Planta
Planta 136, 103-114 (1977)
9 by Springer-Verlag 1977
Differentiation of Stomatal Meristemoids and Guard Cell Mother Cells into Guard-like Cells in Hgna sinensis Leaves after Colchicine Treatment An Ultrastructural and Experimental Approach B. Galatis Institute of General Botany, University of Athens, Athens 621, Greece
Abstract. The temporary development of Vigna sinensis seedlings in the presence of colchicine results in the inhibition of stomata generation and the formation of numerous persistent stomatal meristemoids (P-SM) and guard cell mother cells (P-GMC). Before dividing differentially or becoming GMC, the untreated meristemoids undergo a 'preparatory' differentiation, during which a synthesis of new densely ribosomal cytoplasm, an increase of nuclear size, and a detectable proliferation of all the organelles are observed. The same process appears depressed and delayed in treated meristemoids; the cells have usually undergone only part of it when they reach the C mitosis. After the inhibition of their division, the bulged meristemoids II and GMC increase further in size, synthesize new nonribosomal cytoplasm, and start vacuolating slowly. The plastids also increase in size, change in shape, and become able to synthesize large quantities of starch. The cells retain a ribosomal cytoplasm, rough ER membranes, and active dictyosomes for a long time. At the advanced stages of differentiation, the microtubules reappear in the cells even when the plant remains under colchicine treatment. When mature, the P-GMC and P-SM are quite similar to the guard cells and possess considerably thickened periclinal walls, numerous mitochondria, and small vacuoles, while the nucleus, the plastids, and the cytoplasm occupy significant parts of the cell volume. In the epidermis displaying open stomata in light, significant K + quantities are detectable in guard cells and P-GMC or P-SM, while they are almost absent from their surrounding cells. When the stomata close in darkness, K § is accumulated primarily in the subsidiary or typical epidermal cells surrounding these idioblasts and only minimally inside them. Besides, the P-GMC and P-SM, like the Abbreviations: P-GMC=persistent guard cell mother cell; PSM= persistent stomatal meristemoid; ER:endoplasmic reticulum
guard cells, retain the starch for a long time and build up considerable starch quantities from exogenously supplied sugars.
Key words: Colchicine - Meristemoids -
Stomata
Vigna.
Introduction The ontogeny of the guard cells is the outcome of a complicated process, including: (1) a number of divisions, the first one(s) of which are differential and sometimes obviously asymmetrical and the last of which always results in the symmetrical formation of a pair of guard cells (Bfinning and Sagromsky, 1948 ; Btinning and Biegert, 1953 ; Stebbins and Shah, 1960; Stebbins and Jain, 1960), and (2) a divergent differentiation sequence in the young guard cells expressed structurally by conspicuous protoplasmic changes, the most prominent of which are related to a peculiar microtubule organization, an intense dictyosome activity, and a characteristic plastid differentiation (Landr6, 1969a, b, 1970, 1972; Kaufman et al., 1970; Srivastava and Singh, 1972; Singh and Srivastava, 1973; Calatis, 1974). During the extensive light microscope studies on stomatal ontogeny, a wide range of developmental stomatal anomalies, naturally occurring or experimentally induced, has been illustrated. Naturally occurring P-GMC were observed by Kenda and Weber (1950), Brat and Weber (1951), and Dehnel (1961). Besides, it has been reported that similar cells are formed after different treatments i.e., colchicine (Gavaudan, 1938 ; Weber, 1943 ; Tonzig and Ott-Candela, 1946; Weissenb6ck, 1949; Reese, 1950; Guyot, 1964; Guyot et al., 1968), ultraviolet irradiation (Kropfitsch, 1951a), or ethylene (Kropfitsch, 1951b). The formation of P-GMC in pathological conditions re-
104 lated to galls of fungal or insect etiology has been reported as well (Gertz, 1919), In some o f the preceding studies a great n u m b e r of plastids large in size and containing starch and the thickenings of cell walls have been noted. Both o f these structural characteristics have led to the suggestion that these cells must differentiate toward the direction o f the guard cells. W e b e r (1943) has already questioned whether the P - G M C possess some of the properties o f the guard cells. The present report describes the fine structural differentiation o f meristemoids II and G M C o f Vigna sinensis into guard-like cells under conditions preventing their division. Moreover, presented evidence suggests that these cells possess some o f the functional activities o f the guard cells.
