EFFECTS OF PHENYL-MERCURIC ACETATE ON, STOMATAL AND CUTICULAR RESISTANCE TO TRANSPIRATION

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1 New PhytoL (1975) 75, :: i vi EFFECTS OF PHENYL-MERCURIC ACETATE ON, STOMATAL AND CUTICULAR RESISTANCE TO TRANSPIRATION BY S. MORESHET Agricultural Research Organization, The Volcani Centre, Bet Dagan, Israel {Received 16 September 1974) SUMMARY Phenyl mercuric acetate applied to both sides of the leaves of sunflower plants growing in both wet and dry soil caused a greater closure of the stomates on the upper than on the lower side of the leaves. By contrast, application of PMA increased cuticular resistance to gas diffusion only in the case of leaves of plants growing in dry soil, suggesting that soil moisture stress as well as PMA decreases the permeability of non-stomatal epidermal cells. INTRODUCTION The practical importance of the finding that application of the fungicide phenylmercuric acetate (PMA) reduces transpiration from leaves is mitigated somewhat by a reduction in photosynthesis (Zelitch and Waggoner, 1962; Shimshi, 1963; Slatyer and Bierhuizen, 1964; Squire and Jones, 1971; Sij, Kanemasu and Teare, 1972; Honeycutt and Krogmann, 1972; Bravdo, 1972). A fuller appreciation of the potentialities of PMA for increasing plant water use efficiency therefore requires information on the effect of PMA on the various leaf resistances to gas diffusion. The literature on this is somewhat contradictory. Some authors obtained complete stomatal closure (Zelitch and Waggoner, 1962; Shimshi, 1963; Mansfield, 1967), while others report that stomates were unable to close completely (Waisel, Borger and Kozlowski, 1969; Davenport, Fi^sher and Hagan, 1971). The reported increase in 'apparent' cuticular transpiration (Waisel et al, 1969) may in fact be due to incomplete closure of stomates. Under such circumstances, night transpiration, and to some extent dawn and dusk transpiration, may increase (Davenport et al, 1971). Davenport et al (1971) speculate that PMA decreases the permeability of membranes to solutes and thereby retards the rate of their efflux from the guard cells during closing and the rate of their infiux during opening. In this paper we report on the effect of PMA on both stomatal and cuticular permeability to water vapour and the relative effect of PMA on stomatal resistance of the two leaf surfaces. MATERIALS AND METHODS Plant growth conditions.. Sunffower plants {Helianthus annuus) were grown for 6-7 weeks in pots contammg I kg grumusolic loess soil in a growth room under fiuorescent light of 900 ft-candles at 47

48 S. MORESHET. 25 +1 C air temperature and 50 ± 5% r.h. The plants were irrigated daily to pot capacity (22% w/w soil moisture, equivalent to -o.i bar soil water potential). Treatments Soil moisture.

2 48 S. MORESHET C air temperature and 50 ± 5% r.h. The plants were irrigated daily to pot capacity (22% w/w soil moisture, equivalent to -o.i bar soil water potential). Treatments Soil moisture. Two soil water conditions, 'wet' and 'dry', were obtained by discontinuing irrigation of the 'dry' treatment a few days prior to measurement. The soil water content of the 'wet' treatment at the beginning of the day of measurement was %, equivalent to -0.2±0.1 bar soil water potential. The dry treatment soil contained 10.4 ±0.1%, equivalent to bar. Phenyl-mercuric acetate {PMA). Three concentrations of PMA with 0.2% Triton X 100, a widely used wetting agent, were applied to the leaves in eight experiments, three at a concentration of io~^'^ M, three at io~* M and two at io"''^'^ M. Control plants were treated the same, but without PMA. Four plants were tested in each experiment, representing four treatments: 'wet' soil treated with PMA, 'wet' treated with water and wetting agent, 'dry'+ PMA, and 'dry'+ water and wetting agent. As the measurements showed no significant effect of the three different concentrations of PMA, the results of these were grouped. Experimental chamber An acrylic plastic chamber was used in which the r. h. of the air and the light intensity could be controlled (Moreshet, 1970). During the experiments the r. h. was maintained at %, and light intensity at 30 klx (short wave radiation = 23.2 x io~* erg cm~^ s~^). Air temperature within the chamber increased from 26 to 30 C after the lights were turned on, but thereafter remained constant to within 0.2 C. Pre-experimental plant-preparing procedure Four plants at the desirable soil moisture were selected i day before each experiment. All leaves were removed except for the youngest fully expanded one, which was then immersed either in the PMA solution or in water plus wetting agent, left to dry, and placed in the chamber after sealing the pot's surface to prevent evaporation. The four plants were exposed to the experimental conditions of humidity for 16 h, and of light intensity for 2 h, before the measurements were begun. MEASUREMENTS AND CALCULATIONS Pots were weighed before and after the experiments (4-h interval) on a balance sensitive to O.I g, and transpiration was calculated on the basis of leaf area. Relative humidity of the chamber was measured with an Assmann psychrometer and leaf temperature with a copper-constantan thermocouple attached to the lower leaf surface. The sum of stomatal diffusion resistances of the lower and upper surfaces (r^) was measured with an argon porometer (Moreshet, Stanhill and Koller, 1968). Epidermal resistances to diffusion of water vapour from the upper {rg^^) and lower (r^i) surfaces were measured with a double transpiration porometer (Moreshet, 1970), whence, and r., = I h (I) where r^u and r^, are the stomatal resistances of the upper and lower surfaces of the leaf respectively, and r^ is the mean cuticular resistance, assumed to be the same for the two surfaces.

