MECHANISMS OF iON UPTAKE BY THE LEAVES OF WPHASEWOWS EM m: L ’ 11m; for m. Dogma cf m an MICHiGAN STATE UNIVERSITY Wotan Hang Jyung 1963 ” {meals This is to certify that the thesis entitled chhwnicmfi of ion Untmke bv the IDQVCR of Phnsonius vuidfirifi L. presented by 3’." o n n 7? en 5': Jyu n 0; has been accepted towards fulfillment of the requirements for Ph. D. degree in HortianyLuI'e Major professor LIBRARY Michigan State 5 University *4; J DEDICATION To my late father, Mr. Hack Yang jyung MECHANISMS OF ION UPTAKE BY THE LEAVES OF PHASEOLUS VULGARIS L. By Woon Heng Jyung AN ABSTRACT OF A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1963 ABSTRACT MECHANISMS OF ION UPTAKE BY THE LEAVES OF PHASEOLUS VULGARIS L. by Woon lleng jyung Mechanisms of foliar absorption of phosphate and rubidium in bean leaves were investigated by means of a newly developed leaf immersion and washing technique, and "specific absorption", and by utilizing criteria for active uptake, i. e., time course analysis, temperature, oxygen, and energy dependence, sensitivity to metabolic inhibitors, accumulation against a concentration gradient, irreversibility, and pH response. Classic enzyme kinetics were employed in studying uptake of phosphate and rubidium by bean leaves. The fully expanded primary leaves of bean seedlings constituted the test material. All absorption experiments were conducted in a temperature controlled laboratory. One of the primary leaves of a bean plant was treated by immersion into a solution to a depth of 0. S cm. with the aid of proper . . . . 32 glass rod frames. Treating solutions usually cons1sted of .2 be P /13. 3 )1 moles H3PO4 in 40 ml. for phosphate, and 0. lite Rb86/40 umoles RbCl in 40 ml. for rubidium. Experiments were performed under continuous illumination except for the light-dark experiments. There were two to four single plant replicates for each treatment. Upon harvest, seedlings were Woon Heng jyung — - — 2 separated into the treated leaf and the remainder of the plant. Only the leaf blades of excised leaves were harvested. The non-absorbed residue was re- moved from the leaf surface by washing the treated leaf in three successive 50 ml. portions of distilled water for 15 seconds each. Upon completion of washing, the leaf was blotted with tissue paper for 30 seconds, with one change of tissues after 15 seconds. The ”leaf immersion and washing tech— nique" encompassed the procedures of leaf immersion and washing outlined above. Oven dried plant materials were counted directly by means of an end-window G-M tube and standard scaler circuit. The nutrients retained in the plant materials after the above washing procedure were considered absorbed. Absorption was expressed as "specific absorption" (quantity ab— sorbed per unit area in unit time from an external solution of constant con- centration). , The use of these two approaches for measuring uptake of phosphate and rubidium by primary leaves of bean plants has established that foliar absorp- tion of phosphate and rubidium beginning with zero time, and extending over a 24—hour period, is a metabolic process, subject to temperature, an energy source, and aeration. Metabolic inhibitors, such as 2, 4-dinitrophenol and chloramphenicol, significantly reduced uptake. Exogenously applied adeno— sine triphosphate, however, had little effect on rubidium uptake. Rubidium Vlbon Heng Jyung — - - 3 and calcium accumulated against a concentration gradient. This was true also for phosphate in Chrysanthemum leaves, but nm in bean leaves. Phos- phate and rubidium uptake was essentially irreversible. Kinetlu and N6- benzyladenine tendt-d to enhance phospham and rubidium uptake in dark, but reduce absorption in the light. Ribonuclease (RNase) and S-fluorouracil (S-FU) reduced rubidium uptake, but increased that of phosphate. It was proposed that carriers play an important role in phosphate and rubidium uptake by leaves, and that these carriers are proteinaceous since absorption of both nutrients was inhibited in the presence of chloramphenicol. Specificities of the carriers were demonstrated by the differential responses of phosphate and rubidium absorption to RNase and S-FU. Use of classic enzyme kinetics revealed the following as to the absorp— tion of phosphate and rubidium by bean leaves. The reaction order for both phosphate and rubidium absorption was first order. The binding affinities in terms of apparent Michaelis constant (Km), maximum velocities (Vmax), absorption rate constants (k), times for 50 percent absorption (T .)), acti- 1/- vation energies (E ), "potential carrier concentrations", and the theoretical a molecular activities of the carriers were calculated for foliar absorption of both phosphate and rubidium. These results were evaluated as to contempor— ary and earlier reports for nutrient uptake by other plant parts. MECHANISMS OF ION UPTAKE BY THE LEAVES OF PHASEOLUS VULGARIS L. By Woon Heng Jyung A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1963 CD M‘- ~."”/ I'JQ-QS ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation for sugges— tion of this problem, accurate guidance, help, and especially encouragement given by Dr. Sylvan H. Wittwer, without which the author could not have accomplished the work presented. Also the author is very much indebted to Drs. L. F. Wolterink, R. S. Bandurski, M. J. Bukovac, and D. R. Dilley, for their academic guid- ance and invaluable discussions whenever needed, and to the late Dr. F. G. Teubner for encouragement. The author wishes to extend his appreciation to the Biological and Medical Division of the United States Atomic Energy Commission for finan- cial aid (Contract No. AT(ll-I)-888. - Finally, the author also wishes to extend his appreciation to his wife, Kaphee S. jyung for her interest and enthusiastic encouragement throughout this work. TABLE OF CONTENTS INTRODUCTK»J.. .. .. .. .. .. .. .. . REVHNVOFIJTERATURE. .. .. .. .. . Technology Involved in Foliar Absorption Studies . Methods of Applying Solutions . Methods for Removal of Non-absorbed Residue . Sample Preparation Methods for Assay of Absorption of Foliar Applied Nutrients . . . Indices and Expressions of Foliar Absorption Criteria for Metabolic Foliar Absorption . . . . . . . . . Absorption Rate and Time Course Analysis . . . . . . TknnperatureIDependence . Energy Source Dependence . Sensitivity to Metabolic Inhibitors . Accumulation Against a Concentration Gradient . . Irreversdnlny(MflAbsorpthni. . . . . . . . . ()xygenIDependence . . . . . . . . . . . Specificity and Ion Competition [HTIDependence. MATERIALS AND METHODS . Plant Materials and Culture iii Page 10 13 13 14 CONTENTS CONT' D Page Environmental Conditions . . . . . . . . . . . . . . . . 26 Preparation of Treating Solutions . . . . . . . . . . . . 26 Treatment of Leaves by the Leaf Immersion Technique. . . 27 Harvesting and the Leaf Washing Technique. . . . . . . . 31 Measuring of Variables . . . . . . . . . . . . . . . . . 32 Expression of Absorption and Estimates of Variability . . . 33 RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . 34 Mechanisms of Foliar Absorption of Mineral Nutrients. . . 34 Time Course Studies . . . . . . . . . . . . . . . . 34 Response to Temperature . . . . . . . . . . . . . 37 Response to Energy Sources . . . . . . . . . . . . 38 Responses to Metabolic Inhibitors and Growth Promot— ingAgents. . . . . . . . . . . . . . . . . . . 41 Accumulation Against 3 Concentration Gradient . . . . 51 Irreversibility of Absorption . . . . . . . . . . . . 55 Response to Aeration . . . . . . . . . . . . . . . . 58 pH Response . . . . . . . . . . . . . . . . . . . 60 Kinetics of Foliar Absorption . . . . . . . . . . . . . . o3 Determinations of Reaction Orders, Absorption Rate Constants (k), Times for 5052'". Absorption (Tl/2) and Activation Energies (Ea) . . . . . . . . . . . . . (i3 CONTENTS CONT' D Page Determinations of Michaelis Constants (Km), Maximum Velocities (Vmax), "Potential Carrier Concentrations", and Theoretical Molecular Activities of Carriers. 66 SUMMARY.................... 71 74 LITER'ATURECITED . . . . . . . . . . . . . \l INTRODUCTION An ever-increasing interest in the mechanisms of uptake and the path— ways for foliar absorption of mineral nutrients has developed during the last decade. Yet, present knowledge is limited and fragmentary, and theories are controversial. The delay in progress may be ascribed to the lack of standard methods for accurate appraisal of nutrient uptake by leaves. This thesis will describe, (a) a new and reproducible technique for sub— jecting leaves to nutrient containing solutions, and (b) present a means of standardization for mathematically expressing the rate of nutrient uptake by leaves. The new technique designated as "leaf immersion and washing" consists of submerging leaves into the isotopically labeled nutrient contain- ing solution, which provides a standardizable environment and an easy means of "washing-off' of the non—absorbed residue. The long recognized diffi- culties and limitations of differentiation between the absorption and trans- location systems, and changes in concentration with time of the applied solu- tion are also circumvented. In addition to this new technique, a mathematical expression for foliar absorption designated as " specific absorption” will be introduced and utilized. "Specific absorption" may be defined as the quantity of a substance absorbed per unit area in unit time from an external solution of constant concentration. These two approaches have been efficiently utilized for resolving mechanisms of foliar absorption based upon the criteria proposed by Wittwer and Teubner (129); namely, accumulation, reversibility, absorption rate, sensitivity to metabolic inhibitors, pH dependence, specificity and ion com- petition, oxygen tension, energy source, and the temperature coefficient. Investigations were extended to provide additional insight into the assigned mechanisms in terms of fundamental enzyme kinetics and energetics. The results constitute a sum of accumulating evidences for the presence of a link between nutrient uptake by leaves and metabolism of living cells. REVIEW OF LITERATURE Technology Involved in Foliar Absorption Studies Methods of Applying Solutions — - One of the first considerations with respect to foliar absorption studies of mineral nutrients must be where and how the nutrient enriched solutions are to be applied. The method of appli— cation is often determined by the nature and magnitude of the experiment, as well as the species to be treated. Overall plant spraying has been commonly used, and has constituted the exclusive method for most field experiments, and for application to crops where nutritional disorders were to be corrected. Either the entire above-ground parts have been sprayed (23, 51, 54, 87, 99, 105, 115, 116, 118, 124), single leaves or selected leaves (12, 23, 24, 25, 29, 38, 42, 43, 54, 67, 68, 93, 114, 117, 134), or only a limited part of the leaf surface such as the very young leaves associated with the meristem (10, 11, 13, 24, 26, 88). Specially designed spray equipment was used in many of these studies. Nutrient solutions have also been painted onto leaf surfaces (7, 53, 100, 106, 116, 125, 133, 135, 136, 137, 138). Dipping has been a common method of treat- ment (4, 5, 6, 20, 64, 65, 83, 84, 125, 130). The one drop technique has also been used (7, 19, 20, 37, 43, 47, 63, 64, 67, 125). Other methods, although rarely em- ployed, have been the multiple drop technique (15, 16, 19, 114), injection (11, 1.2, 81), dusting (119), vacuum infiltration (27, 135), agar strip method (2, 3), leaf immersion (37, 70, 131), leaf-piece immersion (1, 2, 3, 71, 72, 73, 107), and " sticking" (91, 92, 132). Moreover, it is important to note that the lanolin ring method, a modification of the one drop technique, was used by Gustafson (47) to retain the drop of treating solution at the site of application where this was desired. This method was adopted later by Massini (81) in his herbicide absorption studies. An earlier method similar to this was the plasticine well method