induction. Both helminthosporol and GA are active in all these tests. This indicates that toxin does not have GA—like activity. In addition, stimulatory effects are not caused by contaminants in the toxin preparation which have GA—like activity. The apparent toxin-stimulation of GA-induced oc-amylase production by nongerminable barley half-seeds is difficult to interpret. Further experiments are required to establish the validity of this phe- nomenon. It is possible that o<-amylase production by poor quality barley seeds is directly related to the number of viable seeds/treat- ment. Toxin at low concentrations did not enhance GA-induced 117 amylase activity in susceptible corn endosperm. High toxin concen- trations inhibited amylase activity. This may be an indirect effect be- cause 48 hr exposure caused only partial inhibition. However, the amylase results with corn are not conclusive because most of the amylolytic activity in corn endosperm results from p-amylase (12) which may not be synthesized EEO—‘33. in seeds (121). Results of experiments with NR support the suggestion that toxin- inhibition of enzyme synthesis is an indirect effect. When axes im- bibed in the presence of toxin for 24 hr, the capacity for NR induction was decreased but not eliminated. By comparison with diseased plants, there is a report that Fusarium infection causes decreased NR activity in roots and increased activity in shoots of rice plants (112). Inducibility on a per axes and on a dry wt basis actually increased with longer toxin exposures. Results suggest that toxin did not directly in- hibit induction but rather inhibited some other function on which induc- tion depends. If long exposure to toxin resulted in disruption of intra- cellular transport systems, materials for enzyme synthesis would not be available. The possibility that toxin causes direct stimulation of protein synthesis was attractive because toxin stimulates growth, and in- creased growth should require increased synthesis. In addition, there is evidence that toxin stimulates incorporation of l4C--labelled amino acids (54). This question could be approached directly by preparing a cell-free amino acid-incorporating system from corn and testing the 118 effect of toxin i_nm However, results of experiments with such systems from plants are not always conclusive because of problems in achieving net protein synthesis (73). Therefore, experiments were de- signed to detect possible effects of toxin on protein synthesis by in- direct means. Enzyme induction was examined using a substrate-in- duced enzyme, NR, as a model. There is evidence that NR is synthe- sized d_e_n_cg9_ in tobacco cells (132). Toxin-treatment during induction enhanced NR activity Evil/p, which at first suggested stimulation of NR induction. However, when tissues were pretreated with toxin, then exposed to inducer (N03), there was increased activity by endogenous NR at 0 induction time. Furthermore, the rate of toxin-stimulated induction did not differ sig- nificantly from the control rate. These observations suggested that toxin had no direct effect on induction of the enzyme. When NR from toxin-treated and control tissues was assayed 12%, little or no difference was observed, providing additional evidence that toxin did not stimulate NR induction by N03. It also showed that toxin itself did not induce NR activity. Several experiments indicated an increased rate of N03 accumu- lation after toxin-treatment. When toxin-treated and control roots were excised and placed in N03 solutions, treated roots accumulated N03 as much as 3 times faster than did control roots. Toxin-treatment stimulated N03 accumulation to different degrees in different experi- ments. However, the effect was observed often enough to provide 119 convincing evidence that it is real. In 30 different experiments, 86 duplicated treatments were compared with 86 duplicated controls (172 individual comparisons). Toxin-stimulation was observed in all cases. The N03 accumulated by toxin-treated roots was retained through 30-60 min desorption, which suggests that the tissues were not leaky to N03. However, these data must be considered within the context of N03 pools in corn roots (Table 13). The metabolic N03 pool is as- sumed to be in the cytoplasm because NR is considered to be a cyto- plasmic enzyme (74, 96, 115). The most likely location for the stor- age NO3 pool is the vacuole. These assumptions form the basis for analyzing data which relate to N03 pools. .When roots are exposed to N03, NR induction begins within 30 min (Figure 11). The amount of NR present after 30 min is capable of reducing N03 at the rate of 850 nmoles/g/hr. Therefore, all N03 taken into the very small metabolic pool in roots during a 30 min absorption period was reduced. When roots were removed from N03 solution, any N03 remaining in the metabolic pool was reduced within a few min (Table 13), i. e. , before roots were killed to terminate the experiment. Thus, measurements of N03 content of roots before and after desorption reflected the N03 content of the storage pool (probably in the vacuole), not the total N03 taken up during 30 min absorption. The fact that little or no N03 was lost from toxin-treated tissues during 30-60 min desorption does not mean that toxin did not affect N03 metabolism or N03 leakage across the plasmalemma. It means that N03 did not leak from the storage pool, probably in the vacuole. 