A CRITICAL EXAMINATION OF THE USE OF DETACHED EPIDERMIS IN STUDIES OF STOMATAL PHYSIOLOGY

Transcription

1 New Phytol. (1969) 68, A CRITICAL EXAMINATION OF THE USE OF DETACHED EPIDERMIS IN STUDIES OF STOMATAL PHYSIOLOGY BY C. M. WILLMER AND T. A. MANSFIELD Department of Biological Sciences, University of Lancaster {Received 15 July 1968) SUMMARY Although several workers have published accounts of stomatal studies with detached epidermis, there has not been general agreement as to whether the stomata continue to respond to environmental factors as they do in the intact plant. This paper reports a critical examination of the value of the technique, experiments having been carried out on material from Vida faba and two species of Commelina. The nature of the incubating medium was found to exert a considerable influence on the behaviour ofthe stomata. In Commelina, the presence of monovalent cations (K'^, Na""") stimulated opening, but divalent cations (Ca^"*", Mg^""") had an antagonistic effect suppressing opening or even preventing it altogether. In Viciafaba cations exerted less noticeable effects. ph changes affected aperture, but in Commelina the changes were of much smaller magnitude than those induced by the different cations. Factorial experiments were performed to determine the effect of detaching epidermis on the stomatal responses to environmental factors. In Vicia faba the light effect was very apparent on attached epidermis, but on detached epidermis the effect was largely obscured by stomatal opening that occurred in darkness; effects of CO2 concentration were detectable on epidermal strips, particularly in darkness, but were of smaller magnitude than those on attached epidermis. On the other hand, in Commelina, effects of both light and COj were of considerable magnitude when strips were immersed in media containing the cations that stimulated opening. The theory that the opening stimulated by monovalent cations is the outcome of their being actively taken up into the guard cells is discussed, and it is concluded that while this might apply to epidermal strips in media, there is inadequate evidence that such a mechanism is responsible for opening in the intact plant; there was no stimulation of opening on attached epidermis when pieces of leaf were immersed in media containing monovalent cations. Attempts are made to explain the difference in behaviour between Viciafaba and Commelina on the basis ofthe course of ion uptake into the cells of the epidermis; evidence of a possible difference in uptake was obtained from studies of the penetration by neutral red. It is concluded that epidermal strips from both species of Commelina are suitable for studies of stomatal responses to Hght and CO2 so long as they are incubated in a suitable medium. Epidermal strips from V. faba are unsuitable for studies of the light responses, and less satisfactory than Commelina for the responses to CO2. INTRODUCTION Progress towards an understanding of the stomatal mechanism has been limited by the difficulty of carrying out specific investigations into the activities of the guard cells. Physiological studies have, in the main, been carried out on intact plants and have yielded much valuable information on the responses of stomata to factors of the environment. However, it has become increasingly clear that if we are to understand the mechanism behind the turgor changes occurring in guard cells, more attention must be paid to their 363