developed by Colwell (27). A plasticine well was set carefully on the middle of the upper surface of the leaf to be treated, into which the treating solution was poured. Drastic and uncontrolled changes in concentration and dehydration of the external solution during the course of the experiment have been the most serious limitations of above methods excluding the plasticine well, immer- sion, and "sticking" methods. Even with the latter, marked changes in concentration often occurred. To be specific, positive correlations between the surface-moisture retention or re-wetting and rate of foliar absorption have been recorded (42, 63, 67). In this regard, leaf immersion methods appear the most promising in terms of reproducibility and control of the external environment by aeration, stirring, and illumination. Kaindl (66), however, has criticized such a procedure in that dipping or immersion of leaves into a nutrient solution precludes normal gas exchange and may modify the structure of the epidermis. Sacher (103), however, 'has reported that the permeability integrity of leaf slices of Mesembryanthemum sp. and Rhoeo discolor remains through five days of immersion. This was shown by the non-occurrence of water-logging of air spaces and the ability of cells to undergo plasmolysis and deplasmolysis. Arisz has also reported (2) that, for immersed Vallisneria leaves, there is no free transport of ions through intercellular spaces and along cell walls. Methods for the Removal of Non-absorbed Residues — - Foliar absorp— tion can be accurately indexed only if, after the designated time interval, the non-absorbed residue is removed efficiently and completely from the leaf surface, or if in leaf immersion techniques absorption from the applied solu— tion is measured by the disappearance of the test nutrient from the ambient medium. The latter is often employed with leaves of aquatic plants. The most common approach has been a washing technique. Details of washing procedures were reviewed by jyung (63). After a predetermined period for foliar absorption the remaining residue was removed from the leaf surface by washing with distilled water (2, 3, 15, 16, 19,26, 37, 43, 63, 70,84, 91, 92, 99, 105, 115, 116, 123, 130, 132). However, acidified water (51), d‘stilled water containing a detergent (7, 29), and acidified-detergent solutions (42, 116, 125) have been used. The effectiveness of different washing solutions for removing the non-ab- sorbed residue remaining from one drop of P32 labeled 25 mM solution Of H PO4 3 applied to the upper surface of the primary leaf of the bean plant was studied by . . '1 jyung (63). Washing solutions consisted of distilled water, H3PO4 at 10 , 10-2, or 103, and CaClz, KH2P04, 0r Na OH at 10‘2 molar. Differences resulting from the various washing solutions were not statistically signifi— cant (P = 0. 05), however, 101M H3PO4 was the most effective. Ten ml of distilled water was sufficient to remove the residue. Wallihan and Heymann-l—lerschberg(125) determined the comparative effectiveness of three different washing solutions for removing the non-ab- sorbed zinc from a single drop of Zn65 solution put on either the lower or upper surface of a navel orange leaf. A detergent solution (Dreft) acidified to 0. 3 N HCl was superior to ivory soap and 10 percent NaEDTA at pH 5. Removal of non-absorbed residues by the washing technique is most efficient if used in conjunction with the leaf immersion method of applying nutrients to leaf surfaces. This was clearly deinonstrated by Fisher and Walker (42). Washing with 0. 1 percent Triton X100 immediately following 32 spray applications of 0. 2 percent KHZP O to the lower surfaces of apple 4 leaves removed 96 to 97 percent of the non—absorbed nutrient. If the treat- ing solutions were allowed to dry, significant amounts of the non-absorbed nutrient were adsorbed on the leaf surface, and were not removed by washing. This may be especially true for phosphate, calcium, and zinc. Limitations of such leaf washing techniques, as recognized by Wittwer and Teubner (129), may introduce a significant positive error for nutrients such as phosphate which are strongly adsorbed, and a negative error for those which are easily leached, such as potassium. The leaf disc removal technique has also been used (20, 47, 48, 63, 81, 125). Details have been reviewed by jyung (63). A limitation with this technique is that absorption is always under estimated. A high percent of the applied nutrient may be absorbed but not translocated beyond the im- mediate absorption site. This is especially true, as pointed out by Wittwer and Teubner (129), for all short term experiments, and for nutrients readily absorbed but relatively immobile such as calcium, magnesium, iron and boron. Another limitation is that, the treated area may be extended by creeping of the applied solution; and its complete removal by the disc tech- nique does not always occur. The lanolin ring method should reduce this error. Sample Preparation Methods for Assay of Absorption of Foliar Applied Nutrients - - Foliar absorption of non-radioactive mineral nutrients may be determined in a gross way by dried or fresh tissue analyses using standard chemical as well as electrometric methods and recording changes in com- position. Tracer techniques, however, have allowed for much greater pre— cision and sensitivity, and, if combined with standard chemical determina— tiOHS, permit a differentiation within the plant of foliar absorbed nutrients and those previously and concomitantly taken up by the roots. There have t. {‘{I ( , II .I‘ .I 'II ‘ ‘l‘ i ll. 1' 1 ‘l I been, however, many variations in assays for radioactivity. Therefore, pertinent literature is reviewed here in which radioactive analyses were employed in studies of foliar absorption of mineral nutrients. Direct counting of oven dried plant tissue samples has been the most simple method and was used by Barrier and Loomis (7) for radioassay of 32 32 P , Boroughs and Labarca (15), Boroughs et al. (16) for P , Bukovac_e_t _a_l_ 28 ~ 42 (19) for Mg , Bukovac and Wittwer (20) for Nazz. P32. 53°, (3136, K , Ca45, Mn55_59, Cu64, ans, 111386, Sr89, M099, and BaLa14O, Dybing and Currier (37) for P32, Jyung (63) for P32 and Ca45, Labarca (74) for P32, Teubnerftiil. (114) for P32, K42, and Rb86, Swanson and Whitney (112) for P32, C8137, and K42, and Wooley_e_t_a_l_. (131) for c136. Often the dried plant tissue was ground and only a portion of it radio-assayed (15, 16, 74, 131). The accuracy of direct counting of plant tissue has been examined. Teubner_e_t al_ (114) found that self-absorption by the tissues of bean seed- 42 9 lings was negligible for P3“, K , and Rb86 when samples were counted directly and then re-counted after ashed in a muffle furnace. Similarly, 45 Swanson and Whitney (112) reported that, with the exception of Ca , self- absorption by stem and hypocotyl parts of bean seedlings was negligible 32 137 . 42 _ for P , Cs , and K , and was usually less than 5 percent. Thus, direct counting appears satisfactory for the estimation of absorption with 9 32 42 86 137 P , K , Rb , or Cs in plant tissues as a consequence of foliar ab- sorption. The beta radiation must be strong and the volume of plant mater- ial relatively constant. Dry ashing of plant materials prior to radioactive assay, has been used in many studies and has the advantage of eliminating considerable self- absorption by the plant material, especially where weak beta emitting p radioactive isotopes (e. g. , 83D and Ca45) are used. This method has been used by Asen St _a_l_: (3) for the radioactivity assay of P32, Higashino and 32 Yatazawa (53) for P32, Koperzhinsky and Sheberstov (68) for P , Mayberry (83) for P32, Okuda and Yamada (92) for P32, Swanson and Whitney (112) for Ca45, Yamada (132) for P32, and Yatazawa (133) for P32. Volatilization of isotopes during the ashing procedure should be minimized. With this concern Mayberry (83) added 3 ml. of 10 percent MgNO to each plant 3 sample before placing it in the furnace. After ashing, the residue is dis- solved in a dilute acid and brought up to a certain volume. An aliquot of this may be dried with the aid of infra-red lamps, and then counted. Wallihan and Heymann-Herschberg (125) for 2116D and Thorne (153) for P32 employed a glass-walled dipping counter for measuring the radioactivity of the ash dissolved in dilute HCl. Kaindl (65) used a different method for 32 radio-assay of P . The ashed or ground sample was placed into a hole on a glass slide as an alcoholic suspension, and covered with a 1 mm thick l 0 layer of paraffin. In most cases a sample density of 60 mg/cm2 was pre- pared and then counted. This density was claimed to have negligible self- absorption. Wet digestion methods have been used by Biddulph _e_t a_1_._ (11) for measuring radioactivity of 835, by Thorne (117) for P32 using sulfuric and nitric acids, by Koontz and Biddulph (67) for P32 using nitric acid, and by Kylin (73) for $35. The digested material is made to a certain volume, aliquots placed in planchets are dried with the aid of infra-red lamps and then counted. As with dry ashing of plant materials pre- cautionary measures should be taken to prevent volatilization of the isotopes. Indices and Expressions of Foliar Absorption - - Wittweretal: (127) has listed the following as indices of foliar absorption: (a) "greening up" as a result of the correction of a nutritional dis-oi der, ('0) increase in growth and yield, (c) changes in plant composition, (d) direct detection or meas— urement by means of radioisotopes. "Greening up" of the nutrient sprayed foliage may be the most obvious evidence of foliar absorption. This criterion, while useful for demonstrat- ing the potentiality of foliar feeding, is not sufficiently accurate for annti- tative investigations. Apparent incorporation of foliar applied nutrients into metabolites within the leaves may fall into this category (7, 10, 26, 132, 134, 136). 11 Increase in plant growth and crop yields was suggested by Wittwer et a_l_. (127) as "the most objective though not always the most reliable, index under field conditions, because of the many variables, other than nutrient supply which may limit growth". Changes in plant tissue composition subsequent? to nutrient spray appli- cations may be used in evaluating the effects of foliar feeding. Mechanisms of foliar absorption may be resolved only when the amount of nutrient ab— sorbed is directly determined. Therefore, literature pertinent to this index and subsequent expression of absorption will be reviewed in a later section. Foliar absorption rates have often been expressed in terms of net radioactivity (c. p. m.) of labeled nutrient retained in the plant, plant parts, or unit weight of plant material (7, 37, 47, 48, 91, 92, 105, 114, 132, 134) at designated intervals of time subsequent to treatment. The rate of absorp— tion has often been recorded in absolute quantity (ug, 11M, mg) per specified material per unit time (2, 22, 24, 25, 72, 107, 108, 114). The most frequently used expression is the percent absorbed of the total applied at defined time intervals (7, 20, 21, 22, 26, 29, 42, 43, 63, 64, 65, 67, 87, 114, 117, 123, 137). Other expressions for identifying the rate of foliar absorption have been the percent of the nutrient in the roots derived from foliar applications (5, 38, 130), the time required for 50 percent (T ) absorption (25, 67, 129); the amount 1/2 absorbed per light energy unit (120) and, as the percent of the dry weight (70, 99), and the concentration in parts per million (84). Comparisons among different experiments, conducted in the same lab- oratory, as well as those with data reported by others, using the above criteria, should be made with great care when absorption is expressed in counts per minute or percent recovered of the total applied. Absorption rate values are subject to variations caused by leaf areas treated, volumes applied, concentrations of applied solution, specific activities of the labeled solutions, and pH. Burr _e_t_ :31 (25) noted, as an example, that the percent absorption