120 In addition to the circumstantial evidence just discussed, experi- mental results also indicate that toxin did not cause N03 leakage from the storage pool (Figure 16). After the metabolic N03 pool in leaves was exhausted, toxin did not cause translocation of N03 from the stor- age to the metabolic pool, as certain other chemicals are known to do (22). Although this point needs further investigation to be conclusive, the data suggest that toxin has no effect on efflux across the tonoplast. One aspect of the N03 pool experiment needs clarification. It is possible that toxin released N03 from the storage pool and all N03 was reduced prior to the end of the 4 hr aerobic toxin-treatment period. This possibility is negated by the large amount of N03 known to be in the storage pool (24 pmoles/g tissue in the experiment described) and by the rate of N03 reduction under these conditions (1 nmole/g tissue/ hr). NR has a half-life of 4 hr; toxin does not affect NR degradation. Assuming no degradation of NR during the 4 hr aerobic period, a maxi- mum of 20% of total storage NO3 could have been reduced in 4 hr, had it been released from the storage pool. Thus, both NR and N03 were present in the cell under these conditions, but toxin did not cause them to interact. There are no data which indicate whether or not toxin affects the N03 pathway between N02 and NH4. However, toxin apparently does not inhibit N03 reduction to N02 (Figures 7-14). This rules out the possibility that toxin blocks N03 metabolism, diverts N03 from the metabolic to the storage pool and thus increases the N03 content of the 121 storage pool. A possible effect of toxin on N02 reduction is not im- portant with respect to stimulation of N03 uptake. Even if toxin blocked N02 reduction, N03 in the metabolic pool would still be con- verted to N02. The N02 would probably be excreted from the cell (24) or otherwise dissipated. Therefore, the increased uptake of N03 into the storage pool is a result of increased uptake across the plasma- lemma, not the result of a block in N03 metabolism. Increased movement of N03 into the storage N03 pool suggested that toxin may alter ion movements across membranes of other organ- elles as do certain antibiotics (76, 87, 109). However, results of ex- periments with mitochondria and chlorOplasts indicated that these or- ganelles are immune to toxin. The negative data may rule out several possible sites of toxin action. Toxin did not uncouple, inhibit electron transport, affect phosphorylation, inhibit the Krebs cycle, or affect the conformational changes of mitochondria or chloroplasts whether or not electron transport occurred. Each of these sites is known to be af- fected by one or more biologically active molecules. Thus, toxin ap- pears to be in a different category, at least with respect to specificity if not mode-of—action. The isolated chloroplasts were actually chloroplast lamellae, that portion of the chloroplasts responsible for light absorption, elec- tron transport, and ATP formation. The effect of toxin.on intact chloroplasts was not tested. It would be of particular interest to test the effect of toxin on 002 fixation in the dark by chloroplasts, because 122 toxin stimulates dark fixation of 002 by whole tissues (56). Methods for isolating a high proportion of intact chloroplasts are available (122). Evidence of toxin-stimulated N03 accumulation and the concept of N03 pools offered an alternative explanation for toxin-stimulated NR activity i_n £119: More N02 was produced because more substrate was available to NR. For this to be true, N03 in the ‘metabolic pool must be the limiting factor in N02 production. This is easily ration- alized with the small metabolic N03 pool in corn root tips, which is ex- hausted in less than 10 min (Table 13). The rate of N03 reduction is thus dependent on the rate at which N03 enters the metabolic pool. If toxin stimulates entry of N03 into the pool, the rate of reduction would increase until the point at which NR activity would be the rate-lirniting factor. Since toxin does not stimulate NR induction, this would result in a systematic increase in N02 production which would be constant throughout the experimental period. The plot of toxin data would then parallel the plot of the control data as seen in my graphs. The explanation must be modified to account for results with leaves. Corn leaves have a large metabolic N03 pool which requires 2 hr for complete reduction (Figure 15). If this pool was full or even partially full, the rate of N03 entry into the pool would not limit the rate of N02 production. Thus, toxin-stimulated