Materials and Methods Six-day-old seedlings of Vigna sinens&,grown in a greenhouse, were further developed for 24, 48, 72, or 96 h in a half-strength Hoagland solution that contained 0.08% colchicine (Sigma). After this treatment the primary leaves were fixed either immediately or after 1, 2, 3, or 14 days of further development in Hoagland solution. Fixation was carried out by immersing small pieces of leaves in 5% glutaraldehyde in 0.025 M phosphate buffer, pH 7, at room temperature, for 2 h. After washing in buffer the samples were postfixed in 1% osmium tetroxide in the same buffer, pH 7 at 4~ C, for 4 h. The specimens were infiltrated in mixtures of Epon 812 or Durcupan ACM (Fluka)/propylene oxide and embedded in the respective resin. Thin sections of material were cut on an LKB Ultrotome III microtome, double-stained with 4% uranyl acetate in 70% ethanol and lead citrate and examined with a Philips 300 electron microscope. Thick sections were cut from the same blocks with an LKB Pyramitome or Ultrotome III microtomes, stained with toluidine blue and photographed with a Zeiss light microscope. K + was localized histochemically in epidermal strips or paradermal sections using Macallum's method (1905) with cobaltinitrite, as has been performed by Fischer (1971).
Observations
Light Microscopy The majority o f the stomata population on the epidermis o f 6-day-old primary leaves o f Vigna sinensis derives f r o m a mesogenous process o f development, which finally forms typical paracytic s t o m a t a (Figs. 1 and 7). This sequence is in brief as follows: A p r o t o d e r m a l cell divides differentially and cuts off the stomatal initial (meristemoid I) and a larger cell that either divides again or differentiates into a typical epidermal cell. The meristemoid I undergoes two successive divisions, the first o f which gives rise to the meristemoid II and the one subsidiary cell, while the
B. Galatis : Differentiation of Stomatal Meristemoids second forms the meristemoid III, which will become the G M C , and the other subsidiary cell. Finally, a symmetrical division taking place in G M C produces the pair of g u a r d cells (Fig. 1). A limited n u m b e r o f s t o m a t a originate f r o m mesoperigenous and perigenous m o d e s of development. The different developmental stages and the completed stomata a p p e a r in a mixed fashion on b o t h epidermides. The terms mesogenous, perigenous and mesoperigenous were proposed by Pant (1965) for the classification of ontogenetic stomatal types. A mesogenous stoma is one in which the guard cell mother cell and all subsidiaries or a single ring-like subsidiary cell arise from the same meristemoid. A mesoperigenous stoma is one in which the surrounding cells are of dual origin, one neighboring cell is mesogenous, and the others perigenous. A perigenous stoma is one in which all neighboring or subsidiary cells derived independently from the guard cell mother cell, which divides only once to form the two guard cells. Examination o f the epidermis o f p r i m a r y leaves, 48-72 h after the onset o f administration o f the drug, revealed that s t o m a t a f o r m a t i o n was noticeably inhibited. M o s t of the developing stomata were blocked at the two- and three-celled stages, which in untreated leaves consist of one or two subsidiary cells, respectively, abuting or surrounding a meristemoid II or I I I or a G M C . A p a r t f r o m these meristemoids II, III, or G M C n o r m a l at the light microscopic l e v e l - n u m e r o u s cells of an idioblastic appearance, larger in size and r o u n d in form, were observed (Fig. 2). The presence of two distinct subsidiary cells confirms that they represent persistent guardcell m o t h e r cells ( P - G M C , Figs. 2, 4 and 5), while the existence of one subsidiary cell and one typical epidermal cell confirms that they must be persistent stomatal meristemoids II (P-SM, Fig. 2). Three different stages of differentiation of G M C into PG M C can be observed in Figures 3-5. Since some mesoperigenous s t o m a t a are f o u n d on the same surface, a small n u m b e r of t h e m m a y represent P - G M C o f mesoperigenous stomata. Nevertheless, the meristemoids II of the mesogenous process are easily recognized because the surrounding cells (one subsidiary and one typical epidermal cell) show p r o m i n e n t differences in size and developmental stage (Fig. 2). The P - G M C and P - S M contain p r o m i n e n t plastids (Figs, 4 and 5) and considerably thickened periclinal walls easily observed in transverse sections (Fig. 6). Conspicuous structural similarities exist between these epidermal idioblasts and the guard cells (cf. Figs. 5 and 6 with Figs. 7 and 8, respectively).