was decreased as the concentration of the applied solution iii- creased. These limitations could be eliminated when absorption is express- ed in absolute amounts and under specified conditions as already proposed. To this end, the most explicit and implicit expression for the rate of foliar absorption is that based on the quantity absorbed per unit weight as adopted by Kylin (72), or of unit area as used by Burr (22), and Burr _e_t 2_1_l_ (24, 25) and Steemann Nielsen (108). Comparative foliar absorption studies in the future should be greatly facilitated by these means of expression, espec- ially when combined with the time for 50 percent absorption (T ) as pro— 1/2 posed by Wittwer and Teubner (129) for comparative absorption rates. l3 Criteria for Metabolic Foliar Absorption Absorption Rate and Time Course Analysis - - Many investigators have reported the typical two-phase time course curves in foliar absorption of mineral nutrients (19, 20, 63, 117). These have been classically denoted as phase 1 and 2. Mechanisms peculiar to these phases in root absorption have been thoroughly discussed by Laties (75). The same interpretations have been applied to those for foliar absorption (9, 126, 127). Non-metabolic processes have been ascribed to phase 1, an initial rapid uptake; whereas metabolic processes are associated with phase 2, a rather steady linear uptake. As pointed out by Laties (75) there has been a general agreement as to the interpretation of phase 2, while that of phase 1 is controversial. Consequently, a linear time course relationship for nutrient absorption has been taken as a criterion for metabolic uptake as emphasized by Epstein and Leggett (40). On this basis Epstein_e_t_al._ (41) claimed no evidence of an initial non-metabolic exchange phase in their time course studies on Rb ab— sorption by barley roots. Absorption was strictly a linear function of time for one hour. Their conclusions were supported by the irreversibility and marked temperature dependence of Rb absorption. According to Kylin (73) active sulphate uptake was a linear function of time in leaf-pieces of Crassula, Vallisneria, and wheat, for 12, 70 and 4 hours, respectively. It is imperative, therefore, that initial absorption and the acute break 14 in time course curves for foliar absorption be re-evaluated. It is proposed that the shoulder in the typical foliar absorption time course curve coincide with the drying, concentration, and crystallization of the applied solution. Biddulph (20), and Koontz and Biddulph (67) reported that the amount ab- sorbed and translocated is a linear function of time until crystallization occurs, and is directly proportional to the drying time of the solution on the leaf surface. The drying of the spray deposit has also been emphasized by Luckwill and Lloyd-jones (79). They (79) found that it was a dominant factor in determining the amount of applied naphthaleneacetic acid (NAA) which entered the leaf. Absorption proceeded at a constant rate (1 percent per minute) until the surface deposits are dry. Thus, the initially rapid ab— sorption of foliar applied materials is not evidence for non—metabolic uptake, whereas, a linear relationship with time is highly suggestive of an active mechanism. Verification, however, must come from an examination of other criterii of metabolic uptake. Temperature Dependence - - Temperature coefficients (Q10) for chem- ical reactions, where an Arrhenius temperature dependence holds, range from 2 to 3 (44). This criterion was suggested for determining if foliar ab— sorption were an active mechanism (129). Values of Q10 greater than 2 have 32, Rb86, been found for foliar absorption of P and K42 by bean leaves (114) over a range of 10. 0’ to 21.1’C., for Mn absorption by soybean leaves (84) over a range of 36° to 64° F., for C060 absorption by bean leaves (47) between a low temperature of 70° to 76° F., and a high temperature of 87° to 100° F. for Br uptake by Nitella cells (57) over a range of 10° to 20° C. , and for dye (cresyl blue solution) by_l_\l_it_e_lla_ cells (61) at 20° to 25° C. Values of Q10 have also been less than 2. For urea absorption and hydrolysis by citrus leaves the Q10 was 1. 28 between 4° and 37° C. (70). Temperature had little effect on P32 absorption by soybean leaves, while the Q10 for translocation was 2. 21 over a range of 15° to 30°C. (7). As an extreme, a negative correlation between the initial rate of urea absorption by apple leaves and temperature (70° to 90° F.) has been reported (29). Pramer (96) has suggested that slowly penetrating molecules may have high Q10 values, and a rapidly entering molecules a low Q10. Also, the magni— tude of Q10 may be influenced by the nature of the molecule being studied, and affected by an alteration of properties of the cell membrane. Stiles (110) has suggested that it may be unwise to draw definite conclusions with respect to the nature of the process of absorption from a Q10 value alone. Energy Source Dependence - - Ion uptake by leaves, if an active pro— cess, would be energy dependent. Light is closely associated with the meta— bolic activity of all living plant protoplasm (76). Light dependence may be the most indicative of all criteria for a diagnosis of energy requirement. To identify the light effect on foliar absorption, independent of its role in photo- 16 synthesis, it may be necessary to control stomatal opening. Phenylmercuric acetate as used by Zelitch and Waggoner (139) offers this possibility. On the other hand, Dybing and Currier (37) reported that phosphate absorption by Zebrina leaves in the absence of a surfactant was about the same whether stomata were open or closed. Light, with few exceptions, has consistently enhanced nutrient uptake. Its accelerated absorption of Cl, NO Br by Nitella, caused Hoagland and 3, Davis (56) to report that absorption is intimately related to growth and meta- bolism. For this suggestion Steward and Sutcliffe (109) gave them credit for originating the concept that salt uptake was a metabolic process. A thorough review has been prepared by Kylin (73) as to the effect of light on ion uptake by isolated green leaves, and by chloroplast—present algal cells and tissues. Pertinent discussions and interpretations have also been presented by Briggs Etil: (17). Lepeschkin (76), in 1930, after reviewing the results of early work on light and nutrient uptake suggested a direct effect of light on the permeability of the plasma membrane. A more general and current opinion, however, is that light affects an energy consuming mechanism for salt uptake associated with photosynthesis. This concept has been substantiated by experimental evidences. van Lookeren Campagne (120) reported that the chlorophyll system absorbs the light active in chloride uptake in Vallisneria leaves, since the action spectrum for chlor- ide absorption was very similar to the transmission spectrum. In a critical 1? experiment, Kylin (73) found that light did not affect sulphate accumulation by leaves of an albino barley. He concluded that the light dependent sulphate accumulation was mediated by the chloroplasts. The light effect appears to be systemic. Arisz and Sol (3) noted in Vallisneria leaves that light exposure of the free part (portion not immersed) increased the chloride accumulation in the absorbing zone. This suggested transport of an accumulation enhancing substance produced by light from the free to the absorbing zone. They also showed that the substance could be stored for several hours. Chloride absorption by Nitella cells was found directly proportional to the duration of illumination (56) indicating a stoichio- metric effect of light on nutrient uptake. Notwithstanding suggestions of a direct light effect (17, 109), it is obvious that sugars produced through photo- synthesis may, in turn, enhance metabolism-dependent absorption processes by providing an energy source for living protoplasm. Consequently, studies as to the effects of exogenously applied sugars on nutrient absorption have been conducted. Results, however, have been variable. Absorption of nutrients by green tissues in the presence of sucrose was increased (2, 106, 135), decreased (29, 54, 114, 123, 132), or varied with the experiment (117). Fermentable sugars (glucose and fructose) increased absorption (135). Variable results with sucrose may relate to a decrease in the rate of diffusion of nutrients toward the absorption surface, or to an inhibition of enzymes critical in the ab- sorption process (123). Thus, sugar applied prior to treatment has reduced uptake during the first few hours and later enhanced it (107). These data would be more convincing if information were available as to possible differ- . ences in utilization of endogenous versus exogenously applied sugars. Thus, it may be concluded that light dependence can be used as a criterion for metabolic absorption of nutrients by leaves. The use of sugars, however, as energy source for accelerating uptake should await further critical re- .evaluation and standardization of methods. Sensitivity to Metabolic Inhibitors - - Metabolic inhibitors provide a means to relate foliar absorption to corresponding process(es) of metabolism. Among commonly used inhibitors are 2, 4-dinitrophenol (DNP) an uncoupliag agent in oxidative phosphorylation, arsenate and iodoacetate, uncouplers of the substrate level phosphorylation. Cyanide and azide inhibit respiration in uncoupled as well as in coupled systems. Gramicidine is an inhibitor of oxi- dative phosphorylation, and fluoride inhibits enolase, and pliloridzine muscle phosphorylase. 2, 4-dinitrophenol (DNP) may be used in a typical diagnosis for the dependence of nutrient uptake on metabolically derived energy. This is because it has been classified as a true uncoupling agent by Racker (97). It eliminates phosphorylation but at the same time, under appropriate condi— tions, stimulates or has no effect on respiration. According to Arisz (1) potassium cyanide, azide, iodoacetate, fluoride, l9 arsenite, and DNP inhibited the transport of both phosphate and asparagine, while phloridzine reduced phosphate transport only. He has, therefore, sug— gested that phosphate is bound by an enzyme system, probably coupled with the process of phosphorylation of adenylic acid, and that enzymatic processes are evidently involved in its uptake and movement. Kylin (73) noted that selenate inhibition of sulphate accumulation in Vallisneria and Crassula was independent of light, whereas the inhibition from DNP and potassium cyanide was greater in the dark. He concluded that both a "light uptake system" and a "dark uptake system" were involved. Also, in his sulphate accumulation studies various degrees of essentially light independent inhibitions were re- corded for arsenate, gramicidine, and arsenite. Uptake of P32 by bean leaves in the presence of DNP, sodium salts of arsenate, fluoride, azide, and iodo- acetate was inhibited according to Boroughs_e_t_al. (16). Sodium arsenite, however, increased absorption. Specific metabolic inhibitors provide a means of ascertaining whether or not foliar absorption of a specific nutrient is likely linked with an energy mobilizing system. One reservation, however, must be imposed on conclusions derived from results with metabolic inhibitors. Any modification of the general activity of protoplasm, such as a change in viscosity, may, in turn, affect net foliar uptake. Accumulation Against a Concentration Gradient - - Ion accumulation re- . quires expenditure of metabolically derived energy and is a criterion for active foliar (129) or root (41) absorption. Arisz (1) reported that accumu- lation of asparagine in Vallisneria leaves was not a diffusion process since it was oxygen dependent. According to Kylin (73), more sulphate was absorbed per unit fresh weight of leaf discs of Thuidium, Crassula, Vallisneria, and wheat than remained per unit volume in the external solution. He concluded that accumulation must be an active process. Accumulation against a concentration gradient suggests metabolic ab- sorption. However, accumulation against a concentration gradient is not evidence for active transport if it occurs along an electrochemical potential gradient as pointed out by Dainty (32). In a recent communication by Briggs S: {L (17) the term, "against a concentration gradient" was dropped from the definition of accumulation. Irreversibility of Absorption - - Irreversibility has been suggested as a criterion for active foliar absorption by Wittwer and Teubner (129). Arisz and Sol (3) noted that there was no loss of chloride previously absorbed by Vallisneria leaves during a 24 hour uptake period when they were subsequently washed with aerated distilled water for 15 and 30 minutes. Leakage of pre- — 35 . . . . absorbed S from leaf tissues of Crassula, Vallisneria, and wheat was negli- gible according to Kylin (73) during a prolonged washing for 0. 