N03 uptake would not be detected by the 13mg assay. However, toxin did stimulate N02 production in leaves. This can be explained by the fact that the experi- ments were started when the pool was empty. The initial rate of N03 123 reduction (by endogenous NR) was limited by the rate of N03 movement into the pool. Since tissues were given a 4 hr pretreatment with toxin, the initial rate of N03 uptake was enhanced and thus N02 production was increased as in root tips. This explanation is supported by the fact that the metabolic pool in corn leaves fills slowly, compared to the time course of my experi- ments. After 9 hr exposure to N03, the N03 content of the metabolic pool in leaves was only 25% of the pool capacity (22). Furthermore, N02 production becomes nonlinear as the final portion of the pool is reduced (22). Below a certain critical N03 concentration, the rate of N03 reduction is dependent on N03 concentration in the pool. Thus, even after N03 enters the pool faster than it is reduced, a time inter- val is required before the N03 concentration in the pool is sufficient to eliminate the rate of N03 entry as the factor limiting N02 production. Since the maximum length of experiments on NR induction in leaves was 2 hr (the experiment shown in Figure 14 was terminated at 40 rnin), it is feasible to propose that the rate of N03 entry into the metabolic pool limits N02 production during the time course of the leaf experi- ments. The rationale presented above predicts that toxin causes an in- creased rate of N03 entry from extracellular sources into the meta- bolic pool. Data in Figure 14 are evidence that this is true. The in- itial rate of N03 uptake was observed in noninduced tissues under an- aerobic conditions in the dark. Uptake of N03 could be observed under 124 anaerobic conditions because leaf tissue does not go anaerobic immed- iately. In leaves, production of N02 is more dependent on darkness than on anaerobiosis (94). Endogenous NR was used to detect N03 as it entered the metabolic pool. At 0 time there was essentially no N03 in either toxin-treated or control tissues. Within 10 min after expo- sure to N03, toxin-treated tissues took up and reduced more N03 than did control tissues. An increased rate of N03 uptake was seen through- out the experimental period. Toxin-induced uptake went into the meta- bolic N03 pool, because NR would not have reduced it at any other lo- cation (22). As noted earlier, toxin-treatment also caused increased uptake of N03 into the storage pool. It is not likely that the linear in- crease in N02 production was complicated by an increase in NR ac- tivity due to induction. There are reports of a lag phase of up to 30 min before NR induction begins (20, 110). Since this experiment was terminated in 40 rnin, N02 production was probably caused by a con- stant amount of endogenous NR. It would be possible to do long term N03 uptake studies in the absence of induced NR activity by suppressing NR induction with tungstate (37). The effect of toxin on the capacity of the metabolic N03 pool was not tested, but it could be by a procedure similar to that described in Figure 16. Cuttings from plants grown in the presence of N03 should be exposed to toxin and N03 for 4 hr. Leaves placed under anaerobic conditions should produce more total N02 if the capacity of the pool had been expanded by toxin-treatment. This would fit with data on the 125 capacity of the soluble leucine pool, which is increased by toxin-treat- ment (Figure 19 and Appendix). An apparent anomaly in the N03 metabolism data is that toxin stimulated N03 accumulation but not NR induction. Increased accumu- lation of inducer without a corresponding increase in induction can be interpreted as evidence for an ”inducer" N03 pool. Such a pool could be defined as that amount of N03 which is capable of inducing NR. When the pool is full, additional N03 uptake has no effect on induction. If such a pool exists, toxin apparently did not affect it. There are other reports of tissues which contained high NO3 concentrations but low NR activity (3). An attempt should be made to explain the decreased NR activity in control tissues after 2 or more hr of induction followed by the i_n_ mg assay (Figures 7, 8, 11, 12). I observed this phenomenon many times in both embryonic axes and root tips. Ferrari (personal com- munication) made the same observation using corn root tips. Induction of NR in apple leaves shows a similar pattern (51). The decreased ac- tivity in my experiments was not caused by exhaustion of N03 in the induction medium. Results of the 33% assay and results of several other workers (20, 110) suggest that NR content increases for several hr. In addition, N03 uptake is linear at least 4 hr (Figure 17). If both N03 concentration and NR activity increase, the decrease in N02 production may result from their failure to interact. Perhaps after brief exposure to N03, the cell's immediate need for nitrogen is 126 satisfied and some N03 is diverted from the