FACTORS TNFLU'ENCING THE TONER 2 ” ,1}:.;j_.}:j3‘i7 PENETRATION 0F NAPHTHALENEABETIC ACID AND * NAPHTHA’LENEACETAMI‘DE INTO LEAVES OF PEAR f jlf (PYRUS COMM-UNIS L) w w - ~ . .,4~ . ' ,fi... u.“ -. '1 Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY DUANE WESLEY GREENE 7‘ 1969 .‘...'.L .. . Tl: .'..‘.. .un .‘ 1nd” 7— . v w T T. I L I b‘ H T R Y Michigan "35336 ' 1 University J This is to certify that the thesis entitled Factors Influencing the Foliar Penetration of Naphthaleneacetic Acid and Naphthaleneacetamide into Leaves of Pear (Pyrus communis L.) presented by Duane Wesley Greene has been accepted towards fulfillment of the requirements for Ph.D. degree in Horticulture ’ Ldflwg ('JzI-g’<_/ War professor Date July 24, 1969 ’ 0-169 ABSTRACT FACTORS INFLUENCING THE FOLIAR PENETRATION OF NAPHTHALENEACETIC ACID AND NAPHTHALENEACETAMIDE INTO LEAVES OF PEAR (PYRUS COMMUNIS L.) By Duane Wesley Greene Factors influencing the foliar penetration of naphthaleneacetic acid (NAA) and naphthaleneacetamide (NAAm) into pear leaf disks (21322 Communis L. Cv. Bartlett) were studied to determine the extent to which each factor influenced the amount entering the leaf. An improved meth- od of assessing foliar penetration is described. Small glass cylinders were attached to leaf disks with silicone rubber, placed in Petri dishes lined with filter paper moistened with water, and arranged in a water bath under a light bank. Factors affecting penetration could be more critically assessed because there was maximum control of experimental conditions. The penetration of NAA and NAAm through the upper surface was linear with time. There was rapid initial penetration through the lower surface followed by a reduced rate after 24 or 48 hours. Pene- tration of NAA applied in glass cylinders was similar to that applied as microdroplets, providing the droplets were prevented from drying out. Droplet drying resulted in increased penetration. Penetration was lin- ear with increasing concentration (10'6 to 10'3 M) except for the re- duced rate of NAA penetration through the lower surface at concentrations above 10'4 M. Increasing temperature (5-35 C) resulted in increased NAA or NAAm penetration. Temperature coefficients generally ranged between 1.51 to 5.46. Penetration of NAA and NAAm through the lower Duane Wesley Greene surface increased with increasing light intensity from 0 up to about 600 ft-c. Increased light intensity above this resulted in no addi- tional penetration. Light enhanced penetration through the upper sur- face could only be demonstrated where the cuticle did not greatly re- strict diffusion. The light effect was independent of stomatal opening. Inhibitors of the Hill reaction (Atrazine, Monuron, Terbicil) and oxi- dative phosphorylation (DNP, m~Cl-CCP, p-F-CCP), phenylmercuric acetate, and nitrogen all significantly reduced penetration of NAA. It was con- cluded that penetration into leaf disks is an active process requiring oxygen and products of photosynthesis. No surfactant studied increased NAA or NAAm penetration through the upper surface. At 0.01 and 0.1% concentration Tween 20 and Tergitol 15-8-9 increased NAA penetration through the lower surface. Tween 20, Triton B-l956, and Xe77 signifi- cantly increased NAAm penetration through the lower surface at a 0.1% concentration. X-77 was found to be the most effective. Surfactants increased NAAm penetration to a greater extent than for NAA. Stomatal penetration has been established under conditions where a surfactant in the treating solution sufficiently lowered (42 dynes/cmz) the surface tension. However, entry via stomata is not considered to be a major portal of entry. Penetration of NAA was always greater through the lower surface than through the upper surface. Uptake of NAAm was greater through the upper surface of young leaves but greater penetra- tion occurred through the lower surface after leaves had fully expanded. Penetration was greater in younger leaves than in older leaves with the exception of NAA penetration through the upper surface. Silver nitrate was shown to preferentially pass through the lower cuticle above veins. No preferential pathways of NAA through isolated upper pear leaf cuticle were observed. FACTORS INFLUENCING THE FOLIAR PENETRATION OF NAPHTHALENEACETIC ACID AND NAPHTHALENEACETAMIDE INTO LEAVES OF PEAR.(PYRUS COMMUNIS L.) By Duane Wesley Greene A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Horticulture 1969 M ACKNOWLEDGMENT The author wishes to express his sincere thanks and appreciation to Dr. M. J. Bukovac for counsel, assistance and guidance during the course of my graduate program. I am also indebted to Drs. D. R. Dilley, G. R. Hooper, A. L. Kenworthy, R. E. Monroe, and M. Zabik for service on the Guidance Committee. Gratefully acknowledged are suggestions by Dr. H. P. Rasmussen for the preparation of micro- radioautograms and assistance by Dr. M. M. Robertson in critically reviewing the manuscript. This investigation was supported in part by NIH training grant No. 5 T01 GM 012 63 from General Medical Sciences and by Public Health Service Grant CC 00246 from the National Communicable Disease Center, Atlanta, Georgia. For her assistance and encouragement and her aid in preparation of this dissertation, I express my sincere gratitude to my wife, Barbara. ii INTRODUCTION. . . . . . LITERATURE REVIEW . . . TABLE OF CONTENTS The-Cuticle: Structure, Waxes . . . Cutin . . . Pectin . . . Cellulose . Methods of Studying Types of Plant Methods of Application Method of Detection. Portals of Entry. Foliar Penetration. . . . Systems Used. Anticlinal Walls . Trichomes . Veins . . . Guard Cells and Accessory Cells Pores, Cracks and Fissures . Ectodesmata. Stomatal Penetration. 0 O O O 0 Factors Influencing Foliar Penetration. . . . Environmental Factors. Time-Course. Concentration. Temperature. pH . . . . . Light. . . . Surfactants. Physical and Chemical Properties 0 O 0 Surfactant Effects on Cuticular Penetration. Surfactant Effects on Stomatal Penetration . Active Uptake . . Naphthaneneacetic Acid and Naphthaleneacetamide . iii of a Surfactant O 0 Page to \JO‘O‘L‘ ooooxx 10 10 ll 11 12 12 15 15 l6 l7 17 18 19 20 20 21 22 22 24 MATERIAL AND METHODS. . . . . . . . . . . . . . . . . . . . Growing of Plants . . . . . . . . . . . . . . . . . . . Selection of a Method to Determine Foliar Penetration . (1) Leaf Disk-Planchet Counting. . . . . . . . . . (2) C02 Combustion - Scintillation Counting. . . . (3) Ethanol Extract - Scintillation Counting . . . (4) Leaf Disk - Scintillation Counting . . . . . . General Methods . . . . . . . . . . . . . . . . . . . . Pretreatment Time . . . . . . . . . . . . . . . . . . . Self Absorption . . . . . . . . . . . . . . . . . . . . Leaf Anatomy. . . . . . . . . . . . . . . . . . . . . . Time-Course of Penetration. . . . . . . . . . . . . . . Effect of Concentration on Penetration . . . . . . . . Effect of Temperature on Penetration. . . . . . . . . . Effect of pH on Penetration . . . . . . . . . . . . . . Effect of Light on Penetration. . . . . . . . . . . . . Effect of Light and Dark Treatment on Penetration. Effect of Inhibitors on Penetration . . . . . . . . . . Anaerobiosis . . . . . . . . . . . . . . . . . . . Inhibitors Applied in Solution . . . . . . . . . . Effect of Surfactants on Penetration. . . . . . . . . . Effect of Surfactants on Surface Tension . . . . . . . Stomataerenetration . . . . . . . . . . . . . . . . . Stomatal Aperture Width Measurements . . . . . . . Influence of Surfactants on Stomatal Penetration When Added Subsequent to Treating Solution Application . . . . . . . . . . . . . . . . . . iv Page 26 26 27 27 27 28 28 30 33 34 35 35 36 36 36 36 37 37 37 37 38 38 40 40 40 Effect of C02 Pretreatment on Stomatal Penetration. . Stomatal Penetration of Silver Nitrate . . . . . . Effect of Droplet Drying on Penetration . . . . . . . . Effect of Leaf Age on Penetration . . . . . . . . . . . Statistical Analysis. . . . . . . . . . . . . . . . . . Microradioautography. . . . . . . . . . . . . . . . . . RESULTS Structure of the Pear Leaf. . . . . . . . . . . . . . . Effect of Light and Dark Pretreatment on Penetration. . Self Absorption . . . . . . . . .p. . . . . . . . . . . Effect of Time on Penetration . . . . . . . . . . . . . Penetration Following Glass Cylinder Application . Penetration Following Microdroplet Application . . Effect of Concentration on Penetration. . . . . . . . . Effect of Temperature on Penetration . . . . . . . . . Effect of pH on Penetration . . . . . . . . . . . . . . Effect of pH on Penetration of NAAm. . . . . . . Effect of Light on Penetration. . . . . . . . . . . . Light Intensity. . . . . . . . . . . . . . . . . Effect of Leaf Age and Surface on NAA Penetration in Light and Dark . . . . . . . . . . .». . . . Effect of pH on Penetration in the Light and Dark. Effect of Cycling Light and Dark on Penetration. . ‘Effect of Light and Stomatal Aperture Width on Penetration . . . . . . . . . . . . . . . . . . Effect of Inhibitors on Penetration . . . . . . . . . . Effect of Inhibitors of the Hill Reaction on Penetration. Effect of Anaerobiosis on Penetration. . . . . . . Page 40 41 41 42 42 42 44 44 44 50 50 50 50 55 55 63 63 63 63 69 69 76 76 76 76 85 Stomatal Penetration. . . . . . . . . Effect of Increasing Light Intensity on Penetration and Opening of Stomata . . . Frequency Distribution of Stomatal Aperture Width in the Light . . . . . . . . Effect of Surfactants on Surface Tension . Effect of Tween 20 on Penetration with Stomata Either Open or Closed. . . . Effect of Vatsol 0T on Penetration . . . Effect of_Surfactants on Penetration of 'Treating Solution. . . . . . Effect of C02 Treatment on Penetration . Penetration of Silver Nitrate. . Effect of Leaf Age on Penetration . . Effect of Droplet Drying on Microradioautographic Study of 3H—NAA DISCUSSION. SUMMARY . . LITERATURE APPENDICES. ISOlatEd CUtiCle o o o o o o o o CITED. 0 0 O 0 o O 0 0 O O O O O O O O O O O I O O O O O O O 0 vi Penetration . . . pH 6.0 Penetration Through Page' 92 92 92 92 99 99 104 104 108 108 112 112 120 138 142 157 10. ll. 12. LIST OF TABLES Page Comparison of four methods for determining foliar penetratien. o o o o o o o o o o o o o o o o o o o o o I o 29 Inhibitors used in studying penetration of NAA by the lower surface of pear leaf disks . . . . . . . . . . . . 39 The effect of light and dark pretreatment on subsequent penetration of NAA through the lower surface of pear leaf diSkS in light 0 O O O O O O O O O O O O O O O O O O 47 Temperature coefficients (010) for penetration of NAA and NAAm into pear leaf disks through the upper and lower surfaces 0 O O O O 6 0 O O G O O O O O O O O O O O O O O 60 The effect of pH on penetration of NAAm into pear leaf disks through the upper and lower surfaces . . . . . . . 68 Effect of light on penetration of NAA through the upper and lower surfaces of disks taken from the first and seventh leaf of a terminal shoot . . . . . . . . . . . . 74 Effect of treating solution pH on penetration of NAA through the lower surface in the light and dark. . . . . 75 Penetration of NAA into pear leaf disks through the upper and lower surfaces in atmospheres of nitrogen and air 9 O O O O O O O O O 0 O O O O 0 O O O O 0 O O O 86 The effect of light and an inhibitor of the Hill reaction (Atrazine) on penetration of NAA into pear leaf disks through the lower surface. . . . . . . . . . . . . . . . 87 Effect of Hill reaction inhibitors on penetration of NAA into pear leaf disks through the lower surface and illuminated with 1000 ft-c of light. . . . . . . . . . . 87 The effect of inhibitors on penetration of NAA into pear leaf disks through the lower surface . . . . . . . . . . 88 The effect of inhibitors on penetration of NAA into pear leaf disks through the lower surface . . . . . . . . . . 88 vii Table Page 13. Effect of Tween 20 concentration on penetration of NAA into pear leaf disks through the upper and lower surfaces I O O O O I I O O O O O O I O O O O O O O O O O 90 14. Effect of Tergitol 15- 3-9 concentration on penetration of NAA into pear leaf disks through the upper and lower surfaces. . . . . . . . . . . . . . . . . . . . 90 15.- Effect of surfactants (0.1%) on penetration of NAAm into pear leaf disks through the upper and lower surfaces. . . . . . . . . . . . . . . . . . . . . . . . 91 16. Effect of Tween 20 surfactant on penetration of NAA into pear leaf disks through the lower surface with stomata open (-C02) and closed (+C02) . . . . . . . . . 107 17. Effect of X-77 surfactant on penetration of NAA into pear leaf disks through the lower surface with stomata open (-C02) and closed (+C02) . . . . . . . . . . . . . 107 18. Effect of Vatsol 0T surfactant on penetration of NAA into pear leaf disks through the lower surface with stomata open (-C02) and closed (+002) . . . . . . . . . . . . . 107 19. Effect of leaf age on penetration of NAA into pear leaf disks through the upper and lower surfaces. . . . . . . lll viii 10. 11. 12. 13. LIST OF FIGURES Schematic description of the method used in studying leaf disk penetration. . . . . . . . . . . . . . . . Photomicrographs illustrating the structure and upper and lower leaf surfaces. . . . . . . . . . . . . . . Influence of leaf disk weight on self absorption of 14C_NAA o I o o o o o o o o o o o o o o o o o o o o Time-course of penetration of NAA and NAAm through the upper and lower surfaces of pear leaf disks. . . . . Time-course of NAA penetration through the upper and lower surfaces of pear leaf disks following application in droplet form. . . . . . . . . . . . . Influence-of treating solution concentration on penetra— tion of NAA and NAAm through the upper and lower surfaces of pear leaf disks. . . . . . . . . . . . . Effect of temperature on penetration of NAA and NAAm through the upper and lower surfaces . . . . . . . . Time-course of penetration of NAA through the upper ' surface of pear leaf disks at 2, 17, and 27 C . . . Effect of treating solution pH on penetration of NAA into pear leaf disks through the upper surface and on the dissociation of NAA molecule. . . . The effect of treating solution pH on penetration of NAA into pear leaf disks through the lower surface . Effect of increasing light intensity on penetration of NAA into pear leaf disks through the upper and lower surfaces 0 O O O O O ‘0 O O O I O O l O O O O O O O 0 Effect of increasing light intensity on penetration of NAAm into pear leaf disks through the upper and lower surfaces . . . . . . . . . . . . . . . . . . . Time-course of NAA penetration into pear leaf disks through the lower surface as influenced by light and dark treatments. 0 o o o o e o o o o o o o '0 o 0 ix Page 32 46 49 52 54 57 59 62 65 67 71 73 78 Figure 140 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. Time-course of penetration of NAA into pear leaf disks through the lower surface as influenced by cycling light and dark. 0 O O 0 O O O O I O O l.‘ O O O C O 0 Effect of light and stomatal aperture width on penetra- tion on NAA into pear leaf disks through the lower surface 0 0 O 0 O O O O O O O O O O O O O O O O O Photomicrographs of acetate replicas showing stomata at various periods during the course of NAA pene- tration with different light regimes . . . . . . . Comparative effects of increasing light intensity on penetration of NAA into pear leaf disks through the lower surface and the per cent of stomata Open. Frequency distribution showing per cent stomata with a particular stomatal aperture width falling within a given size class. . . . . . . . . . . . . . . . . Effect of varying concentrations of selected surfac- tants on surface tension of buffered NAA solutions. Effect of Tween 20 on penetration of NAA into pear leaf disks through the lower surface with stomata open and closed. Stomata were opened initially by pretreatment with 1 1/2 hr of light . . . . . . . . Effect of Vatsol UT on penetration of NAA into pear leaf disks through the lower surface after 3 hr exposure to the light . . . . . . . .‘. . . . . . . Effect of Vatsol OT, X-77, and Tween 20 on penetration of a pH 6.0 NAA solution into pear leaf disks through the lower surface after 3 hr pretreatment in light. . . . . . . . . . . . . . . . . . . . .3. Transverse sections of leaf disks treated with silver nitrate for 4 minb O O O O O O O O O O O O O O O 0 Effect of leaf age on penetration of NAAm into pear leaf disks through the upper and lower surfaces . . Time-course showing the effect of drOplet drying on penetration of NAA and NAAm into pear leaf disks through the upper and lower surfaces. . . . . . . Microradioautograms illustrating the localization of 3H-NAA in isolated upper pear leaf cuticle . . . Page 80 82 84 94 96 98 101 103 106 110 114 116 118 LIST OF APPENDICES Table Page Al. Scintillation solvent mixture used for the selection of a detection method and for calibrating treating solutions. . . . . . . . . . . . . . . . . . . . . . . 157 A2. Standard deviation for penetration of NAA and NAAm into pear leaf disks through the upper and lower surfaces from different concentration treating solutions. . . . 158 A3. Chemical Name and Class, Source, and Trade Name of surfactants used . . . . . . . . . . . . . . .‘. . . . 