6 to 20 hours. Irreversibility of absorption for the several nutrients indicated is an additional evidence for an active foliar uptake. Oxygen Dependence - - A specific oxygen requirement is suggestive of a metabolic process (129). Arisz (1) observed that withholding of oxygen inhibited further uptake of asparagine. He used this oxygen dependence as a decisive criterion for "activated transport" even though the transport process follows the rules of diffusion. The foliar absorption requirement for oxygen may be appreciably less than that estimated for root absorption. Saturation of the oxygen requirement for K and Br uptake by barley roots was recorded at 5 percent (55), and about 10 percent for Rb and Br accumulation in potato and jerusalem artichoke discs (109). Illuminated green leaves may preferentially utilize endogenous oxygen (46), and the internal oxygen concentration may even be greater than the external as cited by Kylin (73). Hence, oxygen dependence for foliar absorption of minerals in the light may be a reality but not easily subject to an objective evaluation. Specificity and Ion Competition - - Specificity and ion competition can be explained only by metabolically produced specific carriers. The occurrence of these phenomena suggest an active mechanism for ion uptake (129). Ion competition and specificity studies have not been attempted in the domain of foliar absorption, though they could be conducted utilizing enzyme kinetics as adopted by Epstein and Hagen (39). pH Dependence - - A pH dependence for active foliar absorption was also suggested by Wittwer and Teubner (129). This possibility is tenable since it is known that the activity of most enzyme-catalyzed processes varies with pll. The importance of pH curve studies in such processes was emphasized by Reiner (98). Analysis of pH effects provides information as to the structure of the so-called "active center" of an enzyme, and is, therefore, a bridge between kinetics and mechanism. If it is assumed, as in the carrier concept for nutrient absorption, that the specific carrier is enzymatic, then studies of pH, with the proper interpretations, should be of great importance in re- solving mechanisms of foliar absorption. van Overbeek (121) has suggested the pH effect is on plant proteins rather than on the penetrating chemicals. Some effects of pH on foliar absorption have been recorded with the appropriate curves. Absorption of potassium, sodium, and ammonium phosphates by bean leaves over a pH range of from 2 to 7 was studied by Teubner ital. (114). Maxima were at pH 2 to 3 and 5, with a valley at pH 4. Absorption of Rb and K chlorides, citrates, and phosphates was also consider- ably lower at pH 4 than 2 or 8. Maximum absorption occurred at pH 8. They pointed out that a hypothesis of undissociated molecule penetration of H3PO4 at low pH levels, or exchange reactions involving any particular ionic species—- H2P04 or HPO4--, failed to explain the marked reduction in absorption at pH 4. A pH dependent curve for foliar uptake of phosphate by bean leaves was also reported by Swanson and Whitney (112). The most rapid absorption occurred at pH 2 to 3. 5, and there was a gradual decrease tip to a pH 7. They suggested that the greater absorption at a low pH was related to at least two factors. H3PO4 dissociation was suppressed, and there was a direct effect of pH on the permeability of epidermal and subjacent tissues. Similar results were obtained by Da' Silva Cardoso and Boroughs (31) for phosphate absorption by cacao leaves. There was a peak in their pH curve at 5 to 6 and a very low value at pH 7 and on up to 12. Yamada (132) found that for ammonium phos- phate absorption by bean leaves over a pH range from 2. 6 to 6. 2 there was an appreciable depression at pH 4. 6 in one experiment, and at pH 5. 6 in the other. Such inconsistencies in pH curves for phosphate absorption could be ascribed to differences in experimental conditions since the phosphate carrier was the same. Several studies of the effects of pH on foliar absorption of urea have been conducted. Cook and Boynton (29) reported that urea absorption by apple leaves was highest at pH 5. 6, lowest at pH 7. 2, and intermediate at pH 8. 0. A depression in the curve was always recorded at pH 7. 2 Both phosphate and acetate buffers at 0. 01 M gave similar results. Volk and McAuliffe (123) also studied the effects of pH levels from 5 to 9 on urea uptake by tobacco leaves. They used three different buffer systems; potassium acid phthalate and NaOH for a pH 5, KH2P04 and NaOH for a pH 6, 7 and 8, and boric acid and KCl for a pH 9. Absorption peaked at pH 5 and 8 with a valley at pH 6, and was lowest at a pH of 9. Relatively low absorption of urea, in both cases cited above, was observed at a pH of 6 to 7. Other explanations have been rendered as to pH effects on foliar absorp- tion. Volk and McAuliffe (123) suggested that NaOH increased the permea- bility of the cuticle. Orgell (94) has indicated that at a pH lower than 5, the cuticle would have no charge, while at a high pH above 7, the cuticle would be charged negatively. Consequently, pH regulated electrostatic repulsion and attraction phenomena may affect cuticular sorption, and consequently the rate of foliar absorption of ions. MATERIALS AND METHODS Plant Materials and Culture Phaseolus vulgaris L. , var. Black Seeded Blue Lake, was selected as the primary experimental plant because of its rapid germination and uni- formity (20). Seeds were spaced approximately 2 by 2 inches and germin- ated in coarse quartz sand in the greenhouse during the summer, but in the laboratory during the winter for experiments on the effect of aeration, and reversibility of phosphate absorption. They were sown about one inch deep and tap water was applied as needed. The seedlings emerged after 7 to 10 days and were retained in the sand usually until the 12th or 14th day, until used. This resulted in a typically low salt plant especially suitable for studying nutrient uptake process. At the age for use, the primary leaves were fairly well expanded, and the first trifoliate leaf was just emerging. During the winter seedlings were started in the laboratory under artificial illumination, with the first week in sand and then in aerated tap water (Figure 2). Occasionally, leaves of Chrysanthemum morifolium R. var. Delaware were used. These were obtained from plants grown in aerated solution cultures as described by Asen et al. (5). 25 Environmental Conditions All experiments were performed in the laboratory (Figure l) with the air temperature’ maintained at 21° C. The temperature of the solution into which a leaf was immersed was usually 23° to 25° C. depending on the light intensity. Variation of the solution temperatures during a treatment did not exceed I 0. 5° C. The treated leaves were continuously illuminated by fluorescent lamps at various intensities ranging from 320 to 1400 foot candles. (ft-c. ). Preparation of Treating Solutions Solution concentrations were usually selected from the lower end of the concentration ranges tested where linear relationships existed between the external concentrations and the rates of nutrient absorption (Figures 8 and 9). The concentrations used for phosphate, Rb, and Ca were in most cases 0. 3 mM, 1 mM and 0. 33 mM, respectively. Specific activities of the labeled solutions 32 were usually 0. 15 uc P per umole H P04, 0. 01 uc Rb86 per umole RbCl, 3 and 0. 15 uc Ca40 per umole CaCl pH was adjusted by adding 0. 05 M tris—Cl 2. _ . _9 . . . at pH 7. 5, sodium acetate buffer at 11 :. 10 “M (ii: ionic strength) or tris- 2 phthalate buffer at,u 2: 10- M. 27 Treatment of Leaves by the Leaf Immersion Technique Forty ml of the isotopically labeled nutrient solution was added to a Petri dish containing a leaf held in position by means of suitable glass-rod (3 mm) frames (Figures 1, 3 and 4). For the aeration experiments, 120 ml was applied to a glass-funnel of 10. 3 cm diameter (Figure 5). The leaf was submerged to a depth of about 0. 5 cm from the solution surface. Eva- poration of the treating solution was prevented by a mylar cover, and by adding water to the pan containing the Petri dishes or clay pots fitted with funnels. This also minimized temperature fluctuations. The usual volume change from evaporation of the external solution in the Petri dish experi- ments during a 24-hour period ranged from 5 to 8 percent, while the net loss of nutrient from the external solution induced by leaf absorption was 0. 5 to 5 percent. In buffered solutions, there was no pH change or less than 0. 2 unit, if any, during an 18-hour period. The provision for aeration (Figure 5) was to ascertain limitations, if any, of reduced partial pressures of oxygen and carbon dioxide on nutrient uptake. While limitations are recognized, the advantages of the leaf immersion technique were clearly evident. A constant concentration can be maintained. The environment is standardized. The non-absorbed residue is easily re- moved. There is precise control of temperature. Since the entire leaf is immersed and subject to an absorption condition, interferences from trans- Figure 1 Top - General view of experimental area. Figure 2 Bottom - Plants growing in aerated tap water during the winter. 23 Figure 3 Top — Treatment of primary leaves of intact bean plants by the leaf immersion technique. Figure 4 Bottom - Treatment of excised Chrysanthemum leaves by the leaf immersion technique. 2‘? £2me :oUnCOmam HEEL new :odeoEc: Em: 950.35 mstmfimB: Surname < m 9.2%; .30 mmaahm ZOEBEOmm< ”$430... mo... 205.1922. “Em; Emommd oz_._.4s‘.‘ ._..._.—.. -.— 31 port within, and translocation out of the treated leaf are reduced to a mini— mum in contrast to employing only a portion of the leaf surface. Trans- piration and humidity problems are non-existent. Reproducibility of foliar absorption studies was possible by the leaf immersion technique, and such a procedure' was followed in all experiments herein conducted. Harvesting and Leaf Washing Technique Plants were harvested, at the designated time intervals, and separated into the treated leaf cut at the base of the petiole and the remainder of the plant. For excised leaves, leaf blades only were harvested and the petioles discarded. The treated leaf was then washed in three successive 50—ml por- tions of distilled water for 15 seconds each. Usually six successive momen- tary dippings were made in each washing operation. Upon completion of the third and final washing, the treated leaf was blotted with facial tissue paper for 30 seconds with one change of tissue paper after 15 seconds. Prelimin— ary experiments showed that all washable non-absorbed nutrient residue remaining on the surface of the treated leaf was removed by the above men- tioned washing procedure. A fourth washing removed negligible amounts. The cumulative percentages of removal for each of four consecutive wash- ings with 50 ml each were 87. 5, 93. 7, 97. 3 and 100. 