metabolic pool to the storage pool. The kinetics of induction were followed only in root tips (which have a very small metabolic pool) and embryonic axes (which have a metabolic pool of unknown size). If N03 was partially diverted from a small metabolic pool such as that in root tips, a decrease in the rate of N02 production would soon be evident, regardless of the amount of NR present. There are other possible explanations (3). An increased rate of N03 accumulation by whole tissues and negative results with all other experiments suggested that the cell surface was the most promising area to examine for a site of toxin action. Uptake of several solutes by corn roots was observed for pos- sible effects of toxin-treatment. The data indicate that 4 hr toxin-treat- ment causes a change in the characteristics of the plasmalemma. This change results in increased capacity of the plasmalemma to actively transport certain substances (N03, Na, Cl, MeG, and leucine) into the cell and retain them within the cell. Transport of certain other sub- stances (N02, K, Ca, P04, 504, and glutamic acid) is not affected by toxin-treatment. The evidence can be summarized as follows: 1) Toxin-stimulated uptake is temperature sensitive. 2) Toxin-treated tissues accumulate ions against a concentration gradient, and develop a steeper gradient with some ions than do control tissues. 3) Mech- anism 1 of ion transport is generally thought to be in the plasmalemma (17, 59). Toxin-treatment stimulates uptake of certain ions by mech- anism l. 4) Substances accumulated by toxin-stimulated tissues cannot 127 be washed out of such tissues in short desorption periods. 5) Toxin- treatment enhanced active influx but had no effect on active efflux of MeG. 6) Accumulation of only one ion (N03) from a single-salt solu- tion (Ca(NO3)2) was stimulated by toxin-treatment. Therefore, the altered plasmalemma had a greater affinity for N03 itself. 7) Nega- tive results from tests of intracellular sites support the conclusion that increased uptake is due to a change at the cell surface. 8) There is a change in electrical potential difference across the plasmalemma within 2 min after exposure to toxin (25). Data on desorption are of particular importance. Toxin-treat- ment stimulated uptake of N03, Na, Cl, MeG, and leucine by roots. There was no evidence that toxin-treatment either stimulated or in- hibited leakage of any substance, although variability was great enough to have masked small differences. In addition, toxin did not affect active efflux of MeG. These results contrast with those for HV-toxin and susceptible oats. HV-toxin causes immediate and massive leakage of materials from cells, which indicates that mechanisms-of—action of the 2 toxins are distinctly different. 0f the ions tested with whole tissues, only the accumulation of N03, Na, and Cl was stimulated by toxin. By comparison, reaction mixtures for experiments with isolated organelles contained high con- centrations of Cl and small amounts of Na (pH was adjusted with NaOH). Light-scattering and storage N03 pool data indicate that toxin had no effect on ion fluxes across organelle membranes. This suggests that 128 toxin specifically alters certain characteristics of the plasmalemma, but has no effect on other cellular membrane systems. The ion uptake experiments were modeled after experiments done by other workers. I have tried to relate my results to those al- ready published. Absorption of most ions, including K, is enhanced in the pre- sence of Ca (16). However, there is a report that Ca inhibits K uptake by corn roots in short term experiments (15). This report has been refuted by other workers (60, 69). Thus, my experimental solutions contained Ca unless Ca was a variable. Attempts were made to test tissues under physiological conditions; Ca apparently is required for normal functioning of the plasmalemma (16, 60, 93). In barley, Na uptake by mechansim l, which has high affinity for ions, is essentially eliminated by the presence of K and Ca (93). Uptake of Na by mech- anism l in corn roots was also greatly reduced in the presence of K and Ca. Mechanism 2 in barley roots, which has low affinity for ions, prefers Na over K (93). In contrast, under my experimental conditions there was more absorption of K than Na by mechanism 2 in corn roots. There was greater loss of 45Ca from corn roots when they were desorbed in CaClz than when they were desorbed in water. This in- dicates that at least part of the 45Ca entered the root by exchange. Since roots were grown in the presence of Ca, it is not surprising that 45Ca should exchange for unlabelled Ca. However, Maas reported that corn roots grown in the presence of Ca linearly absorb net Ca for 129 several hr when placed under the described experimental conditions (68). Maas also reported that mechanism 2 of Ca transport is greatly inhibited in the presence of K (68). My results indicate that mechanism 1 is slightly inhibited by the presence of K. There are reports that Cl is absorbed