THE EFFECT OF PRE-EMERGENT TREATMENT OF PEAS WITH TRICHLORACETIC ACID ON THE SUB- MICROSCOPIC STRUCTURE OF THE LEAF SURFACE

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1 THE EFFECT OF PRE-EMERGENT TREATMENT OF PEAS WITH TRICHLORACETIC ACID ON THE SUB- MICROSCOPIC STRUCTURE OF THE LEAF SURFACE BY BARRIE E. JUNIPER Department of Botany, Oxford {Received 16 Jamiary 1957) (With Plate i and i figure in the text) SUMMARY Peas were sown in sand which was treated with varying concentrations of trichloracetic acid. The behaviour of water droplets on the leaves of peas was observed, and it was found that the angle made by the droplet on the surface falls with increasitig concentration of T.C.A. No changes in leaf structure which could account for these changes in behaviour were revealed by ordinary light microscopy. Investigatioti of the sub-microscopic anatomy of the surface by the carbon replica technique under the electron microscope did reveal significant differences in the leaf surfaces. A reduction in the number and a change in form of the minute wax structures occurs with an increased concentration of T.C.A. in the soil. These changes correlate well with an increase in the wettability of the leaf surface as determined by the contact angle of water droplets. INTRODUCTION The application of certain herbicides, trichloracetic acid (T.C.A.) and sodiutn 2,2- dichlorpropionate (Dalapon) to the soil before germination, is known to produce iti some species metabolic and physical changes which increase the susceptibility to selective herbicides such as M.C.P.A. and 2,4-D. It has been suggested that this change in susceptibility is due to a reduction of the wax layer on the leaf. This may be true in species such as the pea, Pisum sativum, with an obvious layer of wax on the surface. But easily wettable species such as the annual nettle, Urtica urens, also become more susceptible to herbicides after pretreatment with T.C.A. Dewey, Gregory and PfeifTer (1956), and Pfeiffer, Dewey and Brunskill (1957), observed in some experiments with T.C.A. and Dalapon that the wax layer of the cuticle of peas was somehow altered and the retention of sprays increased. PfeitTer et al. (1957) also noticed that pretreatment of the soil with T.C.A. brought about a change in the water economy of plants. They suggested that this was probably due to the increased permeability of the cuticle. This paper reports an investigation of the anatomical effects of T.C.A. on cuticle. This was made possible by the development of a technique for resolving the submicroscopic structure of leaf surfaces by electron microscopy (Bradley and Juniper, 1957). N.P. I