0, respectively. Each value was an average of six washings, two each, at absorption periods of 5, 30 and 60 minutes. The same tendency was observed after a 24—hour absorption period. The expression "leaf immersion and washing technique" as used here- after, was coined as descriptive of the immersion treatment and washing procedure described above. Measurement of Variables The fresh weight of the leaf was determined after washing and its out- line traced on a sheet of paper with the aid of a light box or a projection through a hood window. The leaf area tracing was often done before treat- ment. Leaf area was then determined from the tracing, using a planimeter (Figure 1). An average of at least two readings having less than a 3 per- cent error was recorded. The leaf areas for the primary leaves of the bean plants usually ranged from 13 to 17 cmz. There was no change in leaf area even after a 24—hour treatment period. Plant materials, after harvest, were placed in 1 ounce paper cups, and dried in a forced draft oven at 70°C. for 12 to 24 hours. Dry weights were recorded of the oven dried materials after cooling in a desiccator for 20 minutes. Before the dried samples were counted, they were pressed firmly against the bottom of the container with a smasher made of a rubber stopper, which corrected varia- tions in the geometrical placement of the samples before the window of the counter tube. Each plant sample was counted directly for radioactivity using an end-window Geiger—Miller tube and standard scaler circuit. Self-absorp- . . . . 32 7 , 86 tion by bean seedling tissues has been negligible for P (11-, 114) and Rb (114). Expression of Absorption and Estimates of Variability Retention of labeled nutrients in the plant material after washing was considered equivalent to absorption. All counting data. were corrected for background and converted to absolute values by comparing with the average activity of three 1 nil-standards of each treating solution. The total amount absorbed was divided by the area of the treated leaf to give the quantity absorbed per unit area. Unit area reported herein was equivalent to a leaf disc of that area, and included both the upper and lower leaf surfaces. The term of "specific absorption" was coined to specify the amount absorbed per unit area (which included both the upper and lower absorbing leaf surfaces) in unit time, from an external solution of constant concentration. Determina- tions made after this manner were expressed as mumoles/cm2 x 24 hours from an external solution of 1 mM of the nutrient being studied. Data were subjected to t-test where a comparison of two means was necessary, and to Duncan's multiple range test (36) for a comparison of more than two means, unless noted otherwise. Replicates within a treatment and the treatments were completely random i zed. RESULTS AND DISCUSSION Mechanisms of Foliar Absorption of Mineral Nutrients Time Course Studies - - The first absorption studies with phosphate and Rb employing the leaf immersion and washing technique were conducted with time as a variable (Figures 6 and 7). Absorption of both phosphate and Rb'L proceeded at constant rates, that is, 0. 785 and 6. 81 mumoles/cm2 leaf x hour, respectively, over a 24-hour period, beginning with zero time. These absorption rates were obtained as the slope of the linear regression calculated by tae least square method. Absorption as a linear function of time has been used by Epstein and Hagen (40) as a criterion for active uptake. On this basis, Epstein e_t _a_l_. (41) showed no evidence of ab initial non—metabolic ex- change phase. They observed that Rb absorption in barley roots was a strictly linear function of time for one hour. Their argument was corrobor— ated by the irreversibility and marked temperature dependence of the ab- sorption process. Kylin (73) also reported that the active sulphate uptake by leaf pieces of Crassula, Vallisneria, and wheat was a linear function of time over 12, 65, and 4 hour periods, respectively. These results suggested that both phosphate and Rb absorption by bean leaves are metabolic processes. They also prompted further inquiry whereby other criteria were utilized for identifying metabolic uptake. Consideration of the apparent free space (AFS) 34 Figure 6 A. Upper left: Phosphate uptake by intact primary leaves of 32 per 13. 3 umoles 11-day—old bean plants as a function of time from 2 ac P H3PO4 in 40 ml of solution at pH 3. 5. Plants were exposed to 350 ft-c. of light and a temperature of 23° to 25°C. Each point a mean of two replicates. B. Upper right: Rb absorption as a function of time from 0. 4 uc 86 Rb per 40 umoles RbCl in 40 m1 at pH 6. 1. Other conditions as in (A), except l4-day-old plants were used. C. Lower left: Dependence of phosphate absorption by intact primary leaves of 14-day—old bean plants on the concentration in the external solution. Plants were exposed to 500 ft-c. of light and a temperature of 23° to 24° C. All solutions were buffered to pH 3. 6 with tris-phthalate (p = 2. 25 x 103), and contained Tween-20 at 0. 019:... Each point a mean of three repli- Cc’ltCS. D. Lower right: Dependence of Rb absorption on concentration. Conditions as in (C), except the external solutions were buffered to pH 7. 3 by 12. 5 umoles tris-Cl at pH 7. 5, and 13-day-old intact plants were used. b b n Tim(Ma) » m u a ‘8. 15:23.5: ~ 2.33 :93 38.62 bQSsqsoazsocoteo-Q Timlml LS 3 W O O A m a... w. ._. .Bigxoeécgz 8:. 3 .26.. $5.. .3 3%... 8.33.3 - n n - “0'- “0*- - man» ~23: n .32....ux835 35281 coco .0 .26.. :95! so 3293: w 20" b p h IO .7 0. 33 LC? 3.33 V h 3 5 4 3 ~§m¢3€59\nsosa§ 8.53 .38 .o 3:31.05tq 3 £29. 323$ me: (u x no") Pmufiotd HMO") Figure 7 Phosphate uptake by excised primary leaves of 14- day-old bean plants as a function of time. Conditions as in Figure 6 A. 3 G 9-o.m+o.358x tom.~Eu\m2oE 35 2.33 .33 .o 3.63 foe...“ 33.88 .3 £238.... .0 3:88.; 24 l2 Time (hours) 37 concept introduced to explain an initially rapid uptake, and presumably accounted for by a diffusion process, is not considered critical in these studies. It is of interest to note that the time course studies of phosphate ab- sorption by the excised primary leaves of bean plants indicated a linear function for only 12 hours. The absorption curve then began to level off (Figure 7). The rate of phosphate absorption by excised leaves (Figures 7 and 8) was usually one-half that of intact leaves (Figure 6, A), although translocation out of the treated leaf of intact plants amounted to only 5 to 10 percent of total absorbed. Response to Temperature - — Temperature effects on phosphate and Rb absorption by bean leaves were next studied (Figure 8, A and B). Tem- perature coefficients (Q10) for phosphate and Rh were 1. 82 and 1. 55 respect- ively, from 5’ to 25’C. These Q10 values for phosphate and Rb absorption by primary leaves of bean seedlings are lower than those recorded by Teubner (3311' (114), who found 2. 0 and 2. l for phosphate and Rh absorption, respectively, from 10. 0° to 21. 1°C. These greater Q10 values reported by Teubner 3.15.3.1: (114) may be ascribed to translocation as well as absorp- tion because they expressed absorption as percent of total applied recovered in stem 8 hours after treatment. The Q10 is much greater for P32 trans- location than for its absorptio.. by soybean leaves (7). A Q10 value of 2 or above has been set as a criterion for metabolic ».-—. foliar absorption (129). Typical Q10 values for chemical reactions, where an Arrhenius temperature dependence holds, range from 2 to 3 (44), Not- withstanding, the Q10 values, lower than 2 in the present investigations, are still greater than 1. 2 to l. 3 which are characteristic of purely physical pro- cesses, such as diffusion (85). It is therefore suggested that absorption of phosphate and Rb by primary leaves of bean plants may be coupled to meta- bolic process(es). Responses to Energy Sources - - The effect of light on foliar absorp— tion was selected as a diagnostic tool for energy dependence. "Specific absorption" of phosphate by bean leaves was influenced by light (Figure 8, C). There was an initially rapid increase as the light intensity rose from 0 to 320 ft-C. , and then a steady increase with a further rise in intensity. Complete light saturation for " specific absorption" of phosphate was not evident even at 1400 ft-c. Light saturation for ”specific absorption" of Rb occurred, however, at less than 320 ft-c (Figure 8, D). Phosphate absorption is apparently more light or energy dependent, which implies that the acti- vation energy for phosphate uptake may be greater than that for Rb. Arisz and Sol (3) have reported that light saturation for chloride uptake by Vallisneria leaves was 150 ft-c. The mode of action of light in nutrient uptake has not been clearly de- fined. It is generally believed, however, that light controls an energv con- suming mechanism in nutrient uptake through photosynthesis. The accumu- .._ "'9! “—54," F Figure 8 A. Upper left: Temperature dependence of foliar absorption of phosphate. Excised leaves of 12—day-old bean plants immersed in 2 uc 32 P per 13. 3 umoles H P04 in 40 ml of 0. 01% Tween-20 (pH 3. 5), and ex- 3 O posed to 500 ft-c. of light at 5 to 25° C. Each point a mean of three repli- cates. B. Upper right: Temperature dependence of foliar absorption , . 86 of Rb. Exotsed leaves of 16-day-old plants immersed in 0. 4 uc Rb per 40 umoles RbCl in 40 ml 0. 01% Tween-20 (pH 6. 1). Other conditions as described in (A). C. Lower left: Light dependence of foliar absorption of phosphate. Intact leaves of l4-day-old plants immersed in 2 uc P32 per 13. 3 umoles H P0 in 40 ml (pH 3. 5) at 25°C.T 0. 5. Each point a mean of three repli- 3 4 cates. D. Lower right: Light dependence of foliar absorption of Rb. Intact leaves immersed in 0. 4 uc Rb86 per 40 umoles RbCl in 40 ml 0. 01% Tween-20 (pH 6. 1). Other conditions as in (C). 0.1. homunc- honour- “. a. m. m .. ‘ . a. a Li ' b n n p P E w w w m m w o Cox-Euxuo.oEAE~ 2.33 .33 3 33! .35.... 8 .2. 3 5.883 . u A. Anna. O.- LUZ 5501»me- fun. (Mun) b p 4.2 '- C o .8 1‘ 33-ny 3351.58.33 .33 3 .25! .38.... .8 2238.3 3 8.3.83 Timlhuml - u m .M n N .. .m w L a h P b in’ h [‘5’ m m m n a m m o 333 Quin! ~Eo\33§1.52co3 :93 3 .30! bus... 8 .9. 3 53.8.3 2.323 .m m m. m M .m m D)- L b C 3 «2.3.. 3232 ~EQ83E35 8.33 .33 3 331 32...... .3 2282.... 8.3.88 2:88 ~10 lating evidence is that light affects the chlorophyll system (73, 120) which is involved in the production of a transportable and storable substance which enhanced accumulation (3). There is a direct proportionality between the duration of illumination and light-enhanced absorption (56). It is pro- posed that the light dependence of phosphate and Rb absorption by bean leaves is indicative of a metabolic process. - at“. Juno-W There is the possibility that the light imposed effect may be a condi- tion that results in various degrees of stomatal opening at different light -mu-._A -- -— o intensities. This problem could be resolved by the use of phenylmercuric acetate to control stomatal opening, independent of light, as proposed by Zelitch and Waggmner (l39). It has already been reported, however, that in the absence of a surfactant phosphate absorption by Zebrina leaves was about the same whether stomata were open or closed (37). Studies of the energy dependence of Rb absorption by bean leaves were extended by observing the influence of adenosine triphosphate (ATP) exogenously applied as a biochemical energy source. The increase (2. 