equally well in the presence and absence of Ca (70). My data indicate slight stimulation of Cl uptake when Ca is present. Uptake of Cl was said to be faster from NaCl or KCl solutions than from a CaClZ solution (117). My data show the reverse to be true. One of the most lively controversies in plant physiology concerns the location of the low affinity ion transport system, or mechanism 2 (17, 59, 72, 123). Laties and his group claim that mechanism 1 is in the plasmalemma and that mechanism 2 is in the tonoplast, i. e. , the mechanisms are in series (59, 117). Epstein and co-workers hold that both mechanisms are in the plasmalemma and thus are in parallel (123). Both groups have evidence for their claims, but MacRobbie is not convinced by the claims of either (72). This controversy is of special interest with respect to the site of toxin action. If mechanism 2 is convincingly shown to be in the plasma- lemma, it would support the hypothesis that toxin-treatment affects the plasmalemma. If mechanism 2 is in the tonoplast, then toxin af- fects the tonoplast because it affects mechanism 2. Alternatively, mechanism 2 would appear to be in the plasmalemma if toxin is shown to have no effect on the tonoplast. 130 It is possible that toxin enhances transport by binding directly with the solute to form a lipid soluble complex which can easily pass across the plasmalemma. Certain antibiotics, called ionophores, are thought to act in this way (87, 109). This possibility was not eliminated, but there are several reasons to think it unlikely. 1) It does not seem feasible that toxin would specifically complex with species as different from each other as N03, Na, Cl, MeG, and leucine. 2) Toxin does not complex with K. This was tested by monitoring K concentration in buffered solution with a K sensitive electrode. Addition of toxin to the solution did not affect the K concentration. Of course, toxin does not affect K transport either. Similar experiments should be tried with ions whose movement is affected by toxin. 3) Toxin does not in- terfere in the bacterial N03 assay, but because of the nature of the assay, this does not eliminate the possibility that toxin binds reversibly with N03. 4) Toxin does not require exogenous ions of any kind to 101/11 susceptible corn roots in the standard seedling bioassay. This implies that toxin does not act directly on the substance being trans- ported, but rather causes a change in the plasmalemma which in- creases the affinity of certain carriers for their substrates. The ar- gument is weakened by the fact that endogenous ions are present in tissue. 5) Toxin is host-specific and appears to have at least a degree of specificity for the plasmalemma of susceptible plants. Ionophores generally transport ions across any lipid-water interface (87). 131 Grarnicidin D (GD) is known to stimulate ion fluxes across mem- branes of higher plants (40, 76). It is interesting to compare the ef- fects of GD on plant roots (40) with those of toxin. 1) GD affects membranes of both mitochondria and intact roots. Toxin is specific for membranes of intact roots. 2) GD-enhanced influx does not occur through out ends of roots; the same is true for toxin (Table 17). 3) The percentage stimulation of influx by GD is dependent on the portion of the root used; the same is true for toxin (Table 16). GD stimulates ion uptake by squash, cucumber, pea, corn, barley, and oats; oats are by far the most sensitive. Effects of toxin are specific for sus- ceptible corn (57; Table 28). 4) GD stimulates uptake of Na and Cs but not to the degree observed for K and Rb. Other solutes were not tested. Toxin stimulation is most striking for N03, but stimulation is evident for Na, Cl, MeG, and leucine. There was no effect of toxin on any other substance tested (Table 31). 5) GD-stimulated influx is inhibited by anaerobiosis and respiratory inhibitors. Toxin-stimulated influx is inhibited by low temperature (Table 12). 6) GD enhanced transport of K by mechanism 1 more than that by mechanism 2. Toxin enhanced N03 accumulation by mechanism 1 more than by mechanism 2 (Figure 18), but it caused about equal stimulation of uptake of Na and C1 by both mechanisms (Tables 20 and 23). 7) GD appears to stimu- late ion movement across all types of cell membranes (40, 76, 87). Negative results with organelles suggest that toxin is specific for the plasmalemma. 