3 Phenyl-mercuric acetate affecting transpiration The argon porometer effectively measures the sum r^: Combining equations (i) and (2) gives: rs = ^u + ^i (2) ar/+br^-\-c =- o (3) where a = ^eu + ^i-^. b = [^(^eu + ^i)-2reu^ei] and c = -r/^i^eu- After solving for Tp from (3), equation (i) w^as used to calculate r^^ and r^^. RESULTS AND DISCUSSION The duration of our measurements (4 h) w^as too short to cause a significant difference in relative vv^ater content between the PMA-treated leaves and the control leaves (Table i). Leaf temperature differences, although very small, were significant. Davenport (1967) measured leaf temperature of grass and lettuce plants treated with PMA and found a slight but significant increase compared with untreated leaves. Our results (Table i) Table i. Effect of PMA and soil moisture on plant jwater relations Wet Dry PMA Control PMA Control Measured Relative water content (RWC) o±o ! 1.2 Leaf-air temperature i-4 -O-7 -o-6 difference (A C) Total transpiration 3-77±o-3o 4-59±o-O7 o.87±o.o8 i.o5±o.i2 (10"^g cm"^ s~^) Lower epidermal resistance 2.86±o.o8 3.o2±o.o ±o-53 (s cm-^) Upper epidermal resistance (s cm~^) Stomatal resistance (upper 40.8i i i2.b7 plus lower) (s cm"^) Calculated Lower stomatal resistance 5-ii±o i±o i3.99 (s cm~^) _ Upper stomatal resistance 35.7i ii Oi i2.98 (s cm~^) confirm his findings for plants of the 'wet' treatment. A very small decrease in leaf temperature was found in the 'dry' treatment plants. The PMA treatment reduced transpiration equally in wet and dry soil, the respective reductions being 18% and 17%; however, the absolute reduction was much smaller in the case of the dry soil (Table i). The PMA solution was applied equally to the two leaf surfaces of the sunfiower plants, but its infiuence was not the same. Table i shows that whereas the epidermal resistance of the lower leaf surface was not significantly affected by the treatment, both in the wet and dry soil, that of the upper epidermis was doubled in wet soil and trebled in dry soil. Zelitch and Waggoner (1962) found that PMA closed stomata only on the sprayed leaf surface, but Sij et al (1972), in a preliminary test, found a response similar to ours. Measurements of the diffusion of argon gas through the leaves (Table i) show that in the wet soil the sum of stomatal resistances of the two surfaces was increased about three times by the PMA, whilst in the dry soil PMA caused almost complete stomatal closure. Calculation of the various components of leaf resistances (Table i)

4 S. MORESHET shows a small difference between the stomatal resistances of the lower epidermis in the PMA-treated and control plants of the wet soil. The calculated stomatal resistance of the lower epidermis of the control 'dry' plants was twice that of the PMA 'dry' plants, but the latter was similar to that in the PMA 'wet' treatment. On the other hand, the calculated stomatal resistance of the upper epidermis was strongly affected, both by soil moisture treatment and by PMA treatment, the relative effect being somewhat greater in the wet soil. This can also be seen in the ratio of lower to upper stomatal resistance (Fig. I a). This ratio was close to i in 'wet' soil-control, 0.3 in 'wet' soil-pma and in Control PMA Control PMA Wet soil Dry soil Fig. I. The influence of PMA on (a) the ratio between stomatal resistances of the two surfaces, and (b) cuticular resistances to transpiration., i s.e. 'dry' soil-control, and less than o.i in 'dry' soil-pma which indicates closed stomata in the upper epidermis. Cuticular resistance (Fig. ib) was surprisingly low compared with stomatal resistance. In the 'wet' soil-control plants it was of the same order of magnitude as the stomatal resistance of both upper and lower epidermises, but in the 'wet' soil-pma plants it was about one quarter of the stomatal resistance of the upper epidermis, and in fact unaffected by PMA. The cuticular resistance of the control plants growing under dry soil conditions was twice that for the 'wet' soil control plants. In the dry soil, PMA application almost trebled cuticular resistance, but even so, the effect was less than on stomatal resistance of the upper epidermis. Slatyer and Bierhuizen (1964) stated that an increased cuticular resistance caused by treatment with an anti-transpirant would be of significance only when the stomatal resistance reached a similar high order of magnitude. They cited a value of 32 s cm" ^ for cuticular resistance. Our data show, on the contrary, that cuticular resistances may reach the same order of magnitude as stomatal resistances and sometimes may even be lower, indicating that cuticular resistance should not be neglected, either in wet or in dry soil conditions. Using known values of the various diffusive resistances to water loss, measured leaf