2 364 C. M. WILLMER AND T. A. MANSFIELD special activities. Since advances in this field of investigation so obviously depend on the development of reliable techniques to allow stomatal behaviour to be observed on detached epidermis, we have undertaken an extensive study with this in mind. Although several workers have in the past used leaf portions of strips of epidermis in stomatal studies, there has been little or no attempt to compare their behaviour with that on intact leaves. Recently, however. Glinka and Meidner (1968) reported an important critical study of the practice of investigating stomatal behaviour on floating leaf discs. They found that the responses could be very different from those of intact leaves for example, rates of change and final apertures were affected and the carbon dioxide response could be completely obscured if the stomata being investigated were in contact with the water. Attempts to study stomatal behaviour on isolated pieces of epidermis have met with varying degrees of success and this led to doubts about the value of the technique. Heath (1959) reviewed the attempts of different workers and concluded 'the stomata of epidermal strips or surface sections floated on water do not respond normally to light', but Kuiper (1964) used epidermal strips of Senecio odoris and successfully determined an action spectrum for stomatal opening, which agrees well with that found on intact plants of Xanthium pennsylvanicum (Mansfield and Meidner, 1966). This work and the recent study by Fujino (1967) leave little doubt that the technique can be usefully employed. One of the hazards of using epidermal strips seems to arise from the effect of ph of the solution, changes in which can themselves cause stomatal movements. Numerous investigations into this subject were carried out some years ago, and were reviewed by Williams and Shipton (1950). Some workers, for example Iljin (1957) and Fujino (1967), have stressed the importance of the ion species constituting the solution and their concentration. In view of these reports we have given special attention to the nature of the incubating medium. The cells of epidermal strips floating on water or a dilute solution are likely to have a higher turgor pressure than those in the epidermis of the intact leaf, but since stomatal movements occur because of changes in the turgor difference between guard and subsidiary cells, there seems no reason why such differences should not arise in epidermal strips. The most likely effect of a general increase in turgor in the epidermis is a reduction in stomatal aperture resulting from increased hydrostatic pressure upon the guard cells. This effect is known to occur in intact leaves, where over-hydration leads to a reduction in aperture (Stalfelt, 1955). Any disadvantage arising from the fact that overhydration restricts the extent of stomatal opening in epidermal strips is probably balanced by the definite advantage that the hydration of the tissue is kept constant during an experiment, which is a condition difficult to achieve in experiments on intact plants. In this paper we report the findings of a preliminary investigation into the feasibility of using epidermal strips in studies of stomatal physiology. We have concentrated on determining whether the stomatal responses to environmental factors are comparable with those found in intact leaves and on the effects of the ionic content of the medium in which strips are immersed. MATERIALS AND METHODS Plant material. After trials on several species from which epidermis can be readily removed, two species of Commelina, and broad bean (Vicia faba L.) were selected as

3 Stomatal behaviour on detached epidermis 365 providing the most suitable material. Supplies of Commelina communis L. were raised from seed, and plants of C sikkimensis C.B.Cl. were obtained by vegetative propagation. Vicia faba normally proved satisfactory but epidermal strips from some batches of plants failed to give satisfactory results, their stomata showing a reluctance to open under any treatment. So far we have not been able to explain this difficulty which occurred from time to time even though the plants were of the same variety and, indeed, from the same batch of seeds. Generally, the youngest, fully expanded leaves were used. Epidermal strips of V. faba were taken from one leaflet or, when a larger area of epidermis was needed, from one pair of leaflets. In the case of Commelina communis several leaves were needed to provide the required amount of epidermis and they were carefully selected, the main criterion being their age. Commelina plants brought into the laboratory from the glasshouse in the morning had their stomata practically closed; those of Vicia faba were slightly open, but rarely to more than 2-3 //m. In order to keep the stomata in a closed (or nearly closed) condition, the plants were kept in the dark until required for use. Treatment of epidermal strips. Strips from the abaxial surface of the leaves were initially floated on io~^ M phosphate buffer of ph 6.9. They were kept in darkness or low intensity light until the start of the experiments, when they were transferred to incubating flasks. To keep the ph ofthe incubating solutions constant at a physiologically acceptable level, strips were always placed in buffer solutions. A variety of different buffers has been employed by different workers in attempts to study stomatal movements on epidermal strips. A feature common to the work of Kuiper (1964) and Eujino (1967), who hoth succeeded in observing stomatal responses in detached epidermis, was the use of phosphate buffer. In view of their success we decided to employ a phosphate buffer, two potassium salts (K2HPO4 and KH2PO4) being used to keep the number of ion species to a minimum. With this mixture, a useful working range of ph is obtained. The ph was always checked at the end of the period of incubation and a signiflcant change in ph was not observed in any of the experiments reported below. During incubation a steady stream of gas (air, air free of CO2, etc. according to the experiment) was bubbled through the medium at a rate of ml/min. Strips were removed at intervals and the apertures of ten stomata, selected randomly, were measured on each. Generally, three replicate strips were removed from each flask and the total of thirty stomata was measured in less than 5 minutes. Flasks containing the epidermal strips were placed in a water-bath at C. Illumination was from two sources: a water-cooled tungsten lamp (150 W) in a separate container, centrally placed in the water-bath, and six 13 W warm-white fluorescent tubes beneath the glass base of the bath. The light intensity in the region of the flasks was approximately 15 klux. EXPERIMENTS Preliminary observations were made on epidermal strips of Vicia faba and Commelina communis to determine the effect of different incubating media on the rate of stomatal opening and the total aperture achieved in light. The results of such an experiment are shown in Fig. i. The strips were transferred from low intensity light to 15 klux at zero time, and air {c. 400 ppm CO2) was bubbled through the medium throughout the experiment. Stomata of Vicia faba opened in M/IOO phosphate buffer of ph 6.9, but