132 The mechanisms of stimulation are unknown for both GD (40) and toxin. Since they both show certain degrees of specificity for plant species, ions, and (in the case of toxin) membranes, their sites of action may involve the extremely variable protein or lipid compo- nents of membranes. Chemically similar ionophores differ with re- spect to membrane specificity (40). Some antibiotics act only on specific lipid components of the membrane (40). It is now recognized that membranes are not just lipid-containing permeability barriers, but rather are dynamic units which contain catalytic proteins (72). In contrast to HV-toxin, which acts independently of cell functions (101), any model for HC-toxin action must accommodate known effects on metabolic functions of membranes. Antibiotics other than GD have been reported to have ion speci- ficity similar to that of toxin (29). Modes-of-action of 2 synthetic anti- bacterial compounds were compared with those of gramicidin and valinomycin against bacterial protoplasts. "Synthetic 1” increased permeability to N03; "synthetic 2” to N03 and Cl; gramicidin to N03, NH4, K, and Na; valinomycin to N03, NH4, and K. None of the anti- biotics affected membrane permeability to Ca, Mg, Mn, $04, or H2 P04. It is notable that resistance of some bacteria to these antibac- terials is located in the cell wall; when the wall is removed bacteria become sensitive (29). This is in contrast to the case of HV-toxin and oats. Susceptible cells are sensitive and resistant cells are insensi- tive whether cell walls are present or absent (103, 105). 133 Tissues which are excised and allowed to age have altered meta- bolic capabilities (28, 31, 80). Several experiments demonstrated this phenomenon and tested the effect of toxin on altered tissues. Aged corn roots absorbed P04 at a rate twice as fast as that of freshly ex- cised roots (Table 24). The same thing is known to happen in aged potato slices (28) and sweet potato roots (41). Toxin-treatment did not affect P04 absorption by either fresh or aged corn roots (Table 24). The rate of N03 accumulation was about twice as fast in aged as in fresh corn roots (Table 14). The percent stimulation of N03 accumu- lation by toxin was reduced in aged roots. It should be noted that ex- cised roots were aerated during the aging period, whereas comparable intact roots were not. The effect of aeration was not tested, but it seems unlikely that increased uptake is due to aeration because data agree with those from similar experiments by others (28, 41). The rate of leucine uptake was not measured in aged corn roots. However, the size of the soluble leucine pool in nonaerated aged roots was doubled as compared with that in nonaerated fresh roots (Table 35, Appendix). Data of Oaks also show more l4C-leucine in the soluble pool of aged than of fresh corn roots (Figures 1 and 3 in citation 84). Toxin-treatment expanded the size of the soluble leucine pool in fresh roots but had no effect on the size of the pool in aged roots (Figure 19 and Table 35). The data suggest that cell components have maximum capacities beyond which there is little or no stimulation by toxin. Certain systems 134 under normal cellular control in intact tissues can be stimulated by toxin-treatment. An analogous situation has been reported in squash hypocotyls infected with Hypomyces solani (31). Disease caused 96% stimulation of the MeG uptake rate when hypocotyls were freshly ex- cised. As hypocotyls were aged from 1-28 hr, the rate of uptake in- creased in both healthy and diseased tissues. By 28 hr, there was only 3% stimulation of uptake rate in diseased hypocotyls when com- pared with the control rate (31). Based on kinetics of MeG uptake by healthy and diseased squash hypocotyls, Hancock (31) proposed that infection induced a high affinity MeG carrier which was not present in healthy tissue. Since toxin also stimulated MeG uptake, similar analysis should be performed on the kinetics of MeG uptake by corn roots. Rates of accumulation of N03, Na, Cl, and leucine by toxin-treated corn roots could also be analyzed for evidence of possible induced carriers. Perhaps toxin activates new uptake systems instead of enhancing the affinity of constitutive carriers for their substrates. Such a study would require individual analysis of both known uptake mechanisms where applicable. Woolf- Hofstee plots of data on proposed mechanism 1 of N03 uptake (Figure 18) showed no evidence for a toxin-induced system but rather suggested toxin-enhancement of the normal carrier. However, this is specula- tive and additional experiments are required. Permeability changes are characteristic of plants infected with fungi, bacteria, or viruses (124). The most common observation is 135 increased leakiness of tissues after infection. Some examples: 1) Sweet potato roots develop an increased capacity to absorb 32P, 355, and 86Rb as they age. Infection by Ceratocystis fimbriata inhibits the capacity for increased uptake (41). 2) Mung bean hypocotyls infected with Rhizoctonia solani lose electrolytes at a faster rate than do heal- thy tissues (58). 3) For the first 24 hr after inoculation of cucumber leaves with Pseudomonas lachrymans, the rate of electrolyte loss was slower than that from healthy leaves. (One possible explanation is presence of a stimulatory pathogen-produced substance with action similar to that of HC-toxin.) After 24 hr, infected leaves lost more electrolytes than did healthy controls (127). 4) Pepper leaves infected with Xanthomonas vesicatoria lost electrolytes faster than did healthy leaves (8). 