2 a BARRIE E. JUNIPER METHODS The experimental material used was garden pea variety Alaska, grown 5 cm apart in sand in 25 by 25 cm pots. For the present experiment it has been found essential to grow all material under controllable and repeatable conditions in the greenhouse or growth cabinet. Under field conditions dirt is deposited on the leaf surface and the cuticle is damaged. These confuse the electron microscope image. Applications of T.C.A. at the equivalent of 5.4 kg/ha. (5 Ib per acre) which are not normally damaging to peas in the field, were found to cause excessive damage under greenhouse conditions. Leaching and destruction of the T.C.A. is considerably reduced, and the treated soil is exploited by the plant's root system to a much greater extent than in the field. Therefore, an application of T.C.A. was chosen which caused only a very slight browning and curling of the edges of the leaves, and a range of concentrations was obtained by successively halving this value until the morphological effect was negligible. T.C.A. was applied in the form of a solution of the sodium salt to the soil surface i day after sowing. The peas were harvested after about 14 days at the three leaf stage. The second leaf of a plant growing in each different soil concentration was chosen. One of the pair of leaflets was used for observations on the contact angle, the other for the electron microscope study of the leaf surface. The carbon replica technique for the electron microscope is carried out as follows. Portions of the leaf to be examined are placed in an evaporating chamber, which is then evacuated. While pumping is in progress, very little gas is apparently given off from the leaf and a vacuum (io^^ mm of mercury) sufficient to allow the evaporation of carbon is easily obtained. Carbon is evaporated by passing a heavy alternating current (12 volts at between 50 and 60 amp) through the points of two carbon rods lightly pressed together. The points of the rods are mounted 10 cm above the specimen to be coated. A film about 20 mp, thick is deposited on the leaf surface, and the leaf is then removed from the vacuum. In spite of the level of vacuum reached in the chamber, the leaf does not suffer any superficial distortion due to the escape of gas, provided that the pumping time is kept as short as possible. The carbon film is then backed successively with thin layers of Formvar and Bedacryl I22A- allowing each in turn to dry completely. These are quick-setting liquid plastics, which are extremely soluble in certain solvents. Then the combined film of carbon and plastics is backed with an adhesive cellulose tape and stripped from the leaf. The composite film is then immersed in acetone. This removes the Bedacryl from between the cellulose tape and the Formvar, but does not affect the Formvar itself. The Formvar, which is insoluble in acetone, keeps the carbon film intact and flat in the solvent. Into the space made by the removal of the Bedacryl, specimen grids are inserted. These are thin discs of copper mesh, 80 to i cm (200 to i in.) and 3.05 cm in diameter. The Formvar and the carbon film is then lifted on the grid and the Formvar washed away in chloroform. The carbon film, although extremely thin, is remarkably strong, and will remain more or less permanently suspended across the bars of the grid. The stages of this replica process are shown in Fig. i. The carbon film is very easily stripped from the epidermis by this technique, probably because the waxy cuticle acts as a separating layer between the cell wall and the carbon. This would seem to be confirmed by the fact that leaves with little or no surface wax often prove difficult to strip. For such leaves it is helpful to freeze the whole specimen before stripping.

Pre-emergent treatment of peas 3 The angle between the surface of the leaf and the tangent plane of a water droplet at the circle of contact between air, liquid and leaf (the contact angle) is used

3 Pre-emergent treatment of peas 3 The angle between the surface of the leaf and the tangent plane of a water droplet at the circle of contact between air, liquid and leaf (the contact angle) is used as an arbitrary criterion of the wettability of a surface. A zero angle indicates a completely wettable surface, and any angle up to i8o a degree of unwettability. The angles are measured by a technique similar to that employed by Fogg (1947). A microprojector is used to project the outline made by a droplet of distilled water on the leaf surface. Droplets of a uniform size (0.005 ml) are obtained by using a micrometer syringe. Twenty contact angles are measured on each particular leaf surface. c V Fig. I. A, deposition of carbon; B, carbon layer is backed with Formvar, Bedacryl and cellulose tape; C, leaf is stripped from composite film; D, Bedacryl is dissolved away. Specimen grids are inserted between cellulose tape and Formvar, E, Formvar is dissolved away. Carbon film supported on grid is teased away from the surrounding film and lifted clear, a, carbon rods; b, leaf; c, carbon film; d, Formvar, e, Bedacryl; f, specimen grid; g, solvent for Bedacryl; h, solvent for Formvar; i, cellulose tape. RESULTS The electron microscope shows minute structures which are assumed to be carbon replicas of wax projections from the leaf surface. The shape of these structures is peculiar to the species. Where high contact angles are recorded the replica almost always shows under the electron microscope evenly dispersed projecting structures, which form a wax blanket (Plate la). The scale of the wax structures is very much smaller than that suggested by Van Overbeek (1956). In his figure the size of the droplet in proportion to the wax projections would be between i and 3 u. Actual droplet sizes in commercial practice, with low pressure application, have a mass median diameter between 350 and 450 i. A droplet 100 pi in diameter would be in contact with several thousand projections, and the air trapped between them. Leaves so far examined with low contact angles have either no wax at all (Rumex obtusifolius) or fiat platelets of wax {Narcissus pseiidonarcissus). A surprising feature of this investigation was that in all types of leaf examined so far the outline of the epidermal cells, except that of the guard cells, was not apparent in the replica. The effect of low concentrations of T.C.A. in the soil is simply to reduce the number of wax structures (Plate ib-c). At the highest concentration of T.C.A. (Plate id-f) the surface is radically ahered; the wax instead of being in recognizable 'crystalline' forms is scattered in small flecks on the surface. Some areas were found at this concentration which appeared to be totally devoid of wax. The changes in the surface detail would appear to be confirmed by a change m the contact angle (Table i). The change is not radtcal at the two lower concentrations of T.C.A. probably because the structures are still close enough together to support most of the droplets above and out of contact with the leaf surface. The most striking change in the contact angle occurs between the leaf represented in Plate ic and that represented