Electron Microscopy In untreated leaves the y o u n g meristemoids II and III are small lens-shaped cells similar to each other
B. Galatis: Differentiationof Stomatal Meristemoids in shape, structure, and size. Their recognition as meristemoids II or IlI is based on the presence of one or two subsidiary cells. Furthermore, the 'preparatory' differentiation occurring in a young meristemoid I! resembles closely that of a meristemoid III. In addition, the final differentiation of a meristemoid II into a P-SM is identical to that of a GMC into a P-GMC. To avoid duplication of the presented results, illustrations will be given for the meristemoid III and GMC only. The greatest part of meristemoid II and III cell space is occupied by a nucleus, exhibiting a pronounced nucleolus; yet, they are devoid of vacuoles (Fig. 9). In the densely ribosomal cytoplasm a few undeveloped plastids, mitochondria, rough ER membranes, a few very small microbodies, and rare paramural bodies were observed (Fig. 9). A significant number of ribosomes appear arranged in polyribosoreal complexes. Along the anticlinal walls numerous and densely distributed microtubules were detected (Fig. 17). They are anticlinally oriented and crossbridged to the plasmalemma and to one another with fine links (Fig. 17). The nontreated meristemoids III before becoming G M C - i . e . during the time preceding their symmetrical division-and the meristemoids II before dividing differentially usually undergo a 'preparatory' differentiation. This is expressed structurally by the synthesis of a new ribosomal cytoplasm, the increase of nuclear size and of the number of ER membranes, as well as the proliferation of plastids, mitochondria, and dictyosomes (Fig. 10). Vacuolation does not usually participate in the increase of cell size. The action of colchicine results in the disappearance of subplasmalemmal and spindle microtubules and, after the completion of C mitosis, in the formation of a large, irregular shaped and often lobed nucleus (Figs. 14 and 15). After the inhibition of their division (Fig. 16) the GMC and the meristemoids II, which have completed most of their 'preparatory' differentiation, enter a new differentiation process. The 'preparatory' differentiation in treated meristemoids II and III appears to be depressed and advances slowly in relation to the untreated ones. When these cells reach the onset of C mitosis, they have usually undergone only part of this process and have completed it after the restoration of the nuclear membrane. The nondivided GMC increase further in size and tend to become round in shape (Fig. 11). There is pictorial evidence that this increase represents the synthesis of new nonribosomat cytoplasm and the formation of small vacuoles (Fig. 11). The number of plastids and mitochondria remains more or less constant, while the ER membranes and the dictyosomes appear
105 further increased in number (Fig. 11). The first structural indications of the final differentiation of the meristemoids II and GMC into epidermal idioblasts, which actually commence after the completion of C mitosis, are: the development of plastids that begin increasing in size, changing in shape, and accumulating starch, the development of a vacuole, and the dominant dictyosome activity (Figs. 11 and 14). Among these indications, the divergent plastid differentiation is the most reliable. The cell remains in an active differentiating condition for a long time. This is evidenced from the maintenance of a ribosomal cytoplasm, an extensive rough ER membrane system, and an active Golgi apparatus for a prolonged period of time. The cell in Figure 15 illustrates a persistent meristemoid II o f a mesogenous process (in the inset Figure see the difference in size and developmental stage between the subsidiary cell and the other typical epidermal cell abuted on the meristemoid). The meristemoid final differentiation is identical to that of GMC (see plastid differentiation and wall thickening). On the contrary, only rarely did we observe idioblast cells like the ones described above and devoid of subsidiary cells. It seems likely that they are P-GMC of perigenous type of stomata. The dictyosome activity continuously provides a great number of vesicles and yields a detectable thickening of anticlinal walls and a considerable one of the periclinal walls (Fig. 27). Although dictyosomes were observed at every cell region, a number of them were repeatedly found close to the internal periclinal walls, where numerous mitochondria and ER membranes were placed. These organelle accumulations are possibly related to the cell wall deposition. At more