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 Cellulose acetate replicas were made from silicone rubber impressions of the abaxial surfaces of perennial ryegrass leaves. Apertures between i /x and 4 ju (mean 2 ju) were measured in replicas from leaves which had been exposed to conditions expected to close the stomata. This discrepancy is explained by observations of the ventral walis of guard cells in transverse sections of stomata, and by Araldite impressions of the cut ends of ryegrass leaves. These show that when a stoma is closed the ventral walls make contact below the paradermal walls of the guard cells and consequently a small depression is left between the cells. These depressions are similar in width to the apparent apertures in the cellulose replicas which may be wrongly interpreted as slightly open stomata. In some species the silicone impression method is incapable of giving an accurate measurement of stomatal aperture. Consequently, in attempts to relate such measurements to stomatal function or rate of transpiration it should be established that small apertures shown in cellulose acetate replicas are not the result of depressions between the external surfaces of guard cells in closed stomata. INTRODUCTION In studies of stomatal function and in attempts to relate stomatal aperture to rates of transpiration a direct measurement of stomatal aperture is often needed. Impressions of the leaf surfaces have been made with silicone rubber from which a cellulose acetate replica is taken (Sampson, 1961; Zelitch, 1961). In cotton leaves (Shimshi, 1964) it was found that, using a viseous flow porometer, measurements of the drop of pressure applied to one side of a leaf could be correlated over the whole range of apertures from closed to fully open stomatal apertures, measured with the silicone rubber method. In tobacco leaves (Zelitch, 1961; Walker and Zelitch, i963)and in maize leaves(shmueliandleshem, 1967), however, apparent stomatal apertures of about 2 pl were found in impressions where stomata were expected to be closed in leaves kept in the dark in air containing at least 10% CO2 or in leaves under water stress. When maize leaves, treated in these ways, were placed in a viscous flow porometer there was no drop in the pressure of air applied to one side of the leaves, showing that the stomata were in fact closed. A similar inconsistency was found during a study of water loss from isolated ryegrass leaves, in which the present authors are engaged. In an attempt to find an explanation for this inconsistency it was necessary to obtain a direct view of the ventral walls of guard cells to determine whether or not the pores observed in surface impressions represent stomatal apertures which penetrate to sub-stomatal cavities. * Present address: Department of Field Crops, Volcani Institute of Agriculture, Rehovot, Israel. E* 1047
1048 Y. LESHEM AND R. THAINE METHODS, Perennial ryegrass {Lolium perenne) plants were grown for 3 weeks in growth cabinets in which the light intensity was 1800 ft-candles at the level of the leaves, the length of day was 16 hours and the temperature 20 C day/10 C night. Leaves were removed at different times during the day and held in the dark in air containing at least 10% COj to close the stomata. Cellulose acetate replicas were taken from silicone rubber impressions of the abaxial surfaces of these leaves. To prepare replicas giving a cross-sectional view of a stomata, leaves were cut at right angles to the long axis and the cut end was embedded in fluid silicone rubber mixed with a catalyst. The rubber set in 2 or 3 minutes, the leaf was removed and when the mould was filled with the fluid monomer of Araldite it was placed in an oven at 60 C for 2 days to harden. When the rubber impressions were removed replicas of the cells exposed at the cut ends of the leaves remained in the moulded Araldite blocks. The moulded blocks were embedded in a small quantity of fluid Araldite coloured with Janus green B to distinguish it from the moulded blocks. When the mountingfluidhad hardened, the Araldite disc so formed was reduced by grinding on the reverse side with a medium grade emery cloth. Finally, the ground surface was polished on a glass plate in a pool of metal polish. The thin Araldite replicas were mounted in liquid paraffin between a miscroscope slide and a coverslip and observed and photographed in the light microscope with transmitted light. RESULTS AND DISCUSSION The surface impression (Plate i. No. i) and the impressions of a cut end (Plate i, No. 2) gave different measurements of stomatal aperture. In eight replicas of the abaxial surface of perennial ryegrass leaves in which the stomata were expected to be closed, all the stomata appeared slightly open and their apparent apertures varied between i n and 4 p. in diameter (mean 2 ix). On the other hand, in three Araldite replicas of the cut ends of leaves all the stomata were closed. However, a surface depression in these replicas can be detected where the external surfaces of the two guard cells meet which, in Plate i. No. 2, is 3jU wide and within the range of the apparent apertures in surface replicas. When the surface impression was made this depression probably filled with fluid silicon rubber, so that in the cellulose acetate replica it looked like a small aperture. In stomata with large apertures, this artefact is not likely to occur for the fluid silicone easily penetrates the larger pores giving an accurate representation of the stomatal aperture in the cellulose acetate replica. A photomicrograph of the replica of a stoma with an aperture of 17 ^l diameter is shown in Plate i. No. 3. To relate the impressions of stomata from fresh leaves to the anatomy of guard cells and their subsidiary cells, transverse sections were made of leaves which were fixed in absolute ethanol stained azure blue and embedded in Araldite following treatments to close the stomata. Plate i. No. 4, shows a cross-section of a stoma in which the general appearance of the guard cells and the subsidiary cells can be related to the stomata seen in the Araldite impression of the cut end (Plate i. No. 2). The depression between the guard cells of the closed stoma can also be seen in this section. It is concluded that in some species including perennial ryegrass the surface impression method is incapable of giving an accurate measurement of stomatal aperture when the stomata are nearly or completely closed.
THE NEW PHYTOLOGIST, 68, 4 PLATE I Nos. I and 2. Impressions of ryegrass leaves {Lolitimperenne) which were kept in the dark in a high concentration of CO2 to close the stomata. No. i shows a surface replica obtained with the silicone rubber method. The stoma reveals an apparent aperture of 2 /; which is due to a depression formed by the external surfaces of the guard cells (G) while the stoma is actually closed. No. 2 is a photomicrograph of a cross-sectional Araldite replica of a stoma. A depression occurs between two ridges at the external surface of the epidermis and closure occurred by contact of the ventral cell walls (arrow). It is this depression which gives the appearance of a pore shown in No. i. No. 3. A cellulose acetate replica miade from a silicone rubber impression of a stoma with a large aperture (17 /;) fronn a ryegrass leaf kept in light and in air with the CO2 removed. No. 4. A cross-section of a stoma in leaves held in darkness and in air containing at least 10% CO2 to close the stomiata and then fixed in alcohol and embedded with Araldite. The subsidiary cells of the stomata are indicated by the letter S. lo/x Y. LESHEM AND R. MEASUREMENT OF STOMATAL APERTURE {facing page 1048)
Measurement of stomatal aperture 1049 ACKNOWLEDGMENTS We are grateful to Dr H. Meidner for helpful comments and discussion and to G. E. Barton for his skilful preparation of the Araldite replicas. REFERENCES SAMPSON, J. A. (1961). A method of replicating dry or moist surfaces for examination by light microscopy. Nature, Lond., 191, 932. SHIMSHI, D. (1964). The use of a field porometer for the study of water stress in plants. Israel J. agric. Res., 14, 137. SHMUELI, E. & LESHEM, Y. (1967). The response of forage corn to four irrigation regimes. Volcani Inst. agric. Res. {Israel), Pamphlet No. 116. WALKER, D. A. & ZELITCH, I. (1963). Some effects of metabolic inhibitors, temperature and anaerobic conditions on stomatal movement. PI. PhysioL, Lancaster, 38, 390. ZELITCH, I. (1961). Biochemical control of stomatal opening in leaves. Proc. natn. Acad. Sci. U.S.A., 47, 1423-