159 xi INTRODUCTION The use.of.foliar applied plant growth regulators, pesticides and nutrients has increased exponentially in the past 25 years. Contributing factors have been a declining labor supply, increased cost of production, and a shift toward complete mechanization. Numerous contact, preemergence, general and selective herbicides have virtually eliminated the need of hand weed removal and have allowed weed control to an extent never before possible. New plant growth re- gulation compounds and uses are found regularly. Foliar applications of these compounds are used to effect flowering and fruiting, control plant growth, retard or accelerate fruit abscission, to increase market quality, and reduce labor to mention a few. There is a diverse nature of compounds, a wide spectrum of applica— tions and variety of climatic conditions under which these compounds are applied. Results are often not reproducible from year to year. Spray recommendations vary with location and time of application. Penetration information for a class of compounds for a particular set of conditions often is not applicable to other compounds and conditions. All aerial portions of a plant are covered by a cuticle. This cuticle is lipoidal in nature and thus may act as a sink for lipophilic compounds. Other foliar applied compounds may be absorbed, translocated and accumu— lated in.edible portions of the plant making spray residues a problem for present and future generations. These problems involving foliar penetration of compounds must be investigated to eliminate or at least alleviate health problems and difficulties involved in their use. In spite of the considerable amount of work devoted to the study of foliar penetration, large gaps still exist in our knowledge of how a com- pound in a spray droplet migrates from a position on the surface of a leaf to a place in the plant where it elicits a response. Many questions re- main to be answered concerning factors influencing foliar penetration and the exact pathway or pathways a compound follows on entering a leaf. Applications in early foliar penetration studies were made in the form of sprays or droplets. Changes in concentration, buffering capacity and area during.droplet drying and a lack of environmental control limited the kind of data that could be obtained. Precise methods of determining foliar penetration and pathways of foliar penetration are now available, especially through the use of radioactive compounds and improved methods of application. The leaf has been taken apart and its component parts studied; the isolated cuticle and isolated cells. Much information has been gathered in this manner, which has been an invaluable asset contributing to the body of knowledge of foliar pene— tration. However, until critical studies are carried out on an intact system under controlled conditions, voids will still exist in our know- ledge of the process of foliar penetration. LITERATURE REVIEW The ever increasing use of foliar applied plant growth regulators, pesticides, and nutrients has resulted in a large volume of literature pertinent to this subject. A number of reviews have appeared in recent years in an attempt to organize the existing body of information. Re- views.stressing foliar penetration of herbicides have been written by Blackman et a1. (1951), Crafts (1953), Crafts and Foy (1962),Currier and Dybing (1959), Foy (1964), and Woodford et a1. (1958). Information specifically involving foliar penetration of plant growth regulators has been discussed by Sargent (1965). Reviews by Boynton (1954), Jyung and Wittwer (1965), and Wittwer and Teubner (1959), dealt with penetration of nutrients. A more general discussion of foliar penetration of organic compounds has been presented by Franke (1967), Hull (1964), and Van Overbeek (1956). The Cuticle: Structure, Function, and Composition All plant surfaces exposed to the air are covered by a thin lipoidal membrane. the cuticle (Van Overbeek, 1956). This covering over the epider- mal cells is generally considered to be lamellar in form (Crafts and Foy, 1962), although demarcation between layers is often unclear (Martin, 1965). Waxes varying in form and composition are present on the surface of the cuticle (Juniper and Bradley, 1958; Eglinton and Hamilton, 1967). Cutin is the major constituent of the cuticle and is formed by polymerization of long-chain fatty acids and alcohols, (Crafts and Foy, 1962; Frey-wyssling and Muhlethaler, 1959). Highly oriented wax is embedded in the cutin ma- trix (Sitte and Rennier, 1963; Norris and Bukovac, 1968). Pectic substances and cellulose are interspersed with the cutin, in varying amounts, especially in the region where the cuticle merges into the underlying epidermal cell wall (Baker et al.,l962; Martin, 1965, 1966). Franke (1964), Norris and Bukovac (1968) and Roelofsen (1952) have presented schematic diagrams of the cuticle and the underlying epidermal cell wall. Eases Composition of the surface wax may be one of the important factors regulating penetration. Identification of waxes from a number of plants has revealed that they are composed of long-chain paraffins, alcohols, ketones, fatty acids, hydroxy fatty acids, and esters of the alcohols and acids (Martin, 1966; Purdy and Truter, 1963). The relative proportion of the various wax components may vary with species, age, and position within the cuticle (Baker et al., 1964; Kurtz, 1950; Silva-Fernandes et al., 1964). Chain length of the wax components can vary with the species (Eglinton and Hamilton, 1967) and the particular components (Martin, 1966). During expansion of the leaf there is continual extrusion and deposi- tion of surface waxes (Bystrom et al., 1968; Martin, 1960), however, de- position of these usually ceases with leaf expansion but there is a con— tinual deposition of cutin waxes (Schieferstein, 1957). The embedded waxes are usually deposited in lamellae or pockets deeply seated in the cuticle (Roelofsen, 1952; Meyer, 1938) or in lamellae near the cuticle surface (NOrris and Bukovac, 1968). Conflicting reports appear in the literatuneconcerning the existence of pores, which could allow wax or wax precursors to migrate from the epidermal cells through the cuticle to the surface. Schieferstein and Loomis (1956) and Juniper (1960) were unable to show wax canals in the cuticle. Using a modified gold-palladium replica method, Hallum (1964) confirmed these findings. It was suggested that wax migrates between the cuticular lamellae to the surface of the leaf rather than through pores extending directly from the epidermal cell wall to the exterior. Hall and Donaldson (1962) reported wax channels were beneath each wax platelet in Trifolium_repens and Brassica oleracea. Using a modified freeze-etch tech- nique, Hall (1967) was able to confirm these observations in the case of Trifolium repens. The significance of wax pores as possible pathways of penetration for nonpolar compounds has been pointed out by Mitchell et a1. (1960). However, until a technique is developed providing control of experimental variables on a number of species, the existence of wax canals will likely remain a moot question (Hull, 1964). Before entering the plant a spray droplet must come in contact with the cuticle. The chemical groups exposed and the physical configuration of the surface wax influence the degree with which a droplet can come in contact with the cuticle (Fogg, 1948; Silva-Fernandes, 1965; Holly, 1964). Silva-Fernandes (1955) considered the physical configuration of the waxes to be most influential in allowing a spray droplet to come in contact with the surface. Holly (1964) has pointed out that droplets may balance on wax projections and dry out without coming into contact with the cuticle proper. Hall and Jones (1961) have shown that surface wax removal by brushing increases wettability and cuticular transpiration. Increased absorption fo 3-chlorophenoxy-a-propionic acid by peach leaves following brushing has been demonstrated (Bukovac, 1965). Adam (1948, 1958) explored the effect of functional groups on contact angles. Water on paraffin waxes can give a contact angle of 105-1100 because only methyl groups are exposed at the surface. Polyethylene has a smaller contact angle, 94°, because more hydrophilic-CH2 groups are exposed. are Cutin constitutes the structural framework of the cuticle. Procutin has been shown by Frey-Wyssling and Muhlethaler )1959) and Muhlethaler (1961) to migrate through the epidermal wall to the cellulose-cutin inter— face in submicroscopic droplets. Hydrolysis of cutin has shown these cutin- precursors to be fatty acids and hydroxylated fatty acids in the approximate range of C12 to C22 length carbon chains (Baker et al., 1964). Matic (1956) and Baker and Martin (1963) have found the major cutin constituents in Agave americana and cutin from a number of fruits and leaves to be hydroxy- octadecanoic acids containing from one to three hydroxyl groups in the car- bon chain. The lipophilic hydrocarbon chains are oriented toward the out- side and the carbonyl groups oriented toward the more hydrophilic epidermal cell wall (Foy, 1964; Frey-Wyssling, 1953). Polymerization occurs at the surface by esterification of the hydroxyl and carboxyl groups (Matic, 1956; Martin, 1966). These groups impart hydrophilic properties and -CH2 and -CH3 groups give lipophilic properties to the cuticle. Oxidation of cutin at the surface makes the outer surface more lipophilic (Hull, 1964). Cutin is nega- tively charged when the pH of an applied solution is above the pK for dissociable carboxyl groups present (Crafts and Foy, 1962). Pectin Separating the cuticle from the cell wall is a layer of pectic sub- stances, composed of long—chain polygalacturonic acid molecules having side carboxyl groups (Foy, 1964). Pectins are highly hydrophilic, thus, their potential importance as a pathway for the entrance of water soluble com- pounds must be recognized (Crafts, 1964). Roberts et a1. (1948) have ex— amined in cross-section leaves of Malus domestica and have shown a continuous path of pectic substances between the surface of the leaf and the vein extensions or bundle sheaths. Norris and Bukovac (196 ) found no evidence of the extension of pectic substances to the surface in the pear leaf (Pyrus communisln)o Cellulose Cellulose, a£31,4-g1ucose polymer, is a fourth major component of the cutucle.' It is arranged in micelles, about 10 A0 apart allowing penetration of water and halogens (Frey-wyssling, 1953). Micelles are further organized into larger units, microfibrils. The space between these .13 about 100 A°, large enough to allow penetration of larger molecules such as dyes (Frey-Wyssling, 1953). Like pectins, cellulose fishydrOphilic in nature and is no obstacle to the penetration of water soluble compounds (Foy, 1964). Methods of StudyigggFoliar Penetration Types of Plant Systems Used Various approaches have been taken in the study of foliar pene- tration. Among these are: the whole plant (Allen, 1964; Hauser,1955), a leaf attached to the intact plant (Allgren and Sudia, 1967; Jyung and Wittwer, 1964), excised leaves (Kamimura and Goodman, 1964; Luckwill and Lloyd-Jones, 1962), leaf disks (Sargent and Blackman, 1962, 1965), isolated cuticle (Yamada et al., 1964, 1965), isolated cells (Jacoby and Dagan, 1967; Jyung et al., 1965) and leaf strips (Rains, 1967; Smith and Epstein, 1964). Information gained using these methods is invaluable in determining the relative importance of the cuticle and cells in the whole process of foliar penetration. Since the cuticle and cells are associated in the leaf in an interacting system, penetration of one influences the other. Therefore, it would appear that the most suitable way to study foliar penetration, as it actually occurs when a spray drOplet impinges upon the surface of a leaf, is by the use of the leaf disk method. Sargent (1965) has discussed the advantages of studying foliar penetration using this type of method. Methods of Application Numerous methods have been devised to apply substances for penetra- tion study. The most commonly used methods are (a) foliar sprays (Westwood and Batjer, 1960; Edgerton and Haeseler, 1959), (b) leaf immersion (Jyung and Wittwer, 1964; Ahlgren and Sudia, 1967) and (c) microdrOplets (Luckwill and Lloyd-Jones, 1962; Prasad et al., 1967). A more unique approach by Hughes and Freed (1961) and webster (1962) used a lanolin ring to contain a larger amount of liquid on a defined area. Improvements on this method have been made by Kamimura and Goodman (1964) and Sargent and Blackman (1962, 1965) by securing glass tubes to excised leaves or leaf disks and maintaining them in a covered Petri dish. The advantages of this method are: (a) the solution is not allowed to dry out, thus, maintaining a constant external concentration, (b) a defined area is maintained, (c) buffering capacity is not lost due to drying and (d) the tissue is removed from the influence of the rest of the plant, e.g. translocation. Method of Detection Determining the amounts of foliar applied substances entering a plant is often difficult because of the small amount entering the plant or insensitivity of the method chosen to measure penetration. Recording injury caused to a plant may be adequate (Davis, 1956; Hauser, 1955; westwood and Batjer, 1958). Biological responses (Currier et al., 1964; WestwOOd and Batjer 1958) are more sensitive but require movement to a part of the leaf or plant where they elicit a response. Fluorescent dyes.(Dybing and Currier, 1959; Hull, 1964) provide a quantitative estimate of penetration. The use of radioactive tracers provides the most sensitive determination of the amount of a substance entering a plant (Sargent, 1965). If sufficiently high specific activities are used, the sensitivity of this method exceeds that of colormetric (Freed, 1964) or spectrophotometric (Holly, 1956) determinations. Portals of Entry The cuticle is generally considered a barrier for foliar penetration. However, pesticides, plant growth regulators and nutrients have been demon- strated to_permeate the cuticle. Preferential pathways of penetration into leaf cited in the literature are: anticlinal walls, trichomes and epidermal hairs, areas over veins and stomata, guard cells and accessory cells. Anticlinal Walls Preferential penetration has been shown to occur over anticlinal walls (Fogg, 1948; Dybing and Currier, 1961). Increased penetration over anticlinal walls has been attributed to a thinner cuticle. However, Baker and Martin (1963) and Norris and Bukovac (1968) have shown the cuticle to be actually thicker over anticlinal walls. Frey-Wyssling and Hauserman (1941) have shown a decrease in birefringence over anticlinal walls. This would indicate either a reduced amount of embedded waxes or a less highly oriented wax layer; both conditions may result in increased permeability. Eglinton and Hamilton (1967) show electronmicrographs providing evidence that more wax is present on the surface over periclinal walls than over the anticlinal walls. This is attributed to an easier diffusion pathway of waxes “1 ~3 .anNJrLZ . t, .. . .1 (all. 7.1.!— .. 10 from the cell through the periclinal region than the anticlinal region between cells. Bystrom et a1. (1968) have shown surface replicas Of Beta vulgaris which indicate a waxrcoated central plaque-like area of coalesced wax rodlets. The surface over anticlinal walls was nearly devoid of wax. Trichomes Trichomes or epidermal hairs have been cited as pathways of penetration of 2,4-D (Ennis and Boyd, 1964). Hull (1944) has exten- sively reviewed the literature concerning the preferential absorption of dyes. Mitchell et a1. (1960) have emphasized the importance of these structures as possible pOrtals of entry of exogenously applied substances. Hairs which persist most often contain protoplasm (Esau, 1953) and as such provide the shortest pathway into the protoplasm of the plant. Since the cuticle is thinner (Martin, 1966), less wax is present (Bystrom et a1:, 1968).Because of size and geometry they rise above the water re— pellent epicuticular waxes. Trichomes appear physically suited as preferen— tial areas of penetration. Yeiee Preferential absorption of dyes (Currier and Dybing, 1961; Hull, 1964), 2,4-D (Crafts, 1966; Leonard, 1958; Pickering, 1965) and potassium ferrocyanide (Van Overbeek, 1956)have been reported over veins. Anatomical differences of the vein area offer a possible reason for increased penetra- tion. Thin-walled parenchyma cells, the bundle sheath cells, are present over the surface of veins (Esau, 1953). These often extend to the upper and lower epidermis (Van Overbeek, 1956). There is no reduction in cuticle thickness over the veins of pear but discontinuity of the wax layer may be a significant factor influencing penetration (Norris and Bukovac, 1968). 11 water lacking a surfactant is capable of penetration over veinal areas (Van Overbeek, 1956). Guard Cells and Accessory Cells Stomatal pores, which penetrate the epidermis, are regulated byvery specialized epidermal cells, the guard cells. Morphologically and physio: logically they are quite different from the ground epidermal cells. They are the only epidermal cells containing chloroplasts (Esau, 1953; Ketellapper, 1963; Zuker, 1963). There is evidence that guard cells may preferentially absorb exogeneously applied compounds. Sargent and Blackman (1962) con- cluded that penetration of 2,4-D takes place preferentially through guard cells and accessory cells since a close relationship was found between sto- matal density and entrance of 2,4-D into the abaxial and adaxial surfaces of Phaseolus vulgaris and Coleus blumei. Jyung et a1. (1965) found a correlation between stomatal frequency and rate of absorption of Rb in bean and tomato leaves. Guard cells have been shown to preferentially reduce AgNO3 (Hofler, 1939). Microradioautograms of Spinacea oleracea and XEElS. tricolor treated with radioactive sucrose showed the highest density of reduced silver grains above cuticular edges of the guard cells (Franke, 1964b). Under specific conditions Pallas (1966) was able to show neutral red accumulation in guard cells of Vicia faba. Pores, Cracks and Fissures There are reports both supporting and refuting the existence of pores in the cuticle. These have been discussed in the section describing deposition of epicuticular waxes. If wax canals or pores traverse the cuticle, then there is a direct pathway from the surface of the leaf to the cell for lipophilic substances. Fissures, punctures and insect punc- tures undoubtedly allow mass flow of foliar applied substances (Currier 12 and Dybing, 1959; Harley et al., 1956; Orgell, 1957; Scott and Baker, 1947). Ectodesmata Anticlinal walls, trichomes, veins and guard cells have been cited as preferential areas of penetration. Franke (1961, 1964a) has shown, using a mercury-chloride method, that ectodesmata are especially plentiful in these areas of favored foliar penetration. Ectodesmata are considered to be interfibrillar spaces extending through the cell wall to the cuticle (Franke, 1967). The similarity between the distribution of reduced silver grains of microradioautograms over guard cells (Franke, 1964b), anticlinal walls (Franke, 1964c) and veins (Franke, 1964a) has led Franke to conclude that ectodesmata function as pathways of transport in foliar absorption. However, definitive proof is still lacking. One should not disregard the possibility that the cuticle above these areas of preferential absorp— tion is more permeable and thus ectodesmata may be demonstrated only when the cuticle allows sufficient entrance of the foliar applied substance. _§3omata1 Penetration Stomata are present on all leaves and when open provide an avenue to circumvent the external cuticular barrier. They must beconsidered as a potentially important pathway for foliar penetration. However, the question of whether a foliar applied solution can pass through the stomatal pore and enter the substomatal chamber has been in controversy for an number of years. There is general agreement that aqueous solutions in the absence of a surfactant are unable to penetrate into the substomatal chamber (Fogg, 1948b; Teubner et al., 1957; Weaver and DeRose, 1946). However, with a surfactant present, the occurrence of stomatal penetration is a debated 13 question.. Westwood et al. (1960b) concluded that stomatal penetration of dinitro-o-cresol by apple leaves is unimportant since the major portion of the DNOC was absorbed after the spray dried. Middleton and Sanderson (1965) discount mass flow of inorganic ions through the stomatal pores because (a) absorption was linear with time, (b) there was not a linear relationship with applied concentration and (c) per cent uptake fell as the concentration increased. Muzik et a1. (1954) and Wallihan and Heymann- Herschberg (1956) considered stomatal entry unimportant because an equal amount or less of CMU or zinc, respectively, penetrated through the lower surface as compared with the upper surface. Stomata have been indicated as major sites for entry of urea (Cook and Boynton, 1952), NAA (Harley