vwmvw-‘w—w‘v'4 MECHANISMS OF FOLEAR ABSORPIION 224 HBGHER PIANTS WITH SPECIAL REFERiNCE T0 [ION Timsls for flu Dunc oi: pk. D. WCHEGAN 5mm UHEVEHSITY Seshadri Kannan 1966 THESIS This is to certify that the thesis entitled MECHANISMS OF FOLIAR ABSORPTION IN HIGHER PLANTS WITH SPECIAL REFERENCE TO IRON presented by Seshadri Kannan has been accepted towards fulfillment of the requirements for PhD. Horticulture degree in ' /4§;:%¥142647i¢425é&%31Ep/ Marin professor Sylvan . Wittwer Due February 21. 1966 0-169 ABSTRACT MECHANISMS OF FOLIAR ABSORPTION IN HIGHER PLANTS WITH SPECIAL REFERENCE TO IRON By Se shad ri Kannan Iron was probably the first micronutrient used successfully as a foliar spray for the correction of iron chlorosis. Today, more than 100 years later, its successful commercial use as a nutrient spray has spread only to a few species. The value of iron as well as other nutrients applied as sprays to aerial plant parts is dependent first upon the per- meation of a cuticular membrane, which constitutes the first barrier to absorption, secondly, the absorption by living cells, and thirdly, the . . . . S9 extent of translocation beyond the absorption Site. Penetration of Fe 59 . . from Fe labeled FeSO4 and FeEDDHA, With and Without urea, through enzymically isolated cuticular membranes, and of absorption by enzymically separated leaf cells, were studied in detail with the objective of resolving the contributing mechanisms. Cuticular membranes were separated from dorsal and ventral leaf surfaces of Euonymus japonicus, and from ripe tomato fruits, by treating them with an incubating mixture containing 2% pectinase. These were affixed to one end of a glass tube (15mm. diam.) to resemble the normal surface orientation on leaves and fruits, and the edges hermetically sealed with rubber cement. This tube, containing 3 ml. of the labeled solutes, Seshadri Kannan --2 was suspended into 30 ml. of deionized distilled water inside a large test tube (30 mm. diam). Suitable aliquots from the large test tube were periodically radioassayed by scintillation well and gas-flow detectors. The rates of penetration of 59Fe from 0.1mM solutions of 59Fe labeled FeSO4 and FeEDDHA, in the absence and presence of lOmM urea, were expressed as millimicromoles per unit time. The penetration rate of 59Fe was higher for FeSC)4 than from other iron sources, through the 3 types of cuticles. Permeability Was generally higher through cuticles from dorsal (stomatous) leaf surfaces of My, than through others. Permeability coefficients were derived from the re- sults of penetration of l4C-labeled non-polar solutes, namely, ethylene glycol, glycerol, D-ribose, D-glucose, sucrose, EDTA and EDDHA. A s ignificant inverse relationship between molecular weights of these solutes and their penetration rates was obtained, particularly for cuticular mem- branes from tomato fruits and those from ventral leaf surfaces offlon 'mus. Leaf cells consisting of a mixture of pallisade and mesophyll cells were enzymically isolated from tobacco, bean and soybean leaves for studying 59 mechanisms of Fe absorption. Light, Naz-succinate, and high temperature favored absorption. MH- tabolic inhibitors; viz, NaN3 and DNP reduced it. K3-citrate in the incur" - tion mixture also markedly reduced uptake, and NaI-ICO3 was inhibitory, Seshadri Kannan -- 3 especially in the dark. These results suggested that active metabolic pro- cesses were involved in 59Fe absorption by leaf cells. The presence of Ca at concentrations in excess of 5 x 10-4M in the absorption system was inhibitory to uptake of 59Fe and 54Mn by tobacco leaf cells. Fe and Mn mutually antagonized the absorption of each other, but the inhibition of Mn absorption by Fe was higher than that of Fe by Mn. A significant inverse correlation between the age of the leaf and a direct correlation between the chlorophyll content, and the rate of Fe absorption by the tobacco leaf cells was demonstrated. Fe absorp- tion was significantly lower for cells derived from (Fe dificient) chlorotic leaves than for those from healthy leaves of the Pl 54619-5-1 variety. Results Of studies of 59Fe absorption by leaf cells of tobacco and bean, using 0.1mM solutions of 59Fe labeled FeSO4, FeEDTA, and FeEDDHA, revealed a reduction by chelation, and an increase in 59Fe uptake from FeSO4 in the presence of lOmM urea. Urea had no effect on 59Fe absorption from FeEDTA and FeEDDHA. The data on Fe ab- sorption from FeEDTA and FeEDDHA by tobacco leaf cells also re- vealed that Fe was absorbed by the cells after its separation from the chelates, and that the ligands were not absorbed by the cells. 59 For intact bean plants, foliar absorption of Fe was measured by the "leaf immersion technique." Uptake of FeEDTA and FeEDDHA, Seshadri Kannan -- 4 as compared with FeSO4 was greatly reduced but rate of translocation expressed as percentage of absorption was greatly enhanced. Intact leaves absorbed more 59Fe from FeSO4 than from chelated sources, both in the absence and presence of urea, but this iron was translocated the least. The competitive nature of EDDHA on Fe absorption by tobacco leaf cells was revealed from enzyme-kinetic analysis. The synthetic chelate probably competes with an unknown natural chelate present in cells, for Fe. The affinity constant for Fe by the Fe-carrier was low. Adsorption by cuticular surfaces, diffusion through the cuticles, and subsequent metabolic uptake of Fe by leaf cells were analyzed with data on absorption isotherms for excised leaves of Euonymus japonicus. 59 The leaves were floated on Fe labeled FeSO4 solution (0.1mM) under . . O + 0 500 ft. 0. of cool fluorescent white light, at 20 - 2 C, or, in dark at o + o 0 - 2 C, and uptake was measured at predetermined times. The ad— 59 sorption of Fe by the cuticular surfaces was very rapid and instan- taneous. Diffusion through both the dorsal and ventral cuticular surfaces was activated by light and temperature, perhaps prompted by the rapid removal of the diffused ions by the leaf cells. It is concluded from these observations that foliar absorption is a multiphase process - initial adsorption on the cuticular surfaces, dif- fusion through cuticles and absorption, which is energy dependent. MECHANISMS OF FOLIAR ABSORPTION IN HIGHER PLANTS WITH SPECIAL REFERENCE TO IRON By Seshadri Kannan A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1966 Dedicated in memory of late Shri. G. Seshadri Aniyangar and late Shri. M. S. Krishnamachari. ACKNOWLEDGMENTS The author wishes to gratefully record his sincere appreciation for suggestion of this problem, guidance, help, and especially encourage- ment given by Dr. Sylvan Wittwer throughout the work. The author also is very much indebted to Drs. M. J. Bukovac, R. L. Carolus, H. D. Foth, N. E. Good, and A. L. Kenworthy for their valuable guidance and suggestions during the entire period of the graduate program. Sincere thanks are also extended to colleagues and friends who were helpful in various ways, and to Mrs. Geri Burkhardt for contribu- tion to the final preparation of the thesis. The financial support from the Rockefeller Foundation, New York, for the pursuance of the doctoral program, and from the Biological and Medical Division of the Atomic Energy Commission is gratefully acknowledged. Finally, the author wishes to extend his appreciation to his wife, Kalpakam and his son, Sriram, for their great patience and interest in pursuance of this work. ii TABLE OF CONTENTS Page INTRODUCTION. 0 O O O O O O ..... O O O O '0 O O O O O O 0 O O O O ‘ 1 LH‘ERATURE REVIEW 0 O ........... O O O O O O O O O I O 3 General Considerations . .......... . . ..... . . . 3 Iron as an Important Nutrient Element . . . . ....... 3 Functions of Iron in Plants. . . . . . . . . . ........ 3 Iron Supply .. . .......... . . . . . ........ 4 Iron Requirement and Distribution in Plants ........ 5 Nutrient Factors Related to Iron Chlorosis . . . ........ 6 Physiological Effects of Iron Chlorosis ............. 8 Correction of Iron Chlorosis ................... 8 Effectiveness of Foliar Sprays of Iron Salts and Iron Chelates.... ..... 8 Use of Other Chemicals for Iron Sprays .......... 9 Mechanism of Absorption of Iron and Chelates by Roots . 10 Mechanism of Foliar Absorption ................. 11 General .................. . ........ 11 Penetration of Ions Through the Cuticles of Leaves . . . . 13 Mechanism of Ion Uptake by Leaf Cells .......... 16 Mechanisms of Absorption of Iron by Cells and Cell Organelles .......... . . . . . . . . ..... 17 Foliar Absorption and Transport . . . . . . ....... 18 EXPERIMENTAL METHODS ..................... 19 Procedures for Isolating Cuticles Enzymically ..... 19 Studies on Penetration Through Enzymically Isolated Cuticles ................ . . ...... 20 Studies on Fe Absorption by Enzymically Isolated. . Leaf Cells ......... . . . . . . ....... 22 ill Measurement of Specific Absorption and Transolocation of 59Fe in Intact Bean Plants (Leaf Immersion Technique) ....................... Expression of Results and Estimates of Variability . . RESULTS. . . . . ...... . ..................... Penetration of Solutes Through Cuticular Membranes ..... Penetration of 59FeSO4 and 59FeEDDHA with and without Urea Through Cuticular Membranes ..... Ion Binding by Tomato Fruit Cuticles . . . . . . . . . Effects of Molecular Weight of Solutes on the Rate of Penetration Through Cuticles ............. Permeability Coefficients . . . . . ............ Mechanism of Absorption of Fe by Leaf Cells ......... Absorption of Fe as Related to Cell Frequency in the Incubation Solution ....... . . . . . ...... Factors Influencing the Absorption of Fe by Tobacco Leaf Cells . . . . . ................. External Factors . . . . . . . .......... Light, succinate, and bicarbonate ...... Citrate, malate, and succinate ........ Temperature . . . . . . ........... Metabolic inhibitors ..... . ....... Effects of Ca on ab orption of Fe and Mn . . Internal Factors ................. 59 Absorption of Fe from FeSO4, FeEDTA, FeEDDHA. and F8504 in the Presence of Urea . . . . . ...... Absorption of 59Fe by Leaf Cells ............ Uptake and Transport of 5 Fe in Intact Plants ..... Effects of Pretreatments with EDDHA and Urea on 9Fe Absorption by Bean Leaf Cells . . . . . . . . Absorption of 5 Fe from FeSO4 in Alisence and Pre- sence of Urea, 14C -EDDHA, and 4C -Urea by Tobacco Leaf Cells ....... . ........ iv Pa ge 25 25 27 27 27 27 29 35 36 36 39 39 39 39 42 42 42 51 54 54 54 54 58 Determination of Affinity Constants, and Aé) lication of Enzyme- -Kinetics to Absorption of Fe by Leaf Cells ................ . ..... Ion Absorption by Leaves --- A Multistep Process ..... DISCUSSION ............................. Permeability of Fe Through Cuticular Membranes . ..... Penetration of Organic Compounds and the Relations of Molecular Weight ...... . . . . . ...... Mechanisms of Absorption of Fe by Enzymically Isolated Leaf Cells ................. . ....... Influence of External Factors .............. Effects of Internal Factors . . . ...... . ...... The Role of Chelates and Urea . . . . . ........ The Use of Enzyme-Kinetics and the Competition by Chelates ................ . ....... Steps Involved in the Absorption Process by Intact Leaves . . SUMMARY ...................... . ....... LITERATURE CITED .................. . ..... Pa ge 58 62 65 65 66 66 66 7O 7 l 72 72 74 78 INTRODUCTION Iron was probably the first micronutrient used successfully as a foliar spray for the correction of a nutritional disorder (44). Today, more than 100 years later, its successful commercial use as a nutrient spray has spread, however, only to a few species. These include pineapple (164), grain sorghum (81, 162), and gardenia (147). With these exceptions, remedial measures for supplying iron as a spray to deficient foliage have not been entirely satisfactory. The effectiveness of foliar sprays of iron for most plants is limited, first by slow absorption (163) and secondly by a lack of mobility after absorption (16). Much has been done, by contrast, to increase the availability of iron applied to root media. Reports of the experimental and commercial success of the synthetic chelates, ethylenediamine tetraacetic acid (EDTA) and ethyl- enediamine (di o-hydroxphenylacetic acid) (EDDHA) in facilitating the availabil- ity of iron for plants grown in soilless culture and applied as a band in the soil as a component of a complete fertilizer are now legion. Synthetic chelating agents have proven most valuable as a means of increasing the availability of iron absorbed by roots. Comparably successful results have not apparently been obtained with iron Chelates over iron sulfate applied as foliar sprays al- though a few reports (8, 9, 162) show chelated iron to be equally effective. The value of iron, as well as other nutrients, applied as sprays to aerial plant parts is dependent first upon the permeation of a cuticular membrane which constitutes the first barrier to foliar absorption; secondly, the absorption by living cells; and thirdly, the extent of translocation beyond the absorption site. Experimental data are lacking on the first two and are not extensive on the third. This dissertation will describe for iron and the synthetic Chelates of iron (EDTA and EDDHA) some of the mechanisms of penetration through enzymically isolated cuticular membranes, absorption by enzymically iso- lated leaf cells, and translocation in intact plants. LITERATURE REVIEW General Considerations Iron as an Important Nutrient Element Iron is well known as an important nutrient element that is required by and often deficient in agricultural crops. Symptoms of iron deficiency develop on crops grown in soils either lacking iron or having an insufficient reserve of iron in the plant. Chlorosis, a general term denoting the lack of chlorophyll, is also a symptom caused by inadequate iron. Iron Chlorosis specifically refers to a Chlorosis which could be alleviated by providing the plant with suitable iron compounds. Iron chlorotic plants are found on both acid as well as alkaline or calcareous soils. Functions of Iron in Plants Inorganic iron salts are IQ‘lOWI'l to possess properties of an oxidase and an electron transporter depending upon the pH of the medium, although their capacity to function as a catalase or peroxidase is very negligible (42). The role of iron enzymes in respiration has been well established. The cytochromes found in plant and animal cells (40) consist of a number of iron porphyrin enzymes. An adequate supply of iron is essential for the formation of chlorophyll in the plant. Recently, two different actions of iron on plant growth have been recognized (l8, 19). An iron addition to roots in a iron free nutrient solution results in a reduced rate of elongation in light and an enhancement in darkness and this is recognized as the high iron effect. Addition of EDTA strongly inhibited cell multiplication in darkness by chelating iron, and this is the low iron effect, produced by amounts of iron lower than those carried to the plant from the seed. Iron Supply In the natural environment, the inherent supply of iron is usually adequate for plants and an actual deficiency of iron in the nutrient medium is probably found only in sand or water cultures. Since iron is not deficient in most soils, variations in the capacity of plants to absorb and accumulate this element probably relate to factors other than the level of iron in the soil (12). Thus, there appears to be a regulatory mechanism for the absorption and translocation of iron in plants, similar to the mechanism in animals. Most iron compounds occurring naturally in soils are quite insoluble. Ferrous ions are unstable in aerated soils with a pH value of 6 or higher. Alkalinity and aeration favor oxidation of iron and waterlogging favors reduction (85). The extent of oxidation or reduction is also influenced by other factors, such as activity of microorganisms, level of soil organic matter, and the presence of other cations or anions. Bivalent iron is the form of iron readily absorbed by plants (12). It has been recognized that some plants grow well where others will not grow without developing iron Chlorosis and this has been attributed to the differ- ences among plant species. Brown (11) tested the susceptibility to iron Chlorosis of a number of plant species grown on calcareous and organic soils and found that they responded variously. In iron absorption studies, with a few selected plant species, only the soybean (variety PI 54619-5-1) failed to absorb sufficient iron from a calcareous soil to maintain growth (13). Iron Requirement and Distribution in Plants Iron occurs in rather large amounts in most soils, but it is present only in small amounts in plants. Glenister (39) found that the lower (older) leaves of sunflower contained 10 times as much iron as the upper (younger) leaves. A continuous supply of iron was necessary to keep the younger leaves normal. Similar requirement has been indicated for soybean (13). On the basis of leaf analysis, 60 to 90 ppm. of Fe is considered as optimum (12). Mildly chlorotic citrus leaves contained about 50 ppm. (89), and nearly normal leaves contained 80 ppm. (155). It is reported (12) that 82% of the total iron in spinach leaves was found in chloroplasts, and five-sixths of the firmly bound iron was associat- ted with phosphorus containing proteins. The cell nucleus contained only traces while the cell wall had no iron. Chloroplasts represented about one-"third of the dry weight in sugar beet leaves and contained 6l% of the total iron (161). Bennett (6) found that the active iron content of pear leaves built up rapidly early in growth, and then diminished, while residual iron was low at first and then accumulated as the growth progressed. He concluded that ”active" iron can be transformed into residual iron, but that iron once transformed into the re— sidual state cannot be utilized for chlorophyll formation. Brown and Holmes (13) observed that iron was mobile in green leaves of soybeans with one source of iron, but was immobile to chlorotic leaves which developed on the same plant, if the source of iron was changed. in iron de- ficient bacterial cells of Aerobacter indologenes, there appeared to be a selective distribution of iron, and the iron requirement of cytochrome system was met first (157). With regard to the amount of iron required, the Chlorosis susceptible soybean variety needed a higher iron cencentration to produce green plants, than the non-susceptible variety. Nutrient Factors Related to Iron Chlorosis Iron Chlorosis has been observed in both acid and alkaline soils (89). The causes of lime induced Chlorosis have been discussed by Brown (3, 12, 15) and Wallace and Lunt (148). Phosphates might precipitate ircn either in the soil or in plant tissues (7). High manganese in soils or plants might oxidise iron to an inactive state (128). Excessive amounts of potassium in. leaves might displace iron and disrupt metabolic processes (12). Bicarbonate is known to induce iron Chlorosis (53), which decreased the availability of iron at the root surface (29, 92, 115, 145). Increasing concentrations of KHCO3 progressively depressed protein synthesis and oxidase activity with a decrease in respiration and metabo- lism of potato discs (12). Brown (12) suggested that NaHCO3 might increase the concentration 0f HC03, C03, and OH ions, which might compete with phosphate adsorbed on the surface of soil particles. The excess phosphate in soil solution would combine with iron to form insoluble iron phosphate, thus, indirectly inducing iron chlorosis. The macronutrient elements associated with iron chlorosis are usually calcium, phosphorus, potassium and nitrogen. Consistent differences were obtained between chlorotic and green leaves in the Ca/K ratio (53, 101). Phosphorus could influence iron metabolism and plant species and varieties differ in their susceptibility to this effect upon iron (14, 159). Excess phosphate showed no relationship to iron chlorosis in Utah soils (138). Of the micronutrients elements, the effectiveness for inducing iron chlorosis was of the order: N1 > Cu) Co>Cr> Zn>Mo> Mn (535;, 66). Pineapples developed iron chlorosis when grown on manganiferous soils (63). Accumulation of copper in soil caused iron chlorosis in Florida (12). Nickel, cobalt, zinc and copper induced iron deficiency in a number of annual crops (55, 56, 104, 105, 108, 109). High concentrations of calcium in soil may be a major factor in iron chlorosis (12, 150), but the effect of this ion in soils is closely associated with its effect on soil pH. It was suggested (104) that an interfering ion like Mn, Cu, Zn and Ca might take the place of iron in the malic or malonic acid complexes in the plant. These ions could interfere With iron translocation, since most of the iron was translocated to the top in the form of iron malate or malonate. Physiological Effects of Iron Chlorosis Dekock and Morrison (30) showed that the organic acid content of chlorotic plants was often similar whether it was produced by genetic effects, lime, or even virus. The organic acids in chlorotic plants were high (158). Cytochrome oxidase activity was also inhibited (101), the succinic oxidase system was sensitive (4), and the re was an effect on the oxidation and reduction of cytochrome c in the mitochondria of Ricinus. Peroxidase activity was not markedly different in normal and iron chlorotic leaves, whereas catalase activity was several times greater in the green leaves (12, 29). Changes in the malonic acid content in the leaves of Phaseolus vulgaris were associated with lime induced iron chlorosis (114), while an increase in malonic acid marked the onset of iron deficiency (171). In Neurospora crassa, iron deficiency reduced catalase, peroxidase, cyto- chrome c reductase, oxidase, and DNPase activities (2, 110). Correction of Iron Chlorosis Effectiveness of Foliar Sprays of Iron Salts and Iron Chelates The earliest successful commercial use of iron sulfate was on pineapples where a 2. 