5 V.. -,-.,^.:N;,"J.., Phenyl-mercuric acetate affecting transpiration temperatures, assuming saturation at each of the evaporating surfaces, the water vapour concentration in the air and in the leaf, and the measured boundary layer resistance in the chamber (0.45 s cm" ^), we have calculated the transpiration flux via the different pathways. The results are shown in Fig. 2. Application of PMA to plants grown under wet soil conditions reduced transpiration from the upper stomata only; it did not significantly change transpiration from the stomata of the lower epidermis or from the cuticle of the upper or lower surfaces. In dry soil, PMA reduced transpiration very significantly from the upper stomata as well as from the cuticle. Davenport et al. (1971) claimed that the PMA was less effective on plants in dry soil because of prevention of complete closure of the stomata. They suggested that PMA decreased the permeability of membranes to solutes such as potassium salts which are needed for the stomatal opening and closing processes. However, this would not explain the greater sensitivity of the upper epidermis to the PMA. I' o c o Control Wet soil PMA Control Dry soil Fie 2 The influence of PMA on stomatal and cuticular transpiration. Stippled, upper stomatal transpiration; cross-hatched, lower stomatal transpiration; horizontal hatched, cuticular transpiration. PMA It may be concluded that the cuticular resistance to transpiration is both significant and highly variable, being dependent on the moisture content of both the soil as shown here and of the atmosphere, as shown previously (Moreshet, 1970). Moreover, it can be affected by the application of PMA. REFERENCES BRAVDO, B. A. (1972). Effect of several transpiration suppressants on carbon dioxide and water vapour exchange of citrus and grapevine leaves. Physiologia PL, 20, 152. ^u f <,» DAVENPOR? D C ("967). Effects of chemical antitranspirants on transpiration and growth of grass. J. DAVEN?ofT,'b.'c'.,'FiSHER, M. A. & HAGAN, R. M. (1971). Retarded stomatal closure by phenyl-mercuric,'d. W. (1972). Inhibition of chloroplast reactions with phenyl-mercuric MANSFSD" T 46, 815. Q?)s V m behaviour following treatment with auxin-like substances and phenyl-. Eff^cfKn'vfr'onJn^'ntal factors on cuticular transpiration resistance. PL PhysioL,

52 S. MORESHET MoRESHET, S., STANHILL, G. & KoLLER, D. {1968). A radioactive tracer technique for tbe direct measurement of the diftusion resistance of stomata. J. exp. Bot., 19, 460. SHIMSHI, D.

6 52 S. MORESHET MoRESHET, S., STANHILL, G. & KoLLER, D. {1968). A radioactive tracer technique for tbe direct measurement of the diftusion resistance of stomata. J. exp. Bot., 19, 460. SHIMSHI, D. {1963). EfTect of chemical closure of stomata on transpiration in varied soil and atmospheric environments. PL PhysioL, 38, 709. Sij, J. W., KANEMASU, E. T. & TEARE, I. D. {1972). Stomatal resistance, net photosynthesis and transpiration in PMA-treated sorghum: A field study. Crop Sci., 12, 733. SLATYER, R. O. & BIERHUIZEN, J. F. {1964). The influence of several transpiration suppressants on transpiration, photosynthesis and water use efficiency of cotton leaves. Aust. J. biol. Sci., 17, 131. SQUIRE, G. R. & JONES, M. B. {1971). Studies on the mechanisms of action of the antitranspirant phenylmercuric acetate, and its penetration into the mesophyll. J. exp. Bot., 22, 980. WAISEL, Y., BORGER, G. A. & KOZLOWSKI, T. T. {1969). Effects of phenyl-mercuric acetate on stomatal movement and transpiration of excised Betula papyrifera Marsh leaves. PL PhysioL, 44, 685. ZELITCH, I. & WAGGONER, P. E. {1962). EfTect of chemical control of stomata on transpiration and photosynthesis. Proc. natn. Acad. Sci. U.S.A., 48, iioi.

THE EFFECT OF ABSCISIC ACID ON STOMATAL BEHAVIOUR IN FLACCA, A V\^ILTY MUTANT OF TOMATO, IN DARKNESS

$THE EFFECT OF ABSCISIC ACID ON STOMATAL BEHAVIOUR IN FLACCA, A V\^ILTY MUTANT OF TOMATO, IN DARKNESS$ New Phytol (1972) 71, 81-84. THE EFFECT OF ABSCISIC ACID ON STOMATAL BEHAVIOUR IN FLACCA, A V\^ILTY MUTANT OF TOMATO, IN DARKNESS BY M. T A L AND D O R O T I M B E R Division of Life Sciences, Negev Institute