advanced stages of differentiation, patterned pads are deposited at the corners of the internal periclinal walls (Fig. 27). A few plasmodesmata penetrate the anticlinal walls in both meristemoids II and III as well as in GMC (Fig. 10). In exceptional cases, the GMC do not undergo a distinguishable 'preparatory' differentiation and fail to increase in size. Their cytoplasm gains a higher electron opacity and appears densely filled with ribosomes. In such cells the differentiation toward the direction of P-GMC commences and completes regularly (Fig. 14). The plastids start to accumulate starch, while an increase of dictyosome secretory activity yields a pronounced thickening of the cell walls (Fig. 14). It is interesting that in some GMC of minute size, whose failure to develop further leads to their progressive degeneration, differentiation into P-GMC tends to be considerable (Fig. 13). In the 6-day-old primary leaf epidermis a considerable variability in plastid size and complexity exists.
Fig. 1. Light micrograph showing a portion of a differentiating lower epidermis taken from an untreated leaf. All the developmental stages of the mesogenous mode of stomatal development are depicted, a One-celled stage; M~: meristemoid I; h Two-celled stage; Mr~ meristemoid II; e Three-celled stage; G M C guard cell mother cell; d Completed stoma Fig. 2. Surface view of a 6-day-old epidermis after 72 h of continuous treatment with colchicine. P-GMC as well as P-SM can be observed Fig. 3. Typical three-celled stage at the onset of treatment Fig. 4. Nondivided G M C at an early stage of differentiation toward P - G M C Fig. 5. Median paradermal section through a developed P-GMC. Note the prominency of the plastids. P plastid Fig. 6. Transverse section through a differentiating P-CMC. Both periclinal walls appear thickened Figs. 7 and 8. Paradermal and transverse sections of differentiated stomata; compare with the P - G M C in Figures 5 and 6 respectively Fig. 9. Meristemoid III at a young stage. ER endoplasmic reticulum; M mitochondrion; N nucleus Fig. 10. Paradermal section through an untreated GMC, before the onset of mitosis. The comparison with the meristemoid III in Figure 9 reveals that a synthesis of ribosomal cytoplasm, an increase of nuclear size, and a proliferation of the organelles have taken place. Pd plasmodesma Fig. 11. Electron micrograph showing a nondivided G M C at an early stage of differentiation toward P-GMC. It is clear that the plastids have started changing, while the dictyosomes are numerous and highly active. Numerous ER membranes are dispersed throughout the cell space and a vacuole has already been formed. D dictyosome; V vacuole Fig. 12. P-GMC at an advanced stage of differentiation; compare with Figures 10 and 1l. The anticlinal walls are further thickened, while the plastids have changed markedly and are almost filled with starch
B. Galatis : Differentiation of Stomatal Meristemoids
107
108
B. Galatis: Differentiation of Stomatal Meristemoids
B. Galatis: Differentiation of Stomatal Meristemoids
109
Fig. 13. GMC exhibiting early signs of degeneration (see mitochondrial structure and density of cytoplasm). However, the divergent plastid differentiation and the dictyosome activity appear commenced (note the wall thickening) Fig. 14. GMC differentiating toward P-GMC, without recognizable preparatory differentiation (cf. the GMC in Fig. 11). Note the irregular shape of the nucleus, the differentiation of plastids (compare with the one indicated by the arrow in subsidiary cell), and the dictyosome secretory activity Fig. 15. Meristemoid II at first stages of differentiation toward P-SM; compare with the GMC in Figures 11 and 14. The inset shows the two-celled stage of the mesogenous stoma from which the meristemoid lI has been taken Fig. 16. GMC at C mitosis. Note the position of the nucleus. C H chromosome Figs. 17 and 18. Paradermal sections including portions of the anticlinal walls of a meristemoid Ill and a differentiating P-GMC, respectively. In both cells the microtubules show the same orientation. The arrows point to cross-bridges that link them with the plasmalemma Hg. 19. Undeveloped plastid taken frolr! an undifferentiated GMC, 24 h after the onset of treatment Fig. 20. Well-developed plastid of a P-GMC after 72 h of continuous treatment. The increase of plastid size, membrane development, and starch loading is profound. G granum; S starch Fig. 21. Mature plastid taken from a P-GMC after 14 days of recovery from the drug. The internal membrane development has proceeded further. Note the dilated appearance of the thylakoids and the change in plastid shape Figs. 22 and 23. Subsidiary and typical epidermal cell plastids 72 h after the onset of treatment; compare with the plastid of P-GMC in Figure 19 Figs. 24 and 25. Guard cell plastids 72 h after the onset of the treatment and 14 days after the removal of the colchicine, respectively. Note the similarity in plastid structure between P-GMC of Figures 19 and 20 and guard cells of Figures 24 and 25, respectively