8% spray of ferrous sulfate gave temporary recovery from chlorosis (72). Similar results with iron sulfate sprays have been obtained in citrus (47), cacao (44) and with certain vegetables and ornamental plants (160). Guest and Chapman (45) tested 30 iron compounds and found that only with ferrous sul- fate sprays were satisfactory results obtained. Soil applications of chelated iron, though effective, are very expensive (132, 133). Iron chlorosis may be controlled in currants, plums, and pears by spraying 0.1% ferric EDTA, and in peaches by spraying ferric DTPA (Diethylene triamine pentaacetic acid) (8), although ferric DTPA was not translocated from the leaves and soil applications were more effective (58). On the other hand, Wallihan (155) and Wallihan gal. (156) found no benefit from soil application of EDTA or any other chelating agent for citrus. Syn- thetic Chelates have been used effectively to keep iron soluble and available for plant growth when applied to the root medium (21, 54, 87, 88, 129, 130, 131). Tiffin _et_a_1_. (142) found that chlorosis could not be corrected by an application of 20 ppm chelating agent, while foliar applied radio-iron caused greening only at the sites of application. Use of Other Chemicals for Iron sprgx A combination of citric acid with iron sulfate applied as a spray has been advocated in Arizona (147). Since iron is transported in the form of iron malate (140), it is suggested that iron malate might be effective for foliar application of iron. The usefulness of an iron-fructose chelate in biological systems has also been suggested (147). The sprays of iron che- lated with 2,4-pentanedione, which results in a non-ionic form of iron were not effective (148). 10 Since urea penetrates leaves readily, it may influence the permeability of leaves to other nutrients (75). Some citrus growers in Arizona and Cali- fornia claim that the combination of iron chelate and urea in foliar sprays corrected iron chlorosis (147). Iron sulfate combined with glucylglycine and applied as a foliage spray was exceptionally promising on macadamia (148). Triiodobenzoic acid may favor translocation and utilization of subse- quently applied iron (80). Mechanism of Absorption of Iron and Chelates by Roots A chelating agent is considered as an inert metal carrier (124) which delivers the metals to absorbing surfaces, but is not itself absorbed (68). Several reports indicated that the entire chelating molecule may be absorbed by the roots (57, 58, 146, 153). Some (61, 82, 88, 149, 154) have suggested the uptake of the chelating agent or a decomposition product by the plant roots. Tiffin 33 al_. (141), however, demonstrated a differential absorption of metal chelate components by roots. T iffin and Brown (139) further showed a selec- tive absorption of Fe from FeEDTA, FeDTPA, and FeEDDHA by Hawkeye soybeans. However, the increased concentration of iron in plant materials (147) in addition to the presence of 14C suggested the probability that both chelate components (metal and ligand) were absorbed together. It was further found (162) that equivalent uptake of iron and 15N chelating agent was obtained in citrus. In later work (147) the ratio between iron and 11 chelating agent was greater than unity, which suggested that the uptake of these components might not be equivalent. The same lack of equivalence was shown by Krauss and Specht (82) in studies with green algae. Tiffin 53:11. (136) concluded from work with soybeans, zinnia, and sunflower that these plants selectively absorbed the iron, and the EDDHA remaining, for the most part, in the nutrient medium. The studies by Hale and Wallace (50) showed that less chelating agent than iron found its way into the plants via the root system. The effects of pH on the availability of iron from Chelates are signifi- cant (146, 147). FeEDDHA supplies iron to plants reasonably well on any soil, but FeEDTA is effective only on acid and neutral soils. The differ- ential behaviour of the two is related to the stability of these against hydroiy~ sis in the soil. At pH values above 7, FeEDTA is easily hydrolyzed and the iron is precipitated as hydroxide, which is poorly available to plants. At pH 7 and higher, many times as much FeEDDHA remains in solution as FeEDTA. Mechanism of Foliar Absorption Gene ral The foliar absorption of mineral nutrients is an active metabolic process (74, 164). Many have obtained the typical two phase time course curves in 12 foliar uptake of nutrients (16, 17, 73, 137), which correspond to those for root absorption (84). Typical of an active uptake process, foliar absorp- tion is temperature dependent. Temperature coefficient values in excess of 2 were obtained for 32F, 86Rb and 42K in bean leaves (136) over a range of 10 to 21. 10C, for Mn absorption by soybean leaves (102) over a range of 360 to 640 F, for 6 Co absorption by bean leaves between a low tem- perature of 700 to 760F and a high temperature of 870 to IOOOF (46), and for Br uptake by Nitella cells between 100 to 200C (60). Light is closely associated with the metabolic activity of all living plant protoplasm (90), and the positive effect of light on ion uptake by iso- lated green leaves has been thoroughly documented (10, 83). Metabolic inhibitors provide a means to relate foliar absorption to corresponding processes of metabolism. Among commonly used inhibitors are 2,4-dinitrophenol (DNP), an uncoupling agent in oxidative phosphoryla- tion, and arsenate and iodoacetate, uncouplers of the substrate level phos— phorylation. Cyanide and azide inhibit respiration in uncoupled as well as coupled systems (152). Other factors used as criteria for active uptake include accumulation against a concentration gradient, irreversibility, oxygen dependence, speci- ficity, and ion competition (83, 164), and pH dependence (26, 28, 135, 136, 144, 167). 13 Penetration of Ions Through the Cuticles of Leaves The cuticular membrane which sheaths the aerial parts of higher plants is the first barrier to foliar absorption (164). The chemical nature of cutinized outer epidermal cell walls has been summarized by Frey-Wyssling (37). The chemical components of cuticles have been separated by many workers (36, 86, 99, 100, 118, 167). The outer coating of the cuticle is made up of a wax film and plant waxes which consist of free alcohols, hydrocarbons, unsaturated ketones, long chain (C28) aldehydes and glycerol compounds, the main component being neutral or hard wax (64, 65, 117, 118, 123). Differences in the nature of upper and lower leaf cuticles have been recorded for many species. There is generally greater development of cutin in the upper (ventral) than in the lower (dorsal) surface (99). Cuticular membranes have long been described and their chemical properties analyzed, but only recently have their permeability properties been studied. Hurst (67) working with insect cuticles first reported that the rate of movement of organic substances and even water might not be the same when they were escaping (efflux), as when they were entering (influx). Evidence for permeability of the plant cuticle for water is found in the phenomena of cuticular transpiration to polar substances in the salt residues on the leaves of plants in saline habitats, and to the uptake of 14 non-polar substances after foliar application. Gases have been known to diffuse through leaf cuticles more readily than liquids. Schieferstein (125) found in the case of ivy leaf cuticular membranes, the influx of water was 1.44 times of efflux. It has been explained that permeability of cuticles for liquids is based on pores in the cuticle. The lipoid nature of the cuticle enables the penetration of non-polar substances (the "lipoidal pathway" ), and an "aqueous pathway" through which polar substances may penetrate is the result of imbibition by the cutin layers, as well as through the hydrophilic petic layers (27, 32, 95). The identification of pores in the cuticle which served as excretory ducts, and are concerned with the formation of wax protuberances on the cuticular surface, and also of ducts extending up to the surface of the cuticle have also been recorded (51, 95, 106). Whether these pores are actually concerned with permeability of substance is, however, not known. Recent studies of permeation of cations and anions through enzymically cuticular membranes have greatly contributed to the understanding of foliar absorption. Penetration of ions through cuticles has been assumed as a physical diffusion process which has also been recently confirmed (167, 169). It was demonstrated with ripe tomato fruit cuticles (astomatous), and green onion leaf cuticles (stomatous), that ion penetration followed a typical dif- dusion equation. Permeability of cuticular membranes to isotopically labeled 15 , 45 59 _ 32 35 cations ( Ca, Fe), was greater than that for anions ( P, S). Inorganic ions penetrated more rapidly from the "outer" side to the "inner" side (influx), than from the "inner" to the "outer" side (efflux) (169). The greater ion binding capacity of the "inner" surface was proposed as a possible explanation (166). This was found to be how- ever not applicable to urea (170). Using chemically excised apple leaf cuticles, the reverse results for penetration of urea, benzoic acid, glucose, maleic hydrazide, and simazine have been reported (41). Time course studies have revealed that urea penetrates cuticular membranes at a remarkable rate, and this increases with time (75). Furthermore, urea increased the rate of foliar uptake of other nutrients simultaneously applied. Facilitation of absorption by urea occurred at the cuticular level for anions and at both cuticular and cellular levels for cations (75). Many investigators believe that isolated cuticular membranes still maintained their integrity and natural permeability properties. However, it has been found that the permeation rates of substances through isolated cuticular membranes was much less than when they were intact on the leaf surface (41). Jyung and Wittwer (75) have emphasized the importance of molecular size, charge, partition, volatility, solubility and adsorbability, in solute penetration through cuticular membranes. 16 Mechanism of Ion Uptake by Leaf Cells Once the cuticular entry of nutrients into the leaf is accomplished, they may be either absorbed directly by leaf cells or diffuse within a free space volume. Ion uptake studies by isolated leaf cells of various types (spongy, mesophyll, palisade, epidermal, guard cells) may be one of the most useful approaches for the future in resolving mechanisms of foliar absorption (75, 76). Living cells removed from leaf tissues and suspended in solution constitute a useful system for studying the mechanisms of ion uptake. The structural and functional heterogeneity typical of in- tact leaves is minimized, and all concern for differences associated with stomatal and cuticular penetration and movement through cracks on leaf surfaces is eliminated (75). Although extensive work has been reported on the presumed absorp- tion of solutes by plant cells (71), only excised or intact organs, and slices of storage tissues have been used for such studies. Ion uptake studies exclusively by isolated cells is of very recent origin, especially those in- volving enzymically isolated leaf cells (76). Various procedures have been described for isolating single cells from leaves (22, 76, 77, 172, 173). The procedure herein is based on enzymatic degradation of the intercellular pectic substances by pectinase (126), a technique applied previously to root meristems (22). Intact living cells have been isolated by this method from the leaves of Nicotiana glutinosa, 17 Datura stramonium, potato, Chenopodium amaranticolor, Quercus borealis, Crotelaria spectabilis, peach, cherry, cucumber, and Magnolia sp. (173). Mechanisms of Absorption of Ions by Cells and Cell Organelles Yeast cells have been used to study the mechanism of uptake of man- ganese (70, 71). It has been postulated that both phosphate and manganese are transferred into the yeast cell in an essentially irreversible manner by two systems which are coupled in an unknown way to glycolysis at the 3-phospho- glyceraldehyde dehydrogenase step. Yeast cells which ordinarily absorbed phosphate only after a lag period of thirty minutes, absorbed without delay, if pretreated with glucose and potassium. It was suggested therefore that a phosphate carrier was synthesized during glycolysis. Absorption of Rb and phosphate was studied by Jyung it. 31. (76) using enzymically isolated cells from green tobacco leaves. It. was found that absorption of Rh was temperature dependent. Ener gy sources such as light, succinate and bicarbonate as sources of C02 for photosynthesis promoted uptake. Absorption rates in the light for green leaf cells exceeded enormously those isolated from the pit h. NaN3 and DNP reduced the Rb and phosphorus uptake. The inhibitory effect of DNP on Rb uptake was negated by the addition of ATP (76). Absorption of iron by isolated chloroplasts was studied by Hagen it. a}: (48) and the amount absorbed was governed by at least three factors, (a) the 18 mass of chloroplasts, (b) the concentration of iron in the system and (c) iron-binding capacity of the suspension medium. Mitochondria introduced . . . . 