5) When 32Pecontaining carnation leaves were incubated in a bathing solution with Pseudomonas caryophylli, a pathogen, more 32P was lost than when leaves were incubated in the presence of Cory- nebacterium spp. , a nonpathogen (5). 6) When leaves of pepper plants were inoculated with tobacco etch virus, roots, but not leaves, lost more electrolytes than healthy controls or than pepper varieties which were susceptible but did not express the wilt symptom (27). 7) Bac- teriophage infection of Escherichia coli caused 42K and 28Mg to leak from cells within a few 'minutes (111). In addition to infective entities, many microbial toxins purported to be involved in plant disease are thought to alter cell membrane per- meability (2, 83). Most toxic metabolites are nonspecific with respect 136 to plant species (2, 83) and possible roles in disease development have not been demonstrated. However, HV—toxin specifically disrupts mem- branes of only those oat varieties which are susceptible to the pro- ducing fungus (103, 105). There is abundant evidence that the toxin is required for disease initiation and disease development (103, 105). In contrast to disease-induced leakiness, there are several re- ports of increased uptake or translocation in infected plant tissues. The most clearly defined case of increased uptake in diseased tissue is that of the MeG-squash hypocotyl system (31) described above. Tissues adjacent to lesions contained no fungus. They were appar- ently healthy as measured by apparent free space, plasmolysis, leak- age of amino acids, permeation of urea, and uptake of glycerol (30). However, as noted above, these same tissues had an increased capa- city to accumulate MeG (31). Another well-known case is crown gall, in which tumor cells accumulated twice as much K, P04, and atabrine from the growth medium as did normal cells growing at the same rate (128). Under the conditions used, uptake rates by both cell types were insensitive to temperature. Still other examples of disease- induced stimulation of uptake are known: 1) There was greater accu- mulation of 358, 32P, and 14C-sucrose from external media by dis- eased leaves than by healthy leaves in 19 different diseases (131). In 6 diseases accumulation was decreased in diseased leaves and in 6 others there was no change. 2) Infection of tomatoes with curly-top virus caused an increase in Ca uptake and a decrease in P and S uptake. 137 Efflux was not affected by disease (84). 3) Several reports indicate that there is rapid translocation of organic materials from healthy to infected tissues (14, 49, 63). 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However, some of the data are of interest, and are included in an appendix to preserve their record. In all these experiments, roots were exposed to toxin for 4 hr, then were incubated in 14EC-leucine for 2-3 hr. Ethanol soluble mate- rials were extracted and counted. Thus, these experiments measured the size of the soluble leucine pool, as defined by Oaks (80). Results show that 4 hr toxin-treatment caused an increase in uptake of leucine into ethanol-soluble and insoluble root fractions (Table 32). A 2 hr treatment may have caused slight stimulation in the size of the leucine pool. A toxin-induced increase in the size of the soluble leucine pool has been shown kinetically (Figure 19). The data do not show whether increased soluble leucine results from increased uptake from an 149 150 Table 32. Effect of Toxin on Leucine Content in Ethanol-Soluble and Insoluble Fractions from Corn Roots Seedlings were placed in 30 ml White's solution with or without toxin in 15 cm petri dishes. Toxin preparation gave 50% inhibition of susceptible root growth at 1. 0 pg/rnl. Root sections (1 cm) were ex- cised 2 mm behind the root tip and incubated 2. 5 hr in 1 mM leucine labelled with 0. 25 pc l‘l‘C-l-leucine/ml. There were 5 replicates of each treatment. Time required for leucine treatment and handling was 3. 5 hr, which resulted in totals of 5. 5 and 7. 5 hr from beginning of toxin-treatment until experiment was terminated. Toxin- Treatment Time T , 2 hr 4 hr Fraction ox1n Concn. Toxin- Toxin- Activity/ d Activity/ I d d 5 roots Induce. 5 roots .n uce. Stimulation Stimulation 1.1g /m1 cpm % cpm % Ethanol- 0 1316 . . . 1420 Soluble 1 1379 4 1832 23 5 1469 12 2280 61 Ethanol- 0 274 . . . 278 Insoluble 1 274 0 376 35 5 244 . . . 360 29 151 extracellular source or from increased release from an intracellular source. Increased leucine content of the ethanol-insoluble fraction could result from an affect on protein synthesis or from higher con- centration of914C than 12C-leucine in the soluble pool after toxin treatment. Later experiments suggest that increased pool size re- sults from an effect of toxin on uptake rate (Table 31). Increased in- corporation of 14C-amino acid is probably not caused by a direct ef- fect of toxin on protein synthesis (Figure 12), but rather by a higher concentration of l4C-amino acid in the protein precursor pool. Cer- tain growth regulators are known to enhance amino acid incorporation by increasing the size of the precursor pool (116). Data in Table 33 are additional evidence that 2 hr toxin-treat- ment increases the size of the soluble leucine pool. Note that 3. 