et al., 1957), and C060 (Gustafson, 1956) since more penetration occurred through the lower surface where stomata are more numerous. Fluorescent dyes have been used to study stomatal pene- tration in a number of plants. From surface observations Dybing (1958), Dybing and Currier (1961) and Hull (1964) have concluded that stomatal penetration can occur provided that efficient surfactants were used at the proper concentration. Stomatal penetration also varied with the species tested. The most definitive evidence offered in proof of stomatal pene— tration has been presented by Pickering (1965). Microradioautograms 0f Zebrina leaf cross sections clearly show CIA-2,4-D to have moved into the substomatal chamber. There are several reasons why conflicting reports of stomatal pene- tration may appear in the literature. There is still no general agreement on the process of stomatal movement. However, a number of environmental factors are known to influence stomatal opening and closing; among the more important are light, carbon dioxide concentration, temperature, and 14 water stress on the leaf. The effect of these environmental factors on stomatal movement have been discussed by Ketellaper (1963), Meidner and Mansfield (1965), Pallas (1966) and Zelich (1965). Stomata are closed in the dark except for succulents (Levitt, 1967). Opening depends on light intensity and wavelength. The higher the light intensity the wider the opening. Low 002 concentrations cause stomatal opening and high C02 concentrations tend to close the stomata. Intermediate con- centrations partially close stomata. Temperature (5-25 C) may or may not influence stomatal movement. However, temperatures over 30 - 35 C generally cause closing. When the water deficit reaches a certain value, characteristic for a species, stomatal aper ture tend to become smaller, and if severe become completely closed. Therefore, stomatal aperture widths are highly dependent upon a combination of environmental factors. Changes in anycne of these factors may have a profound effect on stomatal width and thus the amount of a solution that potentially can enter through a stomatal pore. Currier et a1. (1964) have pointed out the difficulties involved in controlling environmental conditions in the greenhouse sufficiently to consistently demonstrate stomatal penetra- tion. The size of the stomatal pore varies according to the plant species. Phaseolus vulgris was found to have a maximum stomatal aperture of 7 x 3pm and ebrina pendula 31 x 12pm (Eckerson, 1908). The size of stomata on a plant and the number is influenced by the environmental con— ditions under which the leaves develop (Meyer et al., 1960). Surfactants differ in their ability to reduce surface tension (Fey and Smith, 1965). Dybing and Currier (1961) have found that stomatal penetration is dependent upon concentration and surfactant used and the 15 species being treated. In general, the more effective surfactants caused the greatest reduction in surface tension and the species showing greatest stomatal penetration had large stomatal aperture. If stomatal penetration is a reality then its importance in foliar penetration must be determined. Skoss (1955) considered stomatal penetra— tion the major portal of entry into the leaf. Cook and Boynton (1952) and Pay (1962) regarded stomatal penetration important for initial entry into the leaf but over a longer period of time cuticular penetration was thought to be the most important pathway of entry. Although stomatal penetration may occur (Crafts, 1964), it cannot be considered an important pathway of entry because it is impossible to predict when stomata will be open since opening and closing is a complex process involving many uncontrollable environmental conditions (Crafts, 1961). Further, lining the substomatal chamber is an internal cuticle (Norris and Bukovac, 1968; Scott, 1950; Yamada et al., 1966). Even if a solution can enter the substomatal chamber, it still must traverse this internal cuticle. However, the internal cuticle is hydrated_ much thinner and increases the absorbing surface (Wittwer et al., 1967). Factors Influencing Foliar Penetration Environmental Factors Environmental factors such as light, temperature and humidity prior to spraying, have been implicated as factors that may determine the sub- sequent performance of a spray application (Edgerton and Haeseler, 1959; Westwood and Batjer, 1960). High light intensity has been shown to in- crease cuticle thickness (Orgell, 1954), thus increasing the diffusion pathway of a penetrating molecule. Juniper (1959) has demonstrated a 16 direct correlation between wettability and surface waxes and light in- tensity under which Pisum sativum plants were grown. Low contact angles were associated with low light intensities. The lower the contact angle the larger the area of a spray draplet in contact with the leaf surface. The influence of temperature on foliar penetration may be made manifest through its effect on growth. Apple and peach leaves absorbed more NAA“ 014 when grown under cool air temperatures (60 F) than those developed under warmer temperatures (70 F) (Donoho et al., 1961). It would seem that growth is slowed down, cuticle development is slower and wax pro- duction retarded at lower temperatures. Immature ivy leaves have thinner cuticle and less wax than mature leaves (Schieferstein, 1957). Foliar absorption of 3-CP (Bukovac, 1965) and 2,4-D (Sargent and Blackman, 1962) has been shown to be greater in immature than in mature fully expanded leaves. Donoho et a1. (1961) have shown that peach leaves de— veloped under a high humidity environment absorbed more NAA-C14 than those developed under low relative humidity conditions. Time-Course Following the rate of foliar penetration over a period of time has been one method used by investigators to determine uptake patterns of foliar applied compounds. Ehlig and Bernstein (1959), Jyung and Wittwer (1964) and Sargent and Blackman (1962) have reported foliar pene- tration of sodium and chloride, phosphate and rubidium, and 2,4-D respec— tively, to be linear with time. Other workers have shown two phases (Prasad and Blackman, 1962), three phases (Vickery and Mercer, 1964), and four phases (Allen, 1964) of foliar uptake. These phases differ in length of time and relative importance in the whole process. However, the first phase is generally the shortest and most rapid. Care must be taken in 17 evaluating time-course studies in the literature since application has often been made by spraying. Interpretation is made difficult by changing concentration and buffering capacities as a result of droplet drying. Concentration The influence of external concentration of the applied solution has been determined for a number of foliar applied compounds. Penetration into leaves has been found to be directly correlated with concentration, for 2,4-D (Sargent and Blackman, 1962; Thimann, 1948), NAA (westwood and Batjer, 1958), urea (Kuykendall and Wallace, 1954), and salts (Eaton and Harding, 1959; Middleton and Sanderson, 1965). Bukovac and Norris (1966) showed a linear relationship between applied concentration and the amount of NAA and NAAm bound to the upper surface of pear leaves. Deviations_ from linearity with applied concentration are seldom observed. However, Jyung and Wittwer (1964) have shown uptake of Rh to deviate from linearity at higher concentrations. Temperature As temperature increases, it is generally agreed that there is an increase in foliar penetration. Currier and Dybing (1959) indicated that the complexity of the overall penetration process makes it difficult to determine where temperature exerts its effect. It is becoming eminently clear that temperature influences both accumulation by the cells and penetration through the cuticle. Cuticular penetration is considered to be a physical diffusion process (Franke, 1967). The temperature coefficient (Q10) for ion diffusion through water is about 1.2 (Briggs et al., 1961). Lipid membranes may exhibit high Q10 values (Sutcliffe, 1962). Van Overbeek (1956) has pointed out that wax lamellae at low temperatures are near solidification and have low permeability. As the temperature is raised 18 these fatty substances become less viscous and more permeable. Briggs et a1. (1961) indicated that molecules moving through lipid containing membranes may have to acquire a relatively large amount of kinetic energy to break the number of bonds between the lipid molecules. Norris and Bukovac (1969) have shown that the Q10 for penetration between 15-25 C of NAA through enzymatically isolated upper pear leaf cuticle is 5.5-6.0. This is particularly significant since this high Q10 value was derived over a physiological temperature range. Temperature coefficients of 2.0 and greater have been considered evidence suggestive of metabolically mediated uptake. Temperature co- efficients for foliar penetration in excess of 2.0 led Goodman and Goldberg (1960), Rice (1948), and Sargent and Blackman (1962) to suggest that absorption is governed by metabolic processes. Since cuticular penetration has been shown to be profoundly influenced by temperature and Q10s for active accumulation by cells are known to be 2.0 or greater, it is reasonable to assume that the temperature coefficient for foliar penetration is a result of temperature influence on both metabolic pro- cesses and the cuticle. Therefore, temperature coefficients for foliar penetration should be rejected as a basic criteria for active uptake. pH_ The influence of pH on regulating the amount of a plant growth substance entering a leaf is well documented. This has been especially true for weak acids such as 2,4-D and NAA. In general, there is an inverse relationship between pH of an applied solution and amount entering the leaf (Currier and Dybing, 1959). Crafts (1953, 1956) and Sargent and Blackman ‘ (1962) have found the greatest 2,4-D penetration at low pH values (below 6.0). Bukovac and Norris (1966) reported that sorption of NAA into the 19 pear leaf was pH dependent, being greater below the pK (4.2) than above. Giese (1962) has discussed the influence of pH on the dissociation of weak acids and bases in relation to their ability to pass through lipoidal cell membrances. Abovetle pK (the pH at which there is an equal number of charged and uncharged molecules) most weak acid molecules are dissociated, have a charge and are not lipid soluble. Below the pK more molecules are in the undissociated form, have no charge, and are more lipid soluble. Cutin has a large number of free COOH groups (Foy, 1964; Franke, 1967). The pH and the hydrogen ion concentration of the applied solution may influence the charge on the cuticle. Van Overbeek (1956) has reported a pH of over 5 for the cutin and Bukovac and Norris (1966) have shown the pK for the surface of pear cuticle to be between 2.8-3.2. At low pH values the carboxyl groups would be largely undissociated and thus more permeable to anions than at higher pH values where more carboxyl groups would be dissociated and, thus, negatively charged (Hull, 1964). Orgell (1957) observed that acid substances were absorbed to the greatest extent at acid pH values. Hull (1964) has attributed this to the probable neutralization of negatively charged acid residues. em The influence of light on foliar penetration is less clear than for other factors. Although light has generally been found to increase foliar penetration, there are a number of instances where equal or greater absorp- tion occurred at lower light intensities or in the dark (Herrett and Linck, 1961; Smith et al., 1959; Weintraub et al., 1954). The energy required for active absorption can be derived in green leaves from photosynthetic processes (Franke, 1967). Increases in foliar penetration under the in- fluence of increasing light have been attributed to the production of 20 photosynthetic reserves (Arisz and S01, 1956; Kamimura and Goodman, 1964). Ahlgren and Sudia (1967) and Van Lookeren Campagne (1957) attributed light dependent foliar penetration to energy derived from light directly; presumably photophosphorylation and not a translocatable product. Sargent and Blackman (1962, 1965) have shown that the light effect on increased absorption of 2,4-D in bean leaf disks is very com- plex. 2,4-D passes through the cuticle and cell wall by diffusion. The steepness of the diffusion gradient is determined by light-stimu- lated uptake into the underlying cells and by a conversion into some 2,4—D metabolite. It was concluded that both physical and metabolic factors influenced 2,4-D penetration. Currier and Dybing (1959) and Dybing (1958) considered that light may influence penetration by causing stomata to Open. Light must be regarded as a prerequisite for foliar penetration to occur via stomata because other factors which may open stomata, i.e. low C02 concentrations, would not occur under normal treating conditions. Surfactants The physical arrangement of epicuticular waxes often prevents spray droplets from coming in contact with the leaf surface (Eglington and Hamilton, 1967). Epidermal hairs covering leaves may also prevent spray dropkms from coming in contact with the surface (Holly, 1964). To overcome the problem of incomplete wetting of foliar surfaces a number of surfactants have been used to reduce the surface tension of aqueous solutions. Physical and Chemical Properties of a Surfactant The term surfactant is a general one referring to a molecule containing two Opposing characteristics (Behrens, 1964). One portion of the molecule has an affinity for the solvent sufficient to bring the whole molecule into 21 solution. The other portion of the molecule which has a low affinity for the solvent tends to accumulate at an interface (Osipow, 1964). Surfactants are commonly classified as anionic, cationic, non-ionic or amphoteric, depending on the nature of the electrical charge or absence of ionization on the hydrophilic portion of the molecule (Parr and Norman, 1965). Aqueous solutions of surfactants exhibit a rather abrupt change in their micelle concentration, osmotic pressure, electrical con- ductance, and freezing and boiling points over a narrow concentration range. This point is referred to as the critical micelle concentration (cmc). At concentrations greater than the cmc value, the surface ten- sion of the solution does not decrease with an increase in surfactant concentration (Osipow, 1964). Surfactant Effects on Cuticular Penetration Surfactants, in most cases, have been shown to greatly enhance foliar uptake. Bryan et a1. (1950) and Hauser (1955) attributed enhanced penetration to a reduction of surface tension, resulting in an increase in the area of spray contact (Stanforth and Loomis, 1949). It is becoming increasingly apparent and well documented that other factors in addition to reduction in surface tension are involved in surfactant effects on pene- tration (Bayer and Drever, 1965; Freed and Montgomery, 1958;Hughes and Freed, 1961). Jansen (1964) and Westwood and Batjer (1958) have shown that there is a relationship among surfactants, chemical, and species in- volved in determining the-extent to which a chemical can penetrate. Some surfactants inhibit or are ineffective in increasing absorption (Jansen et al., 1961; Westwood and Batjer, 1960). The amount of spray on a leaf may be reduced by the use of surfactants (Koontz and Biddulph, 1957). Surfactants may be effective only when conditions, created by the environment, 22 are unfavorable for absorption (Thompson et al., 1958; Westwood and Batjer, 1958). The critical micelle concentration. for most surfactants is usually found in the concentration range between 0.01 and 0.1 per cent (Jansen, 1961). Maximum reduction in surface tension occurs at the cmc (Parr and Norman, 1965). However, since many surfactants increase penetration at concentrations far above the cmc, factors other than reduction in surface tension appear to be important (Colwell and Rixon, 1961; Freed and Mont- gomery, 1958; Holly, 1964). Surfactant effects on herbicidal entry pro- bably correlate best with the colloidal state of the surfactant system (Jansen et al., 1961). The ability of surfactant solutions to dissolve or solubilize water-insoluble materials starts at the cmc and increases with the concentratiOn of-micelles (Osipow, 1964). Micelles can apparently dissolve oils and waxes and remove large areas of wax from leaf surfaces, thus enhancing surfactant penetration of the cuticle, resulting in ex- tended phytotoxic effects (Parr and Norman, 1965). Surfactant Effects on Stomatal Penetration The influence of surfactants has already been discussed in reference to stomatal penetration. It may be concluded from the work of Dybing (1958) and Dybing and Currier (1961) that surfactants most effective in reducing surface tension allow greatest stomatal penetration of aqueous solutions. Active Uptake In order for a growth substance to induce a response it must enter the living protoplasm of the plant. There are a chain of events which take place from the time a spray draplet impinges upon the surface of a 23 leaf until it reaches its site of action in the living continuum of the plant. A molecule must first pass through the cuticle and cell wall. This portion of absorption is considered to be by diffusion, a physical process (Franke, 1967). Once inside the leaf a molecule may be taken up actively by a cell or move in the plant in the cell walls and xylem (Crafts, 1961b). Jyung et a1. (1964, 1965) have shown foliar absorption by isolated leaf cells to be an active process. Because uptake by cells in an intact leaf may be modified by the cuticle that covers them, the mechanism of absorption may be ill-defined (Wittwer et al., 1967). Ahlgren and Sudia (1967), Arisz and S01 (1956), Jyung and Wittwer (1964) and Van Lookeren Campagne (1957) have concluded that uptake of nutrients is metabolic. Evidence has been given for metabolic uptake of streptomycin by apple leaves (Kamimura and Goodman, 1964), 2,4-D by bean leaves (Sargent and Blackman, 1965), and sucrose by bean leaves (Vickery and Mercer, l9 4). Criteria often used for active uptake include: time—course analysis, temperature, accumulation against a concentration gradient, irreversibility, oxygen, energy dependence. and sensitivity to metabolic inhibitors (Giese, 1962; Jyung and Wittwer, 1965). Because a molecule must pass through a cuticle present on the intact leaf or leaf disk, it may be necessary to modify some criteria. Temperature is a questionable criterion because penetration through the plant cuticle has been shown to have high tempera- ture coefficients (Norris and Bukovac, 1969). Active uptake by cells is generally considered to be two phases; a rapid initial phase and a slower linear metabolic phase (Jyung and Wittwer, 1965). This criterion may also have to be modified. 