55 59 into a wheat chloroplast suspenSion containing acetate and Fe- Fe, diminished iron absorption. This suggested a possible competition for iron between different particulates, which might a ffect the availability of iron in immature leaves (48). Waring and Werkman (157) studied the uptake of iron by six bacterial cell species and found .a variance according to the cytochrome system contained. Foliar Absorption and Transport Foliar absorption and transport of mineral nutrients have been dis- cussed extensively by Wittwer (163) and by Jyung and Wittwer ('75). The absorption of potassium, calcium, magnesium, manganese, and zinc by leaf surfaces were found to be very rapid, but were not equal to that of the nitrogen from urea. Marked differences occurred in the extent of translocation from the foliage to other parts. Potassium, along with sodium, rub'rlium, and cesium , were the most mobile of all cations. Zinc, copper, and manganese were moderately mobile, with iron, molybdenum, and boron only slightly mobile, and calcium, and magnesium along with strontium and barium were essentially immobile. Iron and molybdenum in addition to being relatively immobile, were absorbed at slow rates. EXPE RIMENTAL METHODS Procedure for Isolating Cuticles Enzymically Penetration of iron and other substances through cuticles was studied with Euonymus japonicus cuticles obtained from both the dorsal and ventral leaf surfaces and from ripe tomato fruits. The method of enzymic separa- tion was essentially that of Orgell (112) as modified by Okuda and Yamada (113). The enzyme solution used for separating cuticles consisted of the following: Acetate buffer (pH 3.5 - 4. 5) 2. OM Pectinase 2. 0% Cellulase 0. 2% Hemicellulase 0.2% The isolation procedure was as follows: 1. Ripe tomato fruits were sliced and the pulp scooped out. 2. The slices were transferred to the enzyme mixture and incubated for a week at 20-250C. 3. The slices were removed, washed with deionized water to elimi- nate the cell debri and further treated with the enzyme solution for 2-3 days if cell remnants still adhered. 4. Cuticles were washed with deionized water, and cleaned carefully with soft tissue paper until free of cells, air-dried, and stored. The following procedure was adopted for the isolation of the cuticles from Euonmus leaves: 19 20 l. Edges of the leaves trimmed and transfer to enzyme mixture. 2. Leaves in solution aspirated repeatedly to permit diffusion of the enzyme solution through the cut edges. 3. Incubated at room temperature (20-250C) for 2 weeks, or until the cuticle separated easily. 4. The delicate cuticles were peeled carefully from both surfaces and washed free of cell fragments with deionized water. 5. Cuticles resuspended in deionized water and stirred slowly with a magnetic stirrer for 1-2 hours. Resultant cuticular membranes were very clean and free from cellular fragments. 6. Cuticles carefully washed and stored dry. The origin of the cuticle was identified with microscope before use. Cuticles from dorsal surfaces were stomatous. While it was rather easy to separate the cuticles from ripe tomato fruits, the separation was more difficult with Euonymus leaves. The tomato fruits were obtained from plants grown in the greenhouse, and the Euonymus leaves were collected during mid-summer from plants grown on the campus. Studies on Penetration Through Enzymically Isolated Cuticles The rates of penetration of iron, with and without EDDHA and urea, 59 were studied using 0.1 mM solutions of FeSO4 labeled with Fe. Equimolar 21 solutions Fe and EDDHA were used for chelation. The urea concentra- tion was 10. 0 mM, and the pH was 6. l4C-labeled organic compounds of different molecular weights in addition to l4C-labeled EDTA and EDDHA were used to ascertain if molecular weight influenced the rate of penetra- tion. Concentrations of all compounds were 0.1 mM. The apparatus used and methods employed were those of Yamada et a_l. (169). Slight modifications such as a metal jacket to hold additional tubes in the water bath, and the use of rubber cement, self sealing latex bandage (The Sealtex Company, Chicago, lllinois), and Parafilm (Marathon Parafilms) for sealing the cuticular membranes over the open end of the glass tubes, were made. A large test tube (35 mm. diam.) with 30 ml. of deionized water was suspended in the constant temperature (20°C) water bath. The cuticular membrane was carefully affixed on one end of a smaller tube (15 mm. diam.) to correspond with surface orientation on the leaf. This smaller tube was held in position inside the large test tube by means of a rubber stopper. The labeled solution (3 ml) was added to the small tube, while it was care- fully lowered into the deionized water in the large test tube, to the level of the meniscus in the small tube. The specific activity of 59Fe was 3 uc/ umole Fe, and 14C was 10 uc/umole. Rates of penetration through the cuticular membranes was determined periodically by radio-assay of 1 ml. or 2 ml. aliquots of the outer solution in the large tube. A gamma scintillation-well 22 59 _ l4 , detector was employed for the Fe solutions. For C an automatic gas flow counter was used after the liquid samples were dried in planchets under a Fisher Infra-Radiator. Studies on Fe Absorption by Enzymically Isolated Leaf Cells The uptake of Fe was studied using enzymically isolated leaf cells of tobacco and bean. The procedure for cell separation was a modifica- tion of methods by Zaitlin (172), Letham (91), and Jyung _et a_l_. (76). Fully expanded young leaves of tobacco, and primary leaves of bean were used. The modified procedure is outlined in Table I. The separation mixture consisted of the nutrient solution of Murashige and Skoog (107) to which was added pectinase and the pH adjusted to 6 (Table 11). Isolated leaf cells were incubated by suspending approximately 50 mg. dry weight equivalent of living cells in 10 m1. of incubation mixture in 50 ml. Erlenmeyer flasks. These were held in a water bath shaker at 20*: 20C and exposed to ca. 500 ft. c. of cool white fluorescent light. The incubation mix- ture contained 1170 umoles of sucrose and 0.5 umoles 59Fe labeled FeSO4 , FeEDTA, or FeEDDHA. 100 umoles of trismaleate, and 20 umoles each of Naz-succinate, K3-citrate, and NaHCO3 were included only in a few experiments where interference or competition with Fe absorption by cells was not suspected. The specific activity of all radioisotopes used in the cell uptake studies 59 -H- 54 +1- 65 «H— 86 + ( Fe , Mn , Zn , or Rb) was 0.1uc/umole. The salts 23 TA BLE I Procedure for Enzymic Separation of Leaf Cells. Slice the leaves into 2'3 mmZ sections after removing the midrib. Treat with the medium for cell separation (see Table II), in the ratio of 20 ml. per 1-2 gms. of leaf sections. Shake vigorously in a reciprocating shaker for 3-4 hours at ca. 500 ft. c.(1ight intensity), and 20'!- 20C. (temperature). Centrifuge at 600 x g. for 15 minutes; wash sediment with 0.25 M sucrose (ice cold) and again centrifuge. Repeat the process. Re-suspend the cells in sucrose and refrigerate until used. 24 Table II I MEDIUM FOR CELL SEPARATION A. Mineral Salts Major elements Trace elements Salts mg/l. Salts mg/l. 10103 1900 MnSO4. H20 22. 3 C3 C12.2H20 440 ZUSO4.4H20 8. 6 MgSO4.7I-I20 370 KI 0.83 KH2P04 l7 0 N82M004. 2H20 0. 25 Na2 EDTA 37 . 3 CuSO4. SHZO 0. 025 FeSO4. 71120 27. 8 CoCIZ. 61120 0. 025 B. Organic Constituents Sucrose 30 g/l. Myo Inositol 100 mg/l. Glycine 2. 0 mg/l. Nicotinic acid 0.5 mg/l. Indoleacetic acid 1-30 mg/l. Pyridoxin . HCI 0.5 mg/l. Kinetin 0.04 - 10 mg/l. Thiamin . HCI 0.1 mg/l. 1 After Murashige and Skoog (107). Pectinase added to the final solution 10 gm/l. and pH adjusted to 6. 25 were FeSO4, MnSO4, ZnSO4, RbCl, and CaSO4- At the end of an experimental period, the contents in the flask were centrifuged and/ or a known aliquot was collected under mild suction, on a weighed filter paper mounted on a precipitation apparatus. It was washed twice with 1.5 m1. ice cold 0.1 M sucrose solution, and finally with deionized water. The filter paper containing the cells was then air-dried, weighed and radioassayed. . . . . 59 . Measurement of Spec1fic Absorption and Translocation of Fe in Intact Bean Plants (Leaf Immersion Technique) (74). Bean seeds were germinated in sand and 15 day old seedlings were used for specific absorption and translocation studies. One of the primary . . 59 . . . . leaves were immersed in the Fe labeled solution kept in a Petri-dish. All experiments were conducted under an illumination of 500 ft. c. and o + o . temperature of 20 - 2 C. The dry weights of the leaves and other plant parts were determined and the radioactivity measured. Specific absorption 59 . . . was expressed as mumoles of Fe per mg. dry weight, per unit time, and translocation on the basis of that recovered in plant parts other than the treated leaf. Expression of Results and Estimates of Variability The results of studies on cuticular penetration of substances are ex- pressed as the absolute quantity penetrated during different time intervals. 26 All treatments were replicated three times and the results statistically analyzed (31, 127). Treatments with enzymically isolated cells were also replicated three times and each experiment was repeated until reproducibility was certain. All data were statistically analyzed. Points in line graphs are averages of replicates. Radioassay was based on methods described by Aronoff (1). Each sample was counted twice with sufficient time for at least 3000 counts. RESULTS Penetration of Solutes Through Cuticular Membranes 59 59 Penetration of F e804 and F eEDDl-IA, With and Without Urea Through Cuticular Membranes The comparative rates of penetration of 59FeSO4 and 59FeEDDHA in the absence and presence of urea, through cuticular membranes iso- lated from tomato fruits, and those from dorsal and ventral surfaces of Euonmus leaves were recorded at different time intervals. The results are summarized in Figure l. The penetration of Fe from FeSO4, far exceeded that from FeEDDHA, FeSO4+urea, or F eEDDHA+urea. Both the chelate and the presence of urea decreased the penetration of iron in the three types of cuticles. The per- meability of 59Fe through the cuticle removed from the stomatous (Euonymus-dorsal) surface was several times greater than that through the cuticles from the tomato fruit surface and astomatous-ventral surface of the Euonymous leaf. Ion Binding by Tomato Fruit Cuticles Ion binding on cuticular surfaces has been related to ion permeability properties (168). This was determined for FeSO4 on disks of tomato fruit cuticles (3.1 cm2) in the presence and absence of 0.1mM EDDHA. The disks were first floated in the 59Fe labeled solutions for 5 seconds and the ex- posed (inner) surface then rinsed quickly in running deionized water for 15 27 28 Figure l . 