4 366 C. M. WiLLMER AND T. A. MANSFIELD in this medium the stomata of Commelina communis hardly opened at all, despite the high light intensity. However, like Fujino (1967) we found that opening took place if M/15 KCl was added, with an increase in the concentration of the buffer to M/15. This incubating medium was referred to by Fujino as the 'opening mixture'. It was not, however, an 'opening mixture' in the case of Viciafaba, indeed if anything, towards the endof the period of incubation, it appeared to exert the opposite effect (Fig. ia). The data recorded in Fig. i clearly indicate a fundamental difference in the two species in the response to salts added to the incubating medium. The effect of the ph of the medium was investigated in another series of experiments. They were performed on the same two species and the media which had proved to be optimal in the above experiment were used, namely M/IOO phosphate buffer for V.faha and M/15 ^C[ in M/15 phosphate buffer for Commelina communis. The results obtained (a) (b) O I 2 3 Hours Fig. I. Stomatal opening in 15 klux in (a) Vicia faha and (b) Commelina communis, as affected by the nature of the incubating medium., M/IOO phosphate buffer; A, M/15 KCl in M/IOO phosphate buffer; A, M/15 KCl in M/15 phosphate buffer. All solutions were of ph Each point is the mean of measurements on ninety stomata, and the standard error for each final aperture is indicated by the adjacent vertical line. from experiments in which ph treatments were applied in light and darkness are summarized in Table i. These experiments were of a 'randomized block' design: treatments were applied in a random sequence, replication being within 'blocks' of time. Analysis of variance revealed that in both species the small ph effect (which appeared as a 'ph X light-dark' interaction in Viciafaba) was just statistically significant at P< 0.05, and the larger light-dark effect was significant at P< As a result of these experiments a ph of w^s selected for all the other experiments recorded below. One important observation which was fully confirmed by subsequent experiments was the fact that after a sufficient period of incubation, the stomata opened in darkness and, although the difference in aperture between light and darkness was highly significant, it was relatively small, especially in the case of V. faha. Strips taken from the plants and kept for a short time in subdued light had closed stomata (see the values at zero time in Fig. i) but during incubation in a favourable medium in darkness they opened slowly. Iljin (1957) reported that stomatal opening responses on epidermal strips occurred

