CHLOROPLASTS IN THE STOMATA OF ALLIUM CEPA L.

Size: px

Start display at page:

Download "CHLOROPLASTS IN THE STOMATA OF ALLIUM CEPA L."

Osborne Shields
5 years ago
Views:

1 [ 344 ] CHLOROPLASTS IN THE STOMATA OF ALLIUM CEPA L. BY M. SHAW Department of Biology, University of Saskatchewan, Canada {Received i6 June 1953) (With Plate 6) The stomata of Allium cepa are fully motile (Loftfield, 1921) and exhibit a genuine response to light and, by inference, to changes in carbon dioxide tension (Heath, 1952). They are, however, completely starch-free, as demonstrated by a number of investigators from Parkin (1899) to Heath. The former, having examined both alcohol fixed and living epidermis of some eight species of onion, including A. cepa and A. schoenoprasum, reported: ' No chloroplasts could be recognized, nor could I certainly detect any green colour in the guard cells.' He noted, however, that refractive granules were sometimes present. Heath also was unable to recognize chloroplasts in the stomata of A. cepa (var. Ebenezer) and obtained negative results with certain microchemical tests for chlorophyll (saturated KOH and the phaeophytin test: Molisch, 1921), but did not refer to the Molisch reaction (reduction of AgNOg by chloroplasts: Molisch, 1918). It may be noted that Sayre (1926) found that the stomatal plastids of Rumex patientia, though obviously green, did not give the microchemical tests {sic) for chlorophyll, a result he regarded as inconclusive in view of the small amount of pigment present. Apparently neither Heath nor Sayre made use of the fluorescence microscope, with which the stomatal plastids of a number of species have been found to exhibit the red fluorescence characteristic of chlorophyll. Moreover, Heath was apparently not aware that chloroplasts have been reported to occur in onion guard-cells. In 1927 Linsbauer wrote of ^. cepa (pp ): 'Die Chloroplasten, ^^ ^^^ Zahl, liegen zumeist der Riickenwand an und sind auffallend klein und bleich. Nichtsdestoweniger geben sie die Molische Chlorophyll Reaktionen und reduzieren AgNOg.' In a footnote he added that the guard-cell chloroplasts showed typical fluorescence under the fluorescence microscope, commenting that Sayre's observations, recorded above, certainly did not apply to Allium. Again, Hofler (1939), in a study of silver nitrate reduction by guard-cell plastids, obtained positive results with A. cepa and A. schoenoprasum (cf. Parkin, above) as well as with Vicia faba, Helianthus, Tradescantia, Galium and Pelargonium zonale. Hofier and also Linsbauer (1926, for Chrysanthemum) observed that localized blackening of the plastids was accompanied by a general reduction (browning) of silver nitrate throughout the protoplast in open but not in closed stomata, suggesting an increase in reducing substances during opening of 'starch' and 'starch-free' stomata alike. Unfortunately, Hofier presented no experimental data for Allium, merely stating that the results obtained were similar to those for Vicia. It is clear, none the less, that the results of Linsbauer and Hofler are not in accord with those of Parkin and Heath. Since the point at issue has a critical bearing on stomatal

Chloroplasts in the stomata of Allium cepa L. 345 physiology and in view of current interest in Heath's paper (see Nature, Lond., 171, p. no) the following observations now seem worth recording.

2 Chloroplasts in the stomata of Allium cepa L. 345 physiology and in view of current interest in Heath's paper (see Nature, Lond., 171, p. no) the following observations now seem worth recording. EXPERIMENTAL The observations to be described (with the exception of those on fluorescence) have been made repeatedly and consistently during the past two years with both seedling and mature leaves of Allium cepa L. (var. Ebenezer) grown from seed obtained locally and, latterly, also from the Division of Botany and Plant Pathology, Science Service, Ottawa, Canada. Identical observations have been made on several other varieties. The plants were grown under greenhouse (summer and winter) as well as garden conditions. (a) Reduction of silver nitrate Living epidermal strips were floated on i % (w/v) silver nitrate brought to ph 4-0 with acetic acid. Reduction was at first strictly confined to the plastids, some fifteen to twentyfive of these being clearly revealed in each guard-cell after about 5 min. treatment in the diffuse light of the laboratory (PL 6, fig. 1). With open stomata, plastid reduction was followed by a browning of the entire protoplast. This was first visible after some 30 min. and gradually increased in intensity, eventually obscuring the plastids (2-4 hr.: PI. 6, fig. 2). With closed stomata protoplast reduction was very much less marked. Identical results were obtained with Viciafaba and the appropriate photographs (PI. 6, figs. 3, 4) are included for comparison with those of Allium (PI. 6, figs, i, 2). (A) Living stomata It was difficult to discover the guard-cell plastids by inspection of living strips mounted in water {^ in. oil immersion, normal or phase-contrast illumination). This was not merely due to their relative lack of colour as compared with the plastids of such genera as Vicia or Pelargonium, but also to the fact that they were usually closely adpressed along the vertical walls of the guard-cells, i.e. were in parastrophe. However, with strips mounted cuticular surface down, a number of them were seen in oblique or surface view, particularly in partly open stomata (PI. 6, figs. 5-7). Many, however, remained in parastrophe, especially in closed stomata (PI. 6,fig.8), though they often became more rounded and thus more conspicuous, yet remaining in contact with the cell wall. The stomatal plastids appeared a pale but distinct green, were biconvex in cross-section and measured ox i-ojl6 approximately, being about one-third to one-quarter the size of the mesophyll chloroplasts. Light vacuolar staining with neutral red a few mmutes in a o-ooi % solution containing a trace of calcium chloride rendered the green colour of the plastids quite unmistakable and sometimes also improved definition (PL 6, fig. 9). In general appearance they resembled the mesophyll chloroplasts. Being starch-free they became completely invisible in phenolic iodine (refractive index c. 1-5) a point noted by Heath (1949, p- 194) in a comment on the use of this reagent. (c) Eluorescence Epidermal fragments of Allium and Vicia were mounted side by side in glycerol-water and examined for stomatal fluorescence by Lloyd's (1923) method, using a dark-field condenser and visible illumination. A blood to orange-red fluorescence was observed in