59 59 Rates of penetration of Fe from Fe labeled FeSO4 and FeDDHA in the absence and presence of urea through cuticular mem- branes, enzymically isolated from the ventral and dorsal leaf surfaces of Euonymus japonicus, and from ripe tomato fruits. Concentration of urea 10 mM, and others 0.1mM. The vertical intercepts are the standard deViations of the means. PENETRATION IN MIL L mmnouoz. 53 PER [.54 cm 2 ‘1 0' 0| 0 N U 5 r E UONYMUS LEAF CUTICLE ( VENTRAL) FeEDDHA FeEDDHA J I I. TOMA T0 FRUIT CUT/OLE FoSO4 FeEDDHA _1’ IO 20 33 £0 50 .75 .50 .25 .5 .25 F FoSO+ UREA F0 EDDHA+ UREA r __I 1 l Fe EDDHA+UREA F F9304 was 1 FeEDDHA+UREA to 2'0 3'0 4'0 50 TIMEH'IOURS) 29 seconds. After blotting the cuticular disks with tissue paper, the bound 59Fe was estimated by radioassay. It is evident from Figure 2 that the binding of 59Fe from 59FeSO4 was considerably reduced by EDDHA, while it increased with increasing concentration in the absence of EDDHA. 9 9 Cuticular penetration and binding of 5 Fe from 5 FeSO4 by inner cuticu- lar surface were both reduced by EDDHA. Effect of Molecular Weight of Solutes on the Rate of Penetration Through Cuticles Since EDDHA greatly reduced cut icular penetration of Fe, the effect of molecular weight of certain organic compounds was next determined. This was suggested earlier ('75) but not studied. Accordingly, l4C-labeled ethylene glycol, glycerol, D-ribose, D-glucose, sucrose, EDTA, and EDDHA were utilized for comparative penetration studies through the 3 types of cuticles. The results (Figures 3, 4, 5) show that glycerol penetrates very rapidly through all the cuticular membranes and this is also true for ethylene glycol except for the cuticle removed from the dorsal surface of the Euonymus leaf. This suggests that substances of small molecular weight diffuse more rapidly than large mole- cules such as sucrose and EDDHA. Correlations between molecular weights and rates of penetration are illustrated by the regression analyses (Figure 6). Higher correlations were obtained for astomatous than stomatous cuti- cles. 30 Figure 2 . . 59 , , Differences In Fe binding by the inner surface of tomato . . . 59 fru1t cuticles as influenced by Fe derived from solutions of FeSO4 in the absence and presence of EDDHA (0.1mM). +EDDHA 4% 0.3 _ p L b b o. 5. o. 5. o. 3 2 2 I I. WNMQ‘QRQ\§\Q.§§ >2 .N30 in. Nquk§n kx§kk 92‘»an KG NQYKQ§Q QN§>§ xm Q>§Q>§Q 3‘90 .5*- 0 0.2 0.4 0.5 59Fe$04 CONCENTRATION (3: IO'4 M) O.l 31 Figure 3 14 Rates of penetration of C-labeled organic substances through cuticular membranes enzymically isolated from the ven- tral leaf surfaces of Euonymus japonicus. The concentration of each was 0.1mM (Figures in parentheses are molecular weights). PENETRATION IN MIL LIMICROMOL 53/ 1.54 cm2 Q 0 .b 01 GLYCEROL(92) v ETHYLENE o GLYCOUGZ) EON-“(362) EDTM297) /‘ o o/ sucnoss<342> .,———L——-j'— , —?c—Lucosfllam 0 so [0 20 3O 4O 5 TIME (HOURS) 32 Figure 4 14 Rates of penetration of C-labeled organic substances through cuticular membranes enzymically isolated from the dorsal leaf surfaces of Euonymus japonicus . The concentration of each was 0.1mM. (Figures in parentheses are molecular weights). PENETRATION IN MILLINICRONOL 53/1-54 cmz I60 23 o» ‘5 10 IO 0 GLYCEROL (92) F I " D-RIBOSEIISOI SUCROSE(342I (62) ETHYLENE GLYCOL A: /./A Moss (I80) EDTAI297I EDDHA‘SGZI of " ‘ ‘3 4 1 l 1 J IO 20 3O 4O 50 60 TIMEIHOURSI PENETRATION IN MILLIMICROMOL ES/l-54 cmz GLYCEROL (92) I50 - I50 - IOO " o 0 5° ’ D-RIBOSE(I50I 30J SUCROSEI342) I69 . I 5 . ’ (52) ETHYLENE GLYCOL x --'"""""" a /./. x GLUCOSE ( ISO) 5 . EDTAI297I EDDHAI362I A. i A 1 l l l I I0 20 30 4O 50 50 TIME ( HOURS) 33 Figure 5 . 14 Rates 0f penetration 0f C-labeled organic substances through cuticular membranes enzymically isolated from ripe tomato fruits. The concentration of each was 0.1mM. (Figures in parentheses are the molecular weights). PENETRATION IN MILLIMICROMOLES/I.54 cm? ETHYLENE GLYCOL (62) I GLYCEROL D-RIBOSE (I50) . . EDTA(297) A EDDHA (362) ‘ fl/maoss (342 /‘ ##ng ‘0 ‘55: 5 eLucoseueoi l l l _L 1 J IO 20 30 40 50 50 TIME I HOURS) 34 Figure 6 The relation of molecular weight of organic substances and penetration through cuticular membranes as revealed by regression analysis. Molecular weight (x axis) and amount of penetration at 50 hours (y axis). Regression lines: a, cuticular membranes from dorsal leaf surfaces of Euonymus japonicus, y = 77.15 - 0.21x; (r = -.45, significant at P = .05); b, cuticular membranes from ripe tomato fruits, y = 7.53 - 0.019 x, (r = -.75, significant at P = .01); and c, cuticular membranes from the ventral leaf surfaces of Euonymus japonicus , y- = 4.82 - 0.012 x, (r = -.61, significant at P = . 01). The regression lines are drawn with the above equations with the y values plotted on the log scale. PENETRATION IN MlLL/AI/CROMOI. ES / IOO [.54 cm: A50 nouns '5 EUONYMUS LEAF CUTICLE (DORSAL) TOMATO FRUIT CUTICLE EUONYMUS LEAF CUTICLE (VENTRAL) ° I00 200 300 MOLECULAR WEIGHT 35 Permeability Coefficients In cuticular penetration studies, it would be useful for comparative data if the rates of penetration could be mathematically expressed in terms of a constant. If penetration of solutes through a cuticular mem- brane is an example of passive transport or free diffusion (165), then Fick's equation applies, namely, ds = —D. _d__c_ dt dx, where ds is the amount of substance diffusing across an area 1 cm. , . . . , dc , , In time dt, for a concentration gradient of a; With D as a constant . By substituting P (permeability coefficient) for D, (71), the equation would then be, (it and the movement or flux across the cuticular boundary would be, Or, P = 93 (C1 - C2). where, (D is the amount moving per unit area per unit time, and Cl and C2 are the internal and external concentrations, respectively (difference in concentration across the boundary). The dimensions Of P are cm./h0ur, prOVided that the concentration is in millimicromoles per cm . 36 The permeability coefficients for different substances were calculated using the above equation, and are given in Table III. These values for different substances suggest the importance of molecular size on penetration. The permeability is generally higher for ethylene glycol, glycerol and ribose than for other compounds. It is also fairly high for the inorganic ion, Fe, which has an atomic weight of 55. As in the case Of Fick's constant, D, the permeability coefficient also is a property Of both the boundary (cuticular membrane) and the solute. Mechanism of Absorption of Fe by Leaf Cells Absorption Of Fe as Related to Cell Frequency in the Incubation Solution The absorption of Fe by leaf cells varied greatly with the concentration of cells in a constant volume of incubation solution. An experiment was designed with various amounts of leaf cells in a given volume of incubation mixture. to determine the Optimum cell mass per unit volume of incubation solution. The absorption time was 4 hours. The incubation mixture, in addition to the cells, . 59 . contained 1170 umoles sucrose, and 0.5 umoles Fe labeled FeSO4 In a final volume of 10 ml. The contents were shaken under a light intensity of O + O . 500 ft. c. and at temperature of 20 - 2 C. The results are summarized in Figure 7. The relationship between the amount (mass or frequency) of cells and 37 Table III 9 Permeability coefficients (cm/ hour) for 5 Fe from 59FeS04 and several organic compounds for enzymically isolated cuticular membranes from the Euonmus leaf and tomato fruit. Cuticle Cuticle Cuticle from ventral from dorsal from ripe leaf surface leaf surface tomato fruits FeSO4 3.3 x 10'4 1.3 x 10'3 8.8 x 10'4 -4 -3 -3 Ethylene glycol 6. 1 x10 1.8 x 10 1. l x 10 -4 -2 -4 Glycerol 7.4 x 10 2. 9 x 10 6.6 x 10 -4 - -4 D-Ribose 2.4 x 10 2. l x 10 3 4.8 x 10 - - -4 Glucose 0.4 x 10 4 1.3 x 10 3 l. l x 10 -4 - -4 EDTA 1.7x10 1.1x103 1.9x10 - - -4 Sucrose 0.4 x10 4 2.0 x 10 3 l. 1 x 10 -4 -4 -4 EDDHA 2.4x10 3.3x 10 1.9x10 38 Figure 7 , 59 . . Absorption of Fe by enzymically isolated cells from tobacco leaves, as a function of the amount of cells in the incubation mixture. For details see text. (Insert). Linear transformation of the curve showing correla- 59 tion between the weight of cells and absorption of Fe by cells. (r = -. . 83, significant at P = . 01). 40 0‘ 0' H O tn I: a O :I: V \ m ..I .I u o n. O 025 . o E \ m :1 o O 20 2 o 1 ' a :00 E . MILLIGIAMS DIV WEIGHT 0' CELLS IN IO III. 3 l5 .‘0 O o z 9 \ A .25 "' o y ' 69.33! a. o I: '0 \— o . - g o o u . < o 00 T O a“ 5 ID i 0 IO 20 30 4O 50 50 70 30 MILLIGRAMS DRY WEIGHT 0F CELLS IN IO ml. 39 Factors Influencing the Absorption of Fe by Tobacco Leaf Cells External Factors Light, Succinate, and bicarbonate: The incubation mixture used in the experiments contained the following umoles in 40 ml.: sucrose 4680, tris-maleate (pl-I 6.4) 400, 59Fe labeled FeSO4 2. 0, and cells equivalent to 200 mgs. dry weight. Naz-succinate (80 umoles) was included in all treatments except the minus succinate. The contents were shaken at 200 t 2 OC and in light ca. 500 ft. 0. in all treat- ments excepting the minus light. The samples were taken at various time intervals and comparative rates of absorption determined. Light and succi- nate substantially increased the absorption of Fe by the tobacco leaf cells (Figure 8). The effects of an interaction between succinate and bicarbonate are shown in Table IV. Unlike in the case of Rb and phosphate absorption by tobacco leaf cells (76), bicarbonate reduced Fe absorption significantly in dark. While succinate counteracted the effect of bicarbonate in dark, it enhanced the inhibitory effect of bicarbonate in light. This difference was however not statistically significant. Citrate, malate, and succinate: Fe forms chelates with some organic acids and is translocated as a chelate in the intact plant (140). Accordingly, the effects of citrate, 40 Figure 8 59 (a) Effects of succinate on the rate of absorption of Fe by cells enzymically isolated from tobacco leaves in the light. 59 (b) Effects of light on the absorption of Fe by cells enzymically isolated from tobacco leaves. Fe ABSORPTION IN mp MOLES/mg CELLS 59 2.5 2.0 I.0 )- 0 0 W O _ /°f:—SUT:CINATE 00 x/x”. / X x/x /x( "SUCCINATE J I I I 20 40 60 TIME (MINUTES) 41 Table IV Effects of succinate and bicarbonate on the absorption 59 of Fe by tobacco leaf cells. 59 1 Treatment * Fe absorption in mumoles/mg. cells/4 hrs.-/ Chemical Light (500 ft. 0.) Dark Naz-succinate (2 x 10-3M) 5.96 a 3.26d -3 "b NaHCO3 (2 x 10 M) 5.71“t 2.05C Na2 -Succinate + NaHCO3 4. 93b 2. 55Cd 1/ Means not followed by the same letters were significantly different at Odds Of 19:1. The basic incubation mixture contained the following in umoles in the firsigl volume of 10 ml. , sucrose 1170, tris-maleate (pH 6.4) 100, Fe labeled FeSO 0.5, and cells 50 mgs. Conteéit's were shaken in water bath in fight at ca. 500 ft. 0. , and at 20 -— 2 c, 42 malate and succinate on Fe absorption were studied. The results are summarized in Table V. There was no enhancement of uptake, and citrate significantly reduced the absorption of Fe. Temperature: The time course of Fe absorption by tobacco leaf cells, as influenced by temperature is illustrated in Figure 9, and data on temperature coeffi- cients and activation energies are summarized in Table VI. Both Q10 and Ba values decrease with increasing temperature. Absorption of Fe is . 