STOMATAL RESPONSES TO CHANGES IN CARBON DIOXIDE CONCENTRATION IN LEAVES TREATED WITH 3- (4-CHLOROPHENYL) -1,1 -DI METHYLUREA

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1 New Phytol. (1967) 66, STOMATAL RESPONSES TO CHANGES IN CARBON DIOXIDE CONCENTRATION IN LEAVES TREATED WITH 3- (4-CHLOROPHENYL) -1,1 -DI METHYLUREA BY W. G. ALLAWAY AND T. A. MANSFIELD Department of Biology, University of Lancaster {Received 12 June 1966) SUMMARY An inhibitor of photophosphorylation, 3-(4-chlorophenyl)-i,i-dimethylurea, was supplied to detached leaves via their petioles. The stomata partly closed after the inhibitor had entered the lamina but opened again if the intercellular spaces were flushed with air free of carbon dioxide. It is suggested that the closing movement was a response to the accumulation of carbon dio.xide in the intercellular spaces, following the inhibition of photosynthesis. The re-opening after removal of carbon dioxide demonstrated that stomata could open while photophosphorylation was strongly inhibited. The conclusions from these experiments are discussed in relation to a recent hypothesis of the stomatal mechanism. INTRODUCTION Stomatal movements are thought to be the result of changes in the turgor difi^erence between guard and subsidiary cells, and it was at one time considered that the photosynthetic production of soluble carbohydrates in the guard cells created the osmotic potential necessary for opening in light. The discovery that stomata open most widely in light in the absence of CO, (Freudenberger, 1940; Heath, 1948) led to a rejection of the original hypothesis, but it has recently been suggested that photophosphorylation plays an important part in the opening response to light. Zelitch (1963, 1965) envisaged a mechanism depending upon the presence of glycouate (which is a major photosynthetic product at low CO2 concentrations) and its oxidation to glyoxylate; glyoxylate reductase would then achieve the oxidation of reduced pyridine nucleotides, enabling non-cyclic photophosphorylation (Arnon, 1959) to proceed. The suggested role of glycollate oxidation is, however, apparently ruled out by the observation that stomata open in COj-free air in the presence of a high concentration of an a-hydroxysulphonate which inhibits glycollate oxidase (Heath, Mansfield and Meidner, 1965; Meidner and Mansfield, 1966; Mouravieff, 1966). This would not necessarily exclude the participation of photophosphorylation leading to adenosine triphosphate being available for operating a 'guard cell pump' to maintain the cells' turgor (Zelitch, 1963, 1965). We therefore considered it desirable to investigate more directly the role of ATP formation in determining stomatal movements. The substance 3-(4-chlorophenyl)-i,i-dimethylurea (CMU) is known to inhibit photosynthesis and the Hill reaction strongly (Wessels and van der Veen, 1956). It has been shown to block in vivo photophosphorylation so that leaf discs fioated on glucose solution were unable to form starch in the light (Krall and Bass, 1962), and at a concentration of 5 X 10"^ M it inhibited glucose uptake by Chlorella (Butt and Peel, 1963). By 57

2 58 W. G. ALLAWAY AND T. A. MANSFIELD using this inhibitor we have been able to carry out a critical test of the role of photophosphorylation in the stomatal light response. When photosynthesis is inhibited respiratory CO2 will accumulate, as it would in darkness, and this will lead to stomatal closure; if this is the only effect on the stomata it should be largely reversed by flushing the intercellular spaces with air free of COj. Consequently, we have studied stomatal responses to CMU while controlling the intercellular space CO, concentration ] 1 2 Rate of 1 3 I, flow (ml 1Tiir Fig. I. Calibration of the porometer. Porometer reading ('degree of opening' in subsequent Figures), plotted against rate of air flow through the porometer cup, is given in the arbitrary scale units of the recorder used for these experiments. JNIETHODS Two species of plant were studied. Plants of Rumex conghmeratus Alurr. were collected near Harlton, Cambridgeshire, and grown in a glasshouse at approximately 20' C. The rootstock of two individuals was di\-ided before potting, to give two clones. Only mature leaves which had expanded in the glasshouse and with laminae between 9.5 and 13.0 cm in length were used. Vicia faba L. cv. 'White Windsor' was raised from seed and grown under similar conditions in the same glasshouse. Both species have stomata on both upper and lower epidermes, the ratio of number per unit area, upper over lower, being: Rumex conghmeratus per mm^; Vicia faba 4660 per mm^. Selected leaves were detached on the morning of the experiment by cutting the petiole under water at about i cm from the base of the lamina. The lamina was not wetted. The leaf was immediately attached to the porometer in a growth cabinet at approximately 23" C, and illuminated at approximately 18,000 lux by fifteen 150 W water-cooled tungsten-filament lamps. Before entering the cabinet, the light passes through 5 cm of water, which is sufficient to remove all the long-wave infra-red radiation. The porometer cup, similar to that described by Heath and Mansfield (1962), was attached to the abaxial surface of the basal part of the lamina between 2.8 and 4.0 cm

3 and stomatal response from the cut end of the petiole, and was left attached to the leaf throughout each experiment. Air was continuously supplied to the cup, passing through the leaf at a rate dependent on the stomatal aperture, as shown in Fig. i. The area of leaf enclosed by the cup was 0.50 cm~. The relative humidity of the air was maintained constant at about 50 by bubbling it through saturated calcium nitrate solution. In the treatments involving CO^-free air the air stream was passed through a soda-lime tower. eg ' '\ 1 N \ -^ c \ \ \ s. -fe^-r K 16 Time (hours G.M.T.) Fig. 2. Rumex conglo?neratus: stomatal closure induced by io""'' M CMU and re-opening following treatment with CO2-free air. Experimental treatments replicated in four randomized blocks, A, B, C and D. The original records have been re-drawn to a smaller scale. Arrows indicate times at which io"" M CMU was fed; vertical lines show when the COifree air treatment commenced. Partial closure in the afternoon in the 'controls' follows the endogenous rhythm. Control: ; CMU and normal air: ; CMU and CO^-free air:. A 'resistance' porometer (Gregory and Pearse, 1934) was used with a new type of recording device. The air pressure in the porometer cup is registered by a Fielden pressure transducer type UT27, and recorded through a Fielden dififerential transformer-transmitter type D5064 into a 0-15 ma recorder. The instrument responds linearly to changes in pressure in the porometer cup over the range observed in these experiments. Air was supplied to the porometer circuit at a constant increased pressure of approximately 11 cm of water (8 mm of mercury).

4 6o W. G. ALLAWAY AND T. A. MANSFIELD RESULTS Preliminary experiments with both Riimex conglomeratus and Vicia faba showed that CMU at concentration of io*"* M fed through the petiole was sufficient to bring about a considerable closing movement of the stomata with normal air (300 ppm CO2) passing through the leaf. However, the closing movement was reversed when air free of COj was passed through the leaf. An experiment was carried out on Rumex conglomeratus in which the following U Time (hours G.M.T.) Fig. 3. Vicia faba: original record re-drawn to a smaller scale. CMU io * M was fed via the petiole of the detached leaf at the time indicated; stomatal closure induced by the inhibitor was reversed by CO2-free air, but recommenced when the supply was changed back to normal air. This movement was also reversed by CO2-free air. The overall closing tendency throughout the day is attributed to the endogenous rhythm which was evident in 'control' leaves. The horizontal line at the top indicates the CO2 concentration, where, normal air;, CO2-free air. treatments were applied in four randomized blocks (periods of time), plants from the same clone being used within each block: (a) control treatment with the petiole in water, and the leaf flushed with normal air; (b) 10"* M CMU fed through the petiole, the leaf flushed with normal air; (c) 10"'^ M CMU fed through the petiole, the leaf initially flushed with normal air, this being replaced by C02-free air after closure was under way. The observations on the twelve different leaves used in the experiment are shown in Fig. 2. Flushing the leaf with C02-free air after closure had commenced in the CMU treatment always caused re-opening of the stomata. Taking the experiment as a whole, the difference in flnal aperture between normal air and C02-free air (in CMU-treated leaves) was statistically signiflcant at a probability of <o.ooi. These experiments suggested that the normal response of stomata to CO2 was retained in CMU-treated leaves, and this was confirmed in an experiment on Vicia faba in which C02-free and normal air were given alternately (see Fig. 3). It was clearly important to know whether CMU effectively gained access and was

5 CO2 and stoniatal response blocking photophosphorylation when fed through the petiole in the manner adopted above. Leaves were de-starched in the dark for 12 hours and then placed in the growth cabinet for 6 hours with their cut petioles in either io~"* M CMU or water (four leaves per treatment). The method of Krall and Bass (1962) was then used to determine whether the leaves were capable of photophosphorylation. Discs, i cm in diameter, were cut with a cork borer and floated on 50 glucose solution, under illumination from six 13 W warm-white fluorescent tubes providing an intensity of about 12,000 lux. After about 24 hours of illumination the discs were de-colorized in boiling ethanol and stained in iodine-potassium iodide. Excess iodine was washed off in w'ater and the starch content was roughly assessed by measuring the transmission of light through each disc, placing it between a fixed light source and a Weston 'Master V lightmeter. The results are shown in Fig. 4 from which it will be seen that considerably more starch was formed in the untreated leaves than in those treated with CMU. Thus there was little doubt that 6i opaque 9 " " Fig. 4. Absorption of light by Rumex leaf-discs after decolorization and staining in iodinepotassium iodide, giving an indication of the amount of starch formed. Each value represents the mean of at least fifteen discs. The 'C leaf-discs were cut from de-starched leaves, and stained straight away. The following treatments were applied to the de-starched 'A' and 'B' leaves before cutting the leaf-discs: A = no CMU, illuminated at 18,000 lux for approximately 6 hours; B = leaves fed with io"'* M CMU via the petiole for approximately 6 hours, under illumination of 18,000 lux. The 'A' and 'B' discs were floated on 5 " glucose solution for 24 hours under illumination of approximately 12,000 lux before staining. CMU was being distributed through the lamina and was effectively blocking photophosphorylation. To check that the inhibitor was being translocated to the guard cells, a piece of a CMU-treated leaf used in one of the experiments recorded in Fig. 2 and a similar piece of a control leaf without CMU, were decolorized and stained in iodine. The guard cells in the upper epidermis were examined under the microscope: those of the CMU-treated leaf were found to contain only small weakly staining starch grains, while those of the control leaf had large and densely staining starch grains. It was therefore concluded that io"'^ M CMU taken up through the cut end of the petiole was effective in inhibiting photophosphorylation in the guard cells of Rumex conglomeratus leaves.

6 62 W. G. ALLAWAY AND T. A. MANSFIELD It was our experience that abundant starch was formed in guard cells of this species in the light. DISCUSSION For stomata to open, the guard cells have to maintain a turgor which enables them to swell against the resistance offered by the suhsidiary cells. This was shown by the further opening of the pore when a subsidiary cell adjacent to a partially open stoma was punctured (Heath, 1938). One must suppose, therefore, that energy is required for the opening process, and presumably also for the maintenance of the open condition. Walker and Zelitch (1963) found that there was an oxygen requirement for opening in light, but lack of oxygen did not induce closure of stomata that were already open. This suggested that sufficient energy was derived from anaerobic photophosphorylation (cf. Butt and Peel, 1963) to maintain the open condition but that the greater amount of energy required for the opening movement could not be obtained anaerobically. Both Zelitch (1963, 1965) and Kuiper (1964) have concluded that photophosphorylation in guard cells is important for opening in light. Zelitch (1965) thought that at low carbon dioxide concentrations more ATP might be available in the guard cells for operating a 'pump' to increase the turgidity of the cells. However, Punnett and Iyer (1964) obtained evidence from in vitro experiments that increases in CO2 concentrations enhance photophosphorylation and, from a consideration of the results of Emerson and Arnold (1932), they suggested that this could also apply in vivo. Kuiper performed some experiments using 3-(3,4-dichlorophenyl)-i,i-dimethylurea (DCMU), the action of which is similar in many respects to the inhibitor used in the present study (Wessels and van der Veen, 1956). It caused complete stomatal closure at a concentration of 10^^ M and Kuiper concluded that this provided evidence of the importance of photophosphorylation for opening. In our experiments CMU was similarly highly effective in producing closure and we could have reached a similar conclusion had we not taken the precaution of flushing the leaf with COj-free air. The re-opening under this treatment leaves little doubt that the main effect of CMU was due to the inhibition of photosynthesis and the accumulation of respiratory CO, within the leaf. Re-opening in CO^-free air did not quite reach the aperture achieved in the 'control' treatments, but this was to be anticipated since flushing the intercellular spaces cannot remove intracellular CO2 as effectively as does photosynthesis. The experiment presented in Fig. 3 demonstrated that the stomata retained their sensitivity to CO2 during treatment with CMU. It appears therefore that stomatal sensitivity to changes of CO2 concentration in light is not due to changes in the rate of ATP formation by photophosphorylation. One must indeed doubt that photophosphorylation plays any essential role in the carbon dioxide responses of stomata since wide opening in the dark can be induced by the removal of CO2 (Mansfield, 1965). Zelitch (1965) casts some doubt on the observations of opening in darkness, suggesting that they may be artifacts due to water strain. However, the recent observation of Mouravieff (1965) of very wide apertures (up to 12 J) in darkness on well-hydrated tissue in CO2-free air leaves no room for doubt. All the available evidence suggests that stomatal sensitivity to changes in CO2 concentration operates in darkness in the same way as in light and no hypothesis to account satisfactorily for this has yet been produced. In future work it may be worthwhile to give more consideration to 'dark' carboxylation reactions which are affected by changes in carbon dioxide concentration (Meidner and Mansfield, 1965).

7 CO2 and stomatal response 63 In conclusion, it appears that photophosphorylation is not essential for stomatal movements in response to changes in CO. concentration. The use by the guard cells of ATP (or a similar substance) produced by another mechanism to provide the energy for the movements is not, however, precluded. ACKNOWLEDGMENT W. G. Allaway is in receipt of a post-graduate award from the Natural En\ ironment Research Council. REFERENCES ARNON, D. I. (1959). Conversion of light into chemical energy in photosynthesi.s. Nature, Lond., 184, 10. BUTT, V. S. & PEEL, M. (1963). The participation of gh collate oxidation in glucose uptake by illuminated Chlorella suspensions. Biochem. J., 88, ;ip. EMERSON, R. & ARNOLD, W. (1932). A separation of the reactions in photosynthesis by means of intermittent light. ^. gen. PhysioL, 15, 391. FREUDENBERGER, H. (1940). Die Reaktion der Schliesszellen auf Kohlensaure- und SauerstofF-Entzug. Protoplasma, 35, 15. GREGORY, F. G. & PE.^RSE, H. L. (1934). The resistance porometer and its application to the study of stomatal movement. Proc. R. Soc, B114, 477. HEATH, O. V. S. (1938)..An experimental inxestigution of the mechanism of stomatal movement, with some preliminary observations upon the response of guard cells to shock. Ne7c PhvtoL, 37, 3S5. HEATH, O. V. S. (194.8). Studies in stomatal action. Control of stomatal movement by a reduction in the normal carbon dioxide content of the air. Nature, Lond., 161, 179. HEATH, O. V. S. & ]\L\NSFIELD, T. A. (1962)..A recording porometer with detachable cups operating on four separate leaves. Proc. R. Soc, B156, i. HEATH, O. V. S., M.WSFIELD, T. A. & MEIDNER, H. (1965). Light-induced stomatal opening and the postulated role of glycollic acid. Nature, Lond., 207, 960. KR.ALL, A. R. & BASS, E. R. (1962). Oxygen dependency of in vivo photophosphorylation. Nature, Lond., 196, 791. KuiPER, P. J. C. (1964). Dependence upon wavelength of stomatal movement of epidermal tissue of Senecio odoris. PI. PhysioL, Lancaster, 39, 952. MANSFIELD, T. A. (1965). Glycollic acid metabolism and the movements of stomata. Nature Lond MEIDNER, H. & MANSFIELD, T. A. (1965). Stomatal responses to illumination. Biol. Rev., 40, 483. MEIDNER, H. & MANSFIELD, T..A. (1966). Effects of supplying leaves with i-hydroxysulphonate and glycouate on stomata, rates of photosynthesis and respiration, jf. exp. Bot., 17, 502. MOUR.WIEFF, I. (1965). Sur les reactions de l'appareil stomatique a l'acide a-hydroxy-2-pyridinemethansulphonique. Cr. hebd. Seaiic Acad. Sci., Paris, 261, MoUR.AViEFF, I. (1966). Recherches sur les inhibiteurs des mouvements stomatiques. Bull. Soc. linn. Lyon, PuNNETT, T. & IYER, R. V. (1964). The enhancement of photophosphorylation and the Hill reaction by carbon dioxide. _7. biol. Chem., 239, WALKER, D. A. & ZEUTCH, I. (1963). Some effects of metabolic inhibitors, temperature, and anaerobic conditions on stomatal movement. PL PhysioL, Lancaster, 38, 390. WESSELS, J. S. C. & VAN DER VEEN, R. (1956). The action of some derivations of phenylurethan and of 3-phenyl-i,i-dimethylurea on the Hill reaction. Biochim. biophys. Acta, 19, 548. ZELITCH, I. (1963). The control and mechanisms of stomatal movement. Bull. Conn, agric. E.\p. Stn, 664, 18. ZELITCH, I. (1965). Environmental and biochemical control of stomatal movements in leaves. Biol. Rev., 40, 463. E NP

STOMATAL RESPONSES TO LIGHT AND CARBON DIOXIDE IN THE HART'S-TONGUE FERN, PHYLLITIS SCOLOPENDRIUM NEWM.

New PhytoL (1969) 68, 63-66.. STOMATAL RESPONSES TO LIGHT AND CARBON DIOXIDE IN THE HART'S-TONGUE FERN, PHYLLITIS SCOLOPENDRIUM NEWM. BY T. A. MANSFIELD AND C. M. WILLMER Department of Biological Sciences,