5 hr elapsed from the time that roots were removed from toxin until the roots were placed in ethanol. Exposure of roots to toxin for 16 hr re- sulted in a 93% increase in amount of ethanol-soluble leucine present. This increase does not necessarily result from active accumulation because roots were given only 1 rinse in 12C-leucine before being placed in ethanol. Brief rinsing may not remove all leucine absorbed passively across a leaky membrane. Later experiments showed that increased absorptive capacity of roots after 4 hr toxin-treatment re- sulted from active accumulation. Root sections (1 cm long) taken 2 mm behind the root tip were used for all these experiments because this is the most active portion 152 Table 33. Effect of Toxin-Treatment Time on Size of the Leucine Pool in Corn Roots Seedlings were placed in White's solution with or without toxin. Root sections (1 cm) were excised and held in 14C-leucine 2. 5 hr. The toxin preparations used in experiments 1 and 2 caused 50% inhibi- tion of susceptible corn roots at 1. 0 and 0. 2 pg/ml, respectively. In experiment 1, concentrations of 5, 25, and 125 pg/ml gave essentially the same results for all treatment times. In experiment 2, toxin con- centration was 2 pg/rnl. There were 4 replicates of each treatment. Time required for handling and leucine treatment was 3. 5 hr, giving total elapsed times from initial toxin exposure of 4. 0-19. 5 hr. Toxin- Treatment Time Expt. No. hr 1 0.5 16.0 Activity/5 Root Sections Stimulation by C ont r 01 Toxin Toxin cpm cpm '70 1099 1071 1196 1301 8 1 125 1409 25 903 1274 41 701 1093 56 694 1339 93 153 of the root (130). It is not likely that the physiological state or toxin- sensitivity of this area of the root would change with seedling age, but these possibilities were checked experimentally, with the results shown in Table 34. Seedling age did not affect the percentage stimulation of leucine taken up by toxin-treated roots. As plant tissues age, they are known to become more active in uptake and other physiological functions (28, 31, 80). Infection has been reported to have a similar effect (31). Results of an experiment (Table 35) show that control roots excised prior to toxin-treatment time contained twice as much soluble leucine than did roots excised just prior to leucine exposure. These roots had aged 4-5 hr before exposure to 14C-leucine. Toxin-treatment did not increase the soluble leucine content of aged roots, but did increase the leucine content of freshly excised roots. Increased activity after aging was also ob- served in relation to uptake of N03 (Table 14) and P04 (Table 24). Results in Table 35 suggest that a signal from the seed or shoot suppresses the aging process in intact roots and that when this signal is removed by excision, the aging process begins. Such a signal could be required for toxin action. Results of another experiment (Table 36) suggest that the effect of removing the coleoptile or seed is qualitative- ly but not quantitatively the same in the root as excising the root. Roots of seedless or coleoptileless seedlings contained more leucine but were stimulated less by toxin than roots of intact seedlings. It is notable that gibberellins (GA3 and GA4), which normally stimulate 154 Table 34. Age of Seedlings in Relation to Size of the Leucine Pool in Toxin-Treated and Control Corn Roots Seedlings were placed 4 hr in White's solution with or without toxin (2 pg/ml). Toxin ED50 was 0.2 ng/ml. Root sections (1 cm) were held in 14C-leucine 2. 5 hr. Activity/5 Root Sections Stimulation Seedling Age by C ontr 01 T oxin T oxin hr cpm cpm % 48 856 1253 47 60 827 1267 53 72 1031 1605 56 155 Table 35. Effect of Toxin on Size of the Leucine Pool in Aged and Fresh Corn Roots Tissues were placed 4 hr in White's solution with or without toxin. Toxin ED50 was 1. 0 pg/ml. Aged roots were excised before toxin-treatment; fresh roots were excised after toxin-treatment. Root sections were held in 14C-leucine 2. 5 hr. Aged Fresh Toxin Concn. Activity/ Toxin-Induced Activity/ Toxin-Induced 5 Roots Stimulation 5 Roots Stimulation pg/rnl cpm % cpm V % 0 3704 . . . 1624 5 3719 0 2463 52 25 3754 1 2557 57 156 Table 36. Effect of Coleoptile or Seed Removal on Toxin Stimulation of the Leucine Pool in Roots of Corn Seedlings Seedlings were placed 4 hr in White's solution with or without toxin (2 ng/ml). Toxin ED50 was 0.2 pg/rnl. Where indicated, seeds or coleoptiles were removed prior to toxin-treatment. Roots (1 cm) were excised and held in 14C-leucine 2. 5 hr. Activity/5 Root Sections Stimulation Seedling Preparation by Control Toxin Toxin cpm cpm '70 Intact 1151 1469 29 ColeOptile Removed 1402 1606 14 Seed Removed 1333 1532 15 157 cucumber hypocotyl elongation, fail to do so if cotyledons are removed (47). The degree of GA-stimulation was directly proportional to the area of the cotyledon. . ... lily