59 temperature-dependent, and the optimum temperature for Fe uptake by cells appears to be about 25°C. Metabolic inhibitors: The results of a study utilizing metabolic inhibitors viz. , NaN3, and 2,4-DNP (dinitrophenol), in the absence and presence of ATP are summarized in Table VI 1. Absorption of Fe was significantly reduced by NaN3, DNP, and even ATP. It is probable that externally applied ATP might compete with Fe and form a Fe-phosphate complex, which might preclude absorption by cells. Effects of Ca on absorption of Fe and Mn: Esperiments with barley roots on the absorption of, and interactions with Na, K, and Rh (33) have indicated the essential role of calcium in selective cation transport in plant cells. The results in Table VIII show the effects of 43 Table V Effects of tris-maIeate, Naz-succinate, K3-citrate, and Na-bicarbonate on absorption of Fe by tobacco leaf cells. Tre t t 1 Chsmrriice:l* Fe absorption in mumoles/mg. cells/5 hrs.—/ None 5. 20a ' a Tris-maleate 4. 99 ' a Tris-maleate 5. 20 + NaZ-succinate ' b Tris-maleate 4. 48 + K3-citrate ' b Tris-maleate 4. 56 + Nag-succinate + K3-citrate . .13 Tris-maleate 4. 53 + Na -succinate + K3-citrate + Na-bica rbonate _1_/ Means not followed by same letters were significantly different at odds of 19:1. . . 59 The baSic incubation mixture contained sucrose 1170 umoles, Fe labeled FeSO4 0.5 umole, and cells 50 mgs. , in a final volume of 10 ml. The chemicals included in the treatments were tris-maleate (pH 6.4) 100 umoles, and others 20 umoles each. Contents were exposed to light 500 ft. 0. , and temperature 200 t 2°C. (a) (b) 44 Figure 9 59 The effects of temperature on the rate of absorption of Fe by cells enzymically isolated from tobacco leaves. 59 Absorption of Fe by cells enzymically isolated from tobacco leaves, as a function of temperature. 6 cc cc c . . .o .o.o... a ma 3| 0 n. 3 3.4 a m. . :5. _ m 5 o e. m .u. 42 m M ~ ( 0 w E Law m T m in O x m i m p h F LI D b 5 w 0 O 5 4 3 2 .I 2 I mjuu ofixmudoz {E z. 20—Fn—m0mm4 on t30l\%44.w0 uE\m.W403 «\E >2 thltcmmc‘ bk mm TEMPERATURE °C O O 0 3 2 360 933.62 a: 2. 22:33: o... 50 60 40 (MINUTES) 30 TIME 20 IO — L h p D P 5 4 3 2 I taozxmjmu 3330: is 3 zotqmommq um 20 25 30 TEMPERATURE ’0 I5 I0 5 mm 45 Table VI 59 Effects of temperature on Fe absorption by tobacco leaf cells. Temoperature 59Fe absorption Temp. coeff. Active Energyl ( C) (mumoles/mg. cells/hr.) (Q10) (Ea-Kcal.mole ) 5 1.45 1.77 (5°— 150) 9.08 10 1.55 1.65 (10°- 20°) 15 2.57 1.19 (15°- 25°) 20 2.56 0.71 (20°- 30°) 25 3.10 30 1.82 Experimental conditions are the same as those in Table IV excepting for temperature. 46 Table VI II 59 Effects of NaN3, 2,4-DNP and ATP on Fe absorption by tobacco leaf cells. Treatment 59Fe absorption Inhibition Chemical Conc. (M) mumoles/mg. cells/2 hrs.‘ (%) a Control 4. l3 -- -3 b NaN3 10 2. 30 44 2,4-DNP 10'4 2.04b 51 ATP 10'4 1. 75b 58 -4 b 2,4-DNP+ATP 10 1.74 58 1/ Means followed by different numbers are significantly different at Odds of 99:1. Experimental conditions same as Table IV. 47 Table VI I I 59 54 Effects of Ca on Fe and Mn absorption by tobacco leaf cells. Concentration of Absorption in mumoles/mg. cells/4 hrs.-l-/ CaSO4 (M) 59Fe Mn Control 17.4a 5.2a 2 x 10'4 -- 4.95| 5 x 10'4 18.33 3. 6b 8 x 10.4 17,0:11 -- 1 x10.3 8.3b 2,3b 2 x 10'3 4.8b -- l x 10.2 -- 1. 0C 1/ Within each column, means followed by different letters were significantly different at odds of 19:1. Experimental conditions the same as in Table IV. 48 calcium on the absorption of Fe and Mn. Calcium either had no effect or reduced the absorption of both Fe and Mn by leaf cells. The physio- logical concentration of Ca, viz. , 0.5 mM, used in studies of cation trans- port in root tissues by many workers, was perhaps too high for the enzymic- ally isolated leaf cells employed in the present study. Interactions of Fe and Mn in absorption and the role of calcium on Fe-Mn transport are portrayed in Figure 10. These data suggest that absorption of Fe and Mn is mutually antagonistic. The inhibitory effects of Fe on Mn absorption is much greater than that of Mn on Fe uptake. While calcium partially reverses the inhibition of Mn on Fe absorption, it increases the inhibitory effect of Fe on Mn uptake. The inhibition of foliar absorption of Fe, Mn, Zn, and Rb by calcium was further studied using tobacco leaf disks (0.8 cmz). Four disks weighing about 100 mgs. were shaken in a flask containing sucrose 2340 umoles, and l. 0 umole of isotopically labeled FeSO4, MnSO4, ZnSO4 or RbCl in a volume of 20 ml. All experiments were conducted in the light at ca. 500 ft. c. and a temperature 200 1- 20C. At the end of four hours the disks were rinsed in deionized water and radioassayed. The means of 3 values were used to plot the graph in Figure 11. The inhibiting effect of calcium on absorption of Fe, Mn, Zn was observed even with intact leaf disks. There was little effect on Rb. (a) (b) 49 Figure 10 59 Absorption of Fe by cells enzymically isolated from tobacco leaves, as a function of FeSO concentration, and 4 the presence of Mn, and Mn + Ca. (Mn,20mM; Ca, lOmM) . 54 Absorption of Mn by cells enzymically isolated from tobacco leaves, as a function of MnSO4 concentration, and the presence of Fe and Fe + Ca. (Fe,20mM; Ca, lOmM). IZOF ° O 9 .CV E \ {3 I00» ~I =1. 80* E 3 A .2 got /:°:/ TV 01 § /° '0 Q 4!, kl 0\ 2 E L Q It 0 a; Q q I I 5 I0 §§ 60" b Mnonly o V O + In I 20: .4uo——-"” IL; i §( QE '0' z}, Stu 5“ mg 5~ g 3. Mn+Fe A 8 E E $102.: #9 Mn+Fe+QQ O qg 044,2 9 L 0 Lo 5 -4 IO CONCENTRATION ( x IO" M) 50 Figure 11 Effects of increasing levels of calcium on the absorption of 59 54 65 65 , Fe, Mn, Zn, and Rb by tobacco leaf disks. OF LEAF DISKS /4 HOURS ABSORPTION IN mp HOLES/mg % K O C O \"\0 54 L- X ”n y 4—x x °\8§> Zn . 65 0 Eng or 3‘4 ‘aRb 9 0 0.05 .5 5.0 Ca CONCENTRATIONIxIO'3MI 51 Internal Factors The age of the leaf may be a significant factor in foliar absorption Of nutrients. Accordingly, leaf number beginning from the apex to the base of the tobacco plant was correlated with the rate Of Fe absorption by cells isolated from them. With increasing leaf age (leaf number) cell absorption of Fe was significantly reduced (Figure 12). Cells from the youngest leaves absorbed the greatest amount of Fe. The relationship between the chlorophyll content of tobacco leaves and the ability of the leaf cells to absorb 59Fe is portrayed by Figure 13. Total chlorophyll was estimated by the method Of Benne e331. (5). The absorption rate was directly correlated with the chlorophyll content. Fe chlorosis was induced in Hawkeye variety (chlorosis non-sus- ceptible), and P1 54619-5-1 variety (chlorosis susceptible) soybeans by growing the seedlings in minus and plus Fe nutrient solutions. Leaf cells were then isolated from chlorotic and non-chlorotic (normal) first tri- foliate leaves and the rate of absorption of 59Fe recorded (Table IX). Cells from chlorotic leaves of P1 54619-5-1 absorbed less Fe than those isolated from healthy leaves of the same variety. There was, however, no significant difference in 59Fe absorption of cells derived from chlorotic and normal leaves of the Hawkeye soybean. 52 Figure 12 Effects Of tobacco leaf age (designated by leaf number from apex to base) on the absorption of 59Fe by cells enzymically iso- lated from them. Regression equation, y = 4.61 - 0. 18 x, r = -.67, significant at P = . 01. Absorption in millimicromoles of 59Fe/mg. cells/hour is plotted on log scale. Figure 13 Relationship between the rate of 59Fe absorption by enzymically isolated leaf cells and the chlorophyll content of the leaves. Regression equation, 7 =1. 90 + 0.28 x, r = + .61, significant at P = 0.1. Absorp- 59 tion in millimicromoles of Fe/mg. cells/hour is plotted on log scale. MILLIMICROMOLES/IIIO CELLS I HOUR 59:. ABSORPTION -J 000 «b UIO’ I I I I l I __I 2 4 6 8 IO I2 I4 LEAF NUMBER BEGINNING FROM APEX l l I l I 2 3 4 mg OF CHLOROPHYLL/Om FRESH TISSUE 53 Table IX 59 Comparative absorption of Fe by cells removed from chlorotic and healthy leaves of two varieties Of soybean. 59 1 Absorption of Fe in mumoles/mg. cells/l hours] Condition of Hawkeye (chlorosis Pl 54619-5-1 the leaves nonsusceptible) (chlorosis susceptible) b Chlorotic 4. 36a 2. 41 Healthy 3. 64a 5. 10a 1/ Within each column, means followed by different letters were significantly different at odds of 19:1. Experimental conditions are the same as in Table IV. 54 59 Absorption of Fe from FeSO4. FeEDTA, FeEDDHA, and FeSO4 in the Presence of Urea. 59 Absorption of Fe by Leaf Cells The effects of two chelates namely, EDTA and EDDHA, and urea on the absorption of Fe by cells isolated from tobacco and bean leaves are summarized in Table X. Chelation reduced, while urea enhanced uptake . 59 Uptake and Transport of Fe in Intact Plants Intact primary leaves of bean plants were utilized to measure specific . 59 . absorption and subsequent transport of Fe according to methods of Jyung _et al_. (74). The effects of chelation and urea on both uptake and translocation are given in Table XI. Urea enhanced uptake but less of the 59 absorbed Fe as percentage was translocated. On the other hand, che- lation inhibited absorption but similarly enhanced the translocation of the 59 absorbed Fe. 59 Effects of Pre-treatments with EDDHA and Urea on Fe AbsoQtion by Bean Leaf Cells The effects of pre-treatments with EDDHA and Urea, on the subsequent absorption of Fe from different sources, were studied and the results are summarized in Table XI I. Urea combined with FeSO4 was necessary for 55 Table X . . 59 Comparative absorption of Fe from different sources, and in the presence of urea, by cells enzymically isolated from green tobacco and bean leaves. Uptake in mumoles/mg. cells/8 hrs.“ 59 Source of Fe Tobacco leaf cells Bean leaf cells FeSO4 l4. 5:. 33. 8a b .- Fe-EDTA 6.2 29. 3‘1l Fe-EDDHA 4. 3b 5. 8b FeSO4 + Urea 24. 4C 45.2C 1/ Within each column, means followed by different letters were significantly different at odds of 19:1. The incubation mixture contained sucrose 1170 umoles, 391% labeled FeSO4, FeEDTA, and FeEDDHA, 0.5 umoles each, and urea 50 umoles, in a final volume of 10 ml. , in treatments where these are indicated. Contents were exposed to 500 ft. 0. light and to 200 3*: 20C, in a shaking water bath. 56 Table XI 59 Uptake and transport of Fe by intact primary leaves of the bean plant as affected by different carriers (Leaf immersion technique). Uptake” Translocation** Carrier“ mumoles/mg./12 hrs. (‘76,) FeSO4 12. 8b 0. 5C FeEDTA 9. 1C 2. 5b FeEDDHA 1. 3d 6. 0a a c FeSO4 + Urea 30. 7 0. 6 ** Within each column, means followed by different letters were significantly different at odds of 19:1. * The concentration of all the chemicals used was 0.1mM. , excepting urea, which was 10 mM. The leaves were exposed to 500 ft. c. of light, and 200 - 20C. 57 Table XII 59 Effects of pre-treatments with EDDHA on the absorption of Fe from FeSO4, in the absence and presence Of urea, and 59 pre -treatment with urea on the absorption of Fe from FeSO4 and FeEDDHA by isolated bean leaf cells. Absorption of 59Fe 1/ Treatment in mumoles/mg. cells/4 hrs.