5 Stomatal behaviour on detached epidermis 367 when certain metal ions were added to the incubating medium. Accordingly, we have examined the effects of Na +, K"^, Ca^"^ and Mg^"^ ions on V. faba and Commelina communis. The results, which are shown in Fig. 2, again indicate a marked difference in behaviour between the two species. M/15 NaCl and KCl both stimulated opening in C. communis, but NaCl was considerably more effective than KCL Addition of M/IOOO Table i. Effect of ph on stomatal aperture on epidermal strips in light and darkness Vicia faba in M/IOO phosphate buffer ph Light (IS klux) S.I s-7 4- Darkness S Commelina communis in M/IS KCl in M/IS phosphate huffer ph S Light (IS k-lux) s Darkness s.i The data are the means of four experiments, each figure being derived from measurements of a total of 120 stomata. The apertures (jim) were measured after 2 hours incubation. (0) (b) E 5-4 E 2 2 Fig. 2. Stomatal opening in 15 klux in (a) Vicia faba and (b) Commelina communis as affected by the nature of the incubating medium. A, M/ i s KCl; A, M/ i s NaCl; O, M/1S KCl with M/iooo CaCla;, M/IS KCl with M/IOOO MgCI,;, M/IOO phosphate buffer (control). All the salts were in M/IOO phosphate buffer, ph Each point is the mean ot measurements on ninety stomata, and the standard error for each final aperture is indicated by the adjacent vertical line. MgClj depressed the opening induced by M/15 KCl and M/IOOO CaClj virtually prevented it altogether. These findings agree closely with those reported by Iljin. Vicia faba did not show the same pattern of behaviour, as will be seen from Fig. 2(a). The differences in final aperture in different media were not significant; the large and consistent effects found with Commelina communis were not apparent in Vicia faba they were

6 368 C. M. WiLLMER AND T. A. MANSFIELD either absent or obscured by the considerable variability. It is apparent from the standard errors for the mean apertures in Figs, i and 2 that the variation in aperture of individual stomata was always considerably greater with V. faba than with Commelina communis. One question which must inevitably be asked is whether stomatal behaviour, particularly the response to stimuli from the environment, is affected by detaching the epidermis. We have compared stomatal behaviour on detached epidermis with that on small portions of leaf in the same media, using the following experimental procedure. Pieces of epidermis about 5 mm square were carefully separated from the mesophyll but one end was left attached to the leaf. A small square or rectangle containing the leaf tissues intact was then cut, with the piece of epidermis attached. Four factorial experiments were carried out using such material to investigate the stomatal response to light and carbon dioxide. The experiments were of a 'split-plot' design, each piece of material Table 2. Effect of CO2 concentration and of light and darkness on stomatal aperture on attached and detached epidermis [the leaf sections each consisted of an area with the leaf tissues intact and a piece of epidermis detached from the mesophyll) 0% CO2 (a) Victa faba 0.1% CO2 Epidermis Epidermis Detached Attached Detached Attached (i) In M/IOO phosphate buffer Light Darkness S S3 S (ii) In M/15 ^ICl in M/15 phosphate buffer Light Darkness S S % CO2 Epidermis Detached Attached S S (i) In M/IOO Light Darkness (b) Commelina sikkimensis O%CO2 0-1% CO2 Epidermis Epidermis Detached Attached Detached Attached phosphate buffer O.I O.I 0.0 O.I (ii) In M/15 KCl in M/15 phosphate buffer Light Darkness CO, Epidermis Detached Attached The data are stomatal apertures (nm) measured after incubation for 2 hours in the media shown. being regarded as a 'plot' split for the comparison of stomatal behaviour on the detached epidermis and on that remaining attached. This design gave a high level of precision for comparing the 'within plot' treatments, namely attachment and detachment of the epidermis. The response to two environmental factors, light and CO2, was examined. In each experiment replication of treatments was in three randomized 'blocks' of time. The treatment means, each derived from measurements of ninety stomata, are recorded in Table 2. Vtcia faba was again used, together with Commelina sikkimensis which replaced C communis in this experiment because suitable material of the latter was not available (preliminary trials had shown that the stomatal behaviour of these two Commelina species differed very little in experiments of the type described in this paper; anatomically the epidermes were almost identical)