6% in Rb uptake in the presence of tris-ATP at 10-4M was insignificant. A similar result was reported by Kylin (73). ATP at lO-BM slightly inhibited absorption of radiosulphate by leaf pieces of Crassula. Explanations for the above results are not clear. There is the possi— bility of preferential utilization of endogenous ATP or most cells may be . , -3 simply not permeable to ATP. However, a tox1c effect of tris-ATP at 10 M 41 was not noticed in bean leaves as shown as dehydration or decolorization. This implies that ATP or at least a portion of ATP other than the phosphate group entered the leaf tissue, because phosphate itself was not toxic even at IO-ZM. Re-evaluation through more carefully designed experiments appears desirable. Response to Metabolic Inhibitors and Promoting Agents - - 2, 4-dini- trophenol has been known since 1948 as an uncoupling agent (78). It is clas- sified as a true uncoupling agent (97) since it eliminates phosphorylation and at the same time, under appropriate conditions, stimulates or does not affect 02 uptake. . The effects of 2, 4—dinitrophenol (DNP) on the absorption of phosphate and Rb by the primary leaves of bean plant were studied as a classical tool for the diagnosis of dependence on an energy yielding system. The results are summarized .in Tables 1 and 2. A significant reduction was noted in phosphate as well as in Rb absorption in the presence of 10-4M DNP. Hence, the absorption of phosphate or Rb by bean leaves may be linked with energy yielding processes. DNP also inhibited the transport of phosphate and asparagine in Vallisneria leaves (1), the sulphate accumulation in leaf pieces of Vallisneria and Crassula (73), and P32 absorption by bean leaves (16). The study of metabolic inhibitors was also extended to the effects of chloramphenicol on foliar absorption. This antibiotic has been reported as -_‘r TABLE 1. --Effect of 2, 4-dinitrophenol (DNP) on Foliar Absorption of Phosphate. 1 Concentration "Specific Absorption" of PhosphateJ (Molar) (mumoles/cm“ leaf x 24 Hours) 0 (Control) 12.211 10‘5 10.4n -5 5x10 afl 10'4 7.0“l Means with same letter not significantly different at odds of 19:1 (Duncan's multiple range test). The asterisk indicates absorption significantly less than the control at odds of 19:1 (F-test). Absorption 1) primary leaves of intact 11-day-old bean plants from 2 uc P3 per 13. 3 umoles H3PO4 in 40 ml DNP solution. Plants exposed to 350 ft-c. of light and to 23‘ to 25° C. Each value a mean of three replicates. 43 TABLE 2. --Effect of 2, 4-dinitrophenol (DNP) on Foliar Absorption of Rb. "Specific Absorption" of Rb— Concentration (mumoles/cm2 Leaf x 24 Hours) (Molar) n 0 (Control) 162 5 x 10-5 143118 10‘4 1028 1/ — Means with different letters significantly different at odds of 19:1 (Duncan's multiple range test). Absorption by rimary leaves of intact 14-day-old bean plants and from 0.4 uc Rb8 per 40 umoles RbCl and 0.01% Tween-20 in 40 ml DNP solution. Plants exposed to 350 ft-c. of light and to 23° to 25° C. Each value a mean of three replicates. 44 a specific inhibitor of protein synthesis in bacteria (18, 82), in wheat roots (95), and in carrot root slices (62). It has readily entered Nitella cells (96), and leaves of the tomato and broad bean (30). Thus, experiments with chloramphenicol had a dual objective. Relationships between foliar absorption and protein synthesis could be examined, and the possibility that the theoretical specific carrier, if present, may be proteinaceous and of enzymatic nature could be determined. The effects of chloramphenicol on " specific absorption" of phosphate and Rb are tabulated in Tables 3 and 4, respectively. A significant reduc- tion in phosphate absorption occurred at 100 to 1000 ppm (Table 3). The chloramphenicol induced reduction in Rb absorption was significant in the light, but not in the dark (Table 4). These results suggest that (a) foliar absorption of phosphate and Rb is possibly related to protein synthesis, and (b) carriers may be likely proteinaceous. The latter assumption is based on the premise that the primary effect of chloramphenicol is an inhibition of protein synthesis. Brock (18) discussed that chloramphenicol is an inhibitor highly specific in transfer of amino acids from soluble ribonucleic acid (RNA) to protein. He also reviewed the chloramphenicol induced inhibition of the synthesis of following specific proteins, C-galactoside and maltose permeases, which may be equivalent to carriers as called herein. Since a significant reduc- tion of protein synthesis in the presence of chloramphenicol has been TABLE 3. --Effect of Chloramphenicol on Foliar Absorption of Phosphate. Chloramphenicol "Specific Absorgtion" of Phosphatcl/ (Ppm) (mumoles/cm Leaf x 24 Hours) n 0 (Control) 17. 8 ns 10 14. 4 100 10.5S s 1000 9. 0 1/ — Means with different letters significantly different at odds of 19:1 (Duncan's multiple range test). Absorption by primary leaves of intact lO-day-old bean plants and from 2 uc P32 per 13. 3 umoles H PO and 0. 01% Tween—20 in 40 ml chloramphenicol solution- Plants exposed to 500 ft-c. of light and to 24° to 25°C. Each value a mean of three replicates. TABLE 4. --Effect of Chloramphenicol on Foliar Absorption of Rb. Chloramphenicol "Specific Absorption" of Rb-l/ (Ppm) (mumoles/cmz Leaf x 24 Hours) 0 (Control) 143' 250 114 t value 3. 84 t. 99 4. 54 t. 95 2. 35 1/ Absorption by primary leaves of intact 13-day-old bean plants and from 0. 4 uc Rb per 40 umoles RbCl, 40 umoles tris-Cl at pH 7. 5, and Tween-20 at 0. 01% in 40 ml chloramphenicol solution. Plants exposed to 500 ft.-c.. of light and to 24° to 253C. Each value a mean of three replicates, except: two for chloramphenicol in the light. 40 demonstrated even in 60 minutes (82), the above-mentioned premise may be most likely. Chloramphenicol may also affect chloroplast maturation (80). This may modify foliar absorption, as is illustrated in Table 4. It may also be speculated that a chloramphenicol sensitive system operates more in the light than in the dark. After completion of these studies, a very similar hypothesis was proposed by jacoby and Sutcliffe (62). They found that there was about 50 percent reduction in both K uptake and in protein synthesis of carrot root slices in the presence of 2 ppm chlor- amphenicol. Boroughs et a1. (16) also reported that chloramphenicol at . . . 32 . . . . 100, mg/1 inhibited P absorption by bean leaves (about 20 percent inhibi- tion upon calculation). They made no attempt to interpret their results. The above results suggest that foliar absorption for phosphate and Rb are related to protein synthesis. Accordingly, studies with the phyto— kiiiins were initiated. The prevention of protein degradation or stimula- tion of protein synthesis, or both, has been noted with kinetin (69, 88, 111) . 6 . -6 , ,. . . and With N -benzyladenine (N -BA) (104, 122). I\ln(3t1n also favored the accumulation of amino acids, sugar, sulfate, and phosphate (89, 90). . . . 6 The efiects of kinetin and N -BA on phosphate uptake by bean leaves are presented in Tables 5, 6 and 7. "Specific absorption" of phosphate, irrespective of whether by intact or excised leaves, consistently tended to . . . 6 - . . decrease in the presence of kinetin or N -BA at 1.3 ppm, in the light and increase in the dark. No explanation is offered. Phytokinins may inhibit a [V 's‘. v , ~. 47 TABLE 5. --Effect of Kinetin on Foliar Absorption of Phosphate by Intact .r -iw-H'Q‘. w c I. Leaves. , , "Specific Absorption" of Phosphate Kinetin Light (300 ft-c) Dark (Ppm) (mumoles/cm2 Leaf x 24 Hours) 0 (Control 7. 68 4. 10 15 7. 26 6. 56 t valuel/ . 88 1. 53 y - t - ° Where t. 95 — 2. 13, t. 90 —- l. 33, and t. 70 — 0. :37. Phosphate absorption by primaty leaves of intact 13-day-old bean plants and from 2 uc P32 per 13. 3 umoles H PO and Tween-20 at 0. 01% in 40 ml kinetin solution. Plants were expose to 124" C350. 5. Each value a mean of three replicates. TABLE 6. --Effect of Kinetin on Foliar Absorption of Phosphate by Excised Leaves. Kinetin . "Specific Absorption" of Phosphate Light (350 ft-c) Dark (Ppm) (mumoles/Cm2 Leaf x 24 Hours) 0 (Control) 8. 40 3. 64 15 6. 38 6. 60 t value 1. 74 4. 70 t. 2. l 2. r . 95 3 3) t. 90 1. 53 Conditions as in Table 5, except two replicates for the control in the dark, and excised leaves of l3-day-old bean plants were used. 48 TABLE 7. -- Effect of Nb-benzyladenine (Né-BA) on Foliar Absorption of Phosphate by Excised Leaves. "S ecific Abso tion" of Phos hate N6_BA p 1’13 p Light (300 ft-c) Dark (Ppm) (mumoles/cmz Leaf x 24 Hours) 0 (Control) 9. 98 6. 00 15 8. 06 6. 69 t value 2. 73 . 39 t 2. 13 2. 92 . 95 t . 29 . 60 Conditions as in Table 5, except that N6-BA was used instead of kinetin. There were two rather than three replicates for each dark experiment. Excised leaves of 12—day-old bean plants were used. . In l I 1 III. l.lll|lll|‘|..l i tilt I .411' I I III. I‘ .‘II 1.. , fl 49 light-mediated metabolic absorption, while they may stimulate a light- iiidependent metabolic uptake. This explanation seems most unlikely, however, because N6-BA is a respiratory inhibitor (34, 35, 128) which may reduce the energy supply for an absorption process. A possible alternative explanation has been offered by Dedolph (33), based on unpublished data. The slight inhibitory effect of phytokinins on photosynthesis may cause a slight reduction of ion uptake in the light, while an inhibitory effect of phytokinins on the dephosphorylation of ATP may, in turn, increase ion absorption in the dark. Further studies of the effects of metabolic inhibitors were directed toward correlations between foliar absorption and nucleic acid synthesis. 5-Fluorouracil (5-FU) a specific inhibitor of nucleic acid synthesis (8, 14, 60) was utilized. The study was initiated with the concept that a ribonucleo- protein (113) or a ribonucleic acid (52) in the membrane might be involved in ion absorption, transport, or accumulation. The effects of 5—FU on "specific absorption" of phosphate and Rb by bean leaves is summarized in Tables 8 and 9, respectively. It was striking to note that 5-FU tended to reduce Rb absorption in the light and decreased highly significantly in the dark, while it tended to increase phos- phate absorption. The differential responses of phosphate and Rh uptake as related to the presence of 5-FU may be explained by differences in specifi- city of phosphate and Rb in some phase of the absorptive'mechanism. It is TABLE 8. --Effect of 5-fluorouracil (5—FU) on Absorption of Phosphate by Excised Leaves. Molar Concentration "Specific Absorption" of Phosphate of 5- EU (mumoles/cmz Leaf x 24 Hours) 0 (Control) 15. 2 5 x 10‘3 24. 0 t value 1. 64 t. 95 2. 13 t. 90 1. 53 Conditions as in Table 3, except 5—FU instead of chloramphenicol. TABLE 9. --Effect of 5-fluorouracil (5-FU)onAbsorption of Rb by Intact Leaves. Molar Concentration "Specific Absorption" of. Rb of S-FU Light (500 ft-c) Dark (mumoles/cm7 Leaf x 24 Hours) 0 (Control 189 82. 4 _ —3 5 x 10 181 60. 9 I value . 96 2. 40 t 2. 13 . 95 C t. 80 . )4 Conditions as in Table 4, except 5-FU instead of chloramphenicol, intact 9-day—old bean plants, and three replicates. proposed that carriers are necessary for both phosphate and Rb absorption and that these carriers are different, or, at least, they are synthesized through different pathways or from different genetic information. The above results were re—evaluated using a ribonuclease (RNase). "Specific absorption" of phosphate and Rb by bean leaves as modified by RNase are summarized in Table 10 and 11, respectively. Rb absorption was significantly decreased by the presence of RNase at 100 ppm, while phosphate uptake was increased at a pH of 5. 7, and tended to be increased at pH 7. 3. Two-way analysis of variance of the data, assuming that the buffer effect wasonot significant, showed no significant pH effect, and there was no significant interaction of treatment with pH. The results from RNase corroborated the apparent specificity of phosphate and Rh uptake as sug- gested by the use of 5—FU. One must conclude that the consistent responses obtained with metabolic inhibitors on foliar absorption of phosphate and Rb by bean leaves suggest active uptake mechanisms. Accumulation Against a Concentration Gradient - - Movement against a concentration gradient has been suggested as a criterion for active foliar absorption (1, 73, 129). Accordingly, the accumulation of Rb, Ca, and phos- phate in bean and Chrysanthemum leaves was examined. The results are tabulated in Tables 12 and 13, respectively. Accumu- lation was expressed in terms of a concentration factor; that is, the ratio of the amount of the ion absorbed per unit fresh weight (g) of leaves to the UI 1v TABLE 10. --Effect of Ribonuclease (RNase) on Foliar Absorption of Phosphate. "Specific Absorption" of Phosphate RNase _ pH 5. 7 pH 7. 3 (Ppm) (mumoles/cm2 Leaf x 24 Hours) 0 Control 18. 3 20. 4 100 26. 5 26. 7 I value 4. 02 1. 59 t. 95 2. 13 t 90 1. 53 Conditions as in Table 3, except RNase instead of chlorampheni- col. Seventy-five umoles of tris-Cl at pH 7. 