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

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1 New PhytoL (1969) 68, 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, University of Lancaster {Received 5 May 1968) SUMMARY The stomata of Phyllitis scolopendrium differ from those of angiosperms in that the guard cells contain many chloroplasts, comparable in size and appearance with those of the mesophyll. Experiments are described showing that the stomata of this fern respond to light and darkness and changes in carbon dioxide concentration in the same way as those of angiosperms. The stomata opened widely in conditions in which there could be no net photosynthetic carbon dioxide fixation, and it is therefore concluded that substances produced from carbon dioxide by photosynthesis do not play a major role in the opening mechanism and possibly no direct role at all. INTRODUCTION It has often been stated tbat the chloroplasts of guard cells are poorly developed, and contain less chlorophyll than those of the mesophyll. This is certainly true as far as the angiosperms are concerned. We have made microscopic examinations of the stomata of many species and have invariably found that the guard cell chloroplasts are comparatively few in number and that they certainly justify the above description. The guard cells of onion are exceptional, for no chloroplasts can be seen under the normal light microscope, although very small chloroplasts showing typical fluorescence have been recorded with a fluorescence microscope (Drawert, 1952). The stomata of many of the ferns differ from those of angiosperms in at least one respect : their guard cells contain abundant chloroplasts which are of normal size and appearance. In Phyllitis scolopendrium the guard cells are quite remarkable in that they seem to be almost entirely occupied by chloroplasts. The density of occurrence of the chloroplasts is greater in the guard cells than in tbie other epidermal cells, and is at least equal to that in the mesophyll cells. We became interested in the stomata of Phyllitis because they differ in this respect from those of angiosperms and this paper reports a brief study in which we have observed their response to some environmental factors. Plate I shows a photomicrograph of a stoma of Phyllitis showing the abundant chloroplasts and, for comparison, a stoma of Vicia faba with few chloroplasts, which represents the situation in dicotyledons. EXPERIMENTS The stomata of Phyllitis proved difficult to investigate. They are conflned to the lower surface of the leaf in a discontinuous pattern, and these characteristics make viscous flow E N.P. 63

64 T. A. MANSFIELD AND C. M. WILLMER porometer studies virtually impossible, particularly when the mesophyll resistance is high, as it is in this case.

2 64 T. A. MANSFIELD AND C. M. WILLMER porometer studies virtually impossible, particularly when the mesophyll resistance is high, as it is in this case. Small pieces of epidermis can be stripped off and we tried to observe stomatal movements on epidermis immersed in various media, and under various conditions such as light and darkness and a range of CO2 concentrations between o and 1 %. No statistically significant differences in aperture were found under any of the conditions tried, the stomata being mostly closed, or open to a maximum aperture of about 2 fim. Table i. Mean stomatal apertures on Phyllitis scolopendrium under different conditions 8000 lux, CO2 at Conditions Darkness, room air (c. 400 ppm CO2) 8000 lux, room air (c, 400 ppm CO2) compensation point (<iooppm) Mean stomatal aperture (//m) 0, Standard error of mean Microscopic observations of stomatal aperture on an intact leaf were not possible because of its thickness and the high absorbence of light by the dense mesophyll. However, stomata could be readily observed on paradermal sections consisting of the epidermis and a little underlying mesophyll and with these we eventually achieved success. Leaves, either attached to the plant or detached with their bases immersed in water, were placed in different conditions. After several hours, paradermal sections were taken and examined quickly under the microscope and stomatal aperture was measured. Table 2. Effect of carbon dioxide concentration on stomatal aperture of Phyllitis scolopendrium in light of 8000 lux Initial CO2 concentration (%) o o,ob8 i.o Final CO2 concentration (%) 0.05 o.i Mean stomatal aperture (//m) 5, Standard error of mean Table i gives the mean stomatal apertures found in three sets of conditions, the temperature in each case being 20 C, namely: (a) darkness, in room air containing about 400 ppm CO2; (b) light of 8000 lux, in the same room air; (c) light of 8000 lux and low carbon dioxide concentration (< 100 ppm). In the latter case the leaves were detached from the plants and placed in a perspex leaf chamber connected to an infrared gas analyser for measuring CO2 concentration. The system was initially exhausted of CO2, and the leaf was then allowed to establish the CO2 compensation point in the closed circuit. The compensation point varied over the range ppm for different leaves. The stomatal aperture given in Table i is that measured on paradermal strips taken quickly from a leaf removed from the leaf chamber after it had established a steady carbon dioxide concentration, i.e. the compensation point. The difference in aperture between light and darkness, in room air, was statistically significant at P<o.ooi, and the increased aperture at the compensation point, compared with room air, was significant at P<o.o5. The effect of CO 2 concentration is made more apparent by the results summarized in Table 2. In this case the leaves were detached from the plants and placed in a leaf chamber which was immersed in a constant temperature water bath (20 C). Air free of CO2, or containing a known concentration of CO2, was pumped through the chamber.

THE NEW PHYTOLOGIST, 68, i PLATE Photomicrographs of detached epidermis of (a) Vicia faha, with comparatively few chloroplasts per guard cell, and (b) Phyllitis scolopendrium.

3 THE NEW PHYTOLOGIST, 68, i PLATE Photomicrographs of detached epidermis of (a) Vicia faha, with comparatively few chloroplasts per guard cell, and (b) Phyllitis scolopendrium. The abundant well-developed chloroplasts in the guard cells are of similar dimensions and appearance to those in other epidermal cells (which can be seen in the photograph) and mesophyll cells. Note the difference in the size of the guard cells and chloroplasts in the two plants. T A MANSFIELD AND C. M. W I L L M E R S T O M / i r ^ L RESPONSES OE PHYLLITIS {facing page 64)

Stomatal responses of Phyllitis 65 The rate offlowwas adjusted so that the CO2 concentration in the air leaving the chamber was 0.05% in the 0.088% treatment, and o.i % in the 1.0% treatment.

4 Stomatal responses of Phyllitis 65 The rate offlowwas adjusted so that the CO2 concentration in the air leaving the chamber was 0.05% in the 0.088% treatment, and o.i % in the 1.0% treatment. It can be seen that marked closure was brought about in the increased CO2 concentration. The difference in stomatal aperture between o and 0.088% CO2 was signiflcant at P< and that between 0.088% and 1.0% was significant at P<o.ooi. DISCUSSION The stomata of Phyllitis scolopendrium responded to two environmental factors, light and carbon dioxide concentration, in a way comparable with that which has been observed in angiosperms (e.g. Heath and Russell, 1954; Gaastra, 1959). Heath and Russell compared the stomatal aperture in wheat in o and 0.084% CO2 in white light of 8000 lux and found a partial stomatal closure in the latter concentration which was probably similar to that found here in similar treatments. (Their measurements were expressed in terms of log leaf resistance measured with a porometer and consequently a precise comparison is not possible.) Consideration has often been given to the possibility that the chloroplasts of guard cells play an essential part in the stomatal mechanism, von Mohl (1856) first suggested that soluble products of photosynthesis are responsible for the turgor increases which occur in guard cells when the stomata open in light and this view has survived, in part, to the present, having received renewed support from Stalfelt (1964). The observation that stomata can open in the absence of CO2 (Darwin, 1898) and the later discovery that removal of carbon dioxide actually enhances opening (Freudenberger, 1940) did little to support this idea. However, Stalfelt thought that although the openings stimulated by removal of carbon dioxide and by photosynthetic production seem to be directly opposed, they, nevertheless, both contributed significantly, while exclusion of CO2 suppressed opening. The implication of this appears to be that a reduction in CO2 concentration down to a certain point should lead to increased aperture but a reduction beyond this point should cause stomatal closure because of reduced CO2-fixation. This is not, however, borne out by the experimental results of several workers, including Heath and Russell (1954) and Gaastra (1959). Levitt (1967) also stressed the importance of photosynthetic production, stating 'turgor pressure changes are dependent on solute changes, and... photosynthesis (together with the usually small dark CO2-assimilation) is the only known source of these solutes'. Our observations on Phyllitis clearly bear on this question of photosynthetic production and stomatal opening. Since the guard cells of Phyllitis contain well-developed chloroplasts which are almost as densely packed as seems physically possible, one might have expected stomatal opening to have occurred with increased photosynthetic production as CO2 concentration was increased in the light. This is quite clearly not the case. We found, indeed, that the maximum stomatal opening occurred in leaves kept under conditions in which there could be no net photosynthetic CO2 intake, that is, at the CO2 compensation point in Table i, and in air free of CO2 in Table 2. Consequently, we can only conclude that substances produced by photosynthetic CO2-fixation in the guard cells play no essential part in the opening of these stomata in light. To open to their widest aperture in the absence of CO2 seems to be a feature common to all the functional stomata that have been investigated, from onion which has barely visible chloroplasts (Heath, 1959), through normal angiosperms with distinct but poorly developed chloroplasts, to the present example with chloroplasts of normal appearance.

5 66 T. A. MANSFIELD AND C. M. WILLMER The evidence appears to be not inconsistent with the view that the capacity for reversible changes in turgor possessed by guard cells first appeared in chloroplast-containing cells, but that the photosynthetic capacity of the chloroplasts is not an essential part of the mechanism. To a varying extent in different species the chloroplasts have been reduced in number and size. Chloroplasts in guard cells of angiosperms appear to very active in one respect at least as centres for starch deposition and hydrolysis. There is indirect evidence that the large amounts of starch come not only from the photosynthesis within the guard cells but also from other cells in the leaf, e.g. Pallas (1964) found that sugars could be translocated along the epidermis to the guard cells and there converted to starch. Is it possible that chloroplasts whose photosynthetic activities appear to be unimportant are retained because they play another important role, such as being the sites of starch^sugar inter conversion? We were interested to discover that the chloroplasts of Fhyllitis guard cells, although of similar appearance to those of the mesophyll, retained starch quite tenaciously for at least a week in the dark, whereas starch disappeared from mesophyll chloroplasts overnight. The chloroplasts of other epidermal cells also appeared to have a capacity of retaining starch for a long time in the dark, although to a lesser extent than those of the guard cells. We could not detect any disappearance of starch from the guard cells during the day, as has been reported for some angiosperms. ACKNOWLEDGMENT C. M. Willmer is supported by a grant awarded to this Department by the Agricultural Research Council. REFERENCES DARWIN, F. (1898). Observation on stomata. Phil. Trans. R. Soc, B, 190, 531. DRAWERT, H. (1952). Der fluorescenzoptische Nachweis von Chloroplasten in den Schliesszellen von Allium cepa L. Flora, Jena, I3g, 329. FREUDENBERGER, H. (1940). Die Reaktion der Schliesszellen auf Kohlensaure und Sauerstoff-Entzug. Protoplasma, 35, 15. GAASTRA, P. (1959). Photosynthesis of crop plants as influenced by light, carbon dioxide, temperature and stomatal diffusion resistance. Meded. LandbHoogesch. Wageningen, 59, i. HEATH, O. V. S. (1959). Light and carbon dioxide in stomatal movements. Handb. PflPhysioL, 17(1), 4i5' HEATH, O. V. S. & RUSSELL, J. (1954). Studies in stomatal behaviour. VI. An investigation of the light responses of wheat stomata with the attempted elimination of control by the mesophyll. J. exp. Bot., 5, 269. LEVITT, J. (1967). The mechanism of stomatal action. Planta, 74, ioi. MoHL, VON H. (1856). Welche Ursachen bewirken die Erweiterung und Verengung der Spaltoffnungen? Bot. Ztg, 14, 697. PALLAS, J. E. (1964). Guard cell starch retention and accumulation in the dark. Bot. Gaz., 125, 102. STALFELT, M. G. (1964). Reactions participating in the photoactive opening of the stomata. Physiologia PL, 17, 838.

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

New Phytol. (1969) 68, 363-375. 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