7 Stomatal behaviour on detached epidermis 369 The results of these experiments proved to be of great interest in two respects; first, there were major differences in stomatal behaviour between attached and detached epidermis, and secondly, there were considerable differences between the two species used. In Vicia faba (Table 2a) in M/IOO buffer the light effect averaged over all treatments was significant at P<o.ooi. It is clear from examination ofthe data, however, that the effect of light was much more evident on the intact portion of leaf than on the isolated epidermis the analysis of variance showed a significant (P<o.ooi) interaction of attached/detached epidermis (A/D) x light/darkness (lt/dk). Difference of the same type occurred in the experiment where M/15 KCl in M/15 phosphate buffer was the incubating medium. The whole pattern of behaviour was similar in both media, but the interaction of A/D X [CO2] was significant (P<o.ooi) only in the experiment in M/15 phosphate buffer. I2r Hours Fig. 3. The course of stomatal movements in Commelina communis under the following treatments. O, 400 ppm CO2, 15 klux; A, 400 ppm CO2, darkness;, CO2-free air, 15 klux; A, CO2-free air, darkness. In each case the epidermal strips were incubated in M/15 KCl in M/15 phosphate buffer from zero time. Each point is the mean of measurements on ninety stomata and the standard error for each final aperture is indicated by the adjacent vertical line. The observations for Commelina sikkimensis differed quite remarkably from those for Vicia faba (see Table 2b). In detached epidermis large treatment effects were apparent in M/15 K^Cl in M/15 phosphate buffer, but not in M/IOO buffer. On the other hand, in the latter medium distinct stomatal movements were found on the intact leaf portion, whereas in the former virtually no movements were apparent. As with V. faba, some interactions were significant at P<0.001: A/D x lt/dk, in M/IOO buffer; A/D x lt/dk and A/D x[c02], in M/15 KCl in M/15 phosphate buffer. A further experiment provided a good illustration of the suitability of Commelina communis for studies of stomatal responses to light and COj. Epidermal strips were incubated in the favourable medium (M/15 KCl in M/15 phosphate buffer) under four different treatments, namely (a) 15 klux and COa-free air, (b) 15 klux and room air [c. 400 ppm CO2), (c) darkness and COz-free air, (d) darkness and room air {c. 400

8 37 C. M. WiLLMER AND T. A. MANSFIELD ppm CO 2). The experiment was continued for 4 hours and at hourly intervals measurements of stomatal aperture were made. The results clearly show the wider stomatal opening in the absence of CO2, and the light-dark effect (Fig, 3). Finally, we have considered a further question regarding the suitability of detached epidermis for stomatal studies. An experiment was carried out to determine whether the stomata remained functional (as indicated by their responses to alternating light and dark periods) for several hours after detachment. Measurements were made over a Dark E a. J L Light Dark 3 Hours Fig. 4. Effect of i.s-hour alternating periods of light (15 klux) and darkness on stomatal aperture m epidermal strips of (a) Vicia faha and (b) Commelina sikkimemis incubated in M/IOO phosphate buflfer and M/15 KCI in M/IS phosphate bufifer, respectively, ph O, 400 ppm CO2;, zero COj. Each point is the mean of measurements on ninety stomata. period of 6 hours on Vicia faba kept in M/IOO buffer, and Commelina sikkimensis kept m M/15 KCI in M/15 phosphate buffer. The required numbers of epidermal strips were placed in the media, and first illuminated in 15 klux for 1.5 hours, followed by alternating periods of darkness and light of 1.5 hours duration. Strips were removed at 45 minute intervals and measurements made of the stomatal aperture. Some strips were incubated with room air (c. 400 ppm CO2) and some with COj-free air bubbled through the medium. The results, which are presented graphically in Fig. 4, are the means of three replicate experiments, each point being derived from measurements of a total of ninety

9 Stomatal behaviour on detached epidermis 371 stomata. In 400 ppm CO2, the stomata of C. sikkimensis closed in response to darkness, reopened in light, and closed again in the final 1.5 hours darkness; in CO2-free air the light-dark effect was obscured, and the stomata opened to wider apertures than in 400 ppm CO2. In Vicia faba there was a small consistent difference in aperture between the two CO2 treatments, but effects of light and darkness were almost entirely absent, the stomata showing mainly a slow increase in aperture throughout the experiments. DISCUSSION The experimental data presented above indicate that in certain conditions stomata on detached epidermis are able to show responses to environmental factors. However, the effects may differ in magnitude from those found on intact plants. In the account of the results presented in Table 2 we have emphasized the highly, statistically significant interactions between apertures on attached and detached epidermis and the treatments under consideration, namely light and darkness and CO2 concentration. The attached/ detached epidermis x light/darkness effect on Vicia faba provides a good example of the importance of such interactions; the effect of light in stimulating opening was very apparent at all CO 2 concentrations on the attached epidermis, but was small or absent altogether when it was detached. In Commelina sikkimensis, on the other hand, lightinduced opening and C02-induced closure were apparent on detached epidermis in M/15 KCI in M/15 phosphate buffer. In our experience, the isolated epidermis from the two species of Commelina is suitable for studies of stomatal responses, providing that it is maintained in a suitable medium. The nature ofthe incubating medium is clearly ofthe utmost importance. The favourable media for the two Commelina species contained sufficient ions to produce quite a high osmotic pressure and we have considered the possibility that the enhancement of stomatal opening was the result of a reduction in water content of the epidermis leading to a lower pressure on the guard cells. It did not seem very likely that this was the reason, since solutions of similar osmotic pressures but containing different ions behaved dissimilarly. Moreover, we have found that mannitol solutions of up to 3 bars osmotic pressure did not stimulate opening. Our results, like those of Iljin (1957) and Fujino (1967) strongly suggest that the presence of certain metal ions stimulates opening, and that other ions can be antagonistic. An earlier suggestion (Mansfield, 1967) that mannitol added to the medium facilitates the response to environmental factors in Vicia faba has not been fully confirmed by later work. Our more recent experiments have shown that in this species there is a considerable variability of response to solutes added to the medium, as is evidenced by the magnitude ofthe standard errors shown in Fig. 2(a). The effects of the nature of the incubating medium on stomatal movements in Commelina differed completely on attached and detached epidermis. M/15 KCI in M/15 phosphate buffer permitted much wider opening on detached epidermis than did M/IOO phosphate buffer but quite the opposite was true for attached epidermis. At present we can only speculate upon the reason for such behaviour. One possibility is that ions enter the guard cells in detached epidermis more readily because the solution is in contact with their inner walls which are much less cuticularized. It is unlikely that in the intact leaf portions the solution infiltrated the sub-stomatal cavities, since no surfactant was added to reduce the surface tension. The only direct route for uptake into the guard cells would be through any ectodesmata on the outer walls (Franke, 1964), and the only

10 372 C. M. WILLMER AND T. A. MANSFIELD indirect route would be via the epidermal and subsidiary cells, since there are no direct connections between the mesophyll and guard cells. Thus, rapid ion uptake from the medium into guard cells of isolated epidermis could result in the turgor increase leading to the observed opening; the lack of such opening on leaf portions in the same medium might be because the ions are less readily available to the guard cells. The fact that stomatal opening in Commelina was virtually prevented when attached epidermis was immersed in M/15 KCl in M/15 phosphate buffer could perhaps be the result of the turgor increase, following ion uptake, in the other leaf cells, the increased pressure upon the guard cells resisting stomatal opening. We have made a study of the uptake of the vital stain neutral red* into the cells of the epidermis, in an attempt to throw some light on these problems. There were marked differences in the pattern of uptake in Vicia faba on the one hand, and the two Commelina species on the other. When detached epidermis from Vicia faba was immersed in 100 ppm neutral red, uptake began apparently simultaneously into the guard cells and all the epidermal cells (there are no distinctive subsidiary cells in this species). With Commelina the behaviour was different: uptake occurred first of all into the ordinary epidermal cells but the six subsidiary cells (Fig. 5) and the guard cells were not noticeably stained. After about 10 minutes the dye began to accumulate in the guard cells, but the subsidiary cells remained practically unstained. If the epidermis of Commelina was taken out of the neutral red solution after 2-3 minutes, washed, and placed in water, the dye began to accumulate in the guard cells after 10 minutes. This suggested a transport mechanism across the subsidiary cells, even though they themselves did not accumulate the dye. If the uptake of inorganic salts follows a pattern similar to that of neutral red, the difference in the eflfect they exert on aperture in Vicia faba and Commelina might be explained thus: in Vicia faba the uptake occurs in both epidermal and guard cells, both show a resulting increase in turgor pressure, and the net effect is little if any change in stomatal aperture; in Commelina the six subsidiary cells surrounding each pair of guard cells do not themselves increase in turgor, but pass solutes to the guard cells where they accumulate, and the increase in turgor of guard cells relative to subsidiary cells leads to stomatal opening. To explain enhanced opening on detached epidermis in response to certain solutes in the medium, we could accept the view of Fujino (1967) and Fischer (1968), namely that ions are taken up directly from the solution and accumulate in the guard cells by an active uptake mechanism. There is, however, difficulty in accepting Fujino's further suggestion that normal stomatal opening in light in an intact leaf is the result of an active cation transport into the guard cells from other leaf cells, including those of the mesophyll. Our experiment of immersing leaf portions in solutions containing suitable ions should not have prevented this happening, indeed there should surely have been more * Neutrat red chloride is a basic cationic dye having the formula (Gurr, 1965): N 1\ J NH2 cr H Vital staining is not considered to be a process identical with accvimulation of mineral salts (Soran and Lazar, 1965).

11 Stomatal behaviour on detached epidermis 373 cations available for transfer to the guard cells. Even transport of ions for other than short distances along the epidermis to the guard cells seems unlikely, for we found that the totally different behaviour of the stomata on the detached and on the attached epidermis occurred almost immediately to each side of the dividing line; there was not, as might have been expected if transport was occurring, a gradual transition from one pattern of behaviour to the other. Neutral red was not taken up noticeably into attached epidermis on immersed portions of leaf, suggesting there was no lateral transport from the cut edge. Consequently, while we do not at present reject the suggestion that stomatal opening on detached epidermis is a consequence of ions being actively accumulated in the guard cells, we are cautious about assuming that such accumulation is the cause of stomatal opening in the intact leaf. However, although it may be somewhat removed from what occurs in the intact leaf, the influence of the ionic content of the medium on stomatal behaviour in detached epidermis is of considerable interest and further studies may provide important information about the peculiar activities of the guard cells. 30/im / ^ Fig. 5. The stomatal apparatus of Commelina sikkimensis. a, Guard cells containing chloroplasts; b, inner lateral pair of subsidiary cells; c, outer lateral pair of subsidiary cells; d, terminal pair of subsidiary cells; e, epidermal cells. One feature of the experiments which is of interest is the relatively small response to CO2 concentration; the closure produced by 0.1% CO2 was not large in any of our experiments with epidermal strips. The investigation by Glinka and Meidner (1968) revealed that the stomata of leaf discs floating on water responded differently to CO 2 depending whether they were on the lower side, in contact with the water, or on the upper side in contact with the air. When the stomata were in contact with water, the aperture was the same whether the atmosphere above the disc contained 300 ppm or zero CO2, but if the stomata were in contact with the atmosphere, 300 ppm caused a marked reduction in aperture. Glinka and Meidner suggested that the CO2 concentration in the sub-stomatal cavities when the stomata were in contact with water remained virtually the same irrespective of the concentration in the atmosphere above, because of the slow rate of diflfusion of CO2 in water. In our experiments, although the guard cells were always in contact with water, the CO2 treatment applied should have significantly

12 374 C. M. WiLLMER AND T. A. MANSFIELD affected the concentration in the vicinity of the guard cells, since the gas mixture was bubbled continuously through the medium, producing adequate stirring. Moreover, epidermal strips have a far smaller consumption of COj than a leaf disc; in the experiments of Glinka and Meidner the limitation due to the slow rate of CO 2 diffusion in water was probably accentuated by the high rate of CO2 uptake into the neighbouring epidermal cells. It was particularly noticeable in our experiments (especially those on Vicia faba) that the stomata failed to close completely in those conditions known to be at least favourable for opening on the intact plant, namely darkness and high CO2 concentration. It is known that the stomatal sensitivity to CO2 can increase as a water deficit develops in an intact plant (Heath and Mansfield, 1962) and it may be the case that in fully hydrated epidermis, the closing response to CO2 is minimal. This possibility clearly requires further investigation. CONCLUSIONS If consistent responses to environmental factors such as light and CO2 can be regarded as indicating that the stomata are functioning normally, we feel justified in concluding that epidermal strips of the two species of Commelina are suitable for studies of stomatal physiology. The nature of the incubating medium does, however, exert a major influence and the responses to light and CO2 are affected particularly by the presence of certain cations. The ion effects themselves are of considerable interest and clearly merit further investigation. Stomatal behaviour on detached epidermis of Vicia faba is less predictable than in the Commelina species (note the magnitude of the standard errors shown in Figs, i and 2) and responses to environmental factors are often obscured by a tendency for the stomata to open under any treatment. An effect of CO2 is detectable, but is considerably smaller than that in Commelina. We cannot recommend the use of epidermis of Vicia faba for studies in which predictable and readily detectable stomatal responses to environmental factors are required. ACKNOWLEDGMENT C. M. Willmer is supported by a grant awarded to this Department by the Agricultural Research Council. REFERENCES FISCHER, R. A. (1968). Stomatal opening: rote of potassium uptake by guard cells. Science, N.Y., 160, 784. FRANKE, W. (1964). Role of guard cells in foliar absorption. Nature, Lond., 202, FujiNo, M. (1967). Role of adenosinetriphosphate and adenosinetriphosphatase in stomatal movement Sci. Bull. Fac. Educ, Nagasaki Univ., 18, i. GLINKA, Z. & MEIDNER, H. (1968). The measurement of stomatal responses to stimuli in leaves and leaf discs. J. exp. Bot., 19, 152. GuRR, E. (1965). The Rational Use of Dyes in Biology. Leonard Hill, London. HEATH, O. V. S. (1959). Light and carbon dioxide in stomatal movements. Handb. PflPhysioL, ly 11,415. HEATH, O. V. S. & MANSFIELD, T. A. (1962). A recording Porometer with detachable cups operating on four separate leaves. Proc. R. Soc. B, 156, i. ILJIN, W. S. (1957). Drought resistance in plants and physiological processes. A. Rev. PL PhysioL, 8, 257. KuiPER, P. J. C. (1964). Dependence upon wavelength of stomatal movement in epidermal tissue of Senecio odoris. PL PhysioL, Lancaster, 39, 952. MANSFIELD, T. A. (1967). Stomatal behaviour following treatment with auxin-like substances and phenylmercuric acetate. New PhytoL, 66, 325.

13 Stomatal behaviour on detached epidermis 375 MANSFIELD, T. A. & MEIDNER, H. (1966). Stomatal opening in light of different wavelengths: effects of blue light independent of carbon dioxide concentration. J. exp. Bot., 17, 510. SoRAN, V. & LAZAR, G. (1965). Some data concerning the accumulation of neutral red in various tissues and regions of the maize root. Physiologia PL, 18, 329. STALFELT, M. G. (1955). The stomata as a hydrophotic regulator of the water deficit of the plant. Physiologia PI, 8, 572. WILLIAMS, W. T. & SHIPTON, M. E. (1950). Stomatal behaviour in buffer solutions. Physiologia PL, 3, 479. K N.P.

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