24 Naphthaneneacetic Acid and Naphthaleneacetamide Investigations have been carried out to evaluate the effectiveness of naphthaleneacetic acid (NAA) and naphthaleneacetamide (NAAm) as ab- scission promoting and preventing compounds since Gardner et a1. (1939) reported NAA and NAAm as active in delaying abscission in ripening apples. southwick et a1. (1953) found NAA to be effective as a fast action stop- drop-cOmpound on McIntosh apples, but other compounds were superior. In recent years the use of NAA and NAAm has been restricted to post-bloom sprays as a thinning agent (Harley et al., 1957, 1958; Hoffman et a1, 1955; Southwickzand Weeks, 1957; Thompson et al., 1958; Westwood and Batjer, 1958). Considerable variation in thinning responses from year to year and location to location has stimulated investigations to deter- mine the factors that may influence the absorption of NAA and NAAm and thus lead to the variable thinning results observed in the field (Donoho et al., 1961; Edgerton and Haeseler, 1959; Thompson et al., 1958; Westwood and Batjer, 1958, 1960). Murneek and Teubner (1953) have attributed the thinning responses of NAA to a relationship between (a) an inhibition of embryo development and (b) a temporary retardation in separation of cells along the abscission 14 NAA was de- zone. Donoho et al.7(l96l) have shown that ring-labeled C tectable in fruits often after one hour. Distribution throughout the fruit was found after 96 hours with some accumulation in vascular areas and the seeds. Luckwill and Lloyd-Jones (1962) reported rapid conversion of NAA in detached apple leaves to a water-soluble addition Compound I, lacking auxin activity. This addition compound was slowly converted into addition Compound II. Zenk (1962) has demonstrated that a large proportion of the 25 NAA entering etiolated pea stems was converted to the glucose or aspartate conjugates. Sudi (1967) has confirmed the formation of l-naphthaleneacetyl- L-aspartate from NAA in the etiolated pea stems and has shown this conjugate formation to be accomplished by an inducible enzyme. MATERIALS AND METHODS Pear trees, Pyrus communis L. Cv. Bartlett, (Hawley Nursery, Hart, Michigan) were selected as the test plant. A description of the pear leaf in relation to foliar penetration (Norris and Bukovac, 1968a), binding to pear leaf cuticle (Bukovac and Norris, 1966), and penetration through the isolated pear leaf (Norris and Bukovac 1968b, 1969) have established a basis for the use of the pear as the test plant. cx-Naph- thaleneacetic acid (NAA) carboxyl 14C and cx—naphthaleneacetamide (NAAm) carboxyl 14C (Tracerlab, Waltham, Mass.) were selected as the model compounds. 3H-NAA for microradioautography was obtained from Nuclear Chicago Corp., Des Plaines, Ill. Growing Plants During the growing season leaves were collected from established trees on the Michigan State University Horticulture Farm. When leaves were not available from orchard trees, 2-year-old trees were grown in the greenhouse. The general method for growing was to plant the trees in plastic containers in a mixture of 2/3 sandy loam and 1/3 peat. Holes were punched in the bottom of the containers for water drainage. Initially all trees were headed back to about 18 inches and then 2 lateral shoots allowed to grow. All other shoots were removed as they appeared. Leaves were seleCted from experimentation when fully expanded and a deep green color had developed. 26 27 Selection of a Method to Determine Foliar Penetration Four methods for determining foliar penetration were evaluated on the basis of variability of results and ease and rapidity of carrying out the procedure. A leaf disk procedure was modified after the method of Sargent and Blackman (1962). This method allowed the greatest con- trol of experimental variables and environmental conditions during the absorption period. Twelve 1.5-cm leaf disks, with attached glass cyl- inders, were placed in each of 10 Petri dishes. Into each glass cyl- inder was pipetted 0.25 ml of a 5 X 10'4 M NAA solution buffered at pH 4.2 with 10'2 M phosphate-citrate and containing 0.5 uc/ml 14C. After a 12-hour treatment time the radioactive treating solution was removed, the leaf disks washed with a stream of water from a wash bottle and wiped with a piece of cotton moistened with xylene. Three disks were sampled at random from each Petri dish for each of the four methods tested and radioactivity determined by the following procedures. .Ll) Leaf Disk-Planchet Counting Leaf disks were placed treated side down in 2.5-cm stainless steel planchets lined with double sticky tape and then dried in a drying oven for 12-hr at 60 C. Radioactivity was then determined in a Beckman Low Beta II proportion counter. Corrections were made for background and efficiency. (2) C02 Combustion - Scintillation Counting Leaf disks were blotted dry then placed in the drying oven at 60 C for 12 hr. C02 combustion flasks (Arthur Thomas Co., Philadelphia, Pa.) were purged with oxygen. The dried leaf disks, wrapped in black combustion paper, were placed in a platinum holder attached to the top 28 then placed in the flask. Combustion was carried out using a Thomas-Ogg Infrared Igniter (Arthur Thomas Co., Philadelphia, Pa.). Combustion time was approximately 45 sec. Ten ml of ethanol-ethanolamine (2:1) was pipetted into the flask before cooling for 30 min in an ice bath. A 56ml aliquot was removed and placed in a glass scintillation vial with 10 m1 of Cab-O-Sil scintillation cocktail (Appendix Table 1). Samples were then counted in a Packard Tri-Carb liquid scintillation spectro- meter Model 574. Corrections were made for background and efficiency. A BBOT-toluene scintillation cocktail was first used but was found to be unsatisfactory because the small amount of residual water in the leaf disks caused considerable quenching. The Cab-O-Sil cocktail was able to accommodate at least 0.5 ml of water without appreciable quenching. (3) Ethanol Extract-Scintillation Counting Leaf disks were placed in a hand homogenizer with 0.5 ml of ethanol and macerated. The macerated leaf disks were washed twice with 3.5 ml of ethanol to make a total volume of 7.5 ml. A 0.5 ml aliquot was taken and pipetted into a vial with 15 ml of Cab-O-Sil cocktail. Samples were counted in the liquid scintillation counter. Corrections were made for quenching, background and efficiency. (4) Leaf Disk-Scintillation Counting Leaf disks were placed treating side down in scintillation vials containing 15 m1 of Cab-O-Sil cocktail and counted in the scintillation counter. Corrections were again made for background and efficiency. ' The results of this evaluation are shown in Table l. The leaf disk method counted on the Low Beta II propOrtional counter was chosen as the method of determining foliar penetration. This was chosen because Table 1. Comparison of four methods for tion. 29 determining foliar penetra- Activity Standard Coefficient Method of detection (cpm) deviation of variation Leaf disk- Planchet counting 3721 379 10.2 CO combustion- Scintillation counting 5388 1106 20.5 Ethanol extract- Scintillation counting 5151 2145 41.6 Leaf disk- Scintillation counting 3830 517 13.5 30 it had the lowest coefficient of variation and the least amount of manipulation of the tissue. General Methods Initially the method of Sargent and Blackman.(l962) was chosen for application of the treating solution. However, for several reasons it was desirable to modify this procedure: (a)'The melting range of the petroleum jelly is 36-41 C. At temperatures even as low as 30 C, leaks at the bottom of the glass cylinders became a problem. .(b) Glass cylinders fixed to the leaf surface with petroleum jelly would not re- tain solutions containing surfactants. (c) Browning of the leaf disk below the petroleum jelly became apparent after 36 hr or more. A diagramatic representation of the modified method used to apply the treating solution is shown in Fig. 1. Glass cylinders, 1-cm inter- nal diameter, were secured to the leaf by Dow Corning Silastic 68-110-RTV silicone rubber (Dow: Corning Corp., Midland, Michigan) and hardened with T-ll catalyst (Wacker Company, Munich, Germany). General Electric RTV-ll silicone rubber (General Electric Co., Waterford, New York) was also found to be an acceptable adherent. To approximately 7g of sili- cone rubber was added about 0.30 ml of catalyst and thoroughly mixed with a glass stirring rod. Glass cylinders 8 mm in height and 1 cm in diameter were touched to the silicone rubber catalyst mixture so that the bottom edge of the cylinder was coated. The cylinders with the adhering silicone rubber were placed on 1.5-cm leaf disks cut with a cork borer from a leaf. Approximately 30 to 45 min was required for the silicone rubber to harden. Leaf disks were placed in a 15.0 X 2.0 cm Petri dish lined with filter paper and moistened with distilled 31 Figure lJ-—Schematic description of the method used in studying leaf disk penetration. 32 I DRIED IN 60 C DRYING OVEN COUNTER @/ 33 water; usually 10 leaf disks per dish. The radioactive treating solu- tion, 0.25 ml, was pipetted into each glass cylinder, the top placed on the Petri dish and then arranged in a water bath maintained at 25 C and illuminated with 1000 ft-c of light from a light bank of fluorescent tubes. At the end of the treatment period, the glass cylinders con- taining the radioactive treating solution were removed and the leaf disks thoroughly washed with a stream of‘water from a wash bottle. The leaf disks were then blotted dry on a paper towel, placed treating side down in a 2.5-cm planchet lined with double sticky tape, and dried in. a drying oven for 12 hr at 60 C. Leaf disks were then counted on a Low Beta II proportional counter with correction made for background. The standard NAA treating solution was 6.0 X 10.5 M, 1.0 uc/ml - and a specific activity of 16 uc/umole. The NAAm treating solution was~3.5 x 10'4 M NAAm, 0.5 uc/ml and a specific activity of 2.0 uc/ umole. The 3H-NAA treating solution used in the microradioautographic study was 9.3 X 10-4 M NAA, 50 uc/ml and a specific activity of 468 uc/umole. All were buffered at pH 3.0 with 1 X 10’2 M solution of citric acid and dibasic sodium phosphate. All solutions contained 2% ethanol. Pretreatment Time Results of Sargent and Blackman (1962) indicated that light or dark pretreatment could have a profound effect on subsequent penetrar tion of 2,4-D. To evaluate the effect of light-dark pretreatment in the pear leaf system, leaf disks were cut and prepared according to the method described. All Petri dishes were placed in the water bath and.either illuminated with 1000 ft-c of light or kept in the dark by 34 placing aluminum foil over the dish. NAA treating solution was applied at the end of the designated pretreatment times of 0, 4, 8, and 12 hrs. The penetration time was 12 hr. The results of this experiment are shown in Table 3. A 12-hr dark pretreatment was accepted as the stan- dard pretreatment time. Self Absorption 14Carbon is a relatively low energy beta emitter (0.156 Mev) and as such may be absorbed by the tissue quite easily (Wang and Willis, 1965). An experiment was performed to establish to what extent self- absorption was involved in the detection of the radioactivity in pear leaf tissue. Dried pear leaves were ground to 80 mesh in a Wiley mill. Samples were weighed out in planchets on a Metler GramrAtic Balance to provide weights of 0.5 to 24 mg/cmz. 14 Carbon NAA of 0.1 uc/ml activ- ity was pipetted into each planchet, 8880 dpm/mg ground leaf tissue. Ethanol was added to fill the planchet to one—half its height. The ground leaf tissue, ethanol and treating solution were mixed in each planchet to give a uniform coverage of the bottom of the planchet and then air dried. Samples were counted on the Low Beta II proportional counter. The procedure used above deviates from methods more frequently used (Wang and Willis, 1965) but it was found to be most reliable for two reasons. (a) If ground tissue and a radioactive solution were mixed together and then appropriate amounts pipetted into each planchet, it was difficult to get an accurate amount in the planchet because the tissue continued to sediment to the bottom even with continuous agitar tion. (b) If ethanol did not comprise the major volume of solution 35 pipetted, the ground tissue, when dried, cracked and did not provide a continuous layer of tissue which is necessary for self absorption determinations. Leaf Anatomy Tranverse sections were cut on an International-Harris cryostat and stained with Sudan III and Sudan IV according to Norris and Bukovac (1968). Photomicrographs were taken with a Wild M20 research microscope using a 35mm film holder and exposure time determined by a Wild Photo- automat exposure meter. Photomicrographs of the leaf surfaces were taken with a Wild M5 Stereomicrosc0pe fitted with a Polaroid Camera. TimerCourse of Penetration Experiments were conducted according to the general procedure to determine the influence of time on penetration of NAA and NAAm by the upper and lower leaf surfaces. Samples were taken at 12, 24, 48, 72, 96, 120 and 144 hr for the upper surface and at 6, 12, 24, 48, 72, and 96 hr for the lower surface. For long term experiments, it was neces- sary to add water to the filter paper to maintain a saturated atmosphere. and prevent evaporation of the treating solution. The time-course of penetration was followed in one experiment where the treating solution was applied to the leaf as small droplets. Leaf disks were prepared and placed in the Petri dishes. A 20 ul draplet of treating solution was applied to each leaf disk. After 6, 12, 24, 48, 72, 96, and 120 hr the droplet was removed from the leaf disk with a pipette, then the leaf disk was removed, washed and prepared 36 according to the general method. The filter paper was kept moist at all times to prevent drying of the dr0p1et. Effect of Concentration on Penetration The influence of treating solution concentration was evaluated. Concentrations below 6 X 10‘5 M were achieved by dilution of the labeled NAA or NAAm with buffer and an appropriate amount of ethanol. Concen- trations above 6 X 10‘5 M were prepared by the addition of nonlabeled NAA or NAAm. The time of penetration for these experiments was 24 hr because of the low activity of the lower concentrations employed. Effect of Temperature on Penetration Experiments assessing the effect of temperature on penetration were carried out in Percival growth chambers. Water baths were placed in growth chambers maintained at 15, 25 and 35 C and illuminated with 600 ft-c of light with both fluorescent and incandescent lamps. The 5 C temperature was achieved by cooling the water bath in the growth chamber set at 15 C. Effect ofng on Penetration Treating solutions between pH 3.0 and 7.0 were prepared using citrate-phosphate buffer. One of the main advantages of this buffer was that it could be used over a wide range of pH values. Effect of Light on Penetration Experiments determining the influence of light intensity on pene- tration were carried out in a Sherer-Gillett growth chamber Model 37-14. 37 Light intensities between 0 and 1200 ft-c were achieved by varying the distance of the Petri dishes from the light source and by shading with cheese cloth. Honeywell recorder thermocouples were placed in sample leaf disks in each Petri dish and the temperature was monitored and re- corded every 4 min during the course of the experiment. Effect of Light and Dark Treatment on Penetration Several experiments were performed to determine the influence of alternate light and dark periods on penetration. In these experiments light (1000 ft-c) was provided by fluorescent tubes above the water bath and the dark regime was achieved by covering Petri dishes with aluminum foil. Effect of Inhibitors on Penetration Anaerobiosis The influence of anaerobiosis on penetration was established by following penetration of NAA into leaf disks held in specific atmos- pheres in desiccators partially filled with water and placed in water baths. Nitrogen and air were continually introduced into the different desiccators through a tube attached to a hollow glass tube in the top. To maintain a continuous flow of nitrogen or air, the outlet tubes were immersed l/2 in in a beaker of water. Petri dishes were placed in water in the desiccators to minimize any changes in temperature. Leaf disks were pretreated for 12 hr in the dark in an atmosphere of nitrogen or air before the treating solution was applied. Penetration time was 12 hr. Inhibitors Applied in Solution Inhibitors were supplied to the lower surface of leaf disks via 38 glass cylinders and on saturated filter paper in Petri dishes for spec- ific time periods (Table 2). Inhibitor solutions were removed from the glass cylinders with a small polyethylene tube attached to a syringe. Treating solution was then added. Effect of Surfactants on Penetration Surfactant treating solutions were prepared on a v/v basis with dilutions being made with buffer solutions. A series of dilutions, from the initial surfactant solution, was necessary because the high viscos- ity of the undiluted surfactants made accurate pipetting of small volumes difficult. Effect of Surfactants on Surface Tension Several concentrations of selected surfactants were prepared in an identical manner to the radioactive treating solution used except where nonlabeled NAA was used instead of 14C-NAA. Surface tension de- terminations were made using a Cenco-Du Nouy tensiometer. Clean, detergent free,aluminum weighing dishes were used to hold the solution for surface tension determinations. A platinum ring was dipped in ethanol then placed in a flame and heated until red. The platinum ring was placed on the tensiometer and lowered into the solution. The force required to pull the ring from the surface was read directly, in dynes/ cmz, from the tensiometer. Three determinations were made for each concentration. The ring was cleaned after each determination. 