- b FeSO4 28. 8 C FeEDDHA 7. l a FeSO4 + Urea 39 . 3 FeEDDHA + Urea 8.8C Pretreated with b EDDHA F8804 30.8 FeSO4 + Urea 39.4a b Urea FeSO4 29. 0 FeEDDHA 6. 1C 1/ Means followed by different letters were significantly different at odds Of 19:1. Experimental conditions are the same as in Table X. In pre -treat- ments, the cells were treated for 2 hours. in solutions containing sucrose 1170 umoles, EDDHA 0.5 umoles, or urea 50 umoles, in a total volume Of 10 ml. , and treatments were given to the cells after separating them by centrifugation. 58 enhancing the absorption of Fe. Urea had no effect on Fe absorption from F eEDDHA. Pre-treatment with EDDHA had no effect on the subsequent absorption of Fe from FeSO4, either in the absence or presence of urea . 59 Absorption of Fe from FeSO4 in Absence and Presence Of Urea, 14 l4 l4 C-EDTA, C-EDDHA, and C-Urea by Tobacco Leaf Cells 59 The absorption rates of Fe as the sulfate and chelated with EDDHA 9 are portrayed in Figure 14. The enhancement of 5 Fe uptake by urea is 1 also given along with absorption of 4C labeled urea and EDDHA. Absorp- . 14 . 59 59 . . tion of C urea 13 less than Fe from FeSO4; and Fe absorption is 14 greatly reduced by chelation with EDI-1A. Absorption of C-EDDHA by cells is neglibible. Absorption of the ligand is also reduced when combined with the metal (Figure 15). These data reveal that the metal and ligand are absorbed differentially by enzymically isolated leaf cells. Conceivably, the two separate at the cell surface prior to absorption. Determination of Affinity Constant, and Application of Enzyme-Kinetics to 59 Absorption of Fe by Leaf Cells 59 The velocity of Fe absorption by isolated tobacco leaf cells as a function of the concentration of 59Fe labeled FeSO4 is illustrated in Figure 16. The affinity constant l/Km, for the metal-carrier complex was calculated and found of low order. 59 Figure 14 Comparative absorption rates by isolated tobacco leaf cells 59 . . . . Of Fe from FeSO4 alone and in combination With urea; also 9 Fe absorption when chelated with EDDHA, and the absorption of 14C labeled urea and EDDHA. The basic incubation mixture contained Sucrose 1170 umoles, cells 50 mgms, Fe (as in FeSO4) and EDDHA 0.5 umoles, each, and urea 50 umoles, in a final volume of 10 ml. Each point is a mean of three values. - — N N O U) 0 0' 259Fe UPTAKE (myMOLES/mg DRY WI) 0‘ 59 F8 504+ UREA O 59 Fe 804 '4C-UREA 59 Fe -EDDHA !_AL fl '4C-EOOHA 43:: l 8 I2 TIME IN HOURS 60 Figure 15 Absorption rates for 14C labeled EDTA and EDDHA, separately and in combination with Fe, by cells enzymically isolated from tobacco leaves. The incubation mixture contained sucrose 1170 umoles, cells 50 mgms, Fe (as in FeSO4), EDTA, and/or EDHA, 0.5 umoles, in a final volume of 10 ml. Each point represents a mean of three values. t A 0 on n A A 7 H” A H A A D T D T. A 09 nu no 0 D A A E E E E . . _ o . C C C C M M M M . . A A e e — _ F F A a O o no _ _ A O O i L 3455788 p-ppppppp 098765432I .438 558.62 1: mfidxo Lo 22an34 II I2 I0 2 (HOURS) TIME (a) (b) 61 Figure 16 , 59 . . Absorption of Fe by cells enzymically isolated from tobacco leaves as a function of the concentration of 59Fe Lineweaver-Burk plot of 59Fe absorption by cells enzymically isolated from tobacco leaves, in the presence and absence of EDDHA. The incubation mixture contained sucrose 1170 umoles, cells 50 mgs. , and EDDHA 1.0 umole where this was included in a final volume of 10 ml. The absorption was mea- sured at the end of one hour. mp HOLES 59mm CELLS/HOUR V. R? (I 1 I00 - q U I 0 O 1 Kml 0.5 mM vmax' I20 pMOLES IGM/ HR ‘0\ 25 1 LJ 0 5 . IO 3, m M at it CEDDHA 3 I- A 2 _ -EDDI-IA . 62 The Lineweaver and Burk plot (93, 134) of the data (Figure 16b) illustrates a competitive inhibition of Fe absorption by EDDHA. It appears at the cellular level that EDDHA competes with some unknown natural chelate (carrier), for Fe. Ion Absorption by Leaves -- A Multistep Process In the sequence of events leading to ion uptake by the leaf, a number of phenomena participate. Over-all, the process is multistep, in which various phases may be rate-limiting, depending on external and internal conditions. The latter variables are especially related to physiological processes concerning cell activity and energy supply. The phases concerned with the absorption of Fe or ions by the leaf can be distinguished as (1) initial adsorption, which may be on the surface of the cuticle, (2) penetration through the cuticle which is possibly a diffu- sion process, not requiring energy, and (3) the active or metabolic uptake of the ions, which have diffused through the cuticle by living cells. Ad- sorption on and diffusion through the cuticle are related to the physical properties of the leaf which vary with plant species. These processes, however, may be partially controlled by the environment. Euonymus japonicus leaves of uniform size and age were selected from plants growing on the University campus during mid-summer. The cut ends of the petioles were sealed with rubber cement, and floated in a 63 . . . 59 solution containing 0.1 mM Fe labeled FeSO4. They were oriented such that only the dorsal or ventral su rface was in contact with the isotopic solution. Leaves were then removed at different time intervals, . . . . . 2 rinsed in deionized water for 30 seconds. Disks (3. 1 cm ) were then punched by means of a cork borer from the middle of the leaves, and the radioactivity measured. Results are summarized in Figure 17. The . 0+ 0 experiments were conducted both at a temperature of 20 - 2 C and _ o + o , _ light Of 500 ft- C- . and at 0 - 2 C 1n the dark. With leaves exposed to light, the surface in contact with the isotopic solution was opposite the side receiving light, and no orientation was made such that the same surface could be simultaneously exposed to light and to the isotopic solution. Thus, as the dorsal (stomatous) surface was illuminated, uptake was from the ventral (astomatous) surface, and vice versa. (a) (b) 64 Figure 17 59 Absorption isotherm for Fe from a solution of 0.1 mM 59 0f Fe labeled FeSO4 by the ventral surfaces of detached leaves of Euonymus japonicus as a function of time. The same as above, by the dorsal surfaces of the detached leaves of Euonymus japonicus. indicates the probable uptake by adsorption . indicates the probable uptake by diffusion. c indicates the probable uptake by "activated diffusion". d indicates the probable uptake by active absorption. 0‘93 ”Po ABSORPTION IN I», NOLEs / LEAF 0st (3.! cm’l 40 30 20 I0 A VENTRAL SURFACE OF EUONYMUS LEAF LIGHT 8 20’ :1: 2’6 OORsAL'SURPACE OF EUO NYMUS LEAF LIGHT a zo-gz-c TIME (HOURS) II...| I I... ..| 65 DISCUSSION Permeability of Fe Through Cuticular Membranes: Information on the permeability of Fe through cuticular mem- branes is inadequate, hence, penetration rates were studied utilizing three different cuticular membranes. The effects of chelation and urea were included in the study, since facilitation of penetration Of ions has already been indicated, particularly for urea (75), probably owing to its non-ionic properties (147). The results show that the rate of penetration of Fe from FeSO4 was greater than from FeEDDHA. The addition of 10 mM urea also reduced the penetration of Fe through all cuticular membranes. The reductions caused by both EDDHA and urea were significant, especially through the cuticular membranes de- rived from the dorsal surface of Euonymus leaves. The rate of pene- tration Of Fe from FeSO4 was generally comparable to that of Ca and Rh through tomato fruit and onion leaf cuticles (169). Although the re- sults indicated a reduction in Fe penetration by EDDHA, the posibility exists that FeEDDHA, because it is more stable in solution, may be a superior source of Fe for foliar sprays. Experiments with intact plants might reveal a greater availability of Fe over a long period of time from FeEDDHA sprays. Also, Fe applied as FeEDDHA might be transported more expeditiously from leaves than the Fe applied as a FeSO4 spray. 66 Penetration of Organic Compounds and the Relation of Molecular Weight The importance of molecular weight has been indicated by Wittwer £11. (166). The results herein (Figures 3, 4, 5, 6) also suggest a high correlation between molecular weights and permeability, particularly through astomatous cuticles, and for organic solutes. Similar relation- ships between hydrated ion size and penetration rates through astomatous cuticles have recently been obtained for Ca, Sr, Ba, Na, K, Rb, and Cs (49). It has been suggested that permeability for liquids may be related to the pores in the cuticle (95). "Lipoidal" and "aqueous" pathways for cuticular penetration of solutes have often been mentioned (27, 32). Pores extending up to the surface of the cuticle have also been identified, Trifolium repens, Brassica oleracea and Poa colensoi (5i). The permeability properties of cuticular membranes may be analo- gous to those of cell membranes. The clas sical work of Collander‘ (23, 24, 25) showed that the permeation of non-electrolytes intow protoplasts was inversely proportional to the molecular weight x l. 5 which was evidence of a "molecular sieve" principle. Mechanism ofAbsorption of Fe by Enzymically Isolated Leaf Cells Influence of External Factors: The enhancement of Fe absorption by light is evident from Figure 8b. The stimulatory effects of light on ion uptake have been observed in several 67 instances (59, 83), and a direct effect of light on ion absorption by leaves has been suggested (10). An energy source derived from the photosynthetic process might explain the effect of light. Jyung _e_t_ a_l. (76) obtained a similar enhancement of Rb uptake by light. The stimu- lation of Fe absorption by succinate also has as its origin a supply of energy which serves as a respiratory substrate. Inhibition of 59Fe uptake by NaHCO3 is contrary to the promotive effect for Rb uptake (76). Bicarbonate has been attributed as a causative factor in Fe chloro- sis. Its effect is presumably indirect, however (15), in that it increases the amount of phosphate and calcium in solution cultures. The results obtained herein suggest that the bicarbonate effect may also be direct and perhaps at the cellular level. Fe supplied through the roots is transported in soybeans, as the chelate of malate and malonate. Thus, organic acids might function in the translocation of Fe in this and other plants (140). The inhibition of Fe absorption by externally supplied citrate, observed in the present experiments, might be the result of the citrate competing with the cells for Fe. The absorption of Fe by cells is markedly influenced by temperature (Figure 9). Rb and phosphate absorption by tobacco leaf cells was also temperature-dependent (76). Temperature Coefficients (Q10) and energies of activation are used extensively as criteria for enzyme catalyzed reactions. 