110 The more undeveloped cells, like the meristemoids, the GMC, and the young subsidiary cells, have smaller and simpler plastids, since these organelles divide before they develop in size and form a new thylakoidal system. Meristemoids II, III, and GMC contain small plastids with a densely stained stroma, bearing ribosome-like particles, a few thylakoids, and some starch grains (Fig. 19). After the inhibition of cell division, the plastids of meristemoids II and GMC embark on a remarkably divergent pathway of differentiation. They begin increasing in size, developing an internal membrane system, and acquiring starch (Figs. 11, 14 and 15). The elaboration of starch begins early in the development of the P-GMC, P-SM, and the plastid. At the fourth day after the onset of treatment, they appear almost filled with starch deposits and possess a relatively developed internal membrane system that forms some grana (Figs. 12 and 20). Their thylakoids may show a dilated or swollen appearance; they commonly contain plastoglobuli and are indistinguishable from the plastids of the guard cells (Fig. 20; cf. Fig. 24). After 14 days recovery of plants from the colchicine, the plastids of P-GMC and P-SM possess a more developed thylakoidal system and appear identical to those of the guard cells (Fig. 21; cf. Fig. 25). Finally, the plastids of P-GMC and P-SM are larger than those of subsidiary and typical epidermal cells, appear equal to those of the guard cells, but remain smaller than those of the mesophyll. In mature epidermis the plastids of subsidiary and typical epidermal cells reach the same level of membrane development. They show a few grana consisting of an unexpectably large number of thylakoids (sometimes more than twenty), while all epidermal plastids show a grana-fret membrane system adequate to classify them as chloroplasts (Figs. 22 and 23). During the entire developmental process, as well as when they become mature, the plastids of P-GMC and P-SM display spatial associations to mitochondria and microbodies. During the recovery from the administration of the drug, the subplasmalemmal microtubules reappear in both P-GMC and P-SM. In paradermal sections, they are anticlinally oriented and more or less symmetrically distributed along their walls (Fig. 18). Conspicuous bridges connect them with the plasmalemma (Fig. 18). Under the conditions of treatment used here, the colchicine interferes definitely with the subplasmalemmal and spindle microtubules in all epidermal cells but does not affect the subplasmalemmal ones of the differentiated guard cells, P-GMC, and P-SM. Moreover, the microtubules reappear in these cells at the advantaged stages of differentiation,
B. Galatis: Differentiationof StomatalMeristemoids even when the plants remain under colchinine treatment. There are two probable explanations for this microtubular behavior: (1) that the microtubules of differentiated P-GMC, P-SM, and guard cells are more resistant to colchicine or (2) that the plasmodesmata disruption, which seems to occur during the differentiation of these cells (see below), prevents the colchicine penetration. The latter explanation seems more plausible. As the cells approach maturity, the cytoplasm becomes progressively poor in ribosomes, while the activities of the ER membranes and the dictyosomes cease (Figs. 12 and 26). Nevertheless, the mitochondria remain numerous-a fact suggesting high energy requirements for the performance of some cell activities (Figs. 26 and 27). In mature P-GMC and P-SM as well as in guard cells, the nucleus, the plastids, and the cytoplasm occupy significant parts of the cell volume (Figs. 12 and 26 ; cf. Fig. 28). The vacuoles show variable development, but their final size remains limited in comparison to those of subsidiary and typical epidermal cells (Figs. 12 and 27). In most of the examined cases, they exhibit a dissected appearance. During the differentiation process the existent plasmodesmata disappear. Thus, at an adult stage, the P-GMC, P-SM as well as the guard cells, appear isolated from the surrounding cells. The similarity of P-GMC (Fig. 26) and P-SM to the guard cells is obvious in both paradermal and transverse sections (cf. Fig. 26 with Fig. 28 and Fig. 27 with Fig. 29).