39 Table 2. Inhibitors used in studying penetration of NAA by the lower surface of pear leaf disks. Inhibitor Concentration Pretreatment 00 (hr) nupl 1 x 10'3 4 1/2 NaN§ 1 x 10‘3 4 1/2 PMA3 1 x 10'4 4 1/2 Atrazine 4 1 x 10‘5 12 m—Cl-CCPS 1 x 10'5 12 p-F-CCP6 1 x 10"5 12 Terbicil7 1 x 10‘4 12 Monuron8 l X 10.4 12 1 2,4 dinitrophenol 2 Sodium azide 3 Phenylmercuric acetate 4 2-chloro-4-ethylamino-6-190propyl-amino-s-triazine 5 Carbonylcyanide mechlorOphenylhydrazone 6 Carbonylcyanide p-trifluoromethoxyphenylhydrazone ; 3-tert-butyl-5-chloro-6-methyluracil 3-(p-chloropheny1)-l, dimethylurea 4O Stomatal Penetration Stomatal Aperture Width Measurements Measurements of stomatal aperture width were made using two methods. Rhaplex AC-33 (Rohm and Haas Co., Philadelphia, Pa.) was used initially according to the method of Horanic and Gardner (1967). With this method the dried Rhoplex was removed from the leaf and the imprints measured directly. However, since the RhOplex often failed to make good contact with the leaf surface, resulting in incomplete or inadequate coverage, the silicone rubber and cellulose acetate method was more often used (Sampson, 1961; Zelitch, 1961). There appeared to be little difference between the quality of the imprints of these two methods. Influence of Surfactants on Stomatal Penetration when Added Subse- guent to Treatinngolution Application The treating solution was added to glass cylinders, then Petri dishes were placed in the water bath and illuminated with 1000 ft-c of light for 3 hr. Twenty five ul of a concentrated (1.0%) surfactant solution was applied directly to each glass cylinder containing treat- ing solution to give a final volume of 0.25 ml and a 0.1% surfactant solution. In cylinders receiving no surfactant 25 pl of buffered solu- tion were added. Effect of C02 Pretreatment on Stomatal Penetration Leaf disks were prepared according to the general method. The lights were turned on over the water bath. C02 application was made at this time by placing a small hollow piece of Styrofoam in a Petri dish in which was placed about 100 mg of dry ice snow. Petri dish 41 covers were secured in place. Dry ice was added twice at l-hr inter- vals. After 3 hr of pretreatment with C02, the treating solution con- taining 0.1% surfactant was added to control leaf disks and those ex- posed to C02. The treating solution was not washed off with a stream of water. Instead, the disks were blotted dry with paper towels. Omitting the water wash eliminated the possibility of forcing treating solution through the stomatal pore by hydrostatic pressure. Stomatal Penetration of Silver Nitrate Leaf disks were prepared according to the general method and then placed in the water bath under lights for 3 hr to allow stomata to Open completely. A 0.1 M AgNO3 solution containing 0.1% Vatsol OT was added. Penetration was permitted to proceed for 4 min. Leaf disks were infil- trated, fixed in Craf III solution, dehydrated in TBA, embedded in tissue- mat, and cut on a rotary microtome as described by Sass (1958). Ten pm sections were cut. Photomicrographs.were taken on a Wild M20 research microscope as previously described. Effect of Droplet Drying;on Penetration Leaf disks were prepared and droplets applied according to the‘ method described for the time-course of penetration of droplets. At the determined time when droplets were to be dried, both Petri dishes containing the controldwet droplets and those to be dried were removed from the water bath and air dried. The top was removed from the dish where the drOplets were to be dried. After about 40 min the droplets had dried. The cover was replaced and both were returned to the water bath until samples-were to be taken. Draplets applied to the lower 42 surface were dried afterIIZ hr and those applied to the upper surface were dried after 24 hr. Effect of Leaf Agg_9n Penetration Trees used in the study of NAA penetration as influenced by leaf age were planted and grown in the greenhouse as previously des- cribed. When 17 leaves were present on the terminal shoots, the 3rd (apical), 5th, 7th, 9th,.llth, and 13th leaves were removed, leaf disks cut and penetration determined for the upper and lower surfaces accord- ing to the general method. Trees used in the study ofDUMmIpenetration as influenced by leaf age were planted and grown in a Percival walk-in growth chamber. Trees were illuminated with 1800 ft-c of light with a 14-hr day. Night tem- perature was 20 C and the day temperature 24 C. When 17 leaves appeared the 3rd (apical), 5th, 7th, 9th, 11th, and 13th leaves were removed and penetration through the upper and lower surface determined. Statistical Analysis Data were analyzed using analysis of variance. Where means were compared, the Tukey w procedure was used (Steel and Torrie, 1960). Standard errors of the mean are given where data is presented in graphi- cal form. Midroradioautography Pear leaf cuticle was isolated from the leaf according to Norris and Bukovac (1968). Cuticle disks were cut, mounted on glass tubes 43 and treating solution applied as described by Norris and Bukovac (1969). One ml of 50 uc/ml 3 H-NAA was placed in each tube. Cuticle transverse sections were prepared according to Norris and Bukovac (1968). Kodak AerO fine grain stripping film (Eastman Kodak Co., Rochester, New York) was applied, exposed for 17 days and developed as described by Jensen (1962). Photomicrographs were taken with a Wild Me20 research micro- scope as previously described. RESULTS Structure of the Pear Leaf The structure of the pear leaf in cross-section is depiCted in Figure 2 A, C, E. The cuticle is lipoidal in nature and is readily stained with Sudan III and Sudan IV, Figure 2 A, C, E. A transverse section of a pear leaf is shown in Figure 2A. The lower surface is more irregular than the upper. Undulations in the cuticular surface are ap— parent over veins; these being more pronounced on the lower surface.. The cuticle in these areas is thicker because of extensions down between the anticlinal-walls of epidermal cells. Figure 2E demonstrates the rougher nature of the lower surface and extension of the lower cuticle through the stomatal pore onto the epidermal cell wall exposed to the substomatal chamber. An epidermal hair from the lower surface is depicted in Figure 2B. Staining with Sudan III and Sudan IV suggests that epidermal hairs also have a well defined cuticle. Hairs are most frequently found on the lower surface but some may be seen on the midrib and outer edges of the upper surface. Surface photomicrographs of the upper and lower surface are illustrated in Figure 2D and F, respectively. Effect of Light and Dark Pretreatment on Penetration The effect of light and dark pretreatment for 0, 4, 8, and 12 hr was studied to determine the effect on subsequent NAA penetration through the lower surface (Table 3). There was no difference between pretreatment in the light and dark. However, as pretreatment time increased, NAA 44 45 _Figure 2. - Photomicrographs illustrating the structure and upper and lower pear leaf surfaces. Transverse section of a pear leaf Epidermal hair Transverse section of upper surface Upper surface Transverse section of lower surface Lower surface 'TJITIJUOUIIP 46 47 Table 3. The effect of light and dark pretreatment on subsequent penetration of NAA through the lower surface of pear leaf disks in light. Penetration Pretreatment Pretreatment1 time Light. 1".Dark Mean (epm) 0 719 743 731a 4 873 718 796ab 8 945 799 872;b 12 909 901 905 b 1Means within a column followed by a different letter are signifi- cantly different at‘P = 0.05. 48 Figure 3. —- Influence of leaf disk weight on self absorption of SELF ABSORPTION (°/o) 50— 301 I0- 49 IQ \l n *1 RANGE OF WEIGHT OF LEAF DISKS l l I l l I 4 8 l2 I6 20 24 LEAF DISK WEIGHT'(mg/cm2) 50 penetration also increased. A 12-hr dark pretreatment was accepted as the standard pretreatment time. It was found that if the light pretreat- ment exceeded 4 hr, stomata were less responsive to subsequent light treatments. Self Absorption The effect of leaf disk weight on detection of NAA is shown in Figure 3. The maximum and minimum weight of the leaf disks used was 8.4 and 3.6 mg/cmz, respectively. Therefore, data presented in cpm re- presents between 70.5 and 86.5% of the total 14C present in the leaf disk. Effect of Time on Penetration Penetration Followipnglass Cylinder Application The time-course of penetration of NAA and NAAm through the upper and lower surfaces of pear leaf disks was followed (Figure 4). Penetra- tion for both surfaces increased with time. There was a linear relation- ship between time and penetration of NAA and NAAm by the upper surface. Penetration proceeded at a much greater rate and accumulation occurred to a larger extent for both compounds when administered to the lower surface. Penetration was linear for about the first 24 hr for NAA, and 48 hr for NAAm, after which entrance was linear and occurred at a reduced rate for the duration of the experiment. Penetration Followinngicrodroplet Application Penetration was linear with time when NAA was applied as microdrop— lets to the upper surface, whereas movement of NAA through the lower sur- face was initially rapid for the first 12 to 24 hr followed by a slower second linear phase for the duration of the experiment (Figure 5). It 51 Figure 4. —- Time-course of penetration of NAA and NAAm through the upper and lower surfaces of pear leaf disks. . NAA upper surface . NAAm upper surface . NAA lower surface . NAAm lower surface U0tfl> For comparison NAA penetration equals NAAm penetra— tion x 1.48. I400? A 7 I200“ “_I0004 E n G u a _ Z 00 9 '— E P 600“ IJJ 2 U.) 0. 4001 2004 0 r v v’ I ’1 1 O 24 48 72 96 I20 I44 TIME ('1') l0,000-w c 0000‘ E 3 ~’ 60004 2 9 p. < E .I h, 4000 2 U Q 20004 O I Y I I r —‘ 0 24 48 72 96 I20 I44 TIME (hr) 52 PENETRATION (cpm) (cpm) PENETRATION 500 1 400 " 300 -I 200% I00“ 35004 3000‘ 2500~ 2000-‘ I500 'I IOOO‘ 5004 V T 1 40 72 96 TIME (hr) Y l fi 40 72 96 TIME (hr) 53 Figure 5. -— Time-course of NAA penetration through the upper and lower surfaces of pear leaf disks following application in droplet form. 54 coo-I LOWER SURFACE soo-I “@400- O. 3 z 9 ’5 P300- 32’ 33 UPPER SURFACE zoo— . . loo- - o ' ' 1 1 1 1 1 J o 6 :2 24 4s 72 96 120 TIME (hr) 55 appeared that the general penetration pattern of NAA when applied either in microdroplet form or via glass cylinders was similar. Effect of Concentration on Penetration As the treating solution concentration was increased there was a corresponding increase in penetration of NAA and NAAm through the upper and lower surfaces (Figure 6). In general, for each lO-fold increase in concentration there was a corresponding lO-fold increase in penetration. This does not hold true for NAA penetration into the lower surface at concentrations above 10'4 M. It would appear that saturation has occurred at these higher concentrations. Effect of Temperature on Penetration NAA and NAAm penetration through the upper and lower surfaces was generally linear with increasing temperatures between 5 and 25 C and be- tween 25 and 35 C there was a rather sharp increase (Figure 7). Tempera— ture coefficients (Q10) for data presented in Figure 7 are shown in Table 4. Q10 values for NAA penetration range between 1.51 to 3.03 and for NAAm between 1.59 to 5.46. Temperature coefficients of NAAm penetration are generally higher than those for NAA. In most cases, the greatest response of penetration to increasing temperature was between 25 and 35 C (Table 4). The time-course of NAA penetration through the upper surface was linear with time for all temperatures studied: 2, l7, and 27 C (Figure 8). A penetration response to increasing temperature was more dramatic, pos- sibly reflecting an influence of leaves 6 weeks older than those used in the previous temperature studies. 56 Figure 6. —- Influence of treating solution concentration on penetration of NAA and NAAm through the upper and lower surfaces of pear leaf disks. A. NAA upper surface B. NAAm upper surface C. NAA lower surface D. NAAm lower surface (omens) PENETRATION PENETRATION (9 males) 57 m” I .1 A ° 3000] a _ 0 I000 I o .I - nooo-j - - 0 fi ‘ -I ’5 . .2 IOO 1 o E j 1 .9 l g . i 52 .4 m :00— t— d 0 w .4 5 1 ° '01 °* - 1 . d :o/ J ‘ j 4 | I 1 1 I r W n T Y 1 m4 Io-5 no" 10‘3 5: IO‘5 lo" 5: IO" IO'3 CONCENTRATION (It) CONCENTRATION (I) I 1 9 c CI IO.OOO-jI q .1 I 1 ‘5 -I I000~ '5 2 g 1 o 4 0 .I H q 1 z 9 ‘ I- j g IOO-4 b 4 w '1 z '1 no: 3‘ 1 I -I g :3 v I i f r t I '0 1— r T W .04 no" 10“ IO" 51 :0-5 Io" 5. IO“ :0-3 CONCENTRATION (I) CONCENTRATION (I) 58 Figure 7. —— Effect of temperature on penetration of NAA and NAAm through the upper and lower surfaces. . NAA upper surface . NAAm upper surface . NAA lower surface . NAAm lower surface uncut» For comparison, NAA penetration equals NAAm penetra- tion x 1.04. PENETRATION (cpm) PENETRATION (cpm) 59 400 - A . 200 4 300 -1 I50 1 E Q 3 200- z 9 I; loo-4 0: p. u: z w 0. I00 -‘ 30 n o T I I *1 o 1 I Y j 5 I3 25 35 5 I3 25 33 TEMPERATURE (C) TEMPERATURE (C) 0000 " c o 3000 4 4300 . “ET 2000 -‘ a 8 3000 .. g s: < a: p. m z 2 I000 4 I500 -I o I T I 1 o I T T fl 5 I8 23 33 5 I3 23 33 TEMPERATURE (CI TEMPERATURE (C) 60 Table 4. Temperature coefficients (010) for penetration of NAA and NAAm into pear leaf disks through the upper and lower surfaces. Temperature coefficients NAA NAAm Temperature range Upper Lower Upper Lower (C) 5-15 1.87 2.35 1.86 5.46 15-25 1.62 1.51 2.23 1.59 25-35 2.95~ 3.03 3.14 4.43 61 Figure 8. —- Time-course of penetration of NAA through the upper surface of pear leaf disks at 2, 17, and 27 C. PENETRATION (com) 2000 1 I750 - I500 -* I250 - IOOO -+ .4 00 O I 500 - 250 - 62 (-——-27C II HI. 8 l2 I6 20 TIME (hr) 24 63 Effect ofppH on Penetration The pH of the treating solution greatly influenced NAA penetration through the upper surface (Figure 9). As the pH was raised from pH 3.0 to 6.0 there was a progressively lesser amount of NAA entering the leaf disk. The pK of NAA is 4.2. At this pH half of the molecules are dissoci— ated, have a negative charge and are polar and half of the molecules are nondissociated, have no charge and are nonpolar. The more nonpolar a molecule, the more lipid soluble it is. In general, as the pH of the treating solution is lowered more NAA molecules occur in the nondissociated form, and there is a corresponding tendency for NAA to penetrate into the leaf. The response of NAA penetration to an increase in treating solution pH through the lower surface was very similar (Figure 10). The most notable difference between the upper and lower surface was that NAA at pH 6.0, when almost completely dissociated, penetrated to a much greater extent into the lower surface as compared to the upper. Effect ofng on Penetration of NAAm Treating solution pH had no significant influence on penetration of NAAm into either the upper or lower surface (Table 5). Since the pK of NAAm is about 14.0, there would be no change in the charge on the NAAm molecule over the pH range studied (3.0 to 7.0). Significantly more NAAm penetrated through the lower surface than through the upper. Effect of Light on Penetration Light Intensipy Increasing light at low intensities resulted in a significant increase 64 Figure 9. —- Effect of treating solution pH on penetration of NAA into pear leaf disks through the upper surface and on the dissociation of the NAA molecule. 400 -. 300 -~ Pm) PENETRATION (c '3 O I I00- R-cuzcoou 65 o—o PENETRATION (cpm) o——a % IN NONDISSOCIATED FORM pK 4.2 / R- CHZCOO‘ 4.0 5.0 - I00 -50 ~25 PERCENT IN NONDISSOCIATED FORM 66 Figure 10. -- The effect of treating solution pH on penetration of NAA into pear leaf disks through the lower surface. 67 30001 2000‘ I000‘ 253 2925sz 60 5O 4O 50 68 Table 5. The effect of pH on penetration of NAAm into pear leaf disks through the upper and lower surfaces. Penetration1 PH Upper Lower (CW) 3 21a 2638 b 4 21a 3269 b 5 24a 3424 b 6 19a 3380 b 7 22a 3149 b 1Means within a column or within a row followed by a different letter are significantly different at P - 0.01. 69 in penetration of NAA (Figure 11) and NAAm (Figure 12) through the lower surface. As the light intensity was increased from 0 up to about 600 ft-c there was a 2.75vfold increase in penetration for NAA and a 4.8-fold in- crease for NAAm penetration through the lower surface. Above 600 ft-c little additional penetration occurred with a further increase in light intensity. The influence of increasing light intensity on penetration through the upper surface was not statistically significant for either NAA or NAAm. Effect of Leaf Age,,and Surface on NAA Penetration in Light and Dark Leaf disks used in the light intensity studies were taken from field grown leaves. Since leaves grown in the field were found to be much less permeable than greenhouse grown leaves, an experiment was also performed using greenhouse grown leaves to determine if a light effect could be de- monstrated for the upper surface. Two leaf ages with different permeability characteristics were used. With leaves of both ages, light increased pene- tration through the upper and lower surfaces (Table 6). Light had a much more profound effect on penetration through the lower surface. Although penetration into young leaves was twice as great as that in the older, the largest increase from the dark control was in the older leaves. The young leaves had not fully matured and were light green in color indicating in- complete chlorOphyll development. Effect of pH on Penetration in the Light_and Dark Greater penetration of NAA through the lower surface occurred in the light and from a pH 3.2 treating solution (Table 7). The most important factor to note is that pH of the treating solution or charge on the NAA molecule can influence the penetration response in light and dark. Light had a more profound effect on increasing penetration of NAA at pH 5.2 than at 3.2. 70 Figure 11. -- Effect of increasing light intensity on penetration of NAA into pear leaf disks through the upper and lower surfaces. PENETRATION (cpm) 71 3000 1 LOWER SURFACE 2000 -I ° nooo- ,‘ UPPER SURFACE EH 1 fl 0 1 J L l I O 50 I00 300 600 I200 LIGHT INTENSITY (fl-c) 72 Figure 12. - Effect of increasing light intensity on penetration of NAAm into pear leaf disks through the upper and lower surfaces. PENETRATION (cpm) 73 I200 - IOOO - I—‘O-i T - LOWER SURFACE j soo~ 600 1 .5 $ 200 - UPPER SURFACE 0M F :3 0 so I00 300 600 1200 LIGHT INTENSITY (fI-c) 74 Table 6. ‘Effect of light on penetration of NAA through the upper and lower surfaces of disks taken from the first and seventh leaf of a terminal shoot. Leaf Leaf Penetration surface number Light Dark Mean (epm) Upper lst 1844 1134 1054** Upper 7th 763 474 Lower lst 6507 2788 3523 Lower 7th 4161 636 Mean 3319** 1258 ---2 1The lst leaf is from the apical portion of the shoot and the 7th from the basal portion. 2Means for the lst and 7th leaf are significantly different at P - 0.01. ' ' ** Significant at P = 0.01. 75 Table 7. Effect of treating solution pH on penetration of NAA through the lower surface in light and dark. Penetration pH Light Dark Mean (epm) 3.2 3471 506 1989** 5.2 912 77 495 Mean 2192** 292 -—-1 ** Significant at P = 0.01. 1 Interaction of light x pH significant at P = 0.01. 76 Effect of Cycling Light and Dark on Penetration When the leaf disks were illuminated there was an almost immediate increase in penetration in response to light (Figure 13). Conversely, when the lights were turned off, the stimulating effect of light was rapidly lost. Regardless of the order in which light and dark were ad- ministered, penetration after 8 hr was the same. A similar experiment was carried out which showed disks were continually and quantitatively responsive to cycling of light and dark treatments (Figure 14). Effect of Light and Stomatal Aperture Width on Penetration An experiment was performed to evaluate the influence of stomatal aperture width on the response of leaf disks to penetration of NAA in the light (Figure 15). Penetration was followed for 3 hr in light. Acetate replica photomicrographs of the stomata at various stages in the experiment are shown in Figure 16.1 When present in sufficient amount, NAA prevented the opening of stomata following closure during a dark period. It was previously determined that a 3-hr treatment in light with NAA was sufficient to prevent the opening of stomata once closed.a From Figure 15 it may be seen that penetration was actually greater into leaf disks during the second light treatment even though the stomata were essentially all closed. Effect of Inhibitors on Penetration Effect of Anaerobiosis on Penetration The least noxious method of inhibiting metabolic processes in the leaf disks was considered to be by pretreatment in nitrogen and then conducting the experiment in nitrogen. The influence of a complete nitrogen atmosphere on penetration of NAA through the upper and lower ‘~ ’- ‘7 Qm' kin. LL'JJ'J 77 Figure 13. —- Time-course of NAA penetration into pear leaf disks through the lower surface as influenced by light and dark treatments. 2400 '1 (cpm) PENETRATION . I800 - E o 600 - 78 o—-——-o CONTINUOUS LIGHT on“... CONTINUOUS DARK ‘I a..-.——a DARK—9 LIGHT a.__--_a LIGHT—9 DARK TIME (hr) 79 Figure 14. —- Time—course of penetration of NAA into pear leaf disks through the lower surface as influenced by cycling light and dark. (cpm) PENETRATION 8O 5000- "I 40004 IJGHT ----- DARK 30004 g 20007 IOOOA —"’ o l l l l j 0 3 6 9 uz l5 . l‘ J I L“: a V_2'w.-'_1I “ arm-v. as." m' 81 Figure 15. - Effect of light and stomatal aperture width on penetration of NAA into pear leaf disks through the lower surface. PENETRATION (com) I 800-- 3200T 2800-4 2400- 2000- Isoo-J I200- 400- 82 STOMATA-37. OPEN; 7 0.3 pm\) STOMATA -0% OPEN; i 0.2”.» LIGHT DARK ) < a o a ‘o \ a a o 0’ STOMATA- ox OPEN; 2? 01pm STOMATA - 7n OPEN; 3'? 2.8m e—STOMATA- sox OPEN; 3:" 23pm L I I I I I I I L I I 2 3 4 5 6 7 8 9 TIME (hr) E' ’11 ELIE! '1‘ - t}. 19:13“ EMA Ira-r are: 3.1 g 1, 83 Figure 16. - Photomicrographs of acetate replicas showing stomata at various periods during the course of NAA penetration with different light regimes. A. B. C. D. E. F. After 1 1/2 hr light pretreatment After 3 hr light treatment After 3 hr light, After 3 hr light, After 3 hr light, After 3 hr light, 1 1/2 hr dark 3 hr dark 3 hr dark, 1 1/2 hr light 3 hr dark, 3 hr light- 85 surfaces is shown in Table 8. Nitrogen was found to have no effect on decreasing penetration through the upper surface. In two instances there was an increase in penetration into leaf disks maintained in the nitrogen atmosphere. However, penetration of NAA through the lower surface was inhibited when leaf disks were kept in an atmosphere of nitrogen prior to and during the course of penetration. In both instances the difference in penetration between nitrogen and air control leaf disks were highly significant. Effect of Inhibitors of the Hill Reaction on Penetration The responsiveness of NAA penetration to light indicated that energy required for this process may be supplied directly by photosynthesis. Atrazine was the first inhibitor chosen because of its relatively high water solubility and since it was known to be taken up more readily by the leaves than other inhibitors. The influence of atrazine on penetra- tion of NAA by the lower surface is shown in Table 9. In the dark there was no difference in penetration between control and atrazine treated plants. At a low light intensity (200 ft-c) penetration was stimulated. This stimulation was partially reversed (21%) by atrazine. At the highest light intensity (1000 ft-c), light stimulation was reversed to an even greater extent (42%). The effect of two additional inhibitors of the Hill reaction, Terbicil and Monuron, are presented in Table 10. Terbicil inhibited NAA penetration by 16% and Monuron inhibited to an even greater extent, 33%. Effect of Various Metabolic Inhibitors on NAA Penetration Penetration of NAA was significantly inhibited by uncouplers of oxidative phosphorylation (Table 11 and 12): carboxylcyanide m-chloropheny- Table 8. Penetration of NAA into pear leaf disks through the upper and lower surfaces in atmospheres of nitrogen and air. Penetration Surface Expt. Nitrogen Air ' (cm) I 554a 195 b Upper II 349a 341a III 357a 213 b Lower I 1017a 4327 b II 1512a 3799 b 1 Means within an experiment followed by a different letter are significantly different at P = 0.01. 87 Table 9. The effect of light and an inhibitor of the Hill reaction (Atrazine)l on penetration of NAA into pear leaf disks through the lower surface. Light intensity Control Atrazine Inhibition (ft-C) (Cpm) (Z) 0 (dark) 614 614 0 200 1139* 896 21.3 1000 3099** 1799 41.9 1 2-chloro-4-ethylamino-6-isopropyl-amino-s-triaxine (l x 10"5 M) * Significant at P - 0.05 ** Significant at P - 0.01 Table 10. Effect of Hill reaction inhibitors on penetration of NAA into pear leaf disks through the lower sur- face and illuminated with 1000 ft-c of light. Treatment Penetration Inhibition (Cpm) (Z), Control 6491 Terbicill 5455 16.0* Monuron2 4346 33.0** 1 3-tert-butyl-5-chloro-6dmethyluracil (l x 10'4 M). 2 3-(p-chlor0phenyl)-l, dimethylurea (1 x 10’4 M). * Significant at P - 0.05. ** Significant at P - 0.01. :1“ pr . .3, . .. .O-NHE'IN a}; fi _ 88 Table 11. The effect of inhibitors on penetration of NAA into pear leaf disks through the lower surface. Treatment Penetration3 Inhibition (Cpm) (2) Control 4527a m-Cl-CCPl 3585 b 20.8 p-F-ccp2 2428 b 47.4 l Carbonylcyanide m-chlorOphenylhydrazone (1 x 10"5 M). 2 Carbonylcyanide p-trifluoromethoxyphenylhydrazone (1 x 10'”5 M). 3 Means within a column followed by a different letter are significantly different at P = 0.05. Table 12. The effect of inhibitors on penetration of NAA into pear leaf disks through the lower surface. Treatment Penetration1 Inhibition (Cpm) (2) Control 5257a ON?2 1219 b 76.9 PMA3 3422 c 35.0 NaN34 5230a 0.7 ,1 Means within a column followed by a different letter are significantly different at P = 0.01. 2 2,4 dinitrOphenol (l x 10"3 M). 3 Phenylmercuric acetate (1 x 10'4 M). 4 Sodium azide (1 x 10"3 M). 89 hydrazone (20.8%), Carboxylcyanide p-trifluoromethoxyphenylhydrazone (47.4%), and 2,4 dinitrophenol (76.9%). Phenylmercuric acetate reduced NAA penetration by 35.0% while sodium azide was found to have no effect (Table 12). Effect of.Surfactants on Penetration Tween 20 and Tergitol 15—S—9 at 0.001, 0.01 and 0.1% concentrations were found to be ineffective in increasing NAA penetration through the upper surface (Table 13 and 14).- However, penetration through the lower surface was.significantly increased when treating solutions containing 0.01 or 0.1% surfactant were used. Tween 20, Triton B-1956, and X-77, differing in ability to reduce surface tension, were evaluated on penetration of NAAm through the upper and lower furfaces. A 0.1% concentration was chosen because the critical micelle concentration for all three surfactants occurred at or slightly below 2L01%. No surfactant significantly increased NAAm penetration through the upper surface (Table 15. Tween 20 and Triton B-l956 were effective in increasing penetration through the lower surface. However, the greatest increase in penetration through the lower surface occurred with X-77. It would appear that surfactant stimulation of NAA and NAAm penetra— tion through the lower surface did not occur to the same extent. Tween 20 (0.01%), the surfactant used for both compounds, increased NAAm penetra- tion to a much greater extent than it did for NAA. This may be seen by comparing data reported in Table 13 and Table 15. Although no other surface active agents were used common to both, surfactants tended to increase pene- tration of NAAm by the lower surface more than of NAA. EmnIh . ._v .I. .fl...r_ns..fls mm“ ‘lea e 90 Table 13.- Effect of Tween 20 concentration on penetration of NAA into pear leaf disks through the upper and lower surfaces. Penetration1 Concentration Upper Lower (%) (Cpm) 0 1323a 2408a 0.001 937a 3129a 0.01 756a 4570 b 0.1 1038a 4375 b 1Means within a column followed by a different letter are significantly different at P = 0.05. Table 14. Effect of Tergitol lS-S-9 concentration on penetration of NAA into pear leaf disks through the upper and lower surfaces. Penetrationl Concentration Upper Lower (Z) (Cpm) 0 530a 4316a 0.001 421a 4467a 0.01 678a 7021 b 0.1 722a 8067 b lMeans within a column followed by a different letter are significantly different at P a 0.05. 91 Table 15. Effect of surfactants (0.1%) on penetration NAAm into pear leaf disks through the upper and lower surfaces. Penetration1 Treatment Upper Lower (epm) Control 263a 329a Tween 20 220a 1941 b Triton B-1956 297a 1917 b X-77 393a 2833 c 1Means within a column followed by a different letter- are significantly different at P - 0.01. 92 Stomatal Penetration Effect of Increasing Light Intensity 0n Penetration and Opening of Stomata There was a close relationship between penetration and stomatal opening (aperture greater than 2Um), as light intensity was increased (Figure l7)° From this, it would appear that the stomata, in some way, may be involved in bringing about the light stimulated NAA penetration. Frequency Distribution of Stomatal Aperture Width in the Lighg It was mentioned previously that stomatal aperture widths are influenced by a variety of environmental factors including quality and duration of light, temperature, C02 conc., water stress, etc. After measuring a large number of stomatal aperture widths it was ob- served that there was also a wide variation in the width of stomatal pores, even within a particular leaf disk. Results of an experiment show aperture widths in leaf disks varied between 0 t0 5 8 pm (Figure 18). The largest number of stomata had aperture widths in the range of 2 to 5 pm and some stomata were closed or had aperture width in excess of 6 pm. Therefore, it is difficult to accurately specify stomatal aperture width even within a given leaf disk. Effect of Surfactants on Surface Tension The reduction in surface tension of a treating solution is neces- sary if penetration through a stomatal pore is to take place. The in- fluence of surfactant concentrations on surface tension is presented in Figure 19. As the surfactant concentration was increased the surface tension of the solution decreased. Surfactants differed in their ability to reduce surface tension. Tween 20 was least effective, Vatsol 0T the . wwul 93 Figure 17. -— Comparative effects on increasing light intensity on penetration of NAA into pear leaf disks through the lower surface and the per cent of stomata open. PENETRATION (Cme 2400- I800- I200- 600- 94 ~I00 T A. ~75 ~50 o————-o PENETRATION '25 r a XSTOMATA OPEN 92me I l o 0 50 I00 300 600 I200 LIGHT INTENSITY (ft-c) STO MATA OPEN (‘loI '0 “‘ln‘ .“ fi-um V g ”x... ‘ :~.gg_;,~.-.u Figure 18. -- Frequency distribution showing per cent stomata with a particular stomatal aperture width falling; within a given size class. .25 “w 20'- I5- IO- O 96 I I l I I I I I 2 3 4 5 6 7 8 STOMATAL APERTURE WIDTH (pm) I t'é‘PT'WIP—I run “and ‘ be: emf-mg; :4 97 Figure 19. -- Effect of varying concentrations of selected surfactants on surface tension of buffered NAA solutions. 80- 70- 98 o-——o TWEEN 20 o——o TERGITOL I5'S'9 0——0 VATSOL OT e——¢ x-77 a__aTRITON x-IOO ~——ATRITON 8 I956 I I) Z 9 (0 Z LIJ '— 8 3o- . g 20... O 1. ‘3 1 1 J L l L 4 OJ 0.5 I.O 0.005 0.0I 005 SURFACTANT CONCENTRATION (°/oI 99 most effective and X-77, TritonB—l956, Triton X-100 and Tergitol 15-5-9 were intermediate in reducing surface tension at the higher sur- factant concentrations. Triton B—1956 was most effective in bringing about a reduction in surface tension at the lowest concentration (0.001%). The critical micelle concentration (cmc) was defined earlier as the con- centration at which additional surfactant would no longer reduce the sur- face tension. The cmc for all surfactants studied was between 0.01 and 0.1%. Effect of Tween 20 on Penetration with Stomata'Either Open or Closed A time-course study showing the effect 'of Tween 20 on penetration of NAA through the lower surface with stomata either open or closed is presented in Figure 20. Surfactant-containing solution (0.1%) was.added initially to one group of leaf disks and penetration was observed to be linear. The sequence of light-dark-light treatments for the second group was given to provide.leaf disks having stomata closed and to provide a similar light treatment. After Tween 20 was added, the penetration response with leaf disks having stomata closed and in light was linear and quanti- tatively similar to leaf disks with the stomata open. Effect of Vatsol OT on Penetration Surface tension data presented in Figure 19 showed that Vatsol OT The was ., the most effective surfactant at 0.1% in reducing surface tension. addition of Vatsol OT to the treating solution at ,3 hr after initiation of the experiment caused a sharp increase in NAA penetration (Figure 21). - This increase was greater than that caused by the addition of Tween 20 (Figure 20). A number of small water soaked areas appeared over the surface of the leaf disk within 1 min after the addition of the Vatsol OT. The water-soaked l w . i k ‘1 cr- mdruxr. .w— 100 Figure 20. -- Effect of Tween 20 on penetration of NAA into pear leaf disks through the lower surface with stomata open and closed. Stomata were opened initially by pretreatment with 1 1/2 hr of light. 4oooJ 3000-1 PENETRATION (cpm) N . I IW‘ 101 .......-.._ LIGHT+TWEEN 20 ' /’ IJGHT j """’".' DARK ’- SIMEAN 'APERTURE WIDTH (mu) /. l I I I I I O I 2 3 4 5 TIME (hr) 1.3.. p 1.. . ifl,.r1(a.|..:,fl butts-[#4 ifiu (g. «Nu-‘,.I§ “I" denim-Eu.” 102 Figure. 21. -- Effect of Vatsol OT on penetration of NAA into pear leaf disks through the lower surface after 3 hr exposure to the light. PENETRATION (cpm) 4000 - 3000 '- 2000 -* IOOO - o I 2 103 O-——O MINUS VATSOL OT H PLUS VATSOL OT VATSOL OT ADDED \ . . e o I I 1 3 ‘ 4 TIME (hr) d 104 areas varied in number from leaf disk to leaf disk but generally between 20 and 60 appeared. This would suggest that Vatsol CT may have decreased the surface tension to the point where stomatal entry occurred. Effect of Surfactants on Penetration of pH 6.0 TreatinLSolution To differentiate between the effect of surfactants on enhanced cuticular penetration and stomatal entry, penetration of NAA from solutions ‘ buffered at pH 6.0 was followed subsequent to the addition of surfactants (Figure 22). At pH 6.0 about 98% of the NAA is in the dissociated form Cuticular penetration of NAA buffered at kn... and thus has a negative charge. I pH 6.0 is very slow but stomatal entry should not be influenced. There was a rapid increase in penetration immediately following addition of all three surfactants. There was a significantly larger increase in penetra- tion after 45 min with Vatsol OT or X—77 compared to Tween 20. Vatsol OT and X-77 were also found to reduce surface tension to the greatest extent (Figure 19). The Tukey w value shown on the graph is the difference re- quired for significance at the 5% level after 45 min° Effect of C09 Treatment on Penetration High C02 concentrations will induce closure or prevent the opening of stomata. Results showing the influence of Tween 20, X-77, and Vatsol OT on penetration of NAA for 10 min into leaf disks maintained in atmospheres of high or low C02 levels are shown in Tables l6, l7, and 18, respectively. In addition, two different NAA concentrations but with the same radioactivity were used for each C02 treatment. Microsc0pic examination of leaf disks confirmed that 002 completely closed stomata, with all three surfactants. Penetration was significantly greater in leaf disks kept in the low C02 atmosphere and having open stomata. It may be further noted that there was 105 Figure 22. —- Effect of Vatsol OT, X-77, and Tween 20 on penetration of pH 6.0 NAA solution into pear leaf disks through the lower surface after ‘3 hr pretreatment in light ,. PENETRATION (0pm) '200- 700 1 600 ~ 500 - 400 - 300 - I00 d 106 o-—-o VATSOL OT a————a x-77 o——o TWEEN 20 TIME (hr) TUKEY Vl- VALUE Imps 107 Table 16. Effect of Tween 20 surfactant on penetration of NAA into pear leaf disks through the lower surface with stomata open (-C02) and closed (+C02). Penetration Treatment 6 x 10‘5 l x 10'3 Mean (epm) -COZ 116 92 104** +C02 62 59 61 Mean 89 76 **Significant at P = 0.01. Table 17. Effect of X-77 surfactant on penetration of NAA into pear leaf disks through the lower surface with stomata open {-002) and closed (+C02). Penetration Treatment 6 X loo-5 1 X 10-3 Mean _ (epm) 9002 230 241 236** +C02 98 62 80 'Mean 164 152 **Significant at P = 0.01. Table 18. Effect of Vatsol OT surfactant on penetration of NAA into pear leaf disks through the lower surface with stomata open (-C02) and closed (+C02). Penetration Treatment 6 x 10’5 l x 10‘3 Mean (epm) -COZ 534 505 520** +C02 112 83 98 Mean 323 294 “Significant at P - 0.01. 108 ‘no difference between penetration of NAA from the two different con- centrations. The 10"3 M solution contained 16.7 times more NAA than 6 x 10‘5 M solution yet the activity (1.0 um/ml) was the same. There- fore, it was the volume of solution entering the leaf and not the NAA concentration that was the important factor in determining a difference in-penetration.with stomata Open and closed. Although experiments were done at different times, it appears that the amount of penetration may be related to the extent to which the surfactant reduced the treating solution surface tension and permitted movement through the stomatal pore. Penetration of Silver Nitrate AgNO3 (0.1 M) was utilized to document whether or not the treating solution entered the substomatal chamber. Treating solution containing 0.1% Vatsol OT was applied for 4 min to leaf disks pretreated for 3 hr in the light to open the stomata. Photomicrographs of the leaf cross sections are illustrated in Figure 23. Reduced silver, photographed as black spots, was‘present in the substomatal cavity to varying degrees (Figure 23 AuD). Reduced silver may also be observed in the stomatal pore (Figure 23 C). Cuticular penetration, even for this short period of time, was observed especially over veinal areas (Figure 23 E, F). Reduced silver was frequently found in the xylem (Figure 23 E). Effect Of Leaf Age_on Penetration As the leaf age increased, penetration through the upper surface tended to decrease but this difference was not significant (Table 19). However, penetration of NAA through the lower surface decreased signifi- cantly with increasing age. The influence of increasing leaf age on penetration was most pronounced in the younger leaves. 109 Figure 23. —- Transverse sections of leaf disks treated with silver nitrate for 4 min. A.- D. Silver nitrate in substomatal cavity E.- F. Silver nitrate in the cuticle over veins 110 111 Table 19. Effect of leaf age on penetration of NAA into pear leaf disks through the upper and lower surfaces. Penetration Leaf number Upper Lower (epm) 3 (youngest) 2106a 7959a 5 1561a 6166 b 7 956a 4875 c 9 1035a 3355 c 11 750a 2649 c ‘13 (oldest) 733a 3246 c 1Means within a column followed by a different letter are significantly different at P = 0.05. F 112 Penetration of NAAm through the Upper and lower surfaces decreased as leaf age increased (Figure 24). There was not a significant difference between penetration through the upper and lower surfaces. However, the. interaction was significant at P = 0.01. More NAAm entered the youngest leaves through the upper surface. As leaf age increased NAAm penetration was reduced to a greater extent through the upper surface so that more I—* penetration occurred through the lower surface in the older leaves. A direct comparison of NAA and NAAm penetration as influenced by leaf age is not possible since the trees used in the NAAm experiment were grown in the growth chamber and the trees used in the NAA experiment were grown under natural conditions in the greenhouse during March. Effect of Droplet Drying on Penetration The effect of drOplet drying on subsequent penetration of NAA and NAAm by the upper and lower surfaces was manifest in all cases as an immediate increase in penetration. Following the initial increase after drying, penetration appears to continue to increase for the upper surface. This was much more apparent for NAAm. However, subsequent to the initial surge in penetration following droplet drying there appears to be little additional penetration for the duration of the experiment through the lower surface. Microradioautographic Study of 3H-NAA Penetration Through Isolated Cuticle The results of a microradioautographic investigation of possible pathways of 3H-NAA penetration through the enzymatically isolated upper cuticle of pear leaves are shown in Figure 26. The cuticle in cross 113 Figure 24. -— Effect of leaf age on penetration of NAAm into pear leaf disks through the upper and lower surfaces- (cpm) PENETRATION 114 IOOO .. . UPPER E 800 d ‘ 600 - . LOWER 400 - . _ . 200 - . (YOUNGEST) (OLDEST) 0 1 1 1 1 J n 3 5 7 9 II I3 LEAF NUMBER .57 "V ‘ lamb . .I‘ 5‘ rim“ ‘ o. nu, 115 Figure 25. - Time-course showing the effect of droplet drying on penetration of NAA and NAAm into pear leaf disks through the upper and lower surfaces. A. B. c. \c. NAA upper surface- NAAm upper surface NAA lower surface NAAm lower surface I § PENETRAM (can) § .4 0 HM?” MW“? h p b 116 h—qmm I—r 76+ 30-1 254 é—MT m o ‘ 4 1 a 1 1 O I! a. fi 4. 7: TIME (hf) 000- o 000- 000‘ gush E E COO-I m “ED 0004 I TIME (hr) 117 Figure 26. —- Microradioautograms illustrating the localization of 3H—NAA in isolated upper pear leaf cuticle in cross section. A. Light field photomicrograph B. Dark field photomicrograph 1...- ha.- 118 ~ 0 ‘ A: . 'cor. ‘ "' J — . . - » '«Q‘Vwa'a'tt :“‘I ' . \O- ' o' 'o. .. ‘.'.-' ’ . . .h.\ at" -.'.‘ t?;':: . Jaggfif :.-‘): 03'...'0 $./§€;‘ .. ‘\ ’ lovyo$.,.‘ ‘+o ' . 0".;|" -.. u ..-'\- ’- . ' f . ‘-.o ' o 3" ' ' I} ll'lllllll. i llcllllll I‘ll-Ill. ‘Ill- 119 section appears to be uniformly labeled with no preferential areas of penetration following a lZ-hr penetration period. DISCUSSION The process by which a foliar applied chemical can pass through the cuticular barrier and enter the living continuum of the plant is cmuflex and may be controlled or influenced by a number of factors. An improved method of studying foliar penetration is described that allows a critical evaluation of the relative importance of the factors and the conditions under which they are most important in affecting penetration. Penetration of NAA and NAAm into pear leaf disks through the up- per surface was found to be linear with time. This is in agreement with results of Bukovac and Norris (1966) for penetration of NAA through the isolated upper cuticle of the pear leaf. Extrapolation of the penetra- tion curve back to zero time reveals that the curve does not pass through the origin but intersects the abscissa somewhat above this point. Rapid initial binding of NAA and NAAm to the upper surface of pear leaf disks has been shown to occur by Bukovac and Norris (1966). This may explain the initial deviation from linearity of NAA and NAAm penetration. Rapid initial absorption for the first 24 to 48 hr followed by a gradual decrease in rate of penetration with time characterized the penetration of NAA and NAAm through the lower surface. Time-course curves sinfllar to this have been reported by Bukovac (1965) for 3-CP penetration into peach leaves, Hughes ,and Freed (1961) for IAA penetra- tion into bean, and Luckwill and Lloyd-Jones (1962) for NAA penetration into apple leaves. The end of rapid initial absorption through the lower surface occured after about 24 hr for NAA and 48 hr for NAAm. 120 121 The amount of NAA penetration at any given time was approximately twice that of NAAm. Therefore, the difference in the shape of the time-course curve may be attributed to different rates of penetration. Penetration curves for NAA.when applied via glass cylinders or in the form of microdroplets were similar. This point is particularly sig— nificant if information gained using glass cylinders is to be applicable to penetration from a spray droplet applied to a leaf. Rapid penetra- tion occurred as the droplet dried. Although penetration was not deter- mined immediately following droplet drying, most of this increased pene- tration was assumed to have occurred during the drying process. Norris and Bukovac (1969) have suggested that increased penetration, as the droplet dried, through isolated pear cuticle may be related to the in- creased concentration resulting from the loss of the aqueous phase. NAA and NAAm penetration into the lower surface appeared negligible from the droplet residue. Some penetration from the residue appeared to occur through the upper surface but the rate was much more pronounced for NAAm. Penetration through the cuticle is thought to occur by diffusion (Currier and Dybing, 1959; Franke, 1967). The linear relationship of penetration with time and concentration observed in this study would tend to substantiate this for leaf disks. At high treating solution concentration (above 10'4 M) for NAA, or after a considerable amount of NAA or NAAm penetration (24 to 48 hr) into the lower surface, devia- tion from linearity occurred probably because of an approach to satu- ration. The rapid increase and decrease in penetration with the cycling of light and dark showed that the underlying cells may play an active 122 role and thus regulate diffusion through the lower cuticle by influenc- ing the diffusion gradient. However, this is unlikely to be true for penetration into the upper surface of older leaves, where in some in- stances penetration was less than 1% of that observed for the lower surface. Zenk (1962) has shown that NAA can influence its own uptake by pea epicotyl sections. There was a lag phase in NAA absorption which repre- sented the time required for sufficient NAA to be taken up by the sec- tions to induce an enzyme which conjugates NAA.with L-aspartate. Fol- lowing this induction phase there was an increase in NAA absorption. No attempt was made to identify an NAA-aspartate conjugate in pear leaf disks. However, if this or other NAA conjugates were formed in the pear leaf disks resulting in increased penetration, this should have been apparent as a deviation from linearity in the time-course study. None was observed. In addition if an inducible enzyme was formed then there should not be a linear increase in penetration with increasing concentration. Some threshold NAA value should be required to bring about induction of the enzyme. Although the cuticle on the lower sur- face may impede NAA penetration, it is not considered a great barrier preventing a sufficient amount of NAA from entering the leaf disk to bring about the induction of an enzyme.' This conclusion is reached for several reasons. (a) Saturation of the leaf disks occurred after about 24 hr. (b) Leaf disks could also be saturated when NAA concentrations above 10'4 M were used. (c) Light-stimulated penetration could be shown up to 7 times greater than penetration in the dark. The temperature coefficients (Q10) of 2.0 or above have been used as criteria for an active uptake process (Franke, 1967; Rice, 1948; 123 Sargent and Blackman, 1962). A Q10 value of 2.0 is representative of many biological processes (Giese, 1962) , forming the basis for the con- clusion that foliar penetration of a compound is an active process. However, recently Norris and Bukovac (1969) observed Qlo values for pen- etration of NAA through isolated pear cuticle as high as 5.5-6.0 between 15 and 25 C. Therefore, this study of temperature influence on leaf disk penetration must be interpreted in light of at least two potential temperature dependent processes as determining the Q10 value, the cuti- cle and the underlying cells. The leaves used in this investigation were fully mature. Photo— micrographs of transverse sections stained with Sudan III and Sudan IV reveal a cuticle present of significant thickness. The‘cuticle is lipoidal in nature. Higher Q values may be e ected for lipoid mem- 10 "P branes (Suttcliffe, 1962). A diffusing molecule must acquire suffi- cient kinetic energy to overcome the large potential energy barrier when passing from a solution into the cuticle and also overcome a series of smaller energy barriers while passing through the cuticle. High Q10 values exist because at higher temperatures more molecules acquire sufficient energy to diffuse in a given time. Further, within the cuticle there is a continuous layer of oriented embedded waxes in the upper cuticle and a discontinuous layer in the lower (Norris and Bukovac, 1968a). It has been suggested (Van Overbeek, 1956) that at low temperatures permeability of this layer of oriented waxes may be low and hydrogen bonding would tend to be extensive. Penetration at all temperatures was higher through the lower sur- faces than the upper surface. This difference may be partially 124 .attributed to the discontinuity of embedded waxes in the lower surface previously mentioned (Norris and Bukovac, 1969). Greater temperature dependence would be expected for penetration through a layer of oriented 'waxes. Creater penetration into guard cells and accessary cells, pres- ent only on the lower surface, may also contribute to the greater pene— tration through the lower surface. If penetration through the lower cuticle is greater than metabolically mediated uptake by the cells, at a given temperature, then the Q10 could be attributed to active uptake. At 25 C penetration through the lower surface of field grown pear leaves was sufficient so that light stimulation of penetration could be demon- strated. Therefore, the Q10 at.this temperature is at least in part metabolically determined. This is not true for the upper surface. Temperature coefficients for penetration of NAA through the upper surface of pear leaf disks would indicate that the high Q10 values for pene— tration are due to the embedded waxes and not the surface waxes. Sur- face wax accumulation usually ceases with leaf expansion but embedded waxes may still be deposited (Schieferstein, 1957). The Q10 value fOr penetration into young mature leaves, between 15 and 25 C, was 1.61. The Q10 value for leaves 6 weeks older, between 17 and 27 C was 7.7. Penetration of NAA through the upper and lower surfaces increased as the pH of the treating solution decreased. The pK of NAA is 4.2. Below the pK more NAA is in the nondissociated form, has no charge and is lipophilic. As the treating solution pH was increased above 4.2 an increasing number of NAA molecules become dissociated, have a nega- tive charge and are more hydrophilic. Since the cuticle is lipoidal in nature, greater penetration would be expected of the more lipophilic 125 nondissociated molecules. Increased penetration of NAA at pH values below the pK is in agreement with other investigators who have reported great- er penetration of weak acids in the nondissociated form (Albert, 1951; Crafts, 1953; 1956; Swanson and Whitney, 1953). It has been suggested that weak acids are taken up largely in the nondissociated form (Weintraub et al., 1954). Diffusion studies of 2,4-D into and out of Chlorella cells led Wedding and Erickson (1957) to conclude that the relative permeability of dissociated and nondisso- ciated species was a constant ratio, independent of each other in the external solution. Penetration of NAA, from solutions buffered at a high pH, especially for the lower surface, leave no doubt that the dis- sociated NAA species can enter the leaf. Differences in the method used in this investigation do not allow direct comparison with results of Wedding and Erickson (1957). However, if the relative permeabili- ties of cells to dissociated and nondissociated species exists then it must be concluded (Table 7) that this does not occur under all condi- tions. Penetration in the light from a pH 5.2 solution was nearly twice as great as that from a pH 3.2 solution when compared with their respective dark controls. It is suggested that in the dark some NAA from the pH 3.2 solution can partition into the cell because 90% of the molecules are undissociated and thus lipophilic. NAA penetration from the pH 5.2 solution in the dark will be less because only 5% are in the nondissociatedlipophilic form. Under conditions where light is present it would appear that energy may be provided to transport the dissociated NAA species across the plasmalemma and thus increase the diffusion gradient across the cuticle. 126 Penetration of NAAm into the upper and lower surfaces was in- dependent of the treating solution pH. Since the pK of NAAm is about 14, the NAAm molecule was undharged over the pH range studied (pH 3.0- 7.0). Therefore, it is concluded that the influence of pH on the pene- tration is related to its effect on the penetrating molecule and not on the cuticular surface. The influence of pH on penetration of NAA and NAAm is in agree- ment with the influence of pH on binding of NAA and NAAm by pear leaves (Bukovac and Norris, 1966). It also substantiates evidence from the above studies that electrostatic binding may not be critical for the uptake of growth substances. The effect of light on increasing foliar penetration of plant growth substances is not well documented in the literature. Sargent Blackman (1962, 1965) were able to show light dependent uptake of 2,4-D by Phaseolus vulgaris leaf disks. This system was complex in that it was dependent upon pretreatment time in the light or dark, 2,4-D con- centration and light intensity above 1000 ft-c. The absence of a light effect or a small light effect (Herrett.and Linck, 1961; Kamimura and Goodman, 1964; Smith et al., 1959; Weintraub et al., 1954) may be due to several reasons: (a) the growth substance was not taken up, thus penetration would be unaffected by energy provided by light, (b) the method of application was such that spray droplets dried soon after application, thus limiting the amount entering the plant, (c) the cuti— cle limited penetration to such an extent that a light effect could not be detected as was demonstrated herein (Figures 11 and 12) for penetration into pear leaf disks through the upper surface. 127 Increasing light intensity was shown to bring about increased NAA and NAAm penetration through the lower surface until a light intensity of about 600 ft-c had been reached (Figures 11 and 12). Bohning and Burnside (1956) have shown that C02 fixation in shade leaves responded in almost an identical manner to NAA and NAAm penetration with increas- ing light intensity. The seemingly greater stimulation of NAAm pene- tration by increasing light intensities as compared with NAA.is mislead- ing. In subsequent experiments where NAA penetration was measured in the light and dark (Tables 6, 7 and Figures 12, 13, and 14) the increase over the dark controls was always at least 6-fold unless young leaves, with incomplete chlorophyll develOpment were used (Table 6). Therefore, the light stimulated uptake of NAA and NAAm was quantitatively similar. The fact that the light effect may be related to photosynthesis because of the similarity of the CO fixation and penetration curves 2 has already been mentioned. Further evidence supporting this is shown in Table 6. Penetration into young leaves, having incomplete chloro- phyll development, was about 3 times less than penetration into mature leaves, with complete chlorophyll development, when compared with their respective dark controls. However, chlorophyll development was an ob- servation and not an actual determination. Further characteristics of the light effect are: it was immediately lost or gained when leaf disks were transferred from light to dark or from dark to light (Figure 12). The effect can be "turned on" or "turned off" repeatedly with quantita- tively related penetration in each case (Figure 13). The light-enhanced uptake of NAA.was not related to stomatal opening (Figure 15). It is therefore pr0posed that the energy for the light effect was provided by photosynthesis. Since the light effect is lost and gained 128 so rapidly, degradation of starch to provide energy is unlikely. The probable source of energy was one or a combination of the primary prod- ucts of photosynthesis such as ATP, NADPH, or simple sugars. The most definitive evidence that photosynthesis was involved in the light effect was provided by studies using photosynthetic inhibitors which specifically block the Hill reaction. Atrazine is readily ab— sorbed by the Leaf (Klingman, 1961) and is a specific inhibitor of the Hill reaction (Moreland et al., 1959). No inhibition by Atrazine oc- curred in the dark, some occurred at low light intensity (Table 9). Two other Hill reaction inhibitors, Terbicil and Monuron (Crafts, 1961), significantly inhibited NAA penetration in the light, which is further evidence implicating photosynthesis in light-stimulated NAA penetration. Difficulties were encountered when metabolic inhibitors were em- ployed. Determination of the amount of each inhibitor diffusing through the cuticle to the underlying cells was not possible. Concentration and length of pretreatment were established so that there was no visible injury to the leaf at the end of the experiment. However, comparison of inhibitor effects on penetration is not valid because the actual amount of each inhibitor reaching the site of action in the cell remains unknown. Infiltration of the leaf disks proved unacceptable because cracks in the cuticle were created during the infiltration process. The most satisfactory method to inhibit metabolic processes in the leaf disks was to conduct penetration experiments in an atmosphere of nitro- gen. Penetration of NAA into the lower surface was significantly re- duced in an anerobic atmosphere (Table 8). This inhibition was probably due to both a lack of oxygen for metabolic processes and insufficient 129 CO2 for photosynthesis. At present there is no explanation for increased penetration in two instances with NAA through the upper surface. A lack of inhibition is most likely due to the cuticle offering such a barrier to NAA penetration that an active component could not be detected. Uncouplers of oxidative phosphorylation, m-Cl—CCP (Heytler, 1963), p-F-CCP (Hopfer et al., 1968) and DNP (White et al., 1964) all signifi- cantly reduced NAA penetration suggesting that catabolism of simple sugars provided some energy stimulating NAA penetration. Heytler (1963) has also shown that m-Cl-CCP can also inhibit photophosphorylation in the chloroplast. Phenylmercuric acetate, an inhibitor of stomatal open- ing, is postulated to act by combining with sulfhydryl groups in or near the membrane (Zelitch, 1963). Since stomatal aperture width has been shown not to be associated with the light effect (Figures 15 and 16), it may be that PMA reacts with the sulfhydryl group of an enzyme or enzymes responsible for transport across the plasmalemma. Stomatal entry in the control leaf disks is precluded because stomata do not open completely when treating solution is present during the period of opening. The lack of inhibition with sodium azide does not necessarily imply that NaN3 does not inhibit NAA penetration. As explained earlier, it may not have entered the cell in sufficient quantities to bring about inhibition. Surfactants reduce surface tension and thus ensure greater con- tact of the treating solution with the plant surface. This is espec- ially true with irregular or rough leaf surfaces. The upper surface of the pear leaf was shown to be relatively smooth. Of the surfactants studied, none significantly increased penetration of NAA and NAAm through 130 the upper surface. However, penetration of both NAA and NAAm was in- creased through the lower surface, which was observed to be rougher and more irregular. There was a significant difference in penetra- tion of NAA through the lower surface at surfactant concentrations between 0.001 and 0.01%. From Figure 18 it is seen that the surface tension of the treating solution must be reduced to between 45 and 42 dynes/cm2 before a significant increase in penetration can be detected. westwood and Batjer (1960) reported that Tween 20 increased penetration of NAA by both surfaces of apple leaves. However, in this study no increased penetration of NAA through the upper surface of the pear leaf was observed. There are numerous reports in the lit- erature of increased penetration through the lower surface by various surfactants. In addition to increased penetration as a result of a reduction of surface tension Freed and.Montgomery (1958), Jansen (1964), and Wéstwood and Batjer (1960) have emphasized that the molecular inter- action between the surfactant and the growth substance may be of equal or greater importance and this would appear to be the case with surfactant influences on penetration of NAA and NAAm into the lower surface. Tween 20 at 0.1% increased NAA penetration by less than 2-fold but increased NAAm penetration by 9-fold. Tween 20 and Triton B-1956 were equally effective in increasing NAAm penetration into the lower surface. However, X-77 was significantly better than these which is in agreement with Westwood and Batjer (1960). The highest surfactant concentration used in this study was 0.1%; a concentration that is at or slightly above the critical 131 micelle concentration. Therefore, the results reported here are not likely to be the result of solubilization of waxes or other water-in- soluble material in the cuticle that is suggesred to occur at surfac- tant concentrations above the critical micelle concentration (Osipow, 1964; Parr and Norman, 1965). Several approaches were taken to determine the importance of stomatal penetration. Surfactants have been shown to differ in their ability to reduce surface tension (Figure 19). Vatsol OT was found to be the most effective in this respect. When Vatsol OT was added to the treating solution in a time-course study (Figure 21) there was a dramatic increase in penetration. This was considered an indi- cation of, but not proof for, stomatal penetration since the treat— ing solution was buffered at pH 3.0. When the surfactant was added, more NAA in the nondissociated form could come in contact with the surface thus increasing penetration. The effect of pH on penetration has also been shown to be dramatic (Figure 10). To differentiate be- tween the effect of surfactants on enhanced cuticular penetration and stomatal entry an experiment was performed with NAA at pH 6.0 and using three surfactants: Vatsol OT, X-77, and Tween 20. Immediately follow- ing addition of surfactants there was a rapid increase in penetration. Stomatal penetration is indicated since essentially all of the NAA molecules were dissociated and cuticular penetration would be expected to be at a minimum. The surfactants which reduced the surface tension to the greatest extent were most effective in causing enhanced pene- tration. Additional evidence for stomatal penetration is shown in Tables l6, l7 and 18, where surfactant-containing treating solutions were 132 added to leaf disks with stomata open, or closed with C02. In all cases penetration was significantly greater with stomata open, and the degree of penetration was related to the surface tension of the treat- ing solution. There was no significant difference in penetration from 1 X 10"3 M and 6 X 10'5 M solutions containing 1.0 uc/ml even though the l X 10‘3 M solution was diluted by a factor of 16.6 with non- labeled NAA. Therefore, differences in penetration must be attributed to stomatal entry since the volume of solution and not the concentra- tion was shown to be the important factor. Calculations were made to preclude the possibility that the in- crease in penetration was due to the filling of the stomatal pore and not actual passage of the solution into the substomatal cavity. The amount of NAA attributed to stomatal entry from a Vatsol OT solution was 422 cpm (Table 18). This was determined by subtracting the cpm entering leaf disks maintained in a high 002 atmosphere with stomata closed (cuticular penetration) from the control leaf disks having stomata open (cuticular plus stomatal penetration). Since the effi- ciency of the Low Beta II counter was 10% and a 1.0 uc/ml solution was used, 1.9 X 109 um3 of treating solution entered each leaf disk. It was determined that 12,500 stomata were exposed to the treating solu- tion. Therefore, if the increased penetration was due to the filling of stomatal pores, the volume of each would have to be 160,000 um3. Approximate pear stomatal pore dimentions were determined: length 15 um, width 5 pm, and depth 6 pm, giving a volume of 450 um3. There- fore, the volume of solution entering the leaf was approximately 350 times greater than the volume of the stomatal pores. Similar calcula- tions for X-77 (Table 17) and Tween 20 (Table 16) indicated that the 133 volume of treating solution entering the leaf was 156 and 44 times as great, respectively, as can be accounted for by stomatal pore volume. It is concluded that NAA passed through the stomatal pore and entered the substomatal cavity. The extent to which the treating solution filled the internal air space was also calculated. Leaf thickness was determined to be about 225 um and the area of the leaf exposed to treating solution approximately 0.5 cm2 giving a leaf volume of 1.1 X 1010 ums. If 20% of the leaf is assumed to be air space, the leaf volume available to accommodate treating solution entering through stomata would be 2.2 X 109 um3. From the previous calculations it was found that 1.9 X 109 um3 of Vatsol OT-containing solution entered the leaf. Therefore, ap- proximately 86% of the available leaf air space was occupied with the treating solution after 10 minutes. Similar calculations for X-77 and Tween 20 show that 32% and 9% of the air space, respectively, was occu- pied by treating solution containing these surfactants. Further evidence that solutions may enter the pear leaf through stomatal pores was demonstrated with localization of reduced silver in the substomatal chamber after a 4 minute treatment time (Figure 23 ArD). Although cuticular penetration was also observed in this short period of time, localization as shown is best explained by stomatal entry. If silver filled the substomatal chamber following passage through the stomatal pore then more reduced silver might be expected to be present. However, immediately following the 4 minute penetration period, the disks were infiltrated with Craf III killing and fixing solution which contained 0.3% chromic acid, a very powerful oxidizing 134 agent. This may have prevented further reduction of the silver already present in the substomatal chamber. Stomata are probably not important as portals of entry of treat— ing solution into the pear leaf because of the rather precise condi- tions required to open stomata fully. After leaf disks were illumi- nated with light for longer periods than 5 or 6 hr, stomatal aperture widths were observed to decrease. Further, stomata did not open fully when the treating solution was present during opening. Penetration of NAA from a treating solution containing 0.1% Tween 20 was similar irre- spective of stomatal aperture width (Figure 20). In this experiment aperture widths were less than if the stomata were allowed to open on moistened filter paper in the Petri dish (Figure 18). Evidence of stomatal penetration provided here is in agreement with the work of Currier, Pickering, and Foy (1964), Dybing (1958), Dybing and Currier (1961), Hull (1964), and Pickering (1965). However, the factors which bring about stomatal penetration are not well docu- mented. Adam (1948) pointed out that the two most important factors influencing the penetration of a liquid through a pore were (a) low contact angles of the liquid and (b) the shape of the pore. The con- tact angle is influenced by the surface tension of the solution and the nature of the surface. The pore shape changes with degree of open- ing. In this study the extent of stomatal penetration was related to the reduction of surface tension, confirming observations made by Dybing (1958) and Dybing and Currier (1961). It was previously men- tioned that stomatal penetration was believed to occur through only 20-60 stomata (less than 1.0%) out of the approximately 12,500 present 135 in a leaf disk. Therefore, if stomatal aperture width is a determin- ing factor and less than 1.0% of the stomata are involved, then aperture width must be larger than 7.0 pm to allow a surfactant-containing treating solution through the pore (Figure 18). Currier, Dybing, and Fay (1964), and Dybing (1958) found stomatal penetration was corre- lated with the degree of stomatal opening. Foy (1962) found a corre- lation between 2,4-D penetration and larger stomatal apertures induced by high humidity. Data presented here and references cited in the literature would indicate that stomatal penetration occurs through stomata because of large aperture widths. However, alternative ex— planations may conform more to the mathematical considerations pre- sented by Adam (1948). The high humidity conditions used in this study and those reported by Foy (1962) would partially hydrate the cuticle around the stomata. Cuticle hydration reduces the contact angle of the solution (Fogg, 1944) below that caused by the surfactant thus increasing stomatal penetration. Since the cuticle is lipoidal in nature and a surfactant has a lipophilic tail, it may be that the surfactant is sorbed from the treating solution into the cuticle around and in the stomatal pore. It this were the case then a cuticle—treating solution interface would not exist but there would be a continuum of cuticle, surfactant and surfactant treating solu- tion. Such a situation would then result in a zero contact angle and passage through the pore would be very much facilitated. It is also probable that the degree of opening could influence the shape of the stomatal pore to make penetration feasible. Recently, Idle (1969) has demonstrated that the silicone rubber- cellulose acetate method of measuring stomatal apertures may be 136 unreliable for plants having stomata with cuticular ledges. This cuticular ledge is apparent on pear stomata (Figure 23 ArD). However, it is also emphasized that relative differences in stomatal aperture widths are valid even though actual um measurements may be in slight error. Increased penetration of NAA and NAAm by young leaves is consis- tent with 3-CP penetration into peach leaves (Bukovac, 1965), NAA and NAAm binding by pear leaves (Bukovac and Norris, 1966), and 2,4-D pene- tration into bean leaves (Sargent.and Blackman, 1962). Surface waxes are continually deposited on the surface until leaf expansion ceases but embedded.waxes are continually deposited (Crafts and Fay, 1962). Increased cuticle thickness with age may contribute to reduced penetra- tion (Richmond and Martin, 1959). Penetration of NAA through the lower surface was always greater than through the upper surface. In general, differences became pro- gressively greater with increasing leaf age. There are numerous re- ports of greater penetration through the lower surface (Fogg, 1958; Sargent and Blackman, 1962; 1965; Schieferstein, 1957). Penetration of NAAm is unique in that it was greatest through the upper surface of young leaves. This condition was reversed with the older leaves. The increased penetration through the upper surface is difficult to ex- plain. However, the leaves developed under relatively low light (1800 ft-c) in the growth chamber and the epicuticular wax layer may have been insufficiently developed to provide a barrier. Penetration through the lower surface of the same leaves may have been less because this surface is rougher and there may have not been complete contact with the treating solution. 137 Microradioautographic studies with the isolated upper pear leaf cuticle indicated that with the resolution possible preferential ab- sorption did not occur over anticlinal walls. This was similar to the uniform binding of 3-CP by peach leaf cuticle (Bukovac, 1965). Fogg (1948) and Dybing and Currier (1961) have indicated anticlinal walls as sites of preferential absorption. However, intact leaves were used and the underlying cells may have influenced the pattern of cuticular pen— etration. Absorption of silver nitrate (Figure 23) showed cuticular pene- ”tration. At least 3/4 of the time when cuticular penetration was ob- served it was associated with veins. Although the cuticle is thicker over veins due to deeper indentations, embedded waxes were shown to be discontinuous (Norris and Bukovac, 1968a), thus perhaps allowing pref- erential penetration. SUMMARY An improved method of studying foliar penetration and the effect of various factors on foliar penetration of NAA and NAAm into leaves of the pear_Pyrus communis L:_is described. 1. Time Course Penetration of NAA and NAAm into the upper surface was lin- ear. Penetration into the lower surface is characterized by rapid in- itial NAA uptake for 24 hours and NAAm for 48 hours followed by a phase where uptake occurred at a reduced rate. Penetration of NAA when ap- plied either via glass cylinders or as microdroplets was similar, pro- vided that the droplet was not allowed to dry. 2. Droplet Drying Droplet drying resulted in an increase in NAA and NAAm pene- tration through both the upper and lower surfaces. Subsequent to drop- let drying NAA penetration into the upper surface proceeded at a slower rate and NAAm penetration was not reduced. Following the initial surge in penetration as the result of droplet drying, little additional NAA or NAAm entered the lower surface. 3. Concentration Penetration of NAA and NAAm was linear with increasing con- centration into the upper and lower surfaces with the exception of NAA penetration into the lower surface at the highest concentrations where uptake started to plateau. 138 139 4. Temperature Penetration of both NAA and NAAm increased with increasing temperature. The greatest increase was between 25 and 35 C. Temper- ature generally had a greater influence-on NAAm penetration. 5. pH. The penetration of NAA into the upper and lower surfaces was related to the degree of dissociation of the molecule. More NAA was taken up from treating solutions buffered at a low pH (3.0) than those at a high pH (6.0). NAAm penetration was not influenced by treating solution pH between 3.0 and 7.0. 6. Light Penetration of NAA and NAAm through the lower surface in- creased with increasing light intensity up to about 600 ft-c. Beyond this light intensity there was little increase in penetration with increasing light intensity. Light stimulation of NAA and NAAm pene- tration could not-be demonstrated in the upper surface of field grown leaves because the cuticle presented such a great barrier to penetra- tion. Light—stimulated penetration of NAA into the upper surface could be shown with greenhouse grown leaves but stimulation was not as great as for the lower surface. The light stimulation of NAA pene- tration was not influenced by stomatal aperture width. The stimula- tion of penetration from light is immediately gained and when the light is turned off the stimulation is also immediately lost. 7. Inhibitors Specific inhibitors of the Hill reaction, Atrazine, Monuron, and Terbicil, can partially reverse the light stimulated increase in penetration of NAA into the lower surface. It was concluded that 140 primary products of photosynthesis were responsible for increased pene- tration in the light. A 100% atmosphere of nitrogen, carbonylcyanide m-chlorophenylhydrazine, carbonylcyanide p—trifluoromethoxyphenylhy- drazine, 2,4-dinitropheriol, and phenylmercuric acetate all inhibited NAA penetration through the lower surface in the light. 8. Surfactants w No surfactant studied increased penetration of NAA or NAAm into the upper surface. A 0.01% or higher Tween 20 or Tergitol 15-8-9 solution increased NAA penetration into the lower surface. Tween 20 and Triton B-l956 at 0.1% solutions increased NAAm penetration into the lower surface, but X-77 was significantly better than either of these. Surfactants increased NAAm penetration to a much greater ex- tent than for NAA. 9. Stomatal Penetration Stomatal penetration of applied treating solutions has been established. However, it is not considered to be a major pathway of entry of foliar applied compounds. 10. Leaf Age Penetration was greater in younger than older leaves with the exception of NAA penetration into the upper surface. Uptake of NAA into the lower surface was always greater than into the upper. NAAm penetration was greater into the upper surface until leaf expansion was completed then the situation was reversed and the greatest absorption was into the lower surface. 11. Pathways No preferential areas of NAA penetration through the iso- lated upper cuticle were observed. Silver nitrate was preferentially 141 absorbed through the cuticle above veins. 12. 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Standard deviation for penetration of NAA and NAAm into pear leaf disks through the upper and lower surfaces from different concentration treating solutions. Standard Deviation (picomoles) NAA NAAm Concentration Upper 1 Lower Upper Lower 10'6 1 2 .. __ s x 10-6 1 7 -- -- 10'5 9 54 -- -- 5 x 10‘5 19 175 9 8 10-4 40 169 18 31 5 x 10‘4 389 614 202 183 10-3 369 932 326 191 Table A3. 159 Trade name Chemical name and class Source Tergitol 15-8-9 Tween 20 Triton B-l956 Triton X-100 Vatsol OT X-77 Polyethylene glycol ether of linear alcohol Polyoxyethylene sorbitan monolaurate Sodium alkylaryl poly- ether sulfate Alkyl phenoxy polyethoxy ethanol Diocytl ester of sodium sulfosuccinic acid Alkylarylpolyoxyethylene glycols, free fatty acids and isopropanol Union Carbide Corporation New York, New York Atlas Chemical Industries Wilmington, Del. Rhom and Haas Company Philadelphia 5, Pa. Rhom and Haas Company Philadelphia 5, Pa. American Cyanamid Co. New York 20, New York Chevron Chemical Company Ortho Division San Francisco, Calif. TY "1111111111[1131111111111s