5 added to adjust to pH 7. 3, and tris-phthalate at pH 5. 7 and u = 10-3M for pH 5. 7 adjustment, and intact 12-day-old bean plants used. RNase (five times crystallyzed) obtained from Nutritional Biochemicals Corporation, Cleveland, Ohio. TABLE 11. ”Effect of Ribonulease (RNase) on Foliar Absorption of Rh. RNase "Specific Absorption" of Rb-l/ (Ppm) (m umoles/cm2 Leaf x 24 Hours) 0 (Control) 182H 1 166“ 10 169” 100 1408 1/ “ Means with different superscript significantly different at odds of 19:1 (Duncan's multiple range test). Absorption bg primary leaves of intact lZ-day-old bean plants and from 0. 4 uc Rb8 per 13. 3 umoles RbCl, 40 umoles tris-Cl at pH 7. 5, and Tween-20 at O. 01% in 40 ml RNase solution. Plants were exposed to 500 ft-c. of light and to 24° to 25° C. Each value a mean of three replicates. 1/ TABLE 12. --Accumulation of Ions in the Intact Primary Leaves of Bean Plants.— External Solution Leaf Tissue Concentration 10“ pH Cone. (A) Conc. (B) Factor (B/A) (umole/ml) (umole/g. Fr. Wt.) Rb 7.4 0.33 3. 62 10. 9 Ca 7. 4 0. 33 2. 24 6. 7 Phosphate (H2P04) 3. 4 0. 33 0. 30 0. 9 1/ _ Uptake from 40 ml 0. 01% Tween-20 containing the following: for Rb 0. 4 u_c Rbsb, 13.3 umoles RbCl, and 40 umoles trisCl at pH 7. 5; for Ca 2 uc C214”, 13. 3 umoles CaCl , and 40 umoles tris-Cl at pH 7. 5; for phosphate 2 uc P32, 13. 3 umoles H3PO4. Plants (17-day-old) exposed to 500 ft-C. of light and to 24. 5° to 25. 5° C. for 24 hours. Each value a mean of four replicates. TABLE 13. --Accumulation of Ions in Excised Leaves of the Chrysanthemum. Ion External Solution Leaf Tissue Concentration pH Conc. (A) Conc. (B) Factor (B/A) (umole/ml) (umole/g. Fr. Wt.) Rh 7. 4 0. 33 4. 73 14. 2 Ca 7. 4 0. 33 2. 55 7. 7 Phosphate (HZPO4) 3. 4 0. 33 0. 44 l. 3 Conditions as in Table 12, except excised leaves from the top 4 inches of Chrysanthemum plants were used. III“ C: .1 ixv‘lll 1| ' [i l I. 'il'lllu i- i ‘. Ul UI amount of that ion contained in a unit volume (ml) of external solution. The density of the leaf tissue was considered equal to the external solution. The concentration factors for Rb, Ca and phosphate in bean leaves were 10. 9, 6. 7 and 0. 9, respectively, while those for Chrysanthemum leaves were 14. 2, 7. 7 and 1. 3, respectively. All ions, except phosphate in bean leaves, were accumulated against a concentration gradient. Consequently, it is suggested that foliar absorption of Rb, Ca, and phosphate may be achieved only through an expenditure of metabolically derived energy. It should, however, be pointed out that accumulation of ions against a concentration gradient may not always be indicative of metabolic absorption. Accumulation along a electrochemical potential gradient, for example, is not evidence for active transport (32). Irreversibility of Absorption - - It has been suggested by Arisz and Sol (3), Kylin (73), and Wittwer and Teubner (129), that irreversibility could be used as decisive criterion for metabolic uptake through plant fol- iage. Hence, reversibility of phosphate and Rb absorption by bean leaves was evaluated. The results are summarized in Tables 14 and 15, respectively. The exchangeable fraction of absorbed phosphate was obtained by measuring that amount recovered in four successive washings with 50 ml each of equimolar phosphate solution for l, 5, 25 and 125 minutes, respectively. The total exchange period was 156 minutes. Treatment of the leaf for exchangeable. 0 TABLE 14. "Reversibility of Phosphate Uptake by Primary Leaves of Bean Plants._1,/ Component Percent of Total Absorbed Total absorbed 100. 0 Non-exchangeable 96. 2 Exchangeable 3. 8 Total absorbed 100. 0 Non-exodiffusible 97. 7 Exodiffusible 2. 3 1/ _ Phosphate uptake from 1. 7 ac P32 Per 13. 3 umoles H P0 in 40 ml 0. 0192 Tween-20. Plants were exposed to 350 ft-c. of light and to423° to 24° C. for 24 hours. Intact 11-day-old plants were used. Exchange and exodiffu— sion. processes occurred in equimolar phosphate solution and distilled water, respectively, for 156 minutes, immediately followed the harvest of the treated leaf by the leaf washing technique. Each value a mean of three replicates. TABLE 15. "Reversibility of Rb Absorption by Primary Leaves of Bean Plants... Component Percent of Total Absorbed Total absorbed 100. 0 Non-exchangeable 99. 2 Exchangeable 0. 8 1/ so . - Rb uptake from 0, 3 uc Rb per 40 umoles RbCl in 40 ml 0. 01% Tween-20. Plants exposed to 350 ft-c. of light and to 23° to 25° C. for 24 hours. Intact 16-day-old plants were used. Exchange deter- mined by washing with three successive 50 ml portions of equimolar RbCl solution for 15 seconds each. Each value a mean of three repli— cates. J J! phosphorus was initiated after a 24-hour absorption period and the usual washing procedure. Exodiffusion determinations were with distilled water. Non-exchangeable and non-exodiffusible amounts were obtained by differ- ence. Similarly, the exchangeable component of Rb absorption was deter- mined by washing with three successive 50 ml portions of equimolar RbCl for 15 seconds each. The results indicate that foliar absorption of phos- phate and Rb is essentially irreversible. This is shown by the exchange and exodiffusion values for phosphate, and by the exchange values for Rb absorption. Thus, phosphate and Rh absorption by bean leaves cannot be attributed to a diffusion process of the ordinary type, but to an active uptake mechanism that is highly irreversible. Response to Aeration - - Oxygen dependence has also been used as a criterion for active foliar absorption (1, 129). The effect of aeration on phosphate absorption by the immersed leaves was accordingly studied. The results are summarized in Table 16. A 52 percent increase in " specific absorption" of phosphate occurred with aeration. It was, how— ever, difficult to identify the true nature of the effect. Aeration introduced three variables; namely, agitation, oxygen, and carbon dioxide. Agitation may not have been significant if a rapid binding of ions to the leaf surface occurred. Dainty (32), however, has suggested that diffusion through an unstirred layer of 100 u thick from an absorbing surface may be rate limit— ing. As indicated in the Review of Literature section, oxygen dependence for . 1/ TABLE 16. - -Effect of Aeration on Foliar Absorption of Phosphate.— Aeration "Specific Absorption" of Phosphate Increase 7 g ’ (mumoles/cm“ Leaf x 24 Hours) 9;, None 7. 1 With aeration 9/ (ca. 8 ml/min.): 10.8 52.3 t value 1. 64 t. 95 2.13 t. 90 1. 53 1/ _ Phosphate uptake from 3. 14 ac P32 per 40 umoles H PO in 120 ml deionized distilled water. Plants exposed to 350 ft-c. of light and to 20. 5° to 21. 5" C. Excised leaves from 15-day-old plants were used. Each value a mean of three replicates. 2/ The flow rate measured with a Matheson flowmeter No. 204. 60 foliar absorption of mineral nutrients may be a reality but not easily sub— ject to an objective evaluation. Therefore, the supply of carbon dioxide may have been a contributing factor through its effects on photosynthesis. A positive response of foliar absorption of phosphate to aeration should be an index of metabolic uptake. pH Response - - Wittwer and Teubner (129) suggested a possible pH dependence for active foliar absorption. No one has, however, correlated pH data with metabolic uptake through the leaves of plants. Accordingly, the effects of pH on phosphate absorption by bean leaves were evaluated using two different buffer systems of the same ionic strength. The results are presented in Tables 17 and 18. With both the tris- phthalate (Table 17) and the sodium-acetate (Table 18) buffer systems, the valley of the pH curve, within the range tested, tended to occur invariably at 4. 7. These results are very similar to those reported by Teubner _c_t__a_l_. (114) and by Yamada (132). Teubner £t_a_l_. (114) have pointed out that the consistent depression of phosphate absorption in bean leaves at pH 4 could not be explained by a hypothesis of penetration of undissociated molecules of H3PO4 at low pH levels, or by an exchange reactidn involving only H2PO4 or HPO4. Orgell (94) has proposed that at a pH of less than 5, the cuticle is not charged, while at a pH above 7, the cuticle is negatively charged. Thus, the pH sensitive step for phosphate uptake may not be located in the cuticle. Therefore, it may be proposed that the pH effect relates to the 61 TABLE 17. --Effect of pH on Foliar Absorption of Phosphate Utilizing Tris— phthalate Buffer System. pH "Specific Absorption" of Phosphate t Valuesi/ t (mumoles/cm‘Z Leaf x 24 Hours) 3. 7 14.4 4. 7 8.0 4. 35 _ 2. 35 5. 7 9. 0 2. 43 l/ - The t value when compared to the result at pH 3. 7. Absorption by primary leaves of intact l4-day-old bean plants and from 32 . ' . . . . -2 2 uc P per 13. 3 umoles H3PO in 40 ml 0. 01% Tween-20 containing 10 M sucrose buffered to the designated pH. Plants exposed to 500 ft-c. of light and to 24° to 25° C. Ionic strength (11) of each buffer solution u 2: 10—3M. Each value a mean of three replicates, except two for pH 3. 7. TABLE 18. --Effect of pH on Foliar Absorption of Phosphate Utilizing the Sodium Acetate Buffer System. PH "Specific Absorption" of Phosphate t Valuei/ (mumoles/cm2 Leaf x 24 Hours) 3. 6 2. 79 4. 7 1. 50 1. 84 3 7 2. 25 73 l/ - The t value when compared to the result at pH 3. 6, where t 93 : 1. 94, t. £)[) ’4' 1. 4’4, 1.. 70 1: 0 DD. - ,) Conditions as in Table 17, except sucrose at 2 x 10 “M, sodium acetate instead of tris-phthalate, four replicates each, and lB-day-old intact bean plants used. 62 probable active centers of a hypothetical phosphate carrier, assuming that the carrier is enzymatic in nature. Finally, it is suggested that either aspartic acid, glutamic acid, or histidine may be a probable active site for the theoretical phosphate carrier. They all have a pKl of 2. 85 to 3. 16 and pK2 of 5. 20 to 5. 85 in an aqueous ethanol solution (58). The pK values in aqueous ethanol were preferred to those in aqueous solution, because the former condition may be closer to that actually occurring. 63 Kinetics of Foliar Absorption Determinations of Reaction Orders, Absorption Rate Constants (k), Times for 503‘; Absorption (T ), and Activation Energies (E ) - - Re- a 1/2 action orders for phosphate and Rb uptake by bean leaves were determined by the differential method (45). The results are shown in Figure 9 A and B. The significance of these findings may be recognized by the definition of the order of reaction. It is, from the standpoint of quantitative consideration of reaction rates, "the number of atoms or molecules whose concentra- tions (or pressures) determine the rate of the reaction" (45). Since the reaction orders for both phosphate and Rb absorption were first order, these data were used in calculating the reaction rate constants or the ab- sorption rate constants (k), and the times for 50% absorption (Tl/2) per unit area (cmz) from a unit volume (ml) of external solution at different concentrations. The equations for a first order reaction and correspond- ing half times (45) were used. The calculations are summarized in Table 19. As the external concentration decreases, k increases, while T1/2 decreases. It was apparent, however, that at external concentrations around 10—4M, these values tended to remain fairly constant. This may imply that con— centrations of about 10-4M are best for foliar absorption studies. Obviously, these constants could serve as indices or expressions of rates of foliar absorption. Figure 9 A. Upper left: Phosphate uptake by primary leaves of intact 32 (r, . l4-day-old bean plants and from 2 uc P per 40 ml 0. 01/0 Tween—20 containing phosphate of different concentrations and tris-phthalate at _ -3 _ . . pH 3. 6 and u : 2. 25 x 10 M. Plants exposed to 500 tt-c. of light and to 24° to 25°C. Each point a mean of three replicates. The reaction order for phosphate uptake determined by the differential method. B. Upper right: Rb uptake by primary leaves of intact 13- day-old bean plants and from 0. 4 tie Rb86 per 40 ml 0. 01% Tween-20 containing RbCl of different concentrations and 12. 5 umoles tris-Cl at pH 7. 5. Plants exposed to 500 ft-c. of light and to 24° to 25°C. Each point a mean of three replicates. The reaction order for Rb uptake de- termined by the differential method. C. Lower left: Phosphate uptake by bean leaves and deter- minations of Km and Vmax. Conditions as in (A). D. Lower right: Rb uptake by bean leaves and determinations of Km and Vmax. Conditions as in (B). 6% '5 A Reaction order Ier m eplebe by primary 3 1.00 _ leeeee el been elenle o e. 3 Q E U o 0 m ,.0 - . A.” ’ -) . :9 Autumn . —,r 0 g .. § 0 0.0 3 3 v 1 l A L ‘3 30.0000}! 7: Wanna ‘5‘” . 3 (' e: ) g 0030‘ f‘K s, -.so #- AleflNflQfl-LO is t \'.. 4.00 b c. Determination of Km end Vmee e! upm- by primary teem e! been plenle " emu. ene leek“ (eeeflee L . .."_"'_.'_., .4. 1 he (II VI- b M v: veleeityetmh nun- (nmelee mew/au- Del-GNU!) Kn:lhneeuem Vele- : "(WM “We! m _, eeIeIe (I): m (manor) Sui " l I T -'—-0- .000 ~ .°. Kea- Le? Wine: 01.0"0" nus/um é . m uo' mu- - no a m" m fim'lanl led-Cleve 0 I.“ "10‘ I’m Isl-0;"! led-w , . V"! j .e 3 e so I '7 ('1': ON“ 0 :7. )(Rele el Rb’uplebe in My”. Rb'kni'leeldbwre) m(- 0. Reaction order '0' Rb' eenbe by printer) Ieevee e! been plenle 0.]... ‘ Alas-i? ' 49m: "LL 3.0 0 0.7! 7 1 A mien-ac l l A 1 1 l A I 2 3 4 9 0 '09 (Rb’l‘mcenttoflon in «umoles/ml) 3|”. - end- ”to." a Delennlneflen e! Km end We: of Rb’ uptake by primary Ieevee of been plan" “the.“ OM w (it... C In I I - ._.—.— e h “I emblem”! he.“ (We. Brushed-Sheen Mum been.“ haze-em velocity“ "me-e «meme-a (mt...) o'. “a 0. melee/liter «- |0”~leelcuev —". 0&5 “0' .‘oh . L, I '0'. am h’len' bet-Shun ' 30 0” he.” Id-Ieu I A C TABLE 19. --Absorption Rate Constants (k) and Times Required for 5057:. Absorption (T ) for Phosphate and Rb Uptake by Primary Leaves of Bean Plants 5 Influenced by Various Concentrations in the Exter- nal Solution. External Solution I on Concentration K Tl/z (mumoles/ml) (per hour) (hour) 1 -4 3 3. 33 x 10 6. 49 x 10 1.03 x10 ,3 2 _ _ -4 3 Phosphate 1. 67 x 10 5. 64 x 10 1. 23 x 10 (H )PO ) .. 4 ' — 3. 33 x 102 5. 64 x 10 4 1.23 x 103 _ _ _ _ _ _ ._ _ _ _ 3973.193 ______ £8.95 .191. _ 5333203 _ _ 1.00 x 102 8.42 x 10‘3 0. 82 x10‘2 now 1.00 x 103 6.36 x10"3 1. 09 x 102 1.00 x .104 2. 98 x 10‘3 2.33 x 102 ’1‘ Conditions as in Figure 15. ** Conditions as in Figure 16. Calculations: Since the absorption processes of phosphate and Rb were found to be reactions of the first order (Figures 15 and 16), the following should be true: (So) 2. 303 10g -—-—- : kt (st) (S ) or k : 2‘303 log ___9__.. 1 (St) where (SO) : initial concentration of nutrient in medium. (St) : concentration at time t of external solution. and T 24 .0. -_ 693 9 l/.. k 6 6 A significant use of absorption rate constants is the calculation of the activation energy (Ea) for foliar absorption, where temperature dependence data are available. This is apparent from the Arrhenius equation (45) given in Table 20. The calculations show that the value of Ea for phos- phate and Rb absorption by bean leaves were 10. 4 and 8. 4 Kcal/mole, respectively. Since Ea is greater for phosphate absorption than for Rb uptake, phosphate absorption should be more temperature dependent. It is of great interest to note that the magnitude of the values of Ea reported herein are of the same order as that obtained from the hydroly— sis of ATP (7 Kcal/mole). Since the activation energy for foliar absorp— tion is that amount of energy which has to be expended to absorb one mole of nutrient, it is proposed that at least one mole of ATP is needed for absorption of one mole of either phosphate or Rb by bean leaves. Determinations of Michaelis Constants (Km), Maximum Velocities (Vmax), "Potential Carrier Concentrations", and the Theoretical Molecular Activities of Carrier - - Epstein and Hagen (39) first employed classical enzyme kinetics of the Michaelis type (86) for studying mechanisms of nutrient uptake by plant roots. Enzyme kinetics also offer potential appli- cability for resolving mechanisms of foliar absorption of mineral nutrients. Therefore, the absorption systems for phosphate and Rh in bean leaves were characterized in terms of kinetic constants. 67 TABLE 20. ——Activation Energies for Phosphate and Rb Uptake by Primary Leaves of Bean Plants. M Activation Energy Ion Calculation 1* Calculation 11M (Kcal/mole) Phosphate . (“21304) 10. 4 10. 4 Rb 8. 3 8. 5 Arrhenius equation: -E RT K : A e 'd/ where k: rate constant of absorption process A: frequency factor Ea: activation energy T: absolute temperature e: base of natural logarithm R: gas constant Calculation I: the estimation of k based upon the slopes of linear re- gression lines in time courses of absorption at 5° and 25° C. (Figure 8A and B). * Calculation II: the estimation of k based upon the measurements at 12 hours and at 5° and 25° C. (Figures 8 A and B), and the first order kinetic equation given in Table 19. The results are summarized in Figure 9 C and D, respectively. A linear transformation of Michaelis type kinetic: according to Lineweaver and Burk (77) was employed in determining apparent Michaelis constant (Km) and the theoretical maximum velocity of absorption (Vmax). The values for Km and Vmax determined after this manner were 1. 67 x 10'3 moles/liter, and l. 33 mumoles/cm2 leaf x hour, respectively, for phosphate absorption; and 4 x 10‘3 moles/liter, and 30 mumoles/cm2 leaf x hour, respectively, for Rb absorption by bean leaves. The rather high Km values reported herein may suggest that the affinity between the nutrient and the carrier may be low and a high nutrient concentration may be necessary to obtain Vmax for both phosphate and Rb absorption by bean leaves. An attempt was made to calculate the "potential carrier concentration" assuming that the reaction order with respect to carrier was first order, and that the absorption process followed the Michaelis type enzyme kinetics. The calculated values for the 'potential carrier concentration” using the ,. _ . _ . . , -10 2 equations detailed in Table 21 were 1. 28 x 10 moles/cm leaf, or 9. 41 x ’6 ,7. . 7 ‘9 ,.2 ,~ . -5 10 moles/Kg fresh weight. and 1. 3- x 10 moles/cm leaf, oi 9. 70 x 10 moles/Kg fresh weight for the phosphate and Rb carrier, respectively. The values for the "potential carrier concentration" thus obtained may be compared with those reported in literature. The concentration of .- ‘ - phosphate carrier in barley roots has been estimated at 10 E"M (49, 50) and ‘4 .— . . - - t r 7 x 10 M (75). The potassnim carrier concentration in yeast was 0. 12 meq/lxg yeast (28). 69 TABLE 21. -- Equations for Calculation of the "Potential Carrier Concentra- tion". 1. A kinetic equation for a carrier mediated absorption. s, : K (80)1 (R)1 = ks k1. (so)l (R)1 where Si : the amount of nutrient absorbed (So): the initial concentration of the external solution (R) : the carrier concentration K : the overall reaction rate constant k . : the rate constant contributed by nutrient (at 10‘4 to 1073M) kr : the rate constant contributed by carrier 2. A conservation equation for the carrier. Rt: R+SR where Rt is total carrier; R, free carrier; SR, a nutrient-carrier complex. 3. The relationship between Vmax and Rt' Vmax : k R or R : Vmax 2 t t k. 2 where k2 is a rate constant in the following k .__.. _.1. ,_-__-.._. k9 So + R .-.-.._-_......., SR» ...... .- 181 + R' k-i assuming k2 '5 kr’ then Rt 2 Vmax kr where kr = K/kS K can be estimated from the anti-logarithm of the ordinate intercept of a linear plot of logarithm of initial rate of absorption vs logarithm of ex- ternal concentration (Figure 9 A and B). kS can be estimated from the first order kinetic equation (Table 19). 70 A sophisticated speculation would be an estimation of the molecular activity (traditionally called turnover number) of the carrier. Assuming that the calculated values for the "potential carrier concentration were the first approximations of true values, the theoretical molecular activity of phosphate and Rb carriers would be 10. 4 mumoles phosphate/mumole‘ carrier x hour, or one molecule phosphate/ one molecule carrier x 5. 8 minutes; and 22. 7 mumoles Rb/mumole carrier x hour, or one molecule Rb/ one molecule carrier x 2. 6 minutes, respectively. These values were obtained by divi— sion of the theoretical maximum velocity by the "potential carrier concen— tration". It should be pointed out that these values are crude approxima- tions at best. Verification is needed. The most convincing evidence for the existence of carriers would be a direct isolation and subsequent identi- fication and characterization. SUMMARY A new technique (leaf immersion and washing) for assessing the rate of foliar absorption of mineral nutrients, along with a precise mathe- 9 matical index designated as 'specific absorption" (quantity absorbed per unit area in unit time from an external solution of constant concentration) for its expression are described. The use of these two new approaches in measuring uptake of phos— phate and rubidium by primary leaves of bean plants has established that foliar absorption beginning with zero time, and extending over a 24—hour period, is a metabolic process, subject to temperature, energy sources, and aeration. Metabolic inhibitors, such as 2, 4—dinitrophenol (DNP) and chloramphenicol, significantly reduced absorption. Exogenously applied adenosine triphosphate (ATP), however, had little effect on rubidium up- take. Accumulation of rubidium and calcium against a concentration gradi- ent was noted. This was also true with phosphate in Chrysanthemum leaves, but not in bean leaves. Phosphate and rubidium uptake were essentially irreversible. Kinetin and N6-benzyladenine tended to enhance phosphate and rubidium uptake in the dark, but reduce absorption in the light. Ribonuclease (RNase) and 5-f1uorouracil (5-FU) reduced rubidium uptake, but increased that of phosphate. It is proposed that the phosphate and rubidium carriers are 71 I proteinaceous substances because absorption was inhibited in the presence of chloramphenicol. The specificities of these carriers were demonstrated by the differential response of phosphate and rubidium absorption to RNase and 5-FU. .The use of classic enzyme kinetics revealed the following as to phosphate and rubidium absorption by bean leaves. The reaction order for phosphate absorption was first order; binding affinity in terms of apparent Michaelis constant (Km) 1. 67 x 10’3moles/liter; maximum velocity (Vmax), 1. 33 mumoles/cm2 leaf x hour; absorption rate constant (k), 6. 49 to 4. 89 x 10‘4/hour in the range of external concentrations of H PO4 of 3. 33 X 10‘5 1:0 3 l. 67 x 10—3M; time for 50% absorption (TI/2) per cm?‘ leaf from 1 ml of . . . -3 external solution in the above concentration range, I. 07 to 1. 43 x 10 hours: activation energy (Ea)’ 10. 4 Kcal/mole; "potential carrier concentration", -10 2 -6 , - . . 1. 28 x 10 moles/cm or 9. 41 x 10 moles/Kg fresh weight; the theoretical molecular activity (turnover number) of phosphate carrier, 10. 4 mumoles phosphate/mumole carrier x hour, or one molecule phosphate/ one molecule carrier x 5. 8 minutes. The reaction order for rubidium uptake was first order: Km, 4. 0 x 10‘3 moles/liter; Vmax, 30 mumoles/cm2 leaf x hour; k, 8. 42 to 2. 98 x 2 10—3/hour in the range of external concentration of RbCl of 10‘4 to 10‘ M: T1/"' 0. 82 to 2. 33 x 10‘2 hours in the above concentration range; E3, 8. 4 ‘I‘ 73 Kcal/mole; "potential carrier concentration", 1. 32 x 10'9 moles/cm2 leaf, or 9. 70 x 10.D moles/Kg fresh weight: the theoretical molecular activity of the rubidium carrier, 22. 7 mumoles rubidium/mumole carrier x hour, or one molecule rubidium/one molecule carrier x 2. 6 minutes. LITERATURE CITED 1. Arisz, W. H. 1952. Transport of organic compounds. Ann. Rev. Plant Physiol. 3: 109-130. 2. . 1958. Influence of inhibitors on the uptake and the trans- port of chloride ions in leaves of Vallisneria spiralis. Acta Botan. Neerl. 7: 1-32. 3. , and H. H. Sol. 1956. Influence of light and sucrose on the uptake and transport of chloride in Vallisneria leaves. Acta Botan. Neerl. 5: 218-246. 4. 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