68 A Q10 of more than 2 is generally associated with an active uptake process. A Q10 of 3 for K accumulation between 0.50 and 200C (59), and 2.2 of 2.45 for K uptake by wheat roots (98) have been reported. Robertson (119) demonstrated that the Q10 varied from 2 to 2. l for ac- cumulation of KCl by carrot tissue. For Ca uptake by pear roots (69) it was 0.9 between 00 and 100C, and 2 between 100 and 200C. A Q10 of slightly less than 2 was obtained for Rb (76) uptake by enzymically isolated tobacco leaf cells. A value of 5 Kcal./mol. for Ea is generally indicative of a diffusion system (38), and about 13 Kcal./mol. for many metabolic reactions (151). The Ea values for Fe absorption are summar- ized in Table VI. This would suggest that the absorption of Fe by enzymic- ally isolated tobacco leaf cells is also essentially an active metabolic pro- cess, involving energy consumption. A relationship between respiration and salt uptake by plants is well established (84, 97, 98, 120) and salt uptake is generally linked with metabolism. Absorption of Fe by tobacco leaf cells is suppressed by metabolic inhibitors, viz. , NaN3, and DNP (Table V11). Inhibition of oxidative phosphorylation caused by DNP (20, 96) increased the rate of cytochrome-mediated electron transport, but simultaneously depressed salt accumulation (121). Briggs _e_t_ a1_. (10) therefore suggested that it was not simply the electron transport system in the cell that was associated with salt accumulation, but that coupled phosphorylation was necessary. 69 Metal oxidase inhibitors like cyanide and azide did not effect Sr uptake and the uncoupler DNP decreased Sr absorption only to some extent (151). The inhibitory effects of DNP on salt uptake and respiration may be a function of rendering the mitochondria "leaky" (62). The significance of Ca for maintaining the integrity of the selective absorption mechanism for K, Rb and Na (33), and on the apparent per- meability of cell membranes (143) has recently been emphasized. On the other hand, Ca does not appreciably promote Fe -Mn absorption by enzymic- ally isolated tobacco leaf cells, and high levels of Ca are distinctly inhibitory to Fe-Mn absorption. Ca decreased Fe uptake by soybean plants from nu- trient solution (94). Calcium stimulation of K uptake in maize roots and a depression in soybean roots, are both reported by Kahn and Hanson (78). Handley _e_t_ 61‘: (52) found that Ca exerted 2 antagonistic effects on ion uptake. The first was inhibitory and the second stimulatory. Perhaps extremely low concentrations of Ca are required to induce the stimulatory effect on Fe and Mn absorption by leaf cells. The inhibitory effects of high concentrations of Ca Were evident on the absorption of Fe, Mn, Zn, and Rb by tobacco leaf disks (Figure 11). Chemically related ions have virtually equal affinities for common carrier sites, and are mutually and almost equally competitive. This has been established for K and Rb (34, 35). Similar affinities between Fe and Mn, which are also chemically related, have been observed in their 7O absorption by tobacco leaf cells (Figure 10). Although they are mutually competitive, they are not equal. The inhibition of Mn absorption by Fe is greater than that of Fe by Mn. Calcium unlike its role in K, Rb and Dev» Na transport (33), has apparently no effect in the Fe -Mn absorption by i tobacco leaf cells. Effects of Internal Factors: Age of the leaf is an important factor affecting absorption of nutrient elements. The cells obtained from younger leaves of the tobacco plant showed greater absorption of Fe than those from the older leaves. Age of the leaf was indexed by leaf number from the apex to base of the plant. Age was reflected by a greater physiological activity of the younger than the older leaves. A reverse effect on absorption as related to leaf age, however, has been observed. Kamimura and Goodman (79) found that apple leaves near the apex absorbed less leucine than those near the base. The presence of discontinuities and cracks in the cuticles of the older leaves is offered as a possible explanation for greater uptake. The amount of chlorophyll in the leaf may be also highly correlated with the rate of Fe absorption by its cells (Figure 13). This was particu- larly striking in the chlorosis susceptible Pl 54619-5-1 variety of soybean. 71 The Role of Chelates and Urea The absorption of Fe by tobacco leaf cells is considerably reduced by EDTA, and EDDHA, as compared to FeSO4 and greatly enhanced by Ft“ combining urea with FeSO4 (Figure 14). Cellular absorption of the ligand from FeEDDHA is negligible with a ratio of 1000:l for Fezligand. The question as to whether or not the ligand is absorbed by plant cells along with the metal, has not been fully resolved. Does FeEDDHA enter the cell as an intact molecule? The following hypotheses have been proposed as mechanisms of absorption of FeEDDHA (147): (i) FeEDDHA + Cell ------ Fe-Cell + EDDHA (EDDHA remains outside the cell) (it) FeEDDHA + Cell ------ EDDHA-Fe-Cell complex ------ Fe-Cell + EDDHA (EDDHA diffuses out of cell). (iii) FeEDDHA + Cell ------ EDDHA-Fe-Protein. The results obtained herein with single cells enzymically isolated from tobacco leaves suggest the validity of the first mechanism. The enhancing effect of urea on 59Fe uptake by separated leaf cells was further evaluated by pre -treatments with urea or chelate. Pre -treatments of EDDHA and urea had no effect on subsequent uptake of Fe. Urea increased the absorption of 59176 only if it was included along with FeSO4. The effect of urea may arise from an activation of carriers of Fe, or on the permeability of cell wall membranes. 72 The Use of Enzyme Kinetics and the Competition by Chelate Analysis of the data of absorption rates by tobacco leaf cells of Fe in the absence and presence of EDDHA, utilizing an enzyme-kinetic model, reveals that the carrier for Fe has a very low affinity constant, and the inhibition of Fe absorption by EDDHA is probably of a competi- tive nature. There appears to be a competition for Fe, between EDDHA and a presumed Fe-carrier, which is likely a natural chelating sub- stance within the cells. Recently some enzymes which could incorporate metals have been identified (111). An enzyme prepared from rat liver mitochondria has been found to catalyze the chelation of iron by protoporphyrin. Porra and Jones (114) have further suggested the presence of more than one fe rrochelatase (pophyrin-iron-chelating enzyme) in Thiobacillus K. These findings suggest that there may be an iron trans- porting mechanism in the plant which is chelate-like. Steps Involved in the Absorption Process by Ientact Leaf Ion absorption by a leaf begins with initial adsorption by the cuticular surface, followed by diffusion through the cuticular membranes and further uptake by the leaf cells underlying the cuticle. In the absence of cellular activity in the leaf, the quantity of ions entering the leaf would essentially be through the process of diffusion. An attempt was made to differentiate these processes, by plotting the absorption isotherms. The results 73 suggest that adsorption was instantaneous, and that it was not possible to distinguish adsorption from diffusion. Diffusion was also very rapid and reached a maximum in 15-30 minutes (Figure 17). Diffusion was markedly influenced by light and temperature, and may be accordingly designated as an activated diffusion, prompted perhaps by a rapid removal of the diffused ions by the cells beneath the cuticle. This was particularly characteristic of uptake by the ventral (astomatous) surface. Under the present experi— mental conditions, the uptake by ventral surface seems to be greatly influenced by the illumination of the dorsal (stomatous) surface. This perhaps is an evidence for the implication of the role of stomates in foliar absorption. SUMMA RY Mechanisms of foliar uptake of Fe were investigated with a view to elucidate the factors relative to the diffusion of Fe through cuticles, absorption by leaf cells, and transport from the leaf to other parts of the plant. Enzymically isolated cuticular membranes from ventral and dorsal leaf surfaces of Euonymus japonicus and from ripe tomato fruits were employed for studying the rates of penetration of Fe. Cuticular membranes from dorsal surfaces of Euonymus japonicus leaves were characteristically stomatous, and those from ripe tomato fruits and from the ventral sur- faces of the Euonymus leaves were astomatous. The course of penetration of 59Fe from 59Fe labeled FeSO4 revealed that Fe penetrated at rate 59 ,. _ comparable to that of Rb and Ca. Fe penetration was reduced by com- bining with EDDHA and/or urea. Penetration rates were gens.;r-a.‘.ly higher through the cuticular membranes isolated from the dorsal (stomatous) leaf surface. The penetration properties of 14 C-labeled organic compounds, varying in molecular weights, were studied using the three types of cuticles. Diffusion was greater for solutes of small molecular weight. The negative correlation between molecular weight and permeability was demonstrated by regression analysis. This correlation was 74 75 especially high for astomatous cuticles, and is in accord with the rela- tionship obtained between hydrated ion size and penetration through cuticles. These observations are in further accord with theories of "aqueous pathways" and "lipoidal pathways" for cuticular penetration. Enzymically isolated leaf cells were employed for resolving mechanisms of Fe absorption at the cellular level. Light and succinate stimulated the uptake, while metabolic inhibitors, viz. , NaN3 and DNP reduced it. Temperature had a significant effect on Fe absorption. These results, along with those of temperature co- efficient (Q10) and activation energies (Ea) , strongly support the involvement of an active mechanism for the absorption of Fe by leaf cells. The effects of Ca on absorption of Fe and Mn were inhibitory, especially at concentrations higher than 0.5mM, which is the phys- iologically optimum concentration for the K-Rb, Na transport mech- anism in other systems. The absorption of chemically similar ions, Fe and Mn, was mutually antagonistic at the cellular level. If there is a common carrier for K-Rb uptake, it is also possible that Fe- and Mn are transported by a common carrier. Results also show that such a carrier has a greater affinity for Fe, than for Mn, as indicated by a more efficient inhibition of Mn uptake by Fe than of Fe by Mn. 76 The chlorophyll content and age of the leaf are vitally related to the Fe uptake mechanism. Healthy condition of the leaf is a prerequisite for efficient absorption of Fe. So, also, younger leaves appeared more efficient in ion uptake. The absorption of Fe from FeSO4 by enzymically isolated cells: and also by intact leaf, was higher than from FeEDTA or FeEDDHA. The enhancing effects of urea on Fe uptake were significant in leaf cells from both tobacco and bean and also in intact leaves. The role of chelates, on the other hand, was evident in the greater transport of Fe from the site of application to other parts of the plant. The ligands, EDTA and EDDHA, from FeEDTA and FeEDDHA, were not absorbed by the enzymically isolated leaf cells from tobacco. The ratio of absorption between the ligand and metal was about 1:1000. This indicated that the absorption of Fe by the cells from Fe-chelates was selective; the metal alone was absorbed and the ligand remained outside the cell. Enzyme-kinetics analysis revealed that the inhibition of Fe absorption by EDDHA was competitive. The affinity constant for Fe was low. The absorption of Fe from FeSO4 by intact Euonymus japonicus leaves involved a number of processes--initial adsorption by the cuti- cular surfaces, diffusion of penetration through stomatous or astomatous 77 cuticular membranes, which might be "activated, " and absorption by the leaf cells. 10. ll. 12. LITERATURE CITED Aronoff, S. 1961. 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