Experimental Data It is well known that the guard cells have an inherent ability to accumulate starch and retain it for a long time (Konagamitsu and Ono, 1959; Pallas, 1964). To examine whether the exogenously supplied sugars are available for starch production in P-GMC and P-SM, like in guard cells (Konagamitsu and Ono, 1959; Pallas, 1964), leaf paradermal sections were floated on a 0.03 M solution of sucrose or 0.02 M glucose in dark for some days. After 24-48 h of incubation the P-GMC and P-SM appeared completely filled with starch, a phenomenon that is common in guard cells and that becomes prominent after staining with Lugol's iodine (Fig. 31). P-GMC, P-SM, and stomata of sections floated on sugar solution obviously bear more starch than those of sections floated on water (Fig. 30; cf. Fig. 31). However, starch acquisition occurs only in the regions of the sections that carry adherent subepidermal tissue, a fact also underlined by Pallas (1964). In cultured epidermal peels it was observed that the P-GMC and the P-SM retain the elaborated starch and remain viable during a long starvation period,
B. Galatis: Differentiation of Stomatal Meristemoids
111
Fig. 26. Mature P - G M C characterized by prominent plastids and n u m e r o u s mitochondria. The inset is a low power electron micrograph of the P - G M C in Figure 26 including its subsidiary cells Fig. 27. Late stages of differentiation of a P - G M C in transverse section. The considerable thickening of the periclinal walls and the deposition of pads at the corners of the internal wall are clearly seen (arrows) Fig. 28. Differentiated stoma in a median paradermal section; compare with Figure 26 Fig. 29. Median tranverse section of a mature stoma; compare with the P - G M C illustrated in Figure 27
even when the surrounding tissue seems dead. It has been suggested by some investigators that an accumulation of potassium in guard cells is immediately involved in the mechanism by which turgor pressure of guard cells is increased, causing stomatal opening (see review by Raschke, 1975).
With a commonly applied histochemical method (Macallum, 1905), it was observed that in epidermis of illuminated leaves that had substantially opened stomata, significant potassium accumulations were localized in guard cells, P-GMC, and P-SM. At this stage, little or no potassium was detected in the sur-
112
B. Galatis: Differentiation of Stomatal Meristemoids Discussion
Figs. 30 and 31. Portions of freehand epidermal sections, floated on water (Fig. 30) and on a solution containing 0.02 M glucose (Fig. 31). Each of them includes one stoma and one P-GMC. After Lugol's iodine staining, the guard cells and the P-GMC of sections incubated on sugar solution are loaded with starch Figs. 32 and 33. Epidermal sections containing one stoma and one P-GMC each, taken from leaves that maintain open stomata in light and closed ones in dark, respectively. In Figure 32 the potassium accumulations are detected in the guard cells of the open stoma and in the P-GMC. In Figure 33 the reaction product is seen in the subsidiary cells surrounding the closed stoma and the closelylying P-GMC
rounding cells (Fig. 32). When epidermal peels or freehand sections of leaves, maintained in dark for 3 h, were stained with cobaltinitrite, the potassium appeared concentrated in subsidiary cells surrounding P-GMC, P-SM, and guard cells of closed stomata (Fig. 33). Large potassium quantities were also seen accumulated in typical epidermal and mesophyll cells, making the definition of the phenomenon more difficult. The foregoing indications strengthen further the similarity of P - G M C and P-SM to guard cells, which will be discussed in detail.