4 4 BARRIE E. JUNIPER in Plate id-f. In the second case the droplet is probably no longer held up by the points of the wax structures and the air between them. It is interesting to note that the contact angle of the leaf surface of young Ritrnex obtitsifouiis, which apparently has no surface wax, is about 70. Although the electron microscope field is small compared with the whole leaf area, there was no significant variation over the surface of a leaf in plants treated with small amounts of T.C.A. At the highest concentration of T.C.A. there was some variation in the surface detail in a single leaf. This variation is shown in Plate id-f. This is what one would expect from a gross metabolic disturbance in leaf development. These data are consistent with the view that pre-emergent treatment with T.C.A. changes the development of wax structures on the cuticle and makes the leaf more easily wettable and therefore increases the plant's susceptibility to damage by herbicides. I should like to express my gratitude to Imperial Chemical Industries Ltd. for financial assistance, and to Dr. F. A. L. Clowes for his helpful criticism and advice in the above research. I should also like to thank Sir Paul Fildes, F.R.S.. Director of the British Empire Cancer Campaign Unit for Virus Research, for electron microscope facilities in the Sir William Dunn School of Pathology, and Miss Joan Sampson for taking the electron micrographs. Table i. The variation in contact angle zvith pre-emergence treatment of peas with T.C.A. at various concentrations Dose of T.C.A. applied to the soil Ib/acre kg/ha, g/m , I.II Contact angle Standard error Leaf surface electron micrograph 144 ±3-5" Plate I a iag' ±5.0" Plate ±5.5 Plate ic 68 ±7-5" Plate id-f REFERENCES BRADLEY, D. E. & JUNIPER, B. E. (1957). Electron microscopy of leaf surfaces. Nature, 180, 330. DEWEY, O. R., GREGORY, P. & PFEIFFER, R. K. (1956). Factors affecting the susceptibility of peas to dinitroherbicides. Proc. yd Brit. Weed Cont. Conf., I, 313. FOGG, G. E. (1947). Quantitative studies on the wetting of leaves by water. Proc. rov. Soc, Ser B 134, 503- PFEIFFEH, R. K., DEWEY, O. R. & BRUNSKILL, R. T. (1957). Further investigations of the effect of preemergence treatment with trichloracetic and dichlorpropionic acids on the subsequent reaction of plants to other herbicidal sprays. 4//; Int. Cong. Crop. Piot. Summarv, p. 74. VAN OVERBEEK, J. (1956). Absorption and translocation of plant regulators. Ann. Rev. PI. PhvsiuL, 7, 355. EXPLANATION OF PLATE i Plate I. Electron micrographs of carbon replicas of the surface of the adaxial epidermis of pea leaves The replicas arc unshadowed. The height of the wax structures was determined from shadowed replicas. «. Untreated plant. Height of wax structures about i M. 6. Treated with 0.56 g/m^ T C A. c Treated with i.ii g/m- T.C.A. d-f. Treated with 2.22 g/m= T.C..^. Height of wax structures less thaii 0.2 M. ('. Shows part of the surface of a stoma.

5 THE NEW PHYTOLOGIST, 58, i PLATE JUNIPER PRE-EMERGENT TREATMENT OF PEAS (Facing page 4)

A NOTE ON THE MEASUREMENT OF STOMATAL APERTURE

New Phytol. (1969) 68, 1047-1049. A NOTE ON THE MEASUREMENT OF STOMATAL APERTURE BY Y. LESHEM* AND R. THAINE The Grassland Research Institute, Hurley, Maidenhead, Berkshire {Received 18 March 1969) SUMMARY