346 M. SHAW occasional guard-cells. As judged by eye alone, the fluorescence colour was identical with, though weaker, than that of the mesophyll chloroplasts.

3 346 M. SHAW occasional guard-cells. As judged by eye alone, the fluorescence colour was identical with, though weaker, than that of the mesophyll chloroplasts. No differences were noted between the two genera, nor was fluorescence any more pronounced with the larger guard-cell plastids of Vicia. In the course of these observations it was found that fluorescence was most readily obtained with chloroplasts which had escaped from ruptured cells and with isolated cells. Once observed, fluorescence is quite unmistakable. Owing to refraction and scattering, it is not readily detected by the method used when the cells under examination lie in a section or sheet of tissue, even though large amounts of chlorophyll may be present. Thus absence of fluorescence would have to be demonstrated with carefully isolated and dissected single stomata before the complete lack of chlorophyll could be assumed in any particular case. DISCUSSION Whatever the exact cytochemical significance of the silver nitrate reaction (Weier, 1938; Rabinowitch, 1945) there can be no doubt that the structures demonstrated in Allium guard-cells are plastids. Rapid blackening in silver nitrate is characteristic of chloroplasts, and according to Weber (1937) only minute traces of chlorophyll are required. Weier (1938) refers to Molisch as stating that only some chromoplasts and those leucoplasts which appear pale green give the reaction. Direct observation in vivo confirmed the occurrence of plastids; moreover, these appeared green and fluoresced red in visible hght. Such fluorescence constitutes an extremely sensitive test characteristic, as far as is known, only of the chlorophylls, bacteriochlorophylls and algal phycobilins. It is, therefore, reasonable to assume that the guard-cells of A. cepa do contain chloroplasts. This conclusion is in full agreement with the observations of Linsbauer and Hofler and stands in opposition to that of Parkin and Heath. The possibility seems remote that this disagreement may be attributed to varietal or environmental difl"erences. Apart from the conclusions of Parkin and Heath, the author is aware of only one other report of the occurrence of (chloro-)plastid-free stomata on normal green leaves. According to Sawyer (1932) this is true of Vaccinium macrocarpon, V. oxycoccus and V. corymbosum. His conclusion was based on the observed absence of both stomatal starch and plastids. Starch-free chloroplasts were present in the subsidiary cells, yet the stomata were stated to respond but sluggishly or not at all to changes in light, temperature and humidity. Thus absence of guard-cell chloroplasts appears to be correlated with insensitivity to light, despite the presence of normal photosynthetic tissue, and therefore, by inference, with insensitivity to carbon dioxide tension. However, paucity of experimental data render difficult any critical evaluation of Sawyer's report, which requires confirmation. Chlorophyllous stomata often occur on albino leaves and appear to be relatively insensitive to light. In such cases the hght response may be largely prevented by the high internal concentration of carbon dioxide, and evidence illustrative of this was obtained by Scarth & Shaw (1951a, table III; 19516, Fig. 5) for the stomata on albino portions of variegated Pelargonium. On the basis of lack of fluorescence (fluorescence microscope) Paetz (1930) and Freudenberger(i94o) reported, respectively, the occurrence of chlorophyll-free stomata on the non-chlorophyllous portions of variegated Caladium and on albino Sanchezia and etiolated Canria leaves. Paetz found also that such Caladium

4 Chloroplasts in the stomata of AUium cepa L. 347 stomata were insensitive to light, but responded to changes in humidity. Freudenberger did not investigate the light response in these particular cases, but found with etiolated Canna that a reduced carbon dioxide tension promoted opening in darkness, an observation which led her to conclude that normal opening did not depend upon the presence of stomatal chlorophyll. However, the stomata of Canna normally contain green plastids, and Freudenberger's data provide no evidence that it is not the effect of reduced carbon dioxide tension at the surface of the guard-cell plastids which initiates the reactions causing light (or low carbon dioxide) opening in this and other species. Recent work (Freudenberger, Heath, Scarth & Shaw) has indicated that light promotes opening mainly, if not entirely, through photosynthetic removal of free carbon dioxide, a process which must be assisted by such stomatal photosynthesis as may occur. It seems probable that the guard-cell chloroplasts are the cellular centres at which carbon dioxide tension exerts its regulatory effect on stomatal metabolism. The observations reported above, together with a consideration of the pertinent literature, strongly suggest that chloroplasts may occur in the guard-cells of all light-sensitive stomata, whether or not these normally contain starch. The essential similarity of the two types from the standpoint of mechanism has been discussed in a recent paper (Scarth & Shaw, 19516, pp ). The author is indebted to Miss M. Klaassen for assistance in reviewing the German literature, to Mr N. Ferrier for help with the photography and to the National Research Council of Canada for financial assistance. REFERENCES FREUDENBERGER, H. (1940). Die Reaktion der Schliesszellen auf Kohlensaure imd Sauerstoffentzug. Protoplasma, 35, 15. HEATH, O. V. S. (1949). Studies in stomatal behaviour. II. The role of starch m the light response of stomata. Part i. Review of literature, and experiments on the relation between aperture and starch content in the stomata of Pelargonium zonale. New Phytol. 48, 186. HEATH, O. V. S. (1952). Studies in stomatal behaviour. II. The role of starch in the light response of stomata. Part 2. The light response of stomata of Allium cepa L., together with some preliminary observations on the temperature response. New Phytol. 51, 30. H6FLER, R. (1939). Silbemitrat-reduktion der Schliesszellen und Offnungszustand der Stomata. Protoplasma, 33, 258. LiNSBAUER, K. (1926). Beobachtungen an Spaltoffnungen. Planta, 2, 530. LiNSBAUER, K. (1927). Weitere Beobachtungen an SpaltofTnungen. Planta, 3, 527. LLOYD, F. E. (1923). The fluorescence of certain lower plants. Nature, Lond., 112, 132. LOFTFIELD, J. V. G. (1921). The behaviour of stomata. PubL Carneg. Instn. no 314. MOLISCH, H. (1918). Das Chlorophyllkom als Reduktionsorgan. S.B. Akad. Wiss. Wien (Math.-Naturw. Kl., Abt. i), 127, 449- MOLISCH, H. (1921). Mikrochemie der Pflanze, 2nd ed. Jena. PAETZ, K. W. (1930). Untersuchungen uber die Zusammenhange zwischen Stomatarer Offnungsweite und b'ekannten Intensitaten bestimmter Spectralbezirke. Planta, 10, 611. PARKIN, J. (1899). Contributions to our knowledge of the formation, storage and depletion of carbohydrates in monocotyledons. Phil. Trans. B, 191, RABINOWITCH, E. I. (1945)- Photosynthesis, vol. i. New York: Interscience Pubhshers. SAWYER, W. H. (1932). Stomatal apparatus of the cultivated cranberry Vaccimum macrocarpon. Amer. J. SAYRE J D! (1926). Physiology of stomata of ijumex ^aft'enfaa. Ohio jf. Sci. 26, 233. SCARTH G. W. & SHAW, M. (1951a). Stomatal movement and photosynthesis in Pe/ar roraum. I. Effects of light and carbon dioxide. Plant Physiol. 26, 207.,, TT c.xr SCARTH G W & SHAW, M. (19516). Stomatal movement and photosynthesis m Fetargorawm. II. Effects of water deficit and of chloroform: photosynthesis in guard cells. Plant Physiol. 26, 581. WEBER, F. (1937). Silber-reduktion der Chloroplasten. Protoplasma, 29, 427- WEIER, E. (193S). The structure of the chloroplast. Bot. Rev. 4, ^,9-7.

5 348 M. SHAW EXPLANATION OF PLATE 6 (Figs. I and 5-8: -j\- in. oil immersion and x 125 ocular. Figs. 2-4 and 9: ^S i"- o'' immersion and x 6-0 ocular.) Figs. I (Allium) and 3 (Vicia). Plastid reduction with i % (w/v) AgNOj (ph 4-0). Figs. 2 (Allium) and 4 (Vicia). Protoplast reduction with i % (w/v) AgNOj (ph 4-0). Fig. 5. Partly open Allium stoma: plastids in surface view. Fig. 6. Partly open Allium stoma: plastids in parastrophe (left-hand cell) and surface view (right-hand cell). Fig. 7. Partly open Allium stoma: plastids in ohlique view, mainly along dorsal walls. Fig. 8. Closed Allium stoma: plastids in parastrophe, mainly along dorsal walls. Fig. 9. Allium stoma lightly stained with o-ooi % neutral red: plastids gathered about nucleus in right-hand cell. Note. Figs. 5-8 are of living, unstained stomata.

6 THE NEW PHYTOLOGIST, 53, 2 PLATE 6 SHAW CHLOROPLASTS IN THE STOMATA OF ALLIUM CEPA L.

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,