The guard cells differ from the other epidermal cells in form, anatomical characteristics, cytoplasmic elements, vacuolar properties, as well as 'in metabolism, particularly as it is affected by light' (Meidner and Mansfield, 1968). These unique structural features and physiological properties make guard cell differentiation quite interesting. The preceding observations deal with the fine structure of differentiating and mature idioblast epidermal cells experimentally induced from guard cell precursors. Of particular interest was the extent to which these cells acquire the structure and some of the unique functional activities of the guard cells. Light microscopic studies have established that the guard cells exhibit a characteristic shape, highly variable thickenings of cell walls, a radial orientation of wall microfibrils (' Radiomicellat': Ziegenspeck, 1955a, b), and prominent plastids. The mechanism of stomatal movement is based on the differential wall thickening of the guard cells and particularly on the microfibrillar orientation in the walls in relation to the ones of their surrounding cells (Schwendener, 1881 ; Ziegenspeck, 1955a, b; Aylor et al., 1973). F r o m the fine structural studies published so far, some generalizations concerning the ultrastructure of guard cells can be made. In most of the examined cases, the mature guard cells are characterized by: (1) plastids with poor (in monocotyledons) or slightly developed (in dicotyledons) grana-fret membrane systems almost filled with starch grains, (2) the rarity or absence of plasmodesmata, and (3) many mitochondria and vacuoles variable in number, size, and appearance (Brown and Johnson, 1962; Landr~ 1969a, b, 1970, 1972; Thomson and De Journett, 1970; Kaufman et al., 1970; Allaway and Setterfield, 1972; Whatley, 1972; Pallas and Mollenhauer, 1972a, b; Humbert and Guyot, 1972; Srivastava and Singh, 1972; Singh and Srivastava, 1973 ; Galatis, 1974; Ziegler et al., 1974). The structural features of the mature guard cells correspond to those of the mature P-SM and P-GMC, except for the shape, the radial orientation of the wall microfibrils, and the deposition of a local thickening on a part of the anticlinal wall. The highly specialized structure and physiological behavior of guard cells is the manifestation of a peculiar differentiation exhibiting an intense dictyosome and E R membrane activity, a divergent differentiation of plastids, and particularly a unique organization of microtubules. The appearance and existence of the latter keep pace with the patterned thickening of the cell wall (Kaufman et al., 1970 ; Landr6, 1970 b; Srivastava
B. Galatis: Differentiationof Stomatal Meristemoids and Singh, 1972; Singh and Srivastava, 1973; Galatis, 1974). All the cytoplasmic activities occurring in differentiating guard cells are observed in P-GMC and P-SM, except for the organization of microtubules, which is similar to that of other meristemoids. In Vigna sinensis the young guard cells undergo an additional early structural differentiation, during which they synthesize new ribosomal cytoplasmic complement, while its organelles appear to be proliferating (Galatis, 1974). It is interesting that the 'preparatory' as well as the final differentiation of meristemoids II and GMC into P-SM and P-GMC resembles processes of differentiation occurring in guard cells. However, the foregoing observations clearly suggest that the 'preparatory' differentiation, i.e., the cell growth, is not necessary for the operation of differentiation of GMC and meristemoids II into P-GMC and P-SM; it seems likely that it is related to its successful completion and to the final size attained by these idioblasts. The larger GMC and meristemoids II become larger P-GMC and P-SM. The interesting question that arises now is why the P-GMC and P-SM do not take the form of the guard cell, while they gain most of the guard cell characteristics. It is well known that the development of an irregular cell shape is closely related to the microfibrillar wall texture. In the case of stomata the kidney shape of the guard cells is possibly determined by the radial orientation of the microfibrils from the rim of the stomatal pore toward the dorsal walls (Palevitz and Hepler, 1976). In Vigna sinensis this patterned microfibrillar deposition seems to be controