ABSTRACT FOLIAR PENETRATION AND TRANSLOCATION OF SUCCINIC ACID 2,2-DIMETHYLHYDRAZIDE (SADH) By Jorg Schonherr Factors affecting foliar penetration of succinic acid 2,2-dimethylhydrazide (SADH) into bean primary leaves (Phaseolus vulgaris L., cv. "Blue Lake") and translocation of foliar applied SADH using apple (McIntosh) and peach (Suncling) trees were studied. A direct approach using lLlC-SADH and an excised leaf disk system and an indirect approach using a bio- logical response as index of penetration were utilized. In the direct approach, 1uC—SADH solution was added to a small glass tube affixed to the surface of leaf disks kept in a controlled specified environment. This allowed penetration studies under constant conditions (light, temperature, humidity, pH and concentration of SADH and surfactant). An indirect measurement of SADH penetra- tion was achieved by applying SADH in various formula- tions to bean primary leaves and inhibition of internode elongation was utilized as an index of penetration. The rate of penetration was studied by detaching the treated leaves after specified time intervals. Jorg Schonherr Translocation of SADH in one-year-old, potted apple and peach trees with two shoots each was studied by spraying one of the two shoots with Alar 85. The growth of the treated and non-treated shoots of the treated trees was compared with each other and also with the growth of comparable shoots of control trees (non- treated). Foliar penetration of lLlC-SADH was increased by increasing light intensity (up to 3,600 ft.c.), tempera- ture (5-40° C) and surfactant (Tween 20) concentrations (up to 0.1%). The effects of light, temperature and surfactant were related and partially additive. Pene— tration was greater with treating solutions of a pH below rather than above the pKa. This pH effect was observed in the light and in the dark, however, it was reversed in light, when Tween 20 was added to the treat— ing solution. Penetration was linear with SADH concen— tration from 10'“ to 10-2. Osmotic stress and inhibi- tors (NaN3, 2,U-dichlorophenoxyacetic acid, phenylmercuric acetate) inhibited penetration of SADH in light but not in the dark. Penetration was related to stomatal density and stomatal opening. From a theoretical treatment of the problem of entry of liquids into the intercellular air space of leaves through open~stomata, and from direct and in- direct evidence, it appeared that treating solution Jorg Schonherr entered the aperture of open stomata but not the inter— cellular space. Sites of entry for polar molecules into bean leaves (polar pathways) were established using AgNO3 as tracer. These sties were localized along the cuticular ledges of guard cells, the walls of the guard cells, glandular trichomes and anticlinal walls. The partici— pation of these pathways appeared to be modified by light and Tween 20. From the data it was concluded that penetration of SADH into bean leaves was not directly linked to metabo- lism. The above factors affected penetration of SADH by affecting (a) the area of the leaf surface in contact with treating solution (effective surface), that is sur- face through which penetration can proceed, and (b) affecting the permeability of the effective surface. The effective surface was increased by all factors which enhanced wetting and led to or increased stomatal opening. The walls of the guard cells inside the stomatal aperature appeared to be much more permeable than the re- mainder of the leaf surface. Furthermore, the permeabil- ity of the cuticular ledges seemed to be greater in open than in closed stomata. Tween 20 and pH of the treating solution influenced penetration of SADH similarly in the experiments using inhibition of internode elongation as an index of Jorg Schonherr penetration. The rate of penetration was greater with a treating solution of pH 3.0 or 5.0, than of pH 7.0. Tween 20 added to the treating solution also increased the rate of penetration with little effect on the total penetration, which was almost complete after 12 hours whether the solution contained Tween 20 or not. Besides enhancing the rate of penetration, surfactants also re- duced retention of SADH solution by leaves (15 — 25%) and promoted internode elongation of bean plants by up to 60%. This promotion was overcome with SADH. SADH was not or only slightly translocated in apple and peach trees as indicated by the lack of growth in— hibition of the non-treated shoots of the treated trees, whereas shoot elongation was completely inhibited on treated shoots. Growth of the non-treated shoots of the treated peach trees was significantly greater than the growth of comparable shoots of control trees. The concept of ectodesmata with respect to their suggested role in foliar penetration of polar compounds was critically reviewed. It was concluded that the demonstrability of ectodesmata is closely linked to the occurrence of polar pathways in the cuticular membrane. Such pathways seem to be necessary for penetration of H Hg ions, which are essential in demonstrating ecto— desmata. sh \ g‘u‘ ~c \\ - UR“ (AQ x“. Q“ KIT} L 1\ \ (2);) FOLIAR PENETRATION AND TRANSLOCATION OF SUCCINIC ACID 2,2—DIMETHYLHYDRAZIDE (SADH) By Jorg Schonherr A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Horticulture 1969 ACKNOWLEDGMENTS The author wishes to express his sincere apprecia— tion to Dr. Martin J. Bukovac for his guidance, sugges— tions and criticism during the course of the investiga— tion and preparation of the manuscript. Appreciation is also extended to the members of my guidance committee Drs. A. L. Kenworthy and K. Lawton and to Dr. Margaret M. Robertson for their helpful suggestions and editing of the manuscript. The assistance of Dr. H. P. Rasmussen and Mr. V. Shull in the use of the microprobe and Dr. K. Raschke in the use of the recording porometers as well as for making his laboratory equipment available to the writer is gratefully acknowledged. The author is greatly indebted to Mrs. Gracie M. Stone, for her c00peration in supplying the labelled succinic acid 2,2-dimethylhydrazide used in this study. The financial assistance by the Public Health Ser- vice (grant CC 002A6 from the National Communicable Disease Center, Atlanta, Georgia) and the Fulbright Commission which made this work possible is greatly appreciated. ii TABLE OF CONTENTS Page ACKNOWLEDGMENTS . . . . . . . . . . . . ii LIST OF TABLES . . . . . . . . . . . . v LIST OF FIGURES . . . . . . . . . . . . vii INTRODUCTION . 1 LITERATURE REVIEW. 3 Succinic Acid 2,2-Dimethy1hydrazide 3 Methods of Application. . . A Foliar Penetration . . . . 5 Translocation. . . . . . . . 6 Stability in Plants. . . . 6 Foliar Penetration of Polar Compounds. 7 Leaf Structure as Related to Foliar Penetration of Polar Molecules 9 Permeability of Isolated Cuticular Membranes . . . 18 Permeability of Cuticular Membranes to Water . . . 22 Polar Routes Across the Cuticular Membrane . 27 EXPERIMENTAL . . . . . . . . . . . . . 32 Foliar Penetration of Succinic Acid 2,2- Dimethylhydrazide: Studies Employing Excised Leaf Disks . . . . . . . . . 32 Materials and Methods . . . . . . . . 32 Analysis of Procedures. . . . . . . . 40 Results. . . . . . . . . M9 Discussion. . . . . . . . 117 Foliar Penetration of Succinic Acid 2,2- Dimethylhydrazide: Studies Employing a Growth Response As An Index for Penetration. 136 Material and Methods . . . . . . . . 136 Results. . . . . . . . . . . . . lUO Discussion. . . . . . . . . . . . 155 iii Page Translocation of Succinic Acid 2 ,2- Dimethylhydrazide in Apple and Peach Trees . 161 Material and Methods . . . . . . . 161 Results. . . . . . . . . . . . . 163 Discussion. . . . . . . . . . . . 175 LIST OF REFERENCES . . . . . . . . . . . 179 APPENDICES . . . . . . . . . . . . . . 191 iv Table 10. ll. 12. LIST OF TABLES Page Loss of 1[AC-SADH from leaf disks into the underlying filter disks. . . . . . . . A9 Density of stomata and glandular trichomes on fully expanded bean primary leaves . . . 53 Calculated changes in penetrating pressure (P1) along the stomatal aperture shown in Figure 9.. . . . . . . . . . . . 68 The effect of Tween 20 on penetration of SADH into bean leaf disks . . . . . . . 77 The effects of temperature, light and Tween 20 on the Q 0 for penetration of SADH into bean leaf d sks . . . . . . . . . . 85 The effect of plant age and leaf size on penetration of SADH into bean leaf disks . . 91 The effect of pH on the penetration of SADH into bean leaf disks and its modification by Tween 20.. . . . . . . . . . 92 The effect of pH and Tween 20 on the distribu- tion (%) of SADH between aqueous phase and octanol . . . . . . . . . . . . 93 The effect of urea (0.1%) on penetration of SADH into bean leaf disks . . . . . . . 9A The effect of osmotic stress on the penetra— tion of SADH into bean leaf disks . . . . 95 The effect of pre—treatment with inhibitors on penetration of SADH into bean leaf disks . 96 The 1Effect of Tween 20 (0.1%) on retention of C— SADH solution and penetration into bean primary leaves . . . . . . . . 1A9 Table Page 13. The effect of surfactants (0.1%) and SADH (5 x 10-“ M) on elongation of the first internode of bean plants . . . . . . . 1&9 1A. The effect of SADH (5 x 10'3 M) and Tween 20 (0.1%) on elongation of the first internode of bean plants. . . . . . . . . . . 151 15. The effect of pH of SADH treating solution (5 x 10-3 M) on elongation of the first and second internode of bean plants . . . . . 155 16. Penetration of SADH (applied as Alar 85) into apple leaves as indexed by inhibition of terminal growth of apple seedlings . . . 156 17. The effect of Alar 85 (5,000 ppm) on shoot growth of apple (McIntosh) trees. . . . . 171 18. The effect of Alar 85 (5,000 ppm) on shoot growth of peach (Suncling) trees. . . . . 173 19. Leaf area and spray retention of apple and peach trees at the time of treatment . . . 17“ vi LIST OF FIGURES Figure Page 1. Photographs illustrating the penetration units consisting of a glass tube affixed to a leaf disk with silicon rubber. . . . 3A. 2. Tracings of radiochromatogram scans of thin-layer plates . . . . . . . . . A3 3. Radioautogram of bean leaf disks following penetration of luC-SADH for 12 hours in light . . . . . . . . . . . . . A7 A. Secondary electron micrographs of the surfaces of bean primary leaves. . . . . 50 5. Relationship between leaf size (plant age) and stomatal density on the upper surface of bean primary leaves. . . . . . . . 5A 6. Kinetics of stomatal opening and closing of bean primary leaves as indexed by porometer flow . . . . . . . . . . 56 7. Cross sections of stomata from bean primary leaves . . . . . . . . . . 59 8. Schematic diagram illustrating a capillary pore of circular cross-section having inclined walls . . . . . . . . . . 63 9. Schematic drawing of a cross-section of a bean leaf stoma . . . . . . . . . 66 10. Time course of penetration of SADH tin bean leaf disks . . . . . . . . . . 75 11. The effect of light on penetration of SADH into bean and pear leaf disks. . . . 78 12. The effect of temperature on penetration of SADH into bean leaf disks. . . . . . 82 vii Figure Page 13. The effect of SADH concentration on pene- tration into bean leaf disks. . . . . . 86 1A. The effect of pH on penetration of SADH into bean leaf disks . . . . . . . . 89 15. The effect of cufiicular transpiration on penetration of 1 C-tryptophan into bean leaf disks. . . . . . . . . . . . 99 16. Sites of preferential entry, reduction and localization of silver in the upper surface of bean primary leaves (surface view). . . 101 17. Sites of preferential entry, reduction and localization of silver in the upper surface of bean primary leaves (surface View). . . 103 18. Sites of preferential entry, reduction and localization of silver in the upper surface of bean primary leaves (surface view). . . 105 19. Sites of preferential entry and localiza- tion of silver in the upper surface of bean primary leaves, as evident from silver X-ray oscillographs. . . . . . . . . 109 20. Sites of preferential entry and localization of silver in the upper surface of bean primary leaves, as evident from silver X-ray analysis . . . . . . . . . . . . 111 21. Sites of preferential entry, reduction and localization of silver in bean primary leaves (cross-section). . . . . . . . 113 22. Sites of preferential entry, reduction and localization of silver in bean primary leaves (cross-section). . . . . . . . 115 23. The effect of SADH concentration on elonga- tion of the first and second internodes of bean plants . . . . . . . . . . . 1&2 2A. The effect of surfactants and surfactant concentration on retention of treating solution by bean primary leaves. . . . . 1AA viii Figure Page 25. The effecfi of Tween 20 (0.1%) on penetra- rtion of l c— SADH into bean primary leaves . . . . . . . . . . 1A6 26. The effect of Tween 20 (0.1%) on penetra- tion of SADH into bean leaves, as indexed by inhibition of elongation of the first internode . . . . . . . . . . . . 152 27. The effect of Alar 85 (5,000 ppm) on shoot growth of apple trees (McIntosh) . . . . 16A 28. The effect of Alar 85 (5,000 ppm) on shoot growth of peach trees (Suncling) . . . . 166 29. The effect of Alar 85 (5,000 ppm) on growth of apple and peach trees. . . . . 168 ix INTRODUCTION The introduction of the polar growth retardant succinic acid 2,2-dimethy1hydrazide (SADH) and its promise in commercial horticulture has created a need to attack anew the problem of foliar penetration of polar compounds in general and the problem of polar pathways across the cuticular membrane specifically. The literature was reviewed with emphasis on (a) leaf structure affecting penetration of polar compounds, (b) permeability of the cuticular membrane to water and other polar molecules and (c) polar pathways across the cuticular membrane. Special attention was given to the participation of stomata and ectodesmata in foliar pene- tration of polar compounds. The experiments were aimed at (a) elucidation of the major factors affecting foliar penetration of SADH, (b) relating these factors to morphological and physio- logical characteristics of the leaves and (c) finding the mechanisms in which these factors affect the polar pathways in the cuticular membrane and the foliar pene- tration of SADH. In addition, translocation of foliar applied SADH in selected woody plants was studied to determine the significance of coverage in application of SADH as a foliar spray. LITERATURE REVIEW Succinic Acid 2,2-Dimethylhydrazide Succinic acid 2,2-dimethylhydrazide (SADH) and maleic acid 2,2-dimethy1hydrazide (MADH) were reported by Riddell gt_al. (90) as broad spectrum plant growth retardants with potential application in the control of plant growth and develOpment. Since MADH was not stable in aqueous solutions, due to intramolecular hydrolysis of the acid (19), SADH has been used almost exclusively in subsequent investigations. SADH is the active ingredient of the commercial products Alar 85 and B-9. In earlier publications SADH was also referred to as experimental compound B-995 and N,N-dimethylaminosuccinamic acid (DMAS). These names have been used interchangeably in the literature. It should be noted, however, that both Alar 85 and B-9 are formulated with a surfactant (121). Since its introduction in 1962, many experiments involving SADH have been performed and an extensive literature has been accumulated. Investigations have centered on two tOpics: (a) effects on growth and development on various plants, and (b) mode of action. Very little is known about other aspects such as foliar penetration and distribution in the plant, both important with respect to the practical application of a foliar applied growth retardant. The SADH literature has recently been reviewed, particularly with respect to growth responses (15, 56, 122) and the mode of action (56). Therefore, this re- view will be confined to publications dealing with foliar penetration of SADH and its translocation and stability in the plant. Methods of Application SADH is most commonly applied as a foliar spray. Other methods of application may also be effective, e.g. trunk injection, root absorption from a SADH solution (76) or soil medium (7), uptake through a severed stem or petiole (75) and application to the plumule (131). Thus, SADH can penetrate or be taken up by both roots and above ground organs. However, most of these methods of applica— tion are feasible only under laboratory conditions and, since SADH is not stable in soil (76, 121), the most effective commercial mode of application is as a foliar spray. SADH has been employed as foliar sprays at concen- trations ranging from 500 to 50,000 ppm. In tree fruit, 1,000 to A,000 ppm were most commonly used and within this range the response increased with increasing concentration (2A, 30, 51). Higher concentrations than U,000 ppm did not induce a greater response, but a second application two weeks after the first prevented resump- tion of growth of apple trees later in the season (30). Even when used in extremely high concentrations (50,000 ppm) SADH usually was not phytotoxic. Petunias showed no leaf injury when sprayed with 5,000 to 50,000 ppm SADH, though a white residue remained on the surface (1“). Similarily, 10,000 ppm applied to the foliage of apple trees did not cause leaf injury (2“). There is one re- port, however, of leaf injury in the peach following application of 5,000 ppm SADH as a foliar spray (25). Foliar Penetration High concentrations of SADH are required to pro— duce a biological response. This may suggest that (a) SADH penetrated into the leaves poorly, (b) transloca- tion to the site of action was limiting or (c) both. There are indications that SADH does not readily penetrate into the leaves. SADH was more effective in inhibiting shoot growth of apple when applied with than without a surfactant (2“). Further, complete inhibition of flower formation in Pharbitis nil was obtained with only 100 ppm SADH when administered to the roots in the nutrient solution (131). A comparable effect was ob- tained by application of 100 ug to the plumule or 300 ug to the cotelydons (131). The growth response of petunias indicated that washing the foliage within 2“ hours after spray application (5,000 ppm) removed most of the chemical (1A). Penetration into fruits also appears limited, 60% of the lLAC-SADH applied to McIntosh fruits penetrated within four days (26). Translocation Martin gt_al. (75) reported that SADH was rapidly translocated in apple trees and seedlings. Since SADH is highly water soluble, these authors suggested a passive translocation in the transpiration stream and concluded that "coverage should be of less importance." However, following stem injection of luC-SADH most of the 1“C accumulated in the leader directly above the point of injection and very little was found in other leaders (75). That SADH might not be readily translocated in pear trees following foliar application was reported by Wertheim and Van Belle (125). When only the top of the tree was sprayed, growth was reduced in the treated shoots but not in those in the non-sprayed lower part of the tree. Stability in Plants SADH appears to be quite stable in plants. Com— paring the rate of breakdown of SADH, 11‘C-labeled in two different positions (succinic acid and methyl groups), Martin and Williams (76) found that degradation was slow and gradual, irrespective of the position of the label. A decrease in radioactivity was found in the ethanol extractable fraction, while the radioactivity in the residue remained constant. The bulk of the label in the extractable portion was SADH. Of the label remaining in the residue after 2A hours extraction with 80% ethanol, 60% was still identified as SADH. Measurements of CO2 evolution from "Red Delicious" seedlings treated with luC-SADH revealed a constant loss of 1”002 over 7 weeks, which amounted to approximately 20% of the original dose. The great persistance of SADH in apple trees was confirmed by Edgerton and Greenhalgh (26). Following foliar application in June or August significant levels of luC-SADH were found in dormant tissue in January, especially in flower buds. Samples taken 6 months later, in July, revealed traces of lLAC-SADH in spur tissue, cluster bases and vegatative buds totaling approximately 10% of the levels found in dormant tissue. Foliar Penetration of Polar Compounds Whenever biologically active compounds are applied to plants, the dose is of critical importance. This applies for the use of growth regulators as herbicides as well as to their application in controlling growth and development of arable crOps. Most commonly, these compounds are sprayed on the leaves, but the amount of the growth regulator admin— istered to the leaf surface often does not bear any strict relationship to the amount penetrating into the tissue. The portion of the spray deposit which pene— trates into the leaf is dependent upon a number of plant related factors; namely, cuticle structure and chemistry, amount and orientation of waxes, the presence of stomata and trichomes and the physiological condition of the plant in general. Environmental factors also alter penetration: by influencing foliar characteristics (e.g. cuticular waxes), the physiological condition of the plant during the penetration period (turgescence, metabolic activity) and the spray performance on the leaf surface (drying time). Physical and chemical pro- perties of the growth regulator as well as of the formu— lation may also be of great importance. The literature on foliar penetration of growth sub- stances has been reviewed on a number of occasions. Van Overbeek (81) discussed foliar characteristics in refer- ence to uptake and translocation of auxins. Foliar pene- tration of herbicides was reviewed by Currier and Dybing (l7) and the role of stomata was stressed by Dybing and Currier (22). The importance of plant surface charac- teristics in relation to foliar penetration was stressed by Crafts and Foy (l6) and Foy (33). More recent reviews were prepared by Hull (60) and Sargent (95). The role of ectodesmata in foliar penetration was discussed by Franke (”3, HA). Since most of the growth regulators studied were relatively lipOphilic, the problem of foliar penetration of polar compounds has received little attention. Alternate polar and apolar pathways across the cuticular membrane have been suggested (16, 91) in an attempt to explain the empirical fact that inorganic ions and other polar compounds penetrate the lipophilic cuticular mem— brane. Little is known concerning the nature of the suggested polar pathways. Insect punctures, cracks in the cuticle, ectodesmata and entry through the open stomatal pore have been suggested, but direct evidence of their participation and relative importance in foliar penetration of polar molecules is scarce. Leaf Structure as Related to Foliar Penetration of Polar Molecules Epidermis The term epidermis designates the outermost layer of cells on the primary plant body (28). The epidermis of leaves may differ in form, composition and function. In pear, for instance, the epidermis of the upper sur— face is composed of one type of cells while the epidermis of the lower leaf surface contains guard cells in 10 addition (79). The amphistomatous leaves of Phaseolus vulgaris have guard cells on both surfaces, which contain chloroplasts while the common epidermal cell is lacking chloroplasts. Bean leaves have in addition glandular and conical trichomes on both leaf surfaces (21). The cuticle covers the entire epidermis. It ex- tends over epidermal cells, guard cells, accessory cells, trichomes (28) and into the substomatal chambers (A, 79, 85). It seems, now, that a cuticle is generally formed where cells are in contact with the atmosphere (103). The cuticle is continuous and non-cellular and its thickness and composition varying widely (28). Associated with its main function, the reduction of uncontrolled water loss to the surrounding atmosphere, it is relatively impermeable to water and consequently other polar mole— cules., Permeability to polar compounds is further re— stricted by epicuticular waxes which occur in some plant species (96). ”.Epicuticular Waxes In some species, the cuticular membrane is covered with epicuticular waxes of varying configurations (3, 63, 6A, 78). The continuity and the physical arrange- ment of the waxes is important with respect to cuticular penetration and spray retention. A continuous wax layer 11 over the entire cuticle may seriously impair penetration of polar molecules, wettability and spray retention, especially if the waxy covering is rough (2, 23). Quantitative differences in waxes exist between the upper and lower surfaces. In pear leaves more surface wax was found on the lower than on the upper surface (79). The structure of the epicuticular wax has been shown and described for a number of species and the problem of the formation of the waxy coverings has been discussed re— peatedly (60, 63, 6A, 68, 96). The subject is still one of great controversy, particularly the question whether or not wax or wax precursers are excreted through pores in the cuticle. With respect to penetration of polar compounds it is, however, immaterial whether or not such pores exist, because they would constitute an apolar rather than a polar pathway. The question of continuity of wax layers remains open, although it appears that most waxy coverings are not continuous films but rather crystallin bodies such as platelets, rodlets, granules or columns (3). Juniper (63) found less wax on the cuticle of guard cells of pea leaves, while Hull (60) showed dense wax platelets on the guard cells of Prosopis juliflora. In tulip leaves even the cuticular_ledges were covered with surface wax (3). 12 The formation of epicuticular waxes of leaves of Pisum sativum was found to be affected by environmental conditions (6A). No surface wax was observed on plants grown in the dark but it formed rapidly on exposure to light. Within 2A hours after transfer to light minute quantities of wax appeared on the adaxial surface and six days later the waxy layer was complete. No changes took place thereafter. Wind velocity also influenced size and density of the wax structures on pea leaves. Parallel with the formation of epicuticular wax was a dramatic increase in contact angle of water droplets. In dark grown plants the contact angle was 680 and reached a value of 11400 after surface waxes were formed. Leaves of Brassica oleracea and Chrysanthemum segetum regenerated the surface wax if wiped off before leaf expansion was complete (6“). This is in agreement with the findings of Schieferstein and Loomis (96). Young expanding maize leaves regenerated their surface wax layer following dewaxing, but fully expanded leaves failed to do so. When the wax was removed from the growing region of a leaf it was replaced along the mar- gins but not in the center (over periclinal wall) of the epidermal cells. The amount of surface wax of apple leaves was found to decrease with increasing leaf age (7“). In "Bramley's Seedling" only half as much wax per unit leaf surface 13 could be removed from old leaves (52 cm2) than from young, not fully expanded leaves (21 cm2). There was more sur- face wax on the lower than on the upper surface. Cuticular Membrane (CM) ’ Using staining techniques, polarized light micro— scopy and, more recently, electron microsc0py, the structure and chemical composition of the cuticular mem- branes of a variety of plants have been investigated. With the exception of the pear leaf cuticle (79) studies have been carried out on plants of little economic im- portance such as Clivia (A6, 77, 93), Hedera helix and Nicotiana glauca (108, 96), Yucca and Dasyliron (77), Ficus elastica (107) and Philodendron scandens (8). Thus, most of the currently available information on the structure and chemistry of CM's is from more or less xerophytic plants. However, most of the economically important plants are mesophytic and plants of this type have been used predominantly in studying foliar penetra— tion. This has led to the situation that results ob— tained from mesophytic species tend to be interpreted on the basis of cuticular characteristics of xerophytic plants such as Clivia or Ficus. With this in mind, cuticular characteristics in reference to permeability will be discussed next. To avoid confusion, the nomenclature suggested by Sitte and 1A Rennier (107) will be adopted. They proposed the term "cuticle" (prOper) for the outermost or oldest layer of the CM. Other cutin containing layers are termed "cuticular layers" whether they contain cellulose or not. Cuticle proper and cuticular layers together represent the cuticular membrane (CM) which can be iso- lated in some species by digesting the pectin layer which separates the CM from the underlying cellulose wall (80, 127). The CM appears relatively homogeneous following staining with Sudan stains or under the electron micro— scope (8, A6, 107), while two or more different layers can be distinguished with polarized light. Of the major components the cutin matrix is isotropic or sometimes weakly negative birefringent (A6, A8, 93, 9A, 107), oriented cuticular waxes are mostly negative and cellu- lose (if it occurs) positively birefringent. Embedded oriented waxes and cellulose micelles render the other- wise isotropic CM birefringent. A special case of birefringence is the so-called form double refraction exhibited by a porous solid body (Wiener'scher Misch- k6rper) (A6). Form double refraction can be eliminated by imbibition with a liquid having the same refractive index as the solid body. The CM of the upper surface of pear leaves ex- hibited a pronounced zone of negative birefringence 15 under a narrow isotropic band, indicating the presence of embedded oriented waxes (79). This double refractive wax layer was continuous and had a definite structure, the wax being layered with intervening bands of isotrOpic material. The degree of birefringence decreased over anticlinal walls and appeared broken over veinal tissue. the embedded wax of the CM of the lower leaf surface showed a lesser degree of orientation. Oriented embedded waxes have been reported to occur in the CM of many species including Fagus silvatica, Betula pendula, Prunus laurocerasus, Agave americana, Ficus elastica, and Hedera helix (107). In some cases some residual negative biregringence remained even after prolonged extraction with wax solvents (93), which led to the hypothesis that perhaps some of the wax molecules are chemically bound in an oriented position to the cutin matrix. There is indirect evidence that embedded waxes are located in pores of the cutin matrix (68). Follow— ing extraction of the embedded waxes, the remaining cutin skeleton either collapsed (107) or exhibited form double refraction which disappeared on imbibition with a suit- able liquid (e.g. benzene). For Fiscus elastica it has been shown (107) that these pore spaces exist prior to the depostition of wax into the CM. The youngest leaf was covered with a very l6 thin isotrOpic membrane. The CM of the next older leaf showed negative form double refraction in its central portion which disappeared on imbibition. During the next stage, the CM gained in thickness by means of inter- position of cutin between the already existing CM and the cellulose wall. Up to this stage the CM contained very little wax. The last step in the development was an intensive excretion of wax into the pore space of the now fully developed CM, rendering the oldest portion of it the richest in wax. Working with Philodendron scandens, Bollinger (8), found that the CM of leaves was already covered with a wax layer when the leaves unfolded and therefore he pro— posed that cutin and wax were excreted simultaneously. The CM of Nicotiana glauca increased in weight per unit area until the leaves were fully expanded, but not thereafter (108). The total wax content of the CM in- creased until the leaves were fully expanded and then decreased. The same was true for Hedera helix and Agave americana (96, 108). Some data are available concerning the relative amounts of surface and embedded waxes. In pear leaves the amount of embedded waxes was the same for the CM of the upper and the lower leaf surface but the amount of epicuticular wax was twice as great on the lower, than on the upper surface (79). In leaves of Nicotiana glauca 17 80% of the total wax was epicuticular and only 20% was embedded in the cutin matrix. In Agave americana 95% of the total wax was embedded in the CM. Agave and Nicotiana differed also in the chemistry of the waxes. One third of the total waxes of N. glauca were volatile while practically no volatile wax occurred in A. americana (96). A distinct polarity gradient exists in the CM. Epicuticular waxes, a high degree of polymerization of the cutin of the cuticle prOper and oriented embedded waxes render the outermost layer of the CM quite lipo- philic. The CM becomes more hydrophilic towards the pectin layer and the cellulose wall, due to a lesser degree of polymerization of cutin and to cellulose fibrils and pectic materials associated with the cutin matrix. The thickness of the CM is variable. It is generally thinnest over the periclinal walls of the epidermal cells and thickest over the anticlinal walls. The CM can extend deeply between the anticlinal walls as shown for Clivia nobilis (A6) and pear leaves (79). Structure and composition of the CM over the anticlinal walls are not well understood. Negative birefrigence due to embedded oriented waxes was reduced compared to the CM over periclinal walls, indicating a lesser degree of orientation or absence of waxes (A6, 79). The l8 anticlinal ribs of the CM of pear leaves stained only weakly with Sudan stains and showed considerable stain— ing with Ruthenium red (79). This staining pattern suggests that fewer non-polar lipids are present in the anticlinal ribs and more polar groups than in the CM over periclinal walls. It should be noted that Ruthenium red is not a specific pectin stain, since hemicelluloses, alginic acid, proteins, cutin precursors and poly- saccharides that contain sulphate may also be stained (9A). Thus, it cannot be concluded from the Ruthenium red staining alone that the anticlinal ribs contain pectins. The upper CM has been considered to be thicker than the lower (33, 80). In pear leaves, however, the CM was thicker on the lower than on the upper leaf sur- face and the CM overlying veinal tissue was also thicker than that over interveinal areas (79). Permeability of Isolated Cuticular Membranes A great advantage in studying the isolated CM is that certain variables can be better controlled. How- ever, if data obtained from such systems are to be extrapolated to "in vivo" systems it must be established that the two systems are strictly comparable. CM's have been isolated using different methods such as chemical isolation with oxalic acid (92) or l9 enzymatic isolation (80, 96, 108, 127, 128, 129, 130). Only for CM's isolated according to Orgell (80) have structure, composition and binding capacity of the isolated CM's been tested against fresh, non-isolated CM's (12, 79, 127, 128). From these data it appears that the CM's are not appreciably altered during enzymatic isolation. The penetration of inorganic ions and other polar organic molecules through astomatous tomato fruit CM's and stomatous onion leaf CM's have been studied (127, 129). In the onion leaf CM the cuticle extended through the stomatal pore thus forming a sac. Penetra— tion of Rb+, Ca++ , C1“, SOH_, SADH, maleic hydrazide (MH) and urea was greater from the outer to the inner side of the CM (influx) than in the Opposite direction (efflux). No difference between influx and efflux were observed using a dializing membrane. With the exception of urea, penetration was a diffusion process following first order kinetics. There was a distinct relationship between ion binding and penetration. Ion binding was always greater on the inner than on the outer side of the membrane (128, 130), whereas more ions penetrated from the outer to the inner side of the CM's than in the opposite direction. Thus, penetration was greater in the 2O direction towards the side with the greater ion bind— ing capacity. Urea penetrated at a much greater rate than any other compound and the penetration rate increased with time. There was little difference between influx and efflux. It appears possible that urea changed the structure and/or composition of the CM by breaking weak bonds and by dissolving components out of it. Yamada (126) reported that an ether soluble fraction could be extracted from CM's with urea. Compared to the dializing membrane, the CM's used in these studies were relatively impermeable. Eighty per cent of the cations and anions penetrated the dializing membrane in A0 hours, while the comparable figure for the CM's was 0.2 to 2%. The permeability data reported for apple leaf CM's (50) are difficult to evaluate since the CM's were chemically isolated. Furthermore, data were reported in terms of cpm penetrated, even though different iso- 22Na), counting procedures, concentrations topes (1A0, and specific activities were used. Silva Fernandes (105) also isolated CM's from apple leaves chemically. Such CM's were practically impermeable to phenylmercuric acetate and copper acetate. The CM of ivy increased in thickness with increasing age (3 years). This was accompanied by an increase in permeability to 21 water and a drastic decrease in the permeability to 2,A-dichlorophenoxyacetic acid (2,A D) (96). It is very difficult to draw a general conclu- sion concerning the permeability of isolated cuticular membranes to polar molecules, except perhaps that permeability was low to all compounds with the major exception of urea. Yamada g§_a1. (129) concluded that cations penetrate more rapidly than anions. This was true for Ca++ vs. 80;- and Rb+ vs. 01-, however, it was also true that more SQ;- than Rb+ penetrated the tomato fruit CM. While penetration of lipophilic compounds through enzymatically isolated astomatous apricot leaf CM's was related to the partition coefficient (chloroform: water), no such relationship was found for polar com- pounds (20). Sucrose, glucose, valerate and a— chloroacetate penetrated at a higher rate than the par- tition coefficient would suggest. The authors suggested that the mechanism of penetration for polar compounds might differ from that for non-polar compounds. The following illustrates the low permeability of the apri- cot leaf CM (20) for polar compounds: (a) only 2-3% of the sugars penetrated in A8 hours whereas almost complete penetration of N-n-hexyl- and N—isopropyl a-chloro-acetamide occurred during the same time period, and (b) it was estimated that a water or agar membrane 22 of comparable thickness is A x 106 times more permeable to sucrose than the apricot leaf CM. Permeability of Cuticular Membranes to Water If the prime function of the CM is to prevent uncontrolled water loss from leaves, one would expect to find a relationship between its morphological and/ or chemical characteristics and permeability to water. The thickness of the CM has been evaluated most often. Kamp (67) has been cited as saying that there is no relationship between thickness of the CM and cuticular transpiration (CT) (61, 107). This is true only if different genera are compared. A comparison of related species, however, reveals a striking relationship be- tween CT and thickness of the CM as shown by the follow- ing data taken from Kamp (67): Species Thickness of CM CT um mg/g fr.wt. x hour Quercus sessiliflora 2.2 6A Q. rubra 3.0 31 Q. coccifera 8.0 35 Q. ilex 9.5 7.5 Rhamgus l f l s 1'2 29 B. glandulosus 2. 25 R. alaternus 3.2 18 Coffea robusta 1.1 3A.5 Q. arabica 1.9 15 Acer platanoides 1.2 56 A. syriacum 3.5 32 Hedera helix (shade) 3.0 6.8 fl. helix (sun) 5.0 2.2 23 This compilation shows that the CT decreased with increasing thickness of the CM. Possibly CM's of related species are similar in structure and composition and adaptation to environment is accomplished primarily by changes in thickness of the CM. The CT of plants of different ecological type was Ineasured by Pisek and Berger (8A). The greatest CT (higher than 50 mg/g fr. wt. x hour) was observed in jImpatiens, Caltha, Stellaria and Veronica followed by a Egroup of plants having an intermediate CT (20-A0 mg/g :fr. wt. x hour) such as Fagus, Quercus, Convolvulus and IBetula and less than 10 mg/g fr. wt. x hour were observed iflor Rhododendron, Sedum, Hedera, Picea and Pings. Thus, zas the ecotype changed from mesophytic to xerophytic tflie CT and hence the permeability of the CM to water dfecreased. From the data of Sitte and Rennier (107) it (man be seen that the thickness of the CM tends to increase iri the same direction, although no representative of the first group was included. There is agreement (67, 96) that, with increasing age, the permeability of the CM to water (CT) increased, even though the thickness of the CM also increased. No Satisfactory explanation has been given for this obser- vation. Cuticular waxes severely restricted water per- meability of the CM (108). Following extraction of the 2A waxes the permeability to water of the astomatous ivy leaf CM increased dramatically. Interestingly, the permeability of the CM from shade—grown plants increased more than those from sun-grown plants. This indicated that the cutin matrix must have been different since (differences in thickness were small. There is some evidence that the degree of hydra— tzion of the CM affects CT. Incipient drying of the crutermost layer of the CM was shown to rapidly reduce (31'(67, 8A, 112). Incipient drying occurred as soon as vvater loss from the CM to the atmosphere by evaporation (exceeded the water diffusion through the CM. A satura- txion deficit has been thought to occur which in turn ffixrther increased the diffusion resistance to water and tflierefore reduced CT as compared to the fully hydrated CNI. The relationship between the degree of hydration Of‘ the CM and CT was investigated by Hartel (53, 5A). CT? was markedly affected by pH and the presence of cer— tlain ions in the membrane. Leaves pretreated with K~phosphate buffers of various pH values showed maximum CT around pH 6 and 7 and CT decreased above and below these pH values. Leaves which had been killed with Chloroform vapor gave the same response to the buffer treatment as intact living leaves. 25 CM pretreated with solutions containing various cations (0.02 N, pH 7.0) increased in the following order: Li+, Na+, K+, Ca++, Sr++, Ba++, and Li+, Ca++, Al+++. The strongly hydrated ions Li+ and Na+ resulted in the lowest CT, while CT increased with decreasing hydration and increasing valence of the ions. The effect of the cations on CT could be reversed by soak- ing (leaching) previously treated leaves in water for a few hours. Anions produced a similar effect, CT was increased in the following order by citrate-_—, tartrate__, SCH“, Cl', Br-, N03, I’, CNS—. The order of effectiveness of cations and anions was reversed at pH values below 6-7, thus coinciding with the pH- dependent maximum of CT. Hartel (53, 5A) explained these results by assum- ing that CT is controlled by an ampholyte (cutin) interspersed by small pores. The diffusion resistance in this system would depend on the degree of imbibition and the size of the pores. In membranes having rela- tively narrow pores (cuticle type) hydration would re- sult in an increase in diffusion resistance, due to the presence of bound hydration water in the pores hampering the diffusion of free water molecules. In presence of strongly hydrated ions in the pores most of the space would be occupied by bound (hydration) water and the movement of free water molecules severely restricted. 26 In order to explain the effect of pH on water perme— ability of the CM one must assume that the CM is an ampholyte with an isoelectric point around pH 6 to 7, so that the net charge would increase at higher or lower pH values. Currently, however, the cutin matrix and the pectins are assumed to have a negative charge on dis- sociation and these groups are completely dissociated at pH 6 or 7. Thus, there is some doubt as to whether the CM behaves as an ampholyte as assumed by Hartel. Water uptake by needles of Abies cephalonica, Abies alba, Picea omorica and Pinus mugo was also in- fluenced by pH and certain ions (27). Water uptake from solutions of different pH values by needles having a water deficit of 15 - 20% was least at low pH values (1.75, A.25) and increased when the pH was increased to 7.0. The velocity of water uptake from salt solutions increased in the following order: Li+, Na+, K+, Ca++. The presence of strongly hydrated ions impeded water uptake. It should be pointed out that the concept develOped by Hartel (53, 5A) is in contradiction to the concept of incipient drying. While incipient drying is thought to reduce CT by reducing the degree of hydration of the CM, the concept put forward by Hartel argues the opposite, namely, that increase in hydration of the CM 27 reduces water permeability because the bound hydration water "plugs up" the diffusion paths for water. Polar Routes Across the Cuticular Membrane It has been shown that water and other polar molecules do in fact penetrate the lipophilic CM. Hartel (53, 5A) assumed the existence of hydrophilic pores in the cutin matrix. The problem of the locali- zation of hydrophilic channels in the CM, through which polar molecules can diffuse, will be considered next. The terms "hydrophilic channel" or "hydrOphilic pore" will be used to denote a region through which polar molecules can permeate the otherwise impermeable lipo- philic matrix of the CM. Strugger (118) using berberin sulphate (BS) as a tracer studied CT in relation to the extrafascicular transport pathways in plants. BS is a fluorochrome which shows little fluorescence in solution but strong fluorescence if adsorbed to tissue. Below pH 11 this dye exists as a cation and is undissociated at higher pH. In the cationic form BS is taken up slowly into the cytoplasm and thus it is suitable for studying water movement in the apOplast. When shoots of Helxine soleirolii were supplied an aqueous solution of BS through the cut stem, the rise of the dye in the vascular bundles could be 28 observed with a fluorescence micrOSCOpe. BS moved rapidly in the vascular system and reached the leaves within a few minutes. In the leaves, BS appeared first in the veins and then spread out into the cell wall system (apoplast). Soon, the cuticular ledges of the guard cells, the walls of the stalk cells of the globular trichomes and the anticlinal walls of epi— dermal cells showed strong fluorescence, while peri— clinal walls exhibited only weak fluorescence, even after prolonged periods. In the astomatous CM of the upper leaf surface the dye accumulated mainly in the anticlinal walls and in the basal cells of trichomes. Generally, the time required for fluorescence to appear was longer for the upper, than for the lower leaf sur— face. Strugger (118) concluded from the pattern of fluorescence that the sites exhibiting strong fluores- cence were preferentially involved in CT, the dye being carried passively in the transpiration stream and accumulated where the water evaporated. The distribu- tion of BS in the tissue was much too fast to be accounted for by a diffusion process. If transpiration was reduced by inducing stomatal closure or by wetting the leaves the dye spread more slowly, indicating that it moved passively with the water in the plant. 29 If an aqueous solution of BS was administered to one leaf of Helxine soleirolii and the other leaves were allowed to transpire freely, B8 was taken up by the leaf submerged in BS solution. Its cuticular ledges, basal cells of trichomes and anticlinal walls soon exhibited fluorescence and the dye was observed to move out of the submerged leaf into the transpiring leaves where it accumulated at the preferential sites previously men- tioned. The identity in the pattern of fluorescence indi- cated that the sites of entry were the same as the pre— ferential sites of CT. The entry of dye into a submerged leaf was greatest in young leaves and when stomata were Open. Strugger pointed out that the dye did not enter through the open stomatal pore (no surfactant used). He considered it more likely that the hydration of the cuticular ledges differed in open and closed stomata. The presence of hydrophilic pores large enough to permit passage not only of water but also of the large, charged BS molecule (MW 822, consisting of four six—membered rings) in the CM of Helxine was conclusively demonstrated (118). After 30 minutes uptake through the cut stem the dye (85) had been taken up and spread into the transpiring leaves. The lower surface was then coated with a mixture of 5% gelatin containing 0,9 M glucose and 0.1 M KSCN immediately before its 3O solidification. Due to the high osmotic pressure in the gelatin mixture, water diffused out of the leaf into the gelatin carrying with it BS where the pores in the mem- brane were sufficiently large. BS reacted with SCN_ ions resulting in an insoluble precipitate which fluoresced. Such a precipitate was observed where the gelatin had been in Contact with cuticular ledges, with the basis of globular trichomes and occasionally with the back wall of guard cells. Thus, these sites have hydrophilic pores sufficiently large to permit passage of BS. Anticlinal walls and conical trichomes which exhibited strong fluorescence themselves were impermeable for BS molecules as evident from the absence of berberinrhodanide in the gelatin negative of these sites. Renner and Kallmeyer (89) reported that the pat- tern of fluorescence differed in leaves having Open or closed stomata. Very little BS accumulated in the cuticular ledges of closed stomata, while accumulation was pronounced in open stomata. This problem was pursued further by Bauer (6). Leaf segments of Rhoeo discolor were floated on 0.1 M NcCl solution which caused their stomata to remain open independent of light. These pretreated segments were then floated on BS solution together with controls having closed stomata. After 2-3 minutes the segments with Open stomata showed strong fluorescence in the cuticular 31 ledges and 2—3 minutes later in the anticlinal walls, whereas the control segments first exhibited fluorescence in the anticlinal walls (A-6 minutes) and only after 10 minutes did weak fluorescence occur in the cuticular ledges. Since only small quantities of fluorescent dye accumulated in cuticular ledges of closed stomata it can be concluded that transpiration through these ledges (peristomatous transpiration) was reduced as compared to Open stomata. Thus, the cuticular ledges of stomata appear capable of changes in permeability as regards stomatal opening. A differential permeability of cuticular ledges of Open and closed stomata was observed also by Maercker (71) using tritiated water and microradioautography. In Sagittaria sagittifolia dense silver grains occurred in the film over the cuticular ledges of open stomata, whereas a uniform blackening of the film over the entire guard cell and adjacent cell was apparent when stomata were closed. Trichomes of a variety of plants have been shown to be able to take up various fluorochromes (13, 61, 115). In glandular trichomes of Vicia faba and Phaseolus coccineus penetration occurred preferentially through the thin CM over the proximal cells (13). EXPERIMENTAL Foliar Penetration of Succinic Acid 242-Dimethylhydrazide: Studies Employing Excised Leaf Disks Materials and Methods Plant Material Primary leaves of bean (Phaseolus vulgaris L. cv. "Blue Lake") were used as test material. Plants were grown in flats containing vermiculite, placed in a growth chamber. Temperatures of 30° C day and 25° C night were maintained. Light from fluorescent and tungsten lamps at an intensity of l 600 ft.c. was sup— plied for 1A hours a day. Relative humidity was not controlled and averaged 50 — 70% during the light and up to 80% during the dark period. Plants were spaced to prevent mutual shading of the leaves. To minimize pos- sible injury, leaves were not handled by the blade and they were kept dry during watering and free of pests and diseases. Under these conditions the primary leaves reached maximum size at the tenth day after planting. Fully expanded leaves (10 - 12 days old) were used in these studied and unless stated otherwise penetration was followed through the upper surface. 32 33 iProcedure Leaf disks, 17 mm in diameter, were punched from ‘the leaves omitting major veins and placed immediately in Petri-dishes (diameter 9 cm) lined with moist filter paper. Eight disks were punched from the two primary leaves of each plant. A small glass tube (10 mm internal diameter, 7 mm in height) was sealed to the center of each leaf disk using silicon rubber (RTV 11, General Electric). Num- erous catalysts had been tested, but only Harter T l and T 11 (Wacher Chemie GMBH, Munich, Germany) proved suitable since all others caused varying degrees of leaf injury. The silicon rubber and the catalyst were mixed thoroughly, then one side of the glass tube was dipped into the fluid mixture and lowered immediately onto the leaf disks without excerting any pressure. Curing occurred in 5 - 10 minutes (T 1) to one hour (T 11) depending on the quantity of catalyst added. Silicon rubber, instead of petroleum jelly (95) had some advantages: the surface of the leaf exposed to treating solution is well defined and of uniform size for each leaf disk (0.5 cm2) (Figure 1), leaf injury is mini— mized since pressure is not needed for obtaining a tight seal, no leaks occurred with surfactant concentrations up to 1% and temperatures up to A0° C and the silicon rubber is chemically inert. 3A Figure l.-—Photographs illustrating the penetration units consisting of a glass tube affixed to a leaf disk with silicon rubber. 35 36 A volume of 0.2 ml treating solution was pipetted into each glass tube. The standard treating solution (as determined by preliminary experiments) consisted of 5 x 10-“ M lLAC-SADH (labelled in carboxyl or methyl groups, s.a. 2 mC/mM) in 0.05 M citrate phosphate buffer (88) at pH 5.0. If a surfactant was incorporated, 0.1% of the non—ionic polyoxyethylene sorbitan monolaurate (Tween 20) was added. The bottom of the Petri—dish containing the leaf disks was then placed in a Petri-dish cover and the unit was covered with a second cover before positioning it in a shallow water bath with overhead illumination from fluorescent tubes (KEN RAD F 30 T8/CW). Unless mentioned otherwise the water bath was maintained at 30° C and the light intensity was 600 ft.c. In dark treatments the dishes were covered with aluminum foil. With the exception of time-course experiments, penetration was determined after 12 hours. Thereafter, the treating solution was drained off and the disks thoroughly washed with distilled water from a wash bottle. Next, the glass tubes with the adhering silicon rubber were separated from the leaf disks. The leaf disks were blotted lightly and mounted on double adhesive tape in stainless steel planchets to keep them flat during dry— ing at 60° C for 2A hours. 37 The radioactivity was determined using a propor- tional gas—flow end—window GM counter. Each sample was counted for 2,000 counts and the radioactivity calculated as cpm. The leaf disks were of finite thickness (density was 3.1 mg x cm-2). A self absorption curve was pre— pared (Appendix II A) using ground leaf tissue labelled with luC-CADH and the counting geometry was evaluated according to procedures outlined by Schweitzer e£_al. (102). The geometry was constant up to 20 mg x cm—2 density, indicating a constant counting efficiency. Presentation of Data Since the l[AC-SADH used was of the same specific activity for all experiments, data are reported on a net 0pm basis. The data are expressed as rate of pene— tration: amount (cpm) of SADH penetrated through unit area (0.5 cm2) in unit time (12 hours) for a 5 x 10_u M SADH solution, except for those experiments in which time or concentrations were experimental variables. Taking self absorption, counting efficiency and specific activity of l[AC-SADH into account a curve was prepared from which cpm data can easily be converted into moles (Appendix II B). Statistical Treatment A randomized block design was utilized. Each treatment consisted of 10 replicate leaf disks. In 38 experiments with up to eight treatments leaf disks from each plant were distributed systematically over all treatments such that each replicate disk originated from a different plant. In experiments with more than eight treatments leaf disks were distributed at random over all treatments. The 10 disks representing a treatment were distributed in groups of 2 or 3 over a number of Petri—dishes in such a manner that each Petri—dish con- tained A — 5 replicates of treatments receiving either light or dark. In some cases, however, randomization of replications was not possible, e.g. in the experiment where the leaf disks were subjected to osmotic stress by adding sucrose solutions to the Petri-dishes. Analysis of variance was performed on the data and comparison among the treatment means was carried out using Tukey's w—procedure (11A). The F—test was employed if only two means were compared. For data presented graphically, the confidence interval was calculated for each mean at a 5% level of significance. The total length of the vertical bar drawn through the mean represents twice the confidence interval (2 t0.05 Si)° Preparation of Leaf Cross-Sections Leaf segments (5 x 3 mm) were killed and fixed using CRAF III fixative (30 ml 1% chromic acid, 39 20 ml 10% acetic acid, 10 ml A0% formalin, A0 ml HOH), dehydrated in a graded series of tertiary butanol and embedded in Tissuemat. Sections 6 - 7 um thick were found most satisfactory. They were lightly stained with Fast Green. Permanent slides were prepared. Leaf segments for observation in surface View were killed, fixed and dehydrated as above and cleared in clove oil. For inspection and photography the sec- tions were mounted in clove oil. Photography Photographs were made using a Wild M 20 research microscope equipped with a 35 mm film carrier and an automatic exposure control unit. Leaf Surface Images and X-ray Analysis An electron microprobe X-ray analyzer (Applied Research Laboratory Inc., EMX — SM) was employed to obtain images of the bean leaf surface and for study— ing the distribution of silver in leaves. Sample pre— paration was similar for both purposes. Leaf disks were freeze-dried, small segments (2 x 3 mm) cut from the disks and mounted on strips of double adhesive tape on a carbon disk of 3.2 cm diameter. The two ends of the leaf segments extending over the adhesive tape were grounded to the carbon disk with Television Tube Koat A0 No. A9-2 (General Electric). Samples were coated with carbon. The instrument was operated at an accelerating voltage of 20 kV and a sample current of 5 nA. Radioautography Radioautograms of leaf disks treated with 1”c—SAOH or l“Ca-tryptophan were prepared as follows: At the end of the penetration period the disks were thoroughly washed with water, frozen immediately on dry ice and freeze-dried to prevent movement of the label. The dried leaf disks were mounted on card board and radio- autograms were prepared using Kodak ”Blue Brand" X—ray film. Analysis of Procedures The quantity of SADH which penetrated was deduced from the radioactivity detectable in the leaf disks after washing the treated surface with water. This deduction was legitimate only if (a) surface binding of SADH is small compared to the amount which penetrated and (b) if no appreciable loss of activity occurred during the l2-hour penetration period. Breakdown of SADH in plant tissue was shown to occur at a very slow rate (26, 75) regardless of the position of the label. The loss of radioactivity (as llA002) during the penetration period was therefore assumed to be negligible. A1 Preliminary experiments revealed a marked increase of penetration in light. This could have been due to assimilation of 1“C02 originating from breakdown of 1[AC-SADH in the treating solution. Identification of the absorbed label after penetration seemed desirable. The separation procedure of Martin g£_al. (75) was followed, except that methanol was used for extrac- tion. Penetration of 1LAC-SADH (carboxyl labelled) was allowed for 12 hours in light and the leaf disks were washed, lyophilized, ground and extracted with methanol for 12 hours in a Soxhlet apparatus. The methanol ex— tract was brought to 10 ml volume in a warm air stream and an aliquot was counted. The total activity in the methanol extract was 19,900 cpm and in the residue A07 cpm. The methanol extract was passed through a Dowex 50 W X8 (50-100 mesh) cationic ion—exchange resin. The column was washed with 60 ml distilled water and eluted with 60 ml 7.5 N NHMOH. The eluant was brought to a constant volume and an aliquot counted. A figure of 16,650 cpm was obtained for the 50 W fraction, that is 8A% of the total activity of the methanol extract. The fraction not bound by the resin gave 1,890 cpm or 9.5% of the total activity of the methanol extract. This resulted in a 93.5% recovery. A2 Both fractions were chromatographed on thin—layer plates (Silica Gel C) using isopropanol, NHuOH and water (20:1:A) as the developing solvent. The plates were scanned for radioactivity using an Actigraph II Model 1036. Both fractions separated into two peaks with RF values of 0.3 and 0.1 respectively (Figure 2). From a comparison with a 1LAC-SADH standard it was clear that the activity at R 0.3 represented SADH. The compound F at R 0.1 was not identified. This small peak also F occurred using the SADH standard and was also observed and considered an impurity by Martin gp_al. (75). The standard is not published here, instead, the standard solution and 50 W fraction were co—chromatographed and the scan was superimposed over the scan of the 50 W fraction alone (Figure 2 A). There was absolute agreement be— tween the peaks of both scans. Therefore, the bulk of the radioactivity recovered from the bean leaf disks was lquSADH. Because of limited quantitites of lLAC-SADH avail— able an attempt was made to recover some of the pre- viously used labelled compound. After termination of the penetration experiments the treating solution was collected from the glass tubes, concentrated in a warm air stream, acidified with HCl and then passed through the Dowex 50 W X8 column. The fraction not bound was discarded and the NHuOH eluent containing 1LAC-SADH and A3 Figure 2.——Tracings of radiochromatogram scans of thin—layer plates. A. Dowex 50 W X8 fraction of the methanol extra*t from bean leaves treated with l C—SADH (dotted line) and the same fraction co—chromato- graphed with SADH standard (solid line). Fraction of methanol extract from bean leaves treated with lac-SADH not bound to the Dowes 50 W X8 column. Methanol fraction of purified treating solution (dotted line) and the same fraction co—chromatographed with SADH standard (solid line). AA 1 \ \‘ ‘ “ 000‘. I:-:--." 0 0--- 0“. LO 0.. 0.6 All..- OOUOIOUUI“.“I~|I‘ LO 0.. ' AS buffer cations was dried in a warm air stream. The residue was extracted with absolute methanol which brought SADH into solution and left NaOH behind. A sample of the recovered lLAC-SADH was co-chromatographed with standard 1[AC—SADH and tracings of the radioscans obtained are presented in Figure 2 C. There was abso— lute agreement with the standard indicating successful recovery of lLAC—SADH. The recovered material was stored under refrigeration until used. Another experiment was designed to determine the most effective washing medium. Since SADH is very water soluble, distilled water, absolute methanol and 0.02 M SADH, buffered at pH 5.0, were evaluated. Primary leaves of bean were gently agitated in a buffered solu- tion (pH 5.0) containing 2A nC/ml lLAC-SADH, removed after 10 seconds and after excess liquid had drained off they were placed each in 50 m1 of the prescribed washing medium and gently agitated. After 10 seconds they were removed, the leaf surface determined with a planimeter and discarded. The total radioactivity contained in each 50 ml washing medium was determined. Methanol re- l[AC-SADH per cm2 leaf area and distilled 2 moved A8 cpm water A2.5 cpm x cm- , the difference being not signifi— cant. With 0.02 M SADH only 1A.A cpm x cm-2 were re— moved. It was therefore decided to adopt washing with distilled water as standard procedure. A6 Since SADH is very water soluble, it may move freely in the water phase (apoplast) inside the leaf. From the radioautogram it was apparent that the label accumulated in the veins and that some transport to the cut edges occurred (Figure 3). With Tween 20 in the treating solution more label accumulated and appeared more uniformly distributed (Figure 3 B). The silicon rubber sealant did not seem to cause any injury to the cuticle since no preferential penetration took place in the area where silicon rubber was in contact with the leaf surface and the treating solution. Loss of l[AC-SADH from the leaf disk by penetrat- ing through the leaf surface and passing into the moist filter paper, could be a serious source of error. To check this possibility, leaf disks were placed on individual filter disks (2.1 cm in diameter) and pene- tration through the upper or lower surface was allowed to proceed for 6 and 12 hours in light. Radioactivity of leaf disks and their respective filter disks were determined (Table 1). There was some loss of radioactivity from the leaf disks. This loss was proportional to the radio— activity in the leaf disk. More radioactivity was lost through the lower surface (5.25 - 6.25%) than through the upper (3.70%). The loss of A - 6% of the total radioactivity into the filter paper was accepted since A7 Figure 3.—-Radioautogram of bea leaf disks follow- ing penetration of 1 C—SADH for 12 hours in light (ca. 1.5 x). A . SADH B. SADH plus 0.1% Tween 20 A8 A9 it appeared to be proportional to the activity in the leaf disks. TABLE 1.—-Loss of l[AC-SADH from leaf disks into the underlying filter disks. Penetration Radioacthlty Radioact. of Penetration period filter in 7 of surface hours Leaf Filter total activity cpm cpm Upper 6 118 7.9 6.25 12 259 1A.3 5.25 Lower 6 187 7.2 3.70 12 AlA 15.9 3.70 Results Bean Primary Leaf Morphology Knowledge of the leaf morphology is essential for a meaningful interpretation of penetration data. Secondary electron micrographs (SEM) of the upper and lower surface of primary leaves of bean are presented in Figure A. The guard cells appear to be elevated above the epidermal cells (A). The entire stomatal apparatus is approximately 25 - 35 um long and 15 - 20 um wide. The aperture between the cuticular ledges measures 7 - 10 by 10 ~ 15 pm (B). The walls of the guard cells inside the aperture can be seen (B). Glandular trichomes occur on both surfaces of 50 Figure A.-—Secondary electron micrographs of the surfaces of bean primary leaves. A. B. Upper surface with stomata. Open stoma showing front cavity and pore. Glandular and hooked trichomes on lower surface. Glandular trichome. Conical trichomes on upper surface. 52 the leaf and tend to be associated with the veins (C). Glandular trichomes are A0 — 50 um long and consist of a foot cell, the stalk cell and A cells forming the "head" (D). The elongated trichomes (conical and "hooked" trichomes) are up to 100 um long, unicellular and occur randomly distributed over both surfaces (C, E, F). The foot cell of the conical thichomes is sur— rounded by a characteristic ring of specialized cells. The number of stomata per unit surface area was estimated using a replica technique (57). The upper and lower surfaces of 10 fully expanded leaves were coated with Rhoplex AC 33. After drying (30 minutes) the thin, transparent replicas were peeled off and mounted on glass slides. Stomata in 10 fields (500 x) of each replica were counted and the stomatal density was calculated (Table 2). The density of glandular trichomes was determined by counting all glandular trichomes on the upper and lower surfaces of 10 leaf disks (0.5 cm2) directly under the microscope. More stomata (2.78 x) and glandular trichomes (3.70 x) were found on the lower than on the upper leaf surface (Table 2). The relationship between stomatal density and leaf size and leaf age was also investigated using 53 Rhoplex replicas (Figure 5). As expected, leaf size and stomatal density were inversly proportional. From the 7th to the 10th day the leaf area increased by a factor of 2.26 while stomatal density decreased by a factor of 2.30. There was no significant change in either leaf size or stomatal density after the 10th day. TABLE 2.--Density of stomata and glandular trichomes on fully expanded bean primary leaves. Stomata Ratio Gland. trichomes Ratio Surface per mm2 L/U per cm2 L/U Upper (U) 10A 15A Lower (L) 289 2.78 570 3.70 The kinetics of stomatal opening and closing as related to light was determined using continuously operating and recording porometers. The method employed was essentially as described by Raschke (86, 87). Leaf disks from eight different leaves were inserted into the porometer chambers which were submerged in a water bath at 27° C. White light was provided by means of a high pressure xenon arc. The radiation intensity was 15.5 mW x cm-2. The porometer flow of three selected leaf disks (Figure 6) showed that between 60 and 100 minutes were required before maximum opening of stomata occurred. When the light was switched off the initial reduction in 5A Figure 5.--Relationship between leaf size (plant age) and stomatal density on the upper surface of bean primary leaves. Growth conditions: 30° C (day), 25° C (night), 1,600 ft.c. of light for 1A hours a day. DENSITY 0F STOMATA (stomata x mm -2) 55 250 r- A O 200- ~30 o—o Leaf size 0—0 Density of siomara " 25 ISO- -20 _ L n 1 1 1 1 PLANT AGE ( days ) LEAF SIZE (cm‘?) 56 Figure 6.-—Kinetics of stomatal opening and closing of bean primary leaves as indexed by porometer flow. Leaf temperature 27.5° C (light), 27° C (dark), radiation energy 15.5 mW x cm"2 57 IOO- s. ‘0. 0 2 ‘ Q 0 3E 3: \ 1 1 1 1 \ O O O O 0 CO V N M0 7:! 83.1 JNOUOd 3A l1V73£I I50 |20 60 30 T IME (minutes) 58 porometer flow was rapid but even after 50 minutes stomatal opening was greater in all leaf disks than at the beginning of the experiment. The plants were kept in the dark for 10 hours prior to the experiment. From transverse sections it appears that guard cells and accessory cells from the upper leaf surface are larger than from the lower surface (Figure 7). Guard cells have outer cuticular ledges, while the inner ledges are missing. Also leaves with stomata open were fixed, the significance of the difference in stomatal aperture is questionable, since organic solvents used for fixing and dehydrating could have affected stomatal Opening (101). Entry of Liquids into the .Intercellular Space Through Stomata, a Theoretical Treatment The question of aqueous solutions entering the intercellular space through the stomatal pore of leaves is a current topic of discussion. While some workers (108) have claimed that squeous solutions can penetrate stomata readily, others question this (A0). Turrell (120) and Dybing g£_al. (22) are of the opinion that stomatal penetration can occur if the surface tension of the liquid is lowered by a suitable wetting agent. Evidence presented in support of stomatal penetration is largely indirect. Examples are, the establishment of a 59 Figure 7.——Cross sections of stomata from bean primary leaves. A and B: From upper surface. C and D: From lower surface. 60 61 relationship between stomatal density or stomatal open- ing (18) and some parameter of penetration, such as, radioactivity or fluorescence of treated leaves or a biological response of treated plants. To the author's knowledge no detailed studies determining the major factors controlling stomatal pene— tration have been reported. The importance of surface tension is often mentioned in this context, although it has been shown that the contact angle was of greater importance in stomatal penetration than surface tension (101). Ursprung (123) concluded from theoretical con- siderations that the cuticular ledges act as a barrier to stomatal penetration. Capillary rise in pores of varying diameter has been treated theoretically by Adam (1). On this basis an attempt will be made to establish factors governing the entry of aqueous solutions into the leaf via stomatal pores and to assess their relative importance. 7 When a liquid enters a capillary of small radius a curved meniscus will be established, unless the con- tact angle (0) is exactly 90°, producing a difference in pressure (Pl-P2) on the two sides of the liquid surface (the concave and convex sides, respectively). P-P=YL(R1;+R_-) (l) 62 YL being the surface tension of the liquid and R and 1 R2 the principal radii of the curvature (in a capillary of sufficiently small bore the meniscuc will be hemi— 1+1 R1 R2 (circular) with radius r the penetrating pressure Pi spherical and ( ) becomes (%)). For a capillary (Pl—P2) is given by p,=al__<=_a§_9, (2) Using the advancing contact angle (6A) for a tube with parallel walls (2) becomes 2y cos 6A Pi = I, . (3) P1 is positive when the meniscus is concave, that is, when 6A is smaller than 90°. Pi is equal to zero when 9A is exactly 90° and P1 becbmes negative when 6A is larger than 90°. Therefore, capillary rise will not occur, when the contact angle 0 is 90° or larger. A A capillary with inclined walls is depicted in Figure 8. The walls have an inclination of A5° to the horizontal axis (¢l=¢2). If the advancing contact angle of a liquid rising in this tube is A5°, the penetrating pressure P1 will be gg-before passing the point of in- flection and zero after passing. If 6A is larger than A5°, P1 will become negative as indicated by the convex meniscus for a liquid having 6A = 65°. 63 Figure 8.—-Schematic diagram illustrating a capillary pore of circular cross— section having inclined walls. O advancing contact angle A ¢ angle of pore walls 6A some, "wow a .. . . was. txrw A. m¢Iecies ledges are found also on the inner boundary (sub— stomatal chamber side) of the aperture. In stomata with- Ollt inner ledges the inner boundary of the aperture is a Continuation of the inner walls of the guard cells where tfley progress more or less parallel to the leaf surface (Zone 8 in Figure 9). The diameter of the aperture is Seuierally largest in the front cavity and smallest in the pore (Figure 9). A stoma from the upper surface of a primary bean 1eaf’(Phaseolus vulgaris) prepared from a camera lucida 66 Figure 9.--Schematic drawing of a cross-section of a bean leaf stoma (drawn to scale). The penetrating pressure of a liquid (0A = 35°,y'=30 dynes x cm‘l) pene- trating the aperture is illustrated by the radius of the meniscus for each zone. I2r- Cuticular ledge 67 Penetration . J(‘ (‘55? ' on 'A&:;{( ll 0 75'; Substomatcl Chamber 68 dccawing is illustrated in Figure 9. The aperture was ssubdivided into 8 zones, each being characterized by the zingle ¢ which the guard cell wall forms with the hori— zontal axis (Table 3). 'TABLE 3. --Ca1cu1ated changes in penetrating pressure (Pi ) along the stomatal aperture shown in Figure 9. SZone Walldangle (0) Radius (r) Penetrating pressure (Pi )+ egrees um dynes x cm 1 A3 1.9 3,800 2 90 2.5 19,650 3 A7 2.5 29,700 A 58 1.6 A5,600 5 90 1.0 A9,000 6 5A 1.0 1A,300 7 2A 1.9 - 6,030 8 00 2.7 —l3,200 +Assumed: 0A = 35°, 7 = 30 dynes x cm—l. For the purpose of illustrating the application of fYIrmulae A and 5 to the problem of stomatal penetration, tile following conditions will be assumed: a stomatal almerture as shown in Figure 9; relatively smooth walls 01‘ uniform composition throughout the aperture; an aQueous solution containing a surfactant and having a -1 surface tension of 30 dynes x cm and forming a contact 69 aJigle of 35° with the walls of the aperture, which is (nonstant throughout the aperture and with time. The first barrier encountered by a penetrating :solution is the constriction formed by the cuticular ledge, however, since ¢ = A3° is larger than 0A (35°) ‘the liquid can enter the front cavity (Figure 9, Table 3). Towards the pore the diameter of the aperture de- ‘ creases and the slope of the walls increases (larger ¢) resulting in an increasing Pi' Maximum Pi is attained :in the pore (zone 5). After zone 5 the diameter of the aperture increases and the slope of the walls decreases (smaller 0), resulting in a decreasing Pi' At the inner t)oundary of the aperture (zone 8) 0 becomes zero. From zaone 5 to zone 8 P1 decreases but the liquid under con— sideration cannot advance beyond zone 6, because in zone 7 0A becomes larger than e2 making P negative as 1 :indicated by the convex meniscus. Only a liquid having 9A,°f less than 2A° could penetrate further into zone 7 élnd only a liquid having 6 = zero could penetrate the A :substomatal chamber. Up to this point the gravitational pressure (Pg) ARES been neglected. Depending on the orientation of the stoma, Pg must either be added or subtracted from Pi. HQwever, P8 is negligible due to the shortness of the St omatal aperture . 70 P = g hdg (6) In equation (6), h represents the height of the capil— lary rise, d the density of the liquid and g the gravi— tational force (approximately 980 gm x sec—2). For an aqueous solution d is approximately 1 and at h = 6.6 pm Pg is 6.5 dynes x cm_2 which is negligible compared to the Pi values calculated (Table 3). From the model discussed it would appear that the major factors which control the entry of liquids into the stomatal aperture are (a) the geometry of the pore, (b) contact angle and surface tension of the liquid and (c) the diameter of the aperture. These factors shall be considered next. Geometry of stomatal aperture.——In a capillary with increasing diameter the relationship between 9A and 0 determines whether Pi will be negative or posi— tive (equation 5), that is to say, whether or not capillary rise will occur. Most stomatal apertures have two zones in which the diameter increases (52), one is generally direct behind the cuticular ledges (zone 1) and the other beyond the pore (zones 6-8). According to equation 5, a liquid can enter the aperture only, if 6A is smaller than 02 of the cuticular ledges. Any liquid capable of entering the front cavity will pene— trate at least up to the pore, due to the increasing 71 P1 gradient. Beyond the pore, Pi decreases and the liquid will advance only up to the point where 0A be— comes equal to 0. At this point P1 becomes zero. Many plants have stomata with apertures of geometry similar to the bean (Figure 9) (28, 52, 70, 120). The aperture tends to widen abruptly at the boundary of the substomatal chamber, thus making ¢ very small or zero (as in zone 8). Only a liquid with 0A of zero can ad— vance from such an aperture into the substomatal chamber, because any finite contact angle will result in a nega- tive P This appears to be the mechanism which prevents i' the intercellular space of leaves from becoming infil- trated when wetted with water. The air in the inter— cellular space probably does not prevent liquids from penetrating into the leaf, because the intercellular space is a continuum and air could escape through some stomata while others are being infiltrated. Contact angle and surface tension.--The contact angle (6) formed by a liquid on a solid surface is dependent upon the surface tension of the liquid (YL) and the solid (Y3) and on the interfacial tension (YSL) according to Y8 = YSL + YL cos 0 . (7) 72 Thus, the contact angle is affected by the free surface energy of the solid. The largest contact angle a water droplet will form on a smooth surface generally does not exceed 105°. Such contact angles have been observed on lipophilic surfaces, such as stearic acid, paraffin and silicon (2). Roughness generally increases the contact angle if the surface material gives a contact angle larger than 90° on a smooth surface. Contact angles as large as 1A0° have been reported for Triticum leaves (32) and pea leaves (6A). The prob— lem of wettability of leaves has been dealth with (2, 31, 32, 23) but these studies are irrelevant for the present purpose, because the contact angle inside the stomatal aperture is the point in question. No such information could be found. The surface of the stomatal aperture is generally believed to be more hydrophilic than the leaf surface. Wax coverings probably do not occur in the stomatal aperture. Turrell (120) reported that the guard cell walls of Citrus leaves stained with Ruthenium red in the pore but not with Sudan stains. This indicates the presence of polar groups. Thus, 0A of a given liquid will probably be smaller inside the aperture than at the leaf surface. However, even with high con- centrations of surfactants the contact angle seldom is less than 20 - 25° (23), unless, perhaps, the surfactant interacts with the wall in a way which permits the 73 solution to penetrate the wall, resulting in a decrease of 9A with time (1). This may occur with surfactants that are toxic. 'With solutions which do not penetrate into the walls of the aperture (which exhibit only small changes in contact angle with time) infiltration of the intercellular space is not likely because ¢ will ulti— mately become smaller than 20 - 25° making Pi negative. Diameter of stomatal aperture.-—Diameters of stomatal apertures for various plants have been pub— lished. Most of them were obtained either by direct microscopic examination of leaves (22) or from silicon rubber replicas (66). Such replicas were shown to be not always reliable in measuring the degree of stomatal opening (61). The diameter of the stomatal aperture is of minor importance for infiltration of the intercellular space, as long as pressure is not involved. According to equations (M) and (5) P increases with decreasing i diameter of the aperture. It is therefore a misconcep— tion to argue that stomatal penetration of bean leaves is difficult to obtain because of their small diameter (22). Since the diameter of the aperture affects only the size of the forces involved (Pi) and not the sign (positive or negative), both entry of a liquid into the aperture and its advancement into the substomatal chamber are not determined by the diameter. 7U Penetration of SADH Time-course.--Penetration of SADH into bean leaf disks was linear with time over a 12 hour period (Figure 10). This was true for both surfaces, in light and in the dark and in both the absence and presence of Tween 20. Penetration was greater in light than in the dark and in the presence of Tween 20. From these results it was decided to adopt 12 hours as standard duration for all experiments. Surfactant concentration.--Tween 20 at concentra— tions higher than 0.001% increased penetration of SADH in the dark and in light (Table M). The largest in- crease occurred when the Tween 20 concentration was raised from zero to 0.01%. A further increase in sur— factant concentration to 0.1% resulted in a significant increase in penetration in light but not in the dark. Tween 20 at 1% suppressed penetration in light as com- pared to 0.1%, and did not significantly affect penetra— tion in the dark. Eight.--For the light intensity studies a light bank providing 3,600 ft.c. was used and lower intensi— ties were obtained by shading the dishes with cheese cloth. The temperature of the leaf disks and the treating solutions in the glass tubes was monitored over a 12-hour period using cooper-constantan thermo— couples with a recording potentiometer. There was 75 Figure 10.--Time course of penetration of SADH into bean leaf disks. A. Penetration through the upper surface in the dark. B. Penetration through upper and lower surface in light. 76 A952: warp OON 00* 000 .3th 1.3.6. Joe»: 5284 4'4 ‘33.“. 692.3. echo.» C'Q 32.: t >6}. .8853 toss o '0 a E‘m.~o8§u 333 0'0 :52: ml; 52.x :33 9'0 tfi‘m 0'0 00. OON con 00? (rule 9'0 x We) NOIJVUJJNJd 77 very good agreement between the temperature of the water bath, the temperature of the leaf disks and treating solutions at low light intensities. A i 0.50 C devia— tion was found in the dark and at light intensities up to 600 ft.c. The temperature of the treating solution rose 2 — U° C over the bath temperature under light intensities of 1,200 to 3,600 ft.c., whereas the differ— ence between leaf and water bath temperature was + 1 to + 2° C. TABLE A.--The effect of Tween 20 on penetration of SADH into bean leaf disks. Tween 20 Penetration conc' Dark Light cpm cpm % 2 % 2 7; 0.5 cm x 12 hrs. 0.5 cm x 12 hrs. 0.000 16.3 a* 100 138.9 a 100 0.001 15.3 a 9A 160.A a 118 0.01 “9.6 b 304 363.7 b 262 0.1 52.0 b 319 50A.7 c 339 1.0 61.7 b 378 3A5.5 b 2A9 *Means within a column followed by different letters are significantly different at P = 0.05. Penetration of SADH into both leaf surfaces in- creased with increasing light intensity (Figure 11 A). 78 Figure ll.--The effect of light on penetration of SADH into bean and pear leaf disks. A. Penetration through the upper and lower surface of bean leaf disks. Penetration through the upper sur- face of bean leaf disks, with and without Tween 20. Penetration through the upper (astomatous) and lower (stomatous) surface of pear leaf disks (with Tween 20). 79 .6 .: . >thth_ .533 .6... . >tmzuhz. .23... .u .2. rtmzmhz. :63 00o OoN i ¥¢.pool 3 k3 (apOplast) (cells) (glass tube) The concentration of SADH in pool 2 will be determined by the penetration rate kl and the rate of uptake k2. If more SADH penetrates into the apoplast than is taken up into the cells (kl greater than k2), then the SADH concentration in pool 2 will increase until equilibrium between pools 1 and 2 is reached (k1 = k3). A process of this nature would follow first order kinetics and the time required to reach equilibrium would 121 depend on the capacity of pool 2 and on the penetration rate k1 provided a constant concentration in pool 1. The concentration of SADH in the leaf disks (pool 2 + pool 3) increased at a linear rate over a l2-hour period (Figure 10). This may occur under two conditions. (a) The capacity of pool 2 (apoplast) is large relative to the penetration rate kl. The total SADH concentration which could accumulate within 12 hours, therefore, re— mained very low and negligible compared to the SADH con— centration in pool 1. Thus, kl remained constant. (b) Pool 2 is small but the concentration of SADH in pool 2 remained negligible compared to pool 1, because SADH was taken up by the leaf cells at a rate k2 which was of a similar magnitude to kl. For the first case, SADH uptake into cells would be immaterial, because pool 2 is so large (relative to kl) that the concentration of SADH in it remains negligible compared to pool 1 even in the absence of uptake into the cells. In the second case uptake of SADH into the cells, or no uptake at all, would result in an increase in SADH concentration in pool 2 and the penetration rate k1 would then decrease with time. Only in the second case could active uptake of SADH into the leaf cells affect the penetration rate k1. It could be argued that the penetration of SADH was greater in light than in the dark, because in the dark 122 energy-dependent active uptake of SADH into cells was limiting, causing an accumulation of SADH in the apOplast, which in turn depressed the penetration rate kl. If this was true, penetration in the dark could not have been linear with time. The penetration rate k at the begin- 1 ning (when the SADH concentration was still low in pool 2) would have been higher than at the end, and the pene— tration curve would level off with time. Furthermore, the assumption that active uptake is rate limiting in the dark could not explain why addition of Tween 20 and decreasing the pH of the treating solution resulted in an increased penetration rate which was constant with time. The data from the concentration experiments also indicate that active uptake was not rate—limiting (Figure 13). Penetration of SADH was linear with concentration over a wide range (up to 10"2 M). Current concepts assume that carriers are involved in active uptake (65). With increasing concentrations of SADH these carriers must ultimately become saturated and an increase in concen— trations beyond this point would not increase penetra- tion. The fact that this saturation was never achieved is a further indication that active uptake was not the rate-limiting factor in penetration of SADH. Finally, the increasing Q10 with increasing tempera— ture was also evidence against active uptake as a 123 rate-limiting factor as well as the occurrence of tempera— ture quotients as high as 7.“. A Q10 of this magnitude is not associated with metabolic processes. It is therefore suggested that SADH penetrated the cuticular membrane by diffusion and that the penetration rate was constant over 12 hours because kl was low com— pared to the capacity of the ap0plast such that the SADH concentration in the apoplast remained negligible rela- tive to the concentration in the glass tubes. Some of the SADH in the apoplast was probably taken up into the cells but it had little or no effect on the penetration rate. This concept of ap0plastic accumulation of SADH is supported by the fact that SADH was lost from the leaf disks into the underlying filter disks (Table 1). This loss was proportional to the concentration of SADH in the leaf disk and specific for each leaf surface. More SADH was lost through the lower surface than through the upper. The ratio of per cent lost through the lower surface/per cent lost through the upper surface (1.69 for 6 hour and 1.“2 for 12 hour period) was almost identical to the ratio of per cent penetrated into the lower surface/per cent penetrated into the upper sur- face (1.60). If penetration of SADH into bean leaf disks was strictly a diffusion process as assumed, then temperature, 12“ light, Tween 20, pH and urea must have affected the pene- tration rate directly by: (a) affecting the surface area of the cuticular membrane in contact with the treating solution (effective surface), (b) affecting the permea- bility of the cuticular membrane or (c) affecting both permeability and effective surface area. Factors Affecting the Effective Surface Area The apparent leaf surface area inside the glass tube was measured as 0.5 cm2. The true surface area, however, was greater due to topography of the cuticular membrane and the presence of trichomes. A distinction between inner and outer surface must also be made. The outer surface may be defined as that area of the cuticular membrane accessible when stomata are tightly closed. The inner surface is defined as the surface inside the stomatal aperture whose size depends on stomatal density, degree of stomatal opening and size of the stomatal apparatus. For the upper surface of bean leaves the inner surface was estimated to be approximately 0.8 to 1.2% of the apparent outer surface. All factors which tend to increase stomatal Opening will increase the inner surface. 0f the experimental variables in- volved in this study, light, temperature and pH can af- fect stomatal opening (55, 113), while water, which is otherwise an important determinant, was not a factor 125 because the leaf disks were in a constant environment of 100% relative humidity. In most plants stomata close in the dark and re— main closed. Light affects the degree and the speed of opening. Using maize leaves, Raschke (87) has shown that the relative stomatal width (%porometer flow) was directly proportional to CO2 assimilation. With increas- ing light intensity, stomatal width increased rapidly at first with a subsequent leveling off as the light intensity approached saturation. The speed of stomatal opening was prOportional to light intensity. The effect of temperature on stomatal opening in Vicia faba leaves was investigated by Stalfelt (113). In leaves, floating on water and exposed to air and light (20,000 lux), stomata were closed at 5° C. With increas- ing temperature stomatal opening increased almost linearly up to “0° C. Small et_al. (109, 110) found that the guard cells of closed stomata (dark) had generally a lower pH than those of Open stomata in light. Stomata of epidermal strips floating on buffer solutions of different pH values remained closed at low pH values (3-“) and opened at higher pH values (5-6). The response differed with the buffer ions used; nevertheless, stomata opened in the same pH range found in guard cells of open stomata in intact leaves. 126 The inner surface will be an effective surface only if the surface tension of the treating solution is low enough to permit entry into the stomatal aperture. Thus, addition of a surfactant tO the treating solution not only enhances wetting of the outer leaf surface but also makes the inner surface available for penetration when stomata are Open. This cannot be viewed as an either-or mechan— ism. Stomata vary in size and degree of opening. Even in the dark some stomata are more or less Open, that is to say, the number of closed stomata will decrease and the degree of opening will increase as the light intensity is increased. Some stomata may be sufficiently large and Of a geometry such that even a treating solution without a surfactant can enter (Figure 17 C, D). Even under these circumstances a surfactant may increase the effective inner surface, because the treating solution could ad— vance deeper into the aperture. Therefore, the effect of stomatal Opening and addition of a surfactant on the effective inner surface is quantitative rather than qualitative. The inner surface is probably not involved in penetration in the dark when no surfactants are em- ployed, whereas, addition of a surfactant may allow the solution to enter those few Open stomata. Solution can probably enter a small number of stomata in light in 127 the absence Of a surfactant, particularly under high light and temperature conditions. In light and with a surfactant the treating solution will penetrate into the aperture of many or all stomata. The effective inner surface will be largest when stomata have attained their greatest aperture. The conclusion, therefore, is that all factors which tend to increase stomatal Opening and wettability will increase penetration by increasing the effective surface of the cuticular membrane. These factors are light, temperature pH and Tween 20. The following observations in this study can be totally or partially explained by the arguments presented above: the greater penetration in light than in the dark, the gradual in— crease in penetration with increasing light intensity and increasing temperature and the relationship between light, temperature and Tween 20. In the dark and without Tween 20 temperature affected penetration Of SADH only slightly (Table 5) because stomata remained closed and there was little change in effective surface. With increasing light intensity and/or temperature penetration of SADH in— creased because the number Of open stomata and their aperture increased. Temperature and light were more effective in increasing penetration in the presence Of 128 Tween 20, because Of increased effective surface, par— ticularly the effective inner surface. There is a possibility that the sudden increase in penetration in the dark at high temperatures (Figure 12) was due to stomatal Opening in the dark. It has been reported (73) that stomata Of Xanthium leaves Open widely in the dark at temperatures between 27 - 36° C. Inhibitors reduced penetration in the light but were ineffective in the dark (Table 11). This would be difficult to understand if the inhibitor affected a metabolically—linked uptake process, but their effect is easily explained on a basis of a reduced effective sur- face, since all inhibitors prevented stomatal opening in light (9, 132). The lack of activity in the dark would be expected since the stomata would be closed. Stomatal closure by plasmolysis (Table 10) affected penetration of SADH in the same way. (An effect of stomatal closure on the permeability Of the cuticular ledges will be con— sidered later). The close relationship between stomatal density and penetration in light and its absence in the-dark (Table 6) is further evidence for the importance Of stomatal Opening in penetration Of SADH. It must be pointed out, however, that the relationship between stomatal density and penetration is not as good when the upper and the lower surfaces are compared. Only 1.6 times (Table l) or 129 1.8 times (minus Tween 20) and 1.“ times (plus Tween 20) (Figure 10) more SADH penetrated into the lower than into the upper leaf surface, although stomatal density is 2.78 times and the density of glandular trichomes 3.7 times greater on the lower than on the upper surface. Stomata and glandular trichomes may well differ anatomically and/ or physiologically between the two surfaces. It is uncertain whether or not the pH of the treat— ing solution can affect Opening of stomata in intact leaves as it does in isolated epidermal strips. It could be speculated that Tween 20 increased penetration Of SADH more effectively at high pH values (5 — 7) than at low pH values (“), because stomatal Opening was inhibited at pH “.0 (Table 7). Since Tween 20 reversed the pH effect in light, but not in the dark, the possible involvement of stomata should be recognized. Factors Affecting the Permeability of the Cuticular Membrane In order to discriminate between different mechan- isms Of penetration Of pharmaceuticals across the lipid membranes of the digestive tract, the terms "non-ionic diffusion" and "filtration" were coined. Partitioning Of undissociated weak electrolytes into lipid membranes is called non-ionic diffusion and penetration Of charged molecules (dissociated) via polar channels in the lipid 130 membrane, a process linked to water movement, is called filtration (12“). This concept will be adopted for the present discussion. Non-ionic diffusion.——Lipophilic molecules may penetrate a lipid membrane by partitioning. In a homologous series there was good agreement between the partition coefficient (chloroform:water) and cuticular penetration (20). Weak acids and bases, although Often quite polar, can also penetrate a lipid membrane by partitioning in the non—dissociated state. Therefore, penetration of weak electrolytes through lipid membranes is markedly affected by pH, while penetration of non- ionizable molecules is not (106). Below the pKa, penetration of a weak acid is pro- portional to the concentration of the weak acid. With increasing pH above the pKa, the portion Of the total concentration (HA + A-) which penetrates becomes smaller and smaller. It was therefore postulated that only the undissociated molecule (HA) can penetrate while the anion (A_) cannot. If this was true, penetration would have to be proportional to the concentration of the molecule (HA) at all pH values and independent of the concentra— tion Of the anion (A’). However, Simon and Beevers (106) have shown that this was not the case. As the pH was increased above the pKa, penetration became greater than one would have expected from the concentration Of HA. 131 Thus, while the penetration Of the anion (A') was low and negligible at pH values below the pKa’ an increasing portion of the anions penetrated as the pH was raised above the pKa. Thus, the contribution of the anion to the total amount penetrated can become substantial 3 to “ pH units above the pKa. Slhun and Beevers (106) did not Offer an explana- tion for how ions, which are not supposed to partition into the lipid membrane, can nevertheless penetrate. This penetration probably proceeds through localized hydrophilic channels which have been shown to occur in lipid membranes (118, 12“). Hartel (53) reported a pH-dependent change in the permeability of cuticular membrane to water, with a maximum permeability around pH 7.0. At this pH most weak acids are completely dis- sociated and the greater penetration of ions observed at higher pH values might thus be the consequence of an pH-dependent permeability change of the polar channels in the lipid membrane. Theoretically, non-ionic diffusion should not be involved in penetration Of SADH through the cuticular membrane, since SADH appeared to be charged at all pH values used. Above the pKa (“.92) SADH is probably largely a zwitter ion and below the pKa it is cationic. Consequently, the solubility Of SADH in octanol was very low (Table 8). Still, more cationic SADH partitioned 132 into octanol than the zwitter ion form and if the cuticu- lar membrane Of bean leaves is Of similar lipOphilic nature as octanol, these pH-dependent differences in par— titioning could account for the greater penetration Of SADH at pH “.0 than at higher pH values (Figure 1“). It is important to note that Tween 20 (0.1%) did not increase the lipid solubility Of SADH at any pH which indicated that the increase in penetration with Tween 20 cannot be attributed to increasing the lipid solubility Of SADH. Filtration.—-Important characteristics Of filtra- tion are: (a) Movement of molecules is confined to localized permeable channels in the otherwise impermeable lipid matrix Of the membrane. (b) Molecular size of a penetrating molecule is inversely related to the penetra— tion rate and very large molecules may not penetrate at all (12“). (c) Filtration appears to be linked to water movement through these channels (12“). (d) Undissociated molecules if sufficiencly polar and not too large in size probably also move by filtration but this fraction is very small compared to movement by non—ionic diffusion. Charged molecules, on the other hand, seem to move entirely by filtration (12“). Sites of polar channels in the cuticular membrane, as revealed by fluorochromes (6, 118) and radioautography (71), are localized in the cuticular ledges of guard 133 cells, glandular trichomes and anticlinal walls. This was confirmed for the bean by using silver ions, which by their nature must penetrate the cuticular membrane through hydrOphilic channels. Silver ions in pure water are not reduced by light, however, Ag+ in contact with organic matter is reduced in light to metallic silver. Ag+ can also be reduced by reducing substances present in the membrane (72) and in the cuticular ledges (71). The latter processes are responsible for reduction in the dark. If many ions are reduced at one site, the reduced silver becomes visible with the light microscope. These data demonstrated that silver entered the cuticular membrane and was reduced preferentially in the cuticular ledges, glandular trichomes and anticlinal walls, particularly around the stomata. Silver was also found in the walls Of the stomatal aperture and, in smaller quantitites, in the periclinal walls. Silver accumulated in defined areas over the anti- clinal walls and in the membrane of the foot cells of glandular trichomes only if Tween 20 was incorporated in the AgNO3 solution. These channels seem to be avail— able for penetration only if the surface tension of the solution was reduced. The Tween effect on SADH penetra- tion in the dark, which could not be explained completely on the basis of surface changes, because stomata remain l3“ closed in the dark,can thus be explained on the basis Of change in permeability. More silver accumulated in all sites in light than in the dark. If Tween 20 was added to the treating solu— tion, silver was also found inside the guard cells. Light reduction may have been involved, but two other factors discussed previously were probably also involved: the greater permeability Of cuticular ledges in Open stomata as compared to closed stomata and water movement, similar to that which accelerated transfer Of fluorochromes (6, 118). The 15-fold increase in penetration of tryptophan brought about by cuticular transpiration is an impressive example as to how important water movement can be with respect to cuticular penetration. The increase in penetration Of SADH with urea is probably the consequence of increased permeability of the cuticular membrane. Urea has been shown to increase the permeability of isolated cuticular membranes from onion leaves and tomato fruits (129). As long as the nature Of the polar channels in the cuticular membrane remains Obscure, the problem of fil— tration can be discussed only in vague terms. With respect to penetration of SADH it would be interesting to know whether a net charge on a molecule will affect filtration, a likely possibility if the matrix Of the polar channels contains dissociable groups. It would be 135 even more important to know the relative permeability Of the walls Of the stomatal aperture. At the present time it is known that these walls are relatively polar in Citrus (120). Since the permeability of the cuticular ledges appears to be greater in Open than in closed stomata, the relative importance of the inner surface and the cuticular ledges for penetration of SADH must remain obscure, because the two factors cannot be studied separately. To fully understand these implications it must be born in mind that the inner surface was estimated to be 0.8 to 1.2% of the apparent outer surface and that penetration of SADH increased up to 10 fold due to light (Figure 11). This dramatizes the need for specific research in this direction. The role Of water movement is also not well understood. It could be that metabolism affected penetration of SADH indirectly via its effect on the water status and movement in the leaf disk. In this case specific research is also required. 136 Foliar Penetration of Succinic Acid 2,2—Dimethylhydrazide: Studies Employing a Growth Response As An Index for Penetration Material and Methods Bean First and Second Internode Assay An indeterminate type of bean (Phaseolus vulgaris L. cv. "Blue Lake") was used as the test plant. Seed— lings were grown in a growth chamber at 28° C during the light (1“ hours) and 22° C during the dark (10 hours) period. Light was provided by fluorescent and tungsten lamps, adjusted to 1,300 ft.c. at the level of the primary leaves. Humidity was not controlled and varied between 60 - 70% (light) and 70 - 80% (dark). Plants were cul— tured in light soil in 3—inch peat pots. One plant was selected and permitted to develop per pot. To obtain a growth response from the internode between the primary leaves and the first trifoliate leaf (subsequently called first internode) plants were treated before elongation Of the terminal bud began. Optimum time of treatment was 7 - 8 days after planting. At this stage the primary leaves were 50 - 80% expanded. Treatment consisted of dipping one primary leaf momentarily in an aqueous solution Of SADH (technical), buffered at pH 5.0 with 0.01 M citrate-phosphate buffer (88). Treated plants were arranged in the assigned 137 experimental design as soon as the treating solution had dried on the leaf. Two general types of experiments were performed, (a) the treated primary leaf either remained attached to the plant or (b) it was detached at specified intervals following treatment. Control plants were similarly treated with buffer or buffer plus surfactant and, if the experiment involved removal Of the treated leaf, this was detached from the control plants at an inter- mediate time interval. Surfactants used in this study were Tween 20 (polyoxyethylene sorbitan monolaurate), Igepal CO 880 (Nonylphenoxypoly (ethyleneoxy) ethanol, here coded P 303), and Buffer X (principal functional agents: alkylarylpolyethoxyethanol, free and combined fatty and phosphatidic acids). Tween 20 and P 303 are both non- ionic and Buffer X is a blend Of non-ionic and anionic surfactants. When the second internode had ceased elongating (ca. 3 weeks after planting), the length of the first and second internode were determined and the experiment terminated. Randomized block experimental design with 5 to 8 replications was utilized and analysis Of variance per- formed on the data. Comparison of the means was by use of Tukey's w—procedure (11“). 138 Details of specific experiments will be presented with the apprOpriate section. Determination Of Retention Of SADH by Bean Leaves Following Dipping Fully expanded primary leaves of lO-day Old plants, grown under conditions mentioned earlier, were detached and dipped for 10 seconds in treating solution (buffered solution of lLlC-SADH at 25 nC/ml concentration, without or with surfactant added). After removal from the treat- ing solution the leaves were held in a vertical position to allow the excess liquid to drain off and then washed in methanol (2 leaves in 50 ml) to remove the SADH adhering to the leaf surface. The leaf area was determined with a planimeter. Residual radioactivity was determined after the methanol wash on disks punched from randomly chosen leaves. NO significant radio- activity could be detected. Five 5-ml aliquots of the methanol wash from two leaves (50 ml) were plated in stainless steel plachets and assayed for radioactivity. Each treatment consisted of 10 leaves with a total surface of ca. 700 cm2. Analysis of variance was performed (randomized block design) and significance determined by the F-test. 139 Determination of Penetra- tion Of luc-SADH Into Attached Bean Leaves Fully expanded primary bean leaves were dipped 1LlC--SADH solution either without or into a 5 x 10‘5 M with Tween 20 (0.1%) added. Zero (= retention), 1/3, 1, 2, “, 8 and 2“ hours after dipping 5 leaves per time-treatment were detached and each washed in 50 ml distilled water. The leaf area was determined with a planimeter. Two 5- m1 aliquots were plated and counted for radioactivity from each wash solution and the radio— activity removed per unit leaf area was computed. The difference in radioactivity removed per unit leaf area between zero time and the subsequent sampling intervals was considered to have penetrated. Penetration of Alar 85 Into Apple Leaves Ten-week-Old McIntosh seedlings, l2 - 13 cm high and having 12 - 1“ fully expanded leaves each, were selected as a second test specimen. The plants had been raised on light soil in peat pots (3 inch) on a green- house bench at 22° - 2“° C night and 25 - 28° C day temperature. These conditions were maintained during the duration of the experiment but supplemental light from fluorescent lamps (1,500 ft.c.) was given to insure vigorous growth during the months of October and November. l“O Plants were sprayed at 8 a.m. with 1,000 ppm (active ingredient) Alar 85 to a point just short of run-off. Control plants were not sprayed. Leaves appeared dry 20 - 30 minutes after spraying. TO prevent the Alar spray from contaminating the soil, the pots were placed in plastic bags which were tightly closed around the base of the stem, with an intermediate layer of soft blotting paper wrapped around the stem. One-third, 1, 6 and 12 hours after spraying the leaves of 10 plants (replications) per time interval were thoroughly washed with tap water to remove the Alar residue which had not penetrated. One group of 10 plants was not washed. The plastic bags were removed from the pots after the wash, plants arranged in the assigned design and cultured for a further “ weeks. The growth increment obtained during these “ weeks was determined and used as a biological index of penetration of SADH during the time interval between spraying and washing. Results Concentration Response To establish the relationship between SADH concen— tration and internode length one primary leaf per plant was dipped into SADH solution. Five concentrations from “ 2 5 x 10— to 5 x 10- M SADH were used. The treated leaves remained attached to the plants. 1“1 SADH inhibited elongation of the first and second internode (Figure 23). The concentration response curves are sigmoidal. The first internode was more sensitive to low concentrations (5 x 10.3 M) while the second internode was more sensitive to high concentra— tions (5 x 10-2 M). The lowest concentration required to attain a significant inhibition of internode elonga— tion was 10'3 M for the first and 5 x 10'3 M for the second internode. Surfactant Effects All surfactants tested reduced retention of the treating solution by bean leaves as compared to the retention of a solution lacking a surfactant (Figure 2“). The reduction in retention was statistically significant at a surfactant concentration of 0.01% and a 10-fOld increase in surfactant concentration did not result in a significant further reduction of retention, although this was considerable for Tween 20. The differ— ences between the surfactants were not significant. At a concentration of 0.1% P 303 reduced retention to 7“%, Tween 20 to 78% and Buffer X to 85% of that quantity of SADH retained by bean leaves dipped in a solution lacking a surfactant (control). 1“ Penetration of C-SADH into bean leaves was en— hanced by Tween 20 (0.1%) (Figure 25). One hour after l“2 Figure 23.--The effect of SADH concentration on elongation of the first and second internodes Of bean plants. Elongation of the first internode was significantly (P = 0.05) inhibited at SADH concentrations of 10-3 M and higher, and the second internode at 5 x 10-3 M and higher. IN TERNODE LE N 6 TH ('16 of Control I IOO 80 60 40 20 O\° l“3 A—A First intomodo o—o Socand Internode A 7 -4 L j_ L I J 5XIO'4 IO"3 5:: IO-3 '0'2 5110' CONCENTRATION (molar) 1““ Figure 2“.——The effect of surfactants and sur- factant concentration on retention of treating solution by bean primary leaves. Differences between control (lacking surfactant) and 0.01% surfactant con— centration are statistically signifi- cant (P = 0.05). Differences between surfactants are not significant. RETENTION I '/o of control) IOO 90 80 70 60 l“5 o—o Tuna 20 I'll—D Buffer x A—A Surfactant P303 0 ~13 A A _ ~A l_ 1 j 0.0l 0.05 DJ CONCENTRATION (Va) 1“6 Figure 25.--The effect OE Tween 20 (0.1%) on pene- tration of 1 C-SADH into bean primary leaves. 1“7 lOO r- 0—0 SA DH o—o SADH + Twun «toteOgou \0 9‘.» >6: BIL Mkhl IS 20 24 l2 TIME (hours) l“8 1“c—smm dipping the leaves approximately 80% of the retained after dipping had penetrated in the plus Tween treatment, whereas the comparable figure for the minus Tween treatment was ca. 30%. The difference between the plus and minus Tween treatments diminished with time, 8 hours after dipping ca. 90% (minus Tween) and 98% (plus luC—SADH had penetrated. Thus, Tween 20 Tween) of the affected the initial rate of penetration rather than the total amount of SADH that penetrated. The relationship between the two effects of sur- factants (reduction of retention, growth promotion) is recorded in Table 12. Tween 20 (0.1%) reduced retention by 25% and enhanced penetration of 11+C-SADH. Six hours after dipping, 29% of the SADH retained could still be washed Off the leaves if the treating solution lacked Tween 20, whereas in the plus Tween 20 treatment only 5% could be removed. Interestingly, the total amount of SADH which penetrated within 6 hours was similar in both treatments. All surfactants increased the internode length Of bean plants, although the effect Of Buffer X was sta— tistically not significant (Table 13). At a concentra- tion of 0.1% P 303 increased internode length by 25% and Tween 20 by 62%. This effect of surfactants prevailed in the 1; presence of 5 x 10— M SADH as the internodes of the 1“9 TABLE l2.-—Ehe effect Of Tween 20 (0.1%) on retention of 1 C-SADH solution and penetration into bean primary leaves. Amount Amount removed Amount penetrated Treatment retained after 6 hours within 6 hours -2 -2 -2 cpm-cm % cpm-cm % cpm-cm - Tween 61.16 a 100 17.81 c 29 “3.3“ + Tween “5.92 b 75 “.29 d 5 “1.63 Means followed by different letters are significantly different at P = 0.05. TABLE l3.--The effect of surfactants (0.1%) and SADH (5 x lO-Ll M) on elongation Of the first internode of bean plants. Internode length Surfactant Control SADH treated mm %1 mm %1 %2 no surf. ' 113.0 a3 100 62.8 a 100 55.5” Buffer X 12“.6 a 110 70.8 a 112 56.8 P 303 1“1.5 b 125 82.8 a 132 58.0 Tween 20 182.8 c 162 80.8 a 129 “3.7 1Per cent Of no surfactant 2Per cent Of control 3Means within a column followed by different letters are significantly different at P = 0.05. “All SADH effects are significant at P = 0.05. 150 SADH treated plants were longer when the treating solu- tion contained a surfactant (Table 13). For Buffer X and P 303 this increase corresponded closely with the increase found in controls. Statistically, however, these differences were not significant. The comparison of the internode length Of SADH treated plants with the appropriate surfactant-control shows that the surfac— tants did not alter the growth response to SADH, since a similar inhibition was obtained whether the treating solution contained a surfactant of not. The independent action of SADH and surfactants on the elongation of internodes is also illustrated in Table 1“. In this experiment, treated leaves were de— tached after specified time intervals such that only those quantities of SADH and Tween 20 which had pene— trated and were translocated out of the treated leaf could affect growth. With control plants one leaf was detached and the remaining one dipped in buffer or in buffer plus Tween 20. The plus Tween 20 control again had longer inter— nodes (31%) than the minus Tween 20 control. But even plants in which penetration and translocation were stopped after one hour had longer internodes if the treating solution contained Tween 20. The plants whose treated leaves were removed 3 or 6 hours after dipping had practically the same internode length irrespective 151 Of the presence of Tween 20, whereas during the 12 hour penetration and translocation period sufficiently more SADH was apparently exported into the plant to counter— act the Tween 20 effect and to inhibit internode elonga— tion even further. TABLE l“.——The effect of SADH (5 x 10—3 M) and Tween 20 (0.1%) on elongation Of the first internode of bean plants. Treated leaf Internode length detached after - Tween + Tween Tween effect hours mm mm % control1 60.6 a2 79.6 a 131 l 5“.5 a 66.0 b 121 3 ““.7 bc “1.1 ed 100 6 “2.5 be “2.2 d 100 12 33.7 c 26.2 e 78 1 One leaf was detached from control plants and the re— maining one being dipped into buffer or buffer plus Tween 20 respectively. 2Means within a column followed by different letters are significantly different at P = 0.05. Inhibition of internode elongation was much stronger when the treating solution contained Tween 20 (Figure 26). In this figure internode length of SADH treated plants is expressed in per cent Of the internode length Of the 152 Figure 26.—-The effect of Tween 20 (0.1%) on pene- tration of SADH into bean leaves, as indexed by inhibition of elongation of the first internode. (Data from Table l“ plotted on a per- centage basis.) Inhibition of internode elongation was significant (P = 0.05) after 1 hour in the SADH plus Tween treatment and after 3 hours in the SADH treatment. 153 IOO O—O SA DH 0—0 SADH 1* TWO!) l2 O 0 8 Cotton \0 c\o\ $556st 4 Nfibkkmkkx 40- TIME (hours) 15“ minus or plus Tween 20 control plants respectively. A 1-hour penetration and translocation period (treated leaf detached) was sufficient to Obtain a significant inhibi- tion of internode elongation in plants treated with SADH plus Tween, whereas 3 hours were required in plants treated with SADH alone. Thus, the enhancement of foliar penetration of SADH by Tween 20 could be demonstrated using a biological response. The Effect of pH of the Treating Solution To test the effect of the pH of the treating solu— tion on foliar penetration Of SADH, one primary leaf per plant was dipped into 5 x 10‘3 M SADH solution buffered at pH value of 3.0, 5.0 and 7.0. The treated leaves were detached after one hour. In control plants one leaf was detached and the remaining one was not treated. Internode elongation was inhibited at all pH values (Table 15) and the degree Of inhibition increased with decreasing pH values. The greatest inhibition was obtained with a solution of pH 3.0 which was significant for both internodes. Inhibition by solutions of pH 5.0 or 7.0 was significant only for the first internode. Penetration of SADH Into Apple Leaves A penetration period of more than 6 hours was needed to significantly inhibit terminal growth of apple 155 TABLE 15.-—The effect of the pH Of SADH treating solution (5 x 10-3 M) on elongation Of the first and second internode of bean plants. Internode length pH i First internode Second internode mm % mm % Control 56.0 a1 100 X 189.3 a 100 7.0 “5.6 b 81 168.2 a 89 5.0 “0.0 c 71 16“.6 a 87 3.0 29.8 d 53 116.8 b 62 lMeans within a column followed by different letters are significantly different at P = 0.05. seedlings (Table 16). Penetration continued even for a longer time as terminal growth of seedlings whose leaves were not washed was significantly less than that of seedlings washed 12 hours after application Of Alar 85. Discussion Factors influencing penetration of SADH into bean leaves have been studied using excised leaf disks. Such a system proves very useful for investigating the mecha— nisms involved in penetration of SADH from an aqueous solution, and may also furnish some insight as to factors influencing SADH penetration under field conditions. However, while the leaf disk method is essentially a static system providing constancy Of concentration of 156 SADH and surfactant, temperature, light and humidity, are factors effecting penetration under field conditions subject to changes, particularly the concentration of SADH and surfactant. TABLE l6.-—Penetration Of SADH (applied as Alar 85) into apple leaves as indexed by inhibition of terminal growth Of apple seedlings. Leaves washed Terminal shoot growth after hours cm % Control1 33.5 a 2 100 1/3 32.7 a 98 1 28.8 ab 86 6 26.1 ab 78 12 2“.l b 72 not washed 15.9 c “8 lnon-treated 2Means followed by different letters are significantly different at P = 0.05. Therefore, it seemed desirable to establish whether or not the effects of certain factors on penetration of SADH observed in the isolated system could be confirmed under conditions similar to those in the field, par- ticularly when a biological response of the plants to the growth regulator is used as an index for penetration. 157 Bukovac (11) reported that the most pronounced growth response Of bean plants to MADH was the reduction in internode length. The inhibition of elongation of the first and second internode was used as a biological response and related to penetration of SADH. Unfortunately, the dosage response curve for the first and second internodes was sigmoidal and the effective concentration range was rather narrow. This limits this test system for quantitative evaluations but it is sufficiently sensitive to establish some general qualitative relationships. The role of surfactants was more complex than in the leaf disk system. They were effective in three different ways: (a) surfactants reduced retention Of treating solution, (b) they enhanced the initial rate of penetration and (c) promoted internode elongation. The reduction in retention Of treating solution by 15 — 25% due to surfactants, that is to say the re— duction of the amount of growth regulator deposited on the leaf surface by 15 to 25%, might be of practical importance. Particularly, when there is no positive effect Of the surfactant on the total amount of SADH which penetrated, as observed in this study with bean. Practically all the SADH deposited on the leaf surface penetrated irrespective of the absence or presence of Tween 20, which affected mainly the initial rate Of 158 penetration. However, this might be different in plants whose leaves are more difficult to wet. The increase in penetration Of SADH by Tween 20 Observed in the leaf disk system could be confirmed. Since the effect of Tween 20 was on the rate of penetra— tion rather than on the total amount, it was necessary to include the time factor in the experiments by removing the SADH pool on the leaf surface after prescribed time intervals. However, by detaching the treated leaf, SADH which had penetrated but was not yet exported out Of the leaf was also removed. This accounts for the relatively small differences between the plus and minus Tween 20 treatments Observed in this study. Following a 3-hour penetration and translocation period an inhibition of internode elongation of 26% was observed in plants treated with SADH alone, while “8% was observed in plants treated with SADH plus Tween 20 (Table 1“, Figure 28). Differ— ences found with the leaf disk system were much greater. Nevertheless, it should be noted that the enhancement of foliar penetration of SADH by Tween 20 could be demon— strated with three different methods: the leaf disk method, by determining the non-penetrated lLlC-SADH following dipping of the leaves (Figure 25) and by utilizing a biological response (Table 1“, Figure 28). Enhancement Of penetration of a variety Of com- pounds by surfactants has been shown. More 32P 159 penetrated into the lower surface Of McIntosh leaves when Triton X 100 was added (29) and penetration of 3-amino-l,2,“-triazole (“5) and IAA (59) into bean leaves was also increased by a variety of surfactants. In these cases, however, the first determination of the amount which had penetrated was made 2“ hours after treatment. Therefore, the effect Of the surfactants on penetration during the drying of the spray deposit was not determined. It was during this time period that Tween 20 had the greatest effect on SADH penetration. Penetration of SADH into apple leaves was strikingly slower than in bean leaves. The SADH was applied as Alar 85 which contains the surfactant P 303. During drying of the spray deposit very little SADH penetrated as indi— cated by the lack Of response. Since the inhibition of terminal growth increased with increasing time of pene- tration up to 12 hours and longer, penetration must have taken place from an apparently dry surface. The fact that SADH could be easily washed from the leaves is Of practical importance. A rain within 12 hours after spraying may remove a large portion of the spray deposit and significantly reduce performance. The third effect Of surfactants was the promotion of internode elongation in bean plants. Growth promoting effects of surfactants have earlier been observed, al— though surfactant toxicity is more common (83). 160 Methylesters of fatty acids, Tween 20 and Tween 80, which are also esters of fatty acids, were shown to enhance the activity of GA and IAA in promoting growth of pea stem sections (116). Growth of young barley seedlings was promoted by 0.01% of fatty acid ester types Of non—ionic surfactants (82). Surfactants were also found to increase growth of corn and soybean plants (62). In the present study it was shown that the growth promoting effect of surfactants and the inhibiting effect of SADH were inde— pendent from each other. The growth promotion by sur- factants could be overcome by SADH. There is no indi- cation whether surfactants will promote the growth Of fruit trees as well. If they do so, some of the growth retardant will be required for compensation of the growth promoting effect of the surfactant. From the factors affecting foliar penetration Of SADH as evident from direct measurements using 114C—SADH and the leaf disk system the role of surfactants and pH as determinants Of penetration was confirmed using inhi- bition of internode elongation as index for penetration. Both Tween 20 and a low pH of the treating solution en— hanced penetration. The role of light and transpiration in this biological system remain to be studied. 161 Translocation of Succinic Acid 2,2- Dimethylhydrazide in Apple and Peach Trees Material and Methods One—year-old apple (Malus sylvestris cv. ”McIntosh") and peacfl (Prunus persica cv. "Suncling") trees, budded on seedling rootstocks, were planted in light soil in 5-gallon plastic containers on April 22, 1968. They were placed on a bench in a greenhouse equipped with a cooling device capable of maintaining a day temperature of approx- imately 25° C. The night temperature was controlled at 20° C. Vigorous growth of the trees was maintained by carefully watering and fertilization and by adequate con— trol of pests and diseases. Two shoots were allowed to develop on each tree approximately 15 cm apart oriented in opposite directions. They will subsequently be referred to as upper and lower shoots. Peach trees were treated on May 25 and again on June 8. Apple were treated on June 1. At the time Of treatment shoots were ca. 25 cm long. Treatment consisted of spraying either the upper or the lower shoot with 5,000 ppm Alar 85. During spray application the entire tree, with the exception of the shoot to be treated, was covered with a plastic sheet. TO prevent spreading of the spray liquid blotting paper was wrapped around the base of the treated shoot. 162 This was removed once the spray dried. Control trees were not treated. During the course of the experiment shoot growth was measured and recorded at weekly intervals. At the end of July shoot growth had ceased and the experiment was terminated. One tree, typical for each treatment, was selected and photographed. Next, leaves which were present at time of treatment were removed from each shoot. A 3.5 cm diameter disk was punched from each leaf after which the disks and the remainder of the leaves were dried at 70° C in a forced draft oven. After attaining con- stant dry weight, the leaf area per gram leaf dry weight was computed from the leaf disks. This factor was used to Obtain an estimate of the leaf area present on each shoot at the time of treatment. The shoots were detached from the stem and sub— divided into the portion grown before treatment (pre- treatment growth) and after treatment (post-treatment growth). The length of each shoot segment was measured and after drying to constant weight at 70° C the dry weight was determined. Since in peach numerous side shoots had developed, these were included in the dry weight of the main shoots but not in the shoot length. Leaves were taken from comparable peach and apple trees and retention of spray liquid per unit leaf sur- face was determined. Detached leaves were dipped into a 163 5,000 ppm Alar 85 solution to which a trace Of luC-SADH had been added. The procedure was essentially as des- cribed for determination of retention by bean leaves. A split plot design was utilized, each tree being considered a whole plot and the upper and lower shoots sub—plots. Each treatment was replicated five times. Analysis of variance was performed on the data and a comparison among the treatment means was made according to Tukey's w-procedure. Results Alar 85 at 5,000 ppm inhibited shoot elongation of the treated shoots on apple and peach trees (Figures 27, 28, 29). On apple, growth of the treated shoot ceased 2 weeks after spray application, while in peach little inhibition was observed following the first treatment. A second spray was required to terminate growth of the treated shoots in peach (Figure 28). In both, apple and peach, growth of the non-treated shoot continued at a rate similar to the shoots of the control trees. Elongation of the upper and the lower shoots was virtually identical in control trees, indicating that apical dominance was not a factor in this study. Apple trees generally did not develop side shoots, while numerous side shoots develOped on peach trees (Figure 29) on both, upper and lower shoots. The number 16“ Figure 27.-—The effect of Alar 85 (5,000 ppm) on shoot growth of apple trees (McIntosh). Arrow indicates the date of treatment. SHOOT GROWTH (cm) I00 80 60 4O 1- A—A Upper Moor o—o Louver Meet 20 '- O IOO 7 Upper shoot treated 80 - 9 so .- 0/0/0 /0/ ‘0 . /X.———-A A—A——a A A I 20 O IOO r' Louver Moor treated 30)- ‘2 so 3 /A A /A/ 40 I- ‘étfo—O—O—o 3 20 b/O/ I O 1 1 1 1 J 1 1 J O l 3 4 5 6 7 8 165 Can" ol :1” 1. / / A/A TIME I weeks) 166 Figure 28.-~The effect of Alar 85 (5,000 ppm) on shoot growth Of peach trees (Suncling). Arrows indicate dates of treatment. SHOOT GROW TH I an) I00 80 60 40 167 Control r A/o—O—‘ / / A—A Upper ehaat 1- 0-0 LOUOT afloat 8 20 '- O IOO 1- Upper ehaat treated - a /v‘—f ' 80 b /° /° A’ 60 b /A/ // 4O '- g/. ’ 20 - / 9 O IOO - Lower eloaat treated A/Afa 80 '- A/ A 60 " / - / /°/ 40 r A 0 /./, 2° ' e/A , 0 1 1 1 1 1 1 1 1 1 1 O I 2 3 4 5 6 8 IO TIMEIeeeke) 168 Figure 29.—-The effect of Alar 85 (5,000 ppm) on growth of apple and peach trees. A. Apple, control. B. Apple, upper shoot treated. C. Apple, lower shoot treated. Peach, control. Peach, upper shoot treated. *IJITIU Peach, lower shoot treated. 169 170 and the length of the side shoots was reduced by Alar 85. More detailed information was obtained by analysis of post-treatment growth increments (Tables l7, 18). Response Of Apple Trees There was no significant difference in the growth increment between the upper and lower shoot Of the con- trol trees, although the lower shoot tended to be lower in dry weight (Table 17). The increment of the treated shoots (length and weight) was significantly less than of the non-treated shoots. Alar 85 drastically reduced the increase in shoot length of the treated shoot to l“.7% (upper) and 11.7% (lower) of the length increase of the corresponding control shoot. The effect on shoot dry weight was similar, 1“.l% and 15.9% for the upper and lower, respectively, in terms of the dry weight increase of the control. Thus, the upper and the lower shoots of apple trees responded similarly to a given dose of Alar 85. The increase in shoot length of the non-treated shoots of the treated trees was less (8“.9% for the upper and 73.1% for the lower shoot) than that of the corres- ponding shoot Of the control trees, but the difference was not statistically significant. Also non—significant was the slightly greater increment in dry weight of the non-treated shoots as compared to control shoots. Thus, .mo.o u m pm pcoamamfio mapcmOflwficmflm ohm mgoppoa pcosomMHo mo UmBOHHOQ Aw so EOV mcmoz a a.ma O om. m.moa m mm.: N.HH o m.m 0.:w m a.mm empmosp .nm sosoa m.moa m ::.m a.ma o em. a.ms m a.mm s.aa o m.m pounds» .cm homo: n 0.02 e mim 0.02 s 3.: 0.03 a. OS: 0.2: He 0.? Hoaoeoo 1 a w a m e 60 & Eo pooch gosoq poonm Loom: poonm aosoq poonm pogo: unwfios ago coapmwcoam uncapmose npzoaw poonm .mooap AstOQHon madam mo cpsosw pooch no AEQQ ooo.mv mm mafia mo pomgmm onanu.sa mqm¢9 172 in apple trees growth of the upper shoot was not affected by 5,000 ppm Alar 85 applied to the leaves of the lower shoot and vice versa. Response of Peach Trees There was no difference in shoot length increment between the upper and the lower shoot of the control trees (Table 18). The dry matter increment, however, was significantly different. The lower shoot weighing only 5.92 g as compared to 8.6“ g for the upper shoot. This difference is largely due to the side shoots, which were more numerous on the upper shoots. The length and dry weight increment of the treated shoots were in all cases significantly less than the increment Of the non-treated shoots for both treatments. The treated shoots remained shorter and gained less in dry weight than the corresponding control shoots. This difference was statistically significant only for the dry weight data. The non-treated shoots of the treated trees grew more vigorously than their respective controls. This difference was apparent both in terms of shoot elonga- tion and shoot weight, but was statistically significant only for shoot elongation. Following treatment of the upper shoot, the lower shoot accumulated 203.5% Of the weight of the lower control shoot and the upper shoot .mo.o u m pm econommfio mapcmOHMflcmwm was whoopoa pQOLOMMHO mo Omsoaaom Aw LO EOV memoza >.mm o mm.m a.mwa 0U ©N.ma m.H© m m.mm w.mHH Om a.mm Umpmopp .Qm Lozoa m.mom om mo.ma m.mm o mw.m o.HmH on o.as a.mm m w.mm Umpmopp .cm poem: 3 N. 0.00H o mm.m 0.00H m :o.m 0.00H Om m.:m o.ooH How m.~m Hospcoo & m s w R BO R Go poozm 90304 pooch ammo: poonm hmzoq pOOSm swoon pnmaoz mam coapmwsoam ucoEumomB cpsopw poonm .mmmpp AwCHHocsmv comma oo zpzomw pooch co AEQO ooo.mv mm mma¢ mo poommo mnBII.mH mamee 17“ showed an increase of 182.“% over the upper control shoot when the lower shoot was treated. Thus, in peach trees treatment with Alar 85 re— sulted in a growth inhibition of the treated shoots and in a growth promotion of the non—treated shoots inde- pendent Of position. Leaf Area at Time of Treatment and Spray Retention The leaf area at the time Of treatment was very similar for both apple and peach trees. Since there was little difference between the upper and lower shoots, the average Of both shoots is presented (Table 19). At the time of the second treatment the leaf area in peach was almost 2.5 times larger than at the first treatment. TABLE l9.-—Leaf area and spray retention of apple and peach trees at the time Of treatment. Relative retention Species Leaf Area Per unit area Total 2 cm Apple “71 3.5 1,6“8 Peach lst treatment “85 1.0 “85 2nd treatment 1163 1.0 1,161 175 Apple leaves retained 3.5 times more treating solution per unit leaf area than peach leaves. This resulted in a much smaller amount Of SADH deposited per shoot treated at the first treatment. The sum Of the total retention from the first and second treatment of peach trees is almost identical with the total retention in apple. Discussion Terminal growth of the treated shoots of apple and peach trees was strongly inhibited by 5,000 ppm Of Alar 85 applied as foliar spray. Growth of the non- treated shoots was not inhibited. The need for a second spray application in peach to stop terminal growth of the treated shoot was prob- ably the consequence Of the smaller spray retention by peach leaves as compared to apple leaves (Table 19). Other factors, such as a differential foliar penetra- tion or response between the two species, may have been involved as well. Growth of the non-treated shoots of peach trees was promoted. The main shoots were longer and had more and longer side shoots, as compared to the comparable shoots of control trees. Although this cannot be ex- plained physiologically, it might be Of practical 176 significance, since Alar applications may reinforce apical dominance if the tOps Of trees are not covered thoroughly. The lack of growth inhibition in the non-treated shoots suggests that SADH was not translocated from the treated shoots to the non-treated shoots in amounts sufficient to produce a biological response. The trans- piration stream cannot serve for passive basipetal transport of SADH, since it is directed upward. Thus, SADH which might accumulate in the apoplast of the leaves following spray application cannot be translocated in this way. Rather, uptake of SADH into the leaf cells and translocation in the symplast is a prerequisite for basipetal movement. However, it was speculated earlier that SADH might not be readily taken up by leaf cells. This could account for the lack Of translocation Ob- served in this study. These results are in contrast to conclusions Of Martin gt_al. (75) that SADH is readily translocated and coverage should be Of less importance in SADH applica— tions to the foliage. 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Environmental and biochemical control of stomatal movement in leaves. Biol. Rev., “0:“63-“82. APPENDI CBS 191 APPENDIX I CRITICAL EVALUATION OF THE CONCEPT OF ECTODESMATA WITH REFERENCE TO FOLIAR PENETRATION OF POLAR COMPOUNDS 192 Introduction Ectodesmata were observed for the first time by Schumacher and Halbsguth (98) and since have been the , subject of numerous investigations, among them three .3 Ph.D. dissertations which dealt mainly with the problem of demonstrability of ectodesmata (69, 97, 10“). Later, ,, Franke made several attempts to demonstrate that ecto- ‘j '- desmata function in foliar penetration of polar compounds (3“, 35, 39, “0, “3, ““). His concept appears to have been widely accepted, although the ideas as to how ectodesmata might be involved, are quite different. Currier and Dybing (17) described ectodesmata as protoplasmic strands which push into and alternately with— draw from pores in the outer epidermal wall. Crafts and Foy (16) suggested that ectodesmata may desorb penetrat— ing molecules from the cutin reservoir. That ectodesmata participate in foliar absorption of mineral nutrients was assumed by Jyung and Wittwer (65) and Hull (60) wrote that the function of ectodesmata as routes of transport between the cuticle and the living parts of the tissue had been demonstrated almost beyond question. Sargent (95) presented a more recent View of Franke on the nature of ectodesmata as pores, filled with reducing agents 193 19“ excreted by the protoplast under conditions in which staining of ectodesmata is possible. In view of the diversity of contradictory Opinions concerning the nature and function of ectodesmata it was decided to critically re—examine the literature dealing with ectodesmata. A clarification was also needed with respect to a possible role of ectodesmata in foliar pene— tration of SADH. The Nature of Ectodesmata Originally ectodesmata were thought to be plasmatic in nature (69, 98, 99) but later, Franke came to favor the View of their being non—plasmatic (“1, “2, “3, ““). The problem of the nature of ectodesmata is closely related to the problem of consistently demonstrating them. All attempts to demonstrate ectodesmata with the electron microscope have failed so far. These structures could not be stained with 050“, the classical protoplasmic stain (10“). Using HgCl2 staining Schnepf (97) produced some electron micrographs of what he considered to be ectodesmata, but they were claimed to be artifacts by Bollinger (8). Further, what Huber et a1. (58) inter- preted as ectodesmata in cross—sections of guard cells of Helleborus niger were considered artifacts by Sievers (10“) and Schnepf (97). Thus, nothing convincing can be said about the ultrastructure of ectodesmata at the present time. 195 The classical method for demonstrating ectodesmata is the Gilson method (98, 69, 97, 10“). The Gilson fixative used by Lambertz (69) was composed of: “0 ml 30% ethanol, 10 ml formic acid, 5 m1 “0% formalin, 1 ml 65% HNO and 2-3 HgCl to saturation. The presence of 3 2 HgCl2 is essential as ectodesmata cannot be demonstrated without it. Changes in other components affected only shape and number of ectodesmata. Whole leaves or leaf segments were fixed in the Gilson misture from 10 minutes up to 2“ hours, sectioned with a cryostat and the sections washed with 50% ethanol to remove excess HgCl Then, sections were transferred 2. to 20% K1 solution saturated with iodine (5—10 minutes) and stained in pyoktannin acidified with H280“. Following staining the sections were washed in water and embedded in glycerin for microscopic examination. Schnepf (97) pointed out that the fixing technique was crucial: ectodesmata could not be demonstrated when the leaves were infiltrated with the Gilson fluid under vacuum, or when cryostat sections were fixed instead of large leaf segments (10 x 10 mm) prior to sectioning. Ectodesmata could not be found along the cut edges of the leaf segments. From the failure of demonstrating ecto— desmata in thin sections (20-“0 um) Lambertz (69) concluded‘ that ectodesmata might be destroyed when the cells are killed during sectioning. However, Schnepf (97) observed 196 that even non-injured cells close to the cut edges failed to exhibit ectodesmata. He argued that a rapid penetra— tion of the fixative through the cut edges might destroy these structures and that they can be demonstrated only if the fixing solution penetrates the tissue slowly and in a special 'orderly' manner, probably through the cuticle. This view is supported by the fact, that after only a short fixing period, short, incomplete ectodesmata occurred which were attached to the cuticle and which seemed to grow towards the plasma membrane (97, 3“). Franke argued this kind of formation of ectodesmata would not occur if the Gilson fluid would spread by diffusion in the cellulose wall under the CM. Three different forms of ectodesmata have been observed. Complete ectodesmata-—those which extend through the entire outer wall from the CM to the plasmalemma. Shortened ectodesmata——those which are cone-shaped and attached to the CM. Their tips are oriented towards the plasma membrane of the epidermal cell but not in contact with it. This type of ectodesmata generally occurred under conditions less favorable for demonstrating ecto- desmata. 'Abbauformen'--those which are also cone—shaped but attached to the plasma membrane, oriented with their tips towards the cuticular membrane without reaching it. This type was observed in old yellow leaves and in wilting leaves. The diameter of all three types of ectodesmata 197 varied from very thin and thread-like (0.5 pm) to broad cylindrical or cone—shaped (5 pm) ectodesmata (from figures by Lambertz (69)). Lambertz (69) observed a distinct diurnal rhythm in the occurrence of ectodesmata. A great number of complete ectodesmata could be demonstrated during the night while during the day the same leaves showed only few and usually shortened ectodesmata. Sievers (10“) established the role of temperature and light in this process. Temperatures between 20 and 300 C caused ectodesmata to disappear or to become shortened, while leaves kept at 3 - 6° C exhibited numerous complete ectodesmata. Light reduced demonstrability and dark improved it. The light and temperature effects were reversible, which was taken as an indication that ectodesmata are plasmatic extensions of the cytoplasm projecting into the wall or withdrawing from it, de— pending on environmental conditions (69, 97). Later it became clear that the structures were always present in the wall, only their demonstrability with the Gilson mixture changed. Schnepf (97) succeeded in demonstrating ectodesmata with an iodine-silver method. Leaf segments were fixed in 5% KI solution saturated with iodine (8—16 hours), rinsed quickly with water and transferred to a 1% AgNO solution (for a few hours) and then sectioned 3 198 with a cryostat. The sections require no further treat— ment. With this method a part of the outer wall under the CM stained a diffuse dark brown. Sometimes, the dark band was very broad and filled the entire space between CM and cytoplasm. In cases where the band remained small and confined to the zone under the CM numerous ectodesmata could be seen extending from the dark band to the plasmalemma. There are striking differences in the demonstra— bility of ectodesmata between the Gilson and the iodine- silver method. Using the Gilson method, light and temperature had a pronounced effect on demonstrability and ectodesmata could not be demonstrated in killed tissue. Treating leaves with C0, HCN and ether seriously impaired demonstrability of ectodesmata. In old and yellow leaves only the 'Abbauformen' type could be demonstrated. All of these factors were without effect on demonstrability of ectodesmata using the iodine— silver method where numerous complete ectodesmata formed under all conditions and even in killed tissue. In spite of these differences, the structures produced with the two methods were considered identical (3“, 97) although the processes leading to their demonstration were quite different. Schnepf (97) suggested the formation of AgI crystallites following treatment of tissue with KI and AgNO3 which lined 199 certain structures in the outer wall, thus making them visible with the light microscope as ectodesmata. Since AgI is not stable in light Ag+ will be reduced and what is visible is probably not AgI. The for— mation of ectodesmata with the iodine-silver method requires nething else but intermicellar spaces large enough to accommodate the crystallites. The existance of such spaces in the cellulose wall has been shown as well as the tendency of copper, silver and gold crystal— lites to line these spaces when cellulose fibers were submerged in the appropriate salt solution and the metal reduced with hydrazinehydrate (“7). The processes which make ectodesmata visible follow— ing fixing with the Gilson mixture must be of a different nature, as suggested by the fact that they could not be demonstrated in dead tissue, after freezing or treatment with CO, HCN and ether. The number of complete ecto— desmata could be increased by administering ascorbic acid to the leaves prior to fixing them with the Gilson mixture (“1, 97). Franke (“1) concluded that a reducing substance might be involved in demonstrating ectodesmata with the Gilson method, and that the natural reductant might be ascorbic acid. This was postulated on the basis of the following observations: (a) the number of complete ectodesmata could be increased with ascorbic acid but not with other naturally occurring reducing 200 compounds such as glutathion and cystein, (b) in varie— gated leaves of Pelargonium zonale the ascorbic acid content was twice as high in the green than in the white portions and the number of ectodesmata was also greater in the green portions of the leaf, and (c) the ascorbic acid content of the epidermis tends to be 2-10 times higher than in other plant tissues (“1). In a subsequent paper (“2) Franke discussed the nature of ectodesmata in which he favors the hypothesis of their being non-plasmatic in nature. The nature of ectodesmata is of importance with respect of their role in foliar penetration. While the function of plasmatic ectodesmata would be relatively straight forward it is more difficult to Justify a postulated participation of non-plasmatic ectodesmata in foliar penetration of polar compounds. Ectodesmata as Plasmatic Structures The effect of environmental conditions on the demonstrability of ectodesmata was considered a strong argument for their vitality. This view was maintained when it became clear that no such changes could be observed using the iodine-silver method. Since demonstration with the Gilson mixture involved a reduction step (Hg++ ———» Hg+), it was hypothesized that the reducing ability of the plasm was affected by 201 all factors influencing the demonstrability of ectodesmata (3“, 38, 100). This, however, is in contradiction with the observed mode in which these changes take place. The shortened ectodesmata, formed under unfavorable conditions, are attached to the CM and they are not in contact with the cytoplasm. Transferring such leaves with shortened ectodesmata to conditions which promote demonstrability (dark, low temperatures) resulted in an elongation of the shortened forms and, if sufficient time was allowed, they finally became complete ecto- desmata. Thus, the reducing ability was apparent first in proximity of the CM and not in proximity of the cytoplasm as one would expect if ectodesmata were plasmatic. The formation of so—called 'Abbauformen' in wilt— ing leaves and the reoccurrence of complete ectodesmata following re—establishment of turgescence was also con— sidered an argument for their vitality (10“). The similarity between ectodesmata and plasmo- desmata in polarized light—-both appearing as dark isotropic structures in the birefringent wa11-—was another indication that ectodesmata might be plasmatic as are plasmodesmata. However, Buer (10) questioned the identity of structures observed in polarized light and those produced with the Gilson method, because the isotropic bands in the outer wall extended through the 202 entire depth of the section (30-50 pm) while ectodesmata are generally thin and thread-like. Furthermore, the isotropic bands disappeared when the outer epidermal wall was separated on one side from the anticlinal wall. This caused the outer wall to relax and become straight, while it was arched before. From this Buer concluded that the isotropic bands were folds in the outer wall. Finally, the failure of ectodesmata to stain with 050“ is another indication that they are probably non— plasmatic in nature. Ectodesmata as Non—Plasmatic Structures If ectodesmata are not plasmatic, then the Hg++ has to be reduced by a substance other than the plasm. As already mentioned, Franke (“1) suggested that ascorbic acid might be the natural reductant. From his research (35, 37, 39, “0) he arrived at the con— viction that ectodesmata can function as transport pathways in the outer epidermal wall and suggested that the vapor pressure gradient was the moving force in transport from the cytoplasm to the CM. The movement of water during CT is thought to follow at least partially distinct pathways through the outer wall and the CM. Solutes such as ascorbic acid are thought to accumulate in these postulated pathways (pores) when the water evaporates from the surface of the CM. 203 The localized ascorbic acid would then result in localized precipitates of Hg+—compounds, when such a leaf is fixed in the Gilson mixture. The following are the main arguments Franke places in support of his hypothesis: (a) Under severe water stress leaves start wilt— ing. A portion of the imbibition water of the outer wall will be lost by CT and an additional portion is thought to be drawn towards the cytoplasm carrying with it dissolved substances. Under these conditions the reducing agent will be concentrated near the protoplast and this would explain why only 'Abbauformen' type ectodesmata can be demonstrated in wilting leaves. (b) Ectodesmata could not be demonstrated in leaves having a very thick cuticle or strongly cutinized walls. In these species CT is low and therefore the localized accumulation of reductant cannot take place. (c) CT and excretion are affected by environ- mental factors. During rainy days only few shortened ectodesmata could be demonstrated. Under these condi— tions CT will be low and the reductant might be leached out. Dry and sunny days will result in increased CT and since no leaching takes place the reductant carried in the transpiration stream can be accumulated in the pores of the outer wall and thus explain the good demonstrability reported under these conditions (10“). 20“ In the dark, stomata are closed and stomatal transpir— ation is low, but CT is maintained or even increased (reference by Franke “1), hence the excretion of reductant and its accumulation in the outer wall can continue. Furthermore, ascorbic acid is more stable in the dark and at low temperatures than in light and high temperatures. With high temperatures incipient drying will reduce the degree of imbibition of the CT and thus reduce CT. Therefore, more complete ectodesmata can be demonstrated in leaves which have been subjected to low temperatures in the dark than in leaves exposed to light and high temperatures. (d) Plants grown in greenhouses tend to have only few and shortened ectodesmata (97, 10“). Franke ex- plained this by pointing out that the high relative humidity found in greenhouses will reduce CT. (e) The pores in which movement of transpiration water and accumulation of the reductant take place, might be brought about by pull forces and pressure exerted against the wall. Areas exposed to mechanical stress, such as guard cells, anticlinical walls, the basis of trichomes and the elevated veins of the lower leaf surface, tend to be densely populated by ecto- desmata. The argument is based on assumptions such as: (a) the occurrence of straight continuous pores in the 205 outer wall of the epidermis perpendicular to the orien— tation of the cellulose fibrils, (b) the excretion of a water soluble reductant and its accumulation in pores which occur only in plants exhibiting ectodesmata following appropriate treatment but not in plants with a thick CM, and (c) the localization of a water soluble reductant in pores. The third assumption deserves some further considerations. The cell wall materials are very polar and the pore spaces (intermicellar and interfibrillar spaces) are generally assumed to be filled with water (9“, “6, “8, 118). Rapid water movement in the cell wall system (apoplast) has been shown to occur (6, 118). Thus, it is not easy to visualize a water soluble reductant localized in certain pores and not diffusing out into the water saturated cell wall environment. In this context, Franke's argument as to why fluorescent dyes cannot be used to demonstrate ectodesmata is in con- flict. Attempts to demonstrate ectodesmata with fluorochromes failed because they diffused out into the wall (“0). Since the outer epidermal wall is composed of polar materials such as cellulose and polyuronides and saturated with water (“6, “8), there is no obvious need for transport channels for polar compounds. Franke (“2) stated that often the outer walls do not 206 exist of a simple cellulose skeleton covered by a cuticle, but, within this skeleton lipoidal materials 'such as wax and cutin are embedded which hamper the free diffusion of hydrophilic materials taken up by the leaf. In this case pores would be required. It can be imggined that these pores could also serve as pathways for precursors of wax, cutin and suberin. It should be pointed out, however, that cutin pre— cursors do not appear to traverse the wall in pores (8, “8). More important, ectodesmata could not be demon- strated in plants having thick cuticles or incrusted outer walls (“2, 10“). In conclusion, Franke's argument in support of his hypothesis (“1) is not convincing. This does not mean to imply that ectodesmata may not be non-plas- matic--they probably are. However, the processes which lead to their demonstration are probably different from what has been suggested. Ectodesmata and Foliar Penetration Diverse substances such as glucose, amino acids, ascorbic acid and caffein were found to affect the demonstrability of ectodesmata when applied to the leaf surface prior to fixation with Gilson mixture (99). This observation led these authors to suggest a relationship between ectodesmata and foliar absorption. 207 This idea has been extensively pursued by Franke (3“, 35, 36, 37, 39, “0, “3, ““)- The discussion is rendered difficult due to the fact that ectodesmata are ill-defined with respect to nature and structure. Indeed, the nature can only be negatively defined as being non-plasmatic. Virtually nothing can be said about the structure of ectodesmata. With respect to their role in foliar penetration the points of origin under the CM and near the cytoplasm are important. Originally it was reported that ecto— desmata do not penetrate the cuticle (69, 97, 10“) and no pores or thin areas were found in the cuticle over ectodesmata. This was confirmed by Franke (““) in writing, however. Figure l of his paper depicts two ectodesmata which appear to penetrate through the cuticular membrane. In accordance with the majority of reports and from examination of appropriate figures in publications cited it appears likely that ectodesmata do not extend through the cuticle. Franke appears to be convinced that ectodesmata perform a function in foliar penetration of polar compounds. The experimental evidence presented is indirect and will be examined next. (a) When leaf segments of Plantago major were A fixed with the Gilson mixture but the subsequent steps were omitted, coarse crystals of unknown nature were 208 formed at the same sites where ectodesmata can be demonstrated. Since these crystals cannot be seen in fresh leaves, it was concluded that they must have formed under participation of the Gilson mixture, which is proof that it had penetrated, possibly through the cuticle over those crystals. There is little doubt that the Gilson mixture containing formic acid, ethanol and formalin can penetrate the cuticle, but there is little proof "that the exchange of materials between the leaf surface and the inner tissue took place through the ectodesmata." (b) When strips of living epidermis of onion bulb scales (Allium cepa) were stained in neutral red and mounted in paraffin oil, the excretion of small drop- lets above the cuticle was observed (5, 37, “9). The droplets formed chain-like rows along the anticlinal walls of the inner epidermis, but were more randomly scattered over the outer epidermis. Ectodesmata were similarly distributed. The nature of the droplets was not determined--they were water soluble when fresh, but water solubility diminished on prolonged exposure to air (“9). Occasionally droplets contained neutral red indicating that not only water but also solutes dissolved therein can be excreted. Since there was good agreement between the sites of droplet excretion and ectodesmata, Franke concluded that the excretion 209 occurred through the ectodesmata neglecting the cuti- cular membrane. (c) Strugger~(ll8) had shown that in Helxine soleirolii CT occurred preferentially from cuticular ledges, anticlinal walls and from the basis of glandu- lar trichomes. Further, berberin sulphate was shown to penetrate the CM at these same sites. Using the same plant species, Franke (35) demonstrated ectodesmata in large numbers in guard cells, along anticlinal walls and around the basis of glandular trichomes. He con— cluded that in Strugger's experiments penetration of BS took place only at those sites corresponding to ectodesmata. He again minimized the fact that ectodes- mata are structures in the cell wall and not in the cuticular membrane. (d) Droplets of a solution containing 1LAC-labeled sucrose, amino acids and phosphate esters were placed on isolated epidermal strips of spinach (Spinacea oleracea) and pansy (Viola tricholor) leaves mounted on glass slides. Penetration was permitted for 10 to 60 minutes. After washing the surface of the strips with distilled water microradioautograms were prepared. Comparable tissue was prepared to demonstrate ectodesmata. More silver grains were found in the film over guard cells and anticlinal walls than elsewhere. Many ectodesmata could be demonstrated in the same areas and this was considered proof that ectodesmata function as pathways 210 in foliar absorption (“0). Unfortunately, Franke failed to make sure that the blackening of the film over the guard cells and anticlinal walls was not due to surface bound material. This is a possibility since it was shown (130) that uSCa and 36Cl were preferentially bound to the cuticle (onion leaf) over anticlinal walls and guard cells. Franke's argument that certain compounds were excreted or penetrated through ectodesmata is not con- vincing since ectodesmata do not seem to perforate the cuticle, which has been shown to be the prime barrier in foliar penetration of polar compounds. All that Franke's evidence shows is, that ectodesmata can be demonstrated where the CM has been shown to be permeable for polar compounds. This is not surprising, since the Hg++ ions essential for demonstrating ectodesmata in the wall are polar themselves and cannot partition through the lipid membrane and depend for penetration on polar pathways which had earlier been shown to occur. From this discussion it is concluded that ecto- desmata can be demonstrated in the wall where the CM permits penetration of polar compounds (including Hg++) provided there is some reductant in the wall. APPENDIX II 211 212 APPENDIX II A. Plot of actual radioactivity vs. measured radioactivity for 1“C—SADH (self—absorption curve). APPENDIX II B. Plot penetration (cpm) vs. penetration (pumoles) (conversion graph). RAD/0407'] VIT Y (Cme 6 000 5 000 4000 3000 2000 l000 ID 000 PENETRATION I cpm I I I IOOO IOO IO 213 0—0 Measured radioacfl'w'fy A—A Actual radioacliw‘ly l 20 sumo: DENSITHM x cm-l) IOO DC!) PENETRATION (ppmolu) 0 A 1.) “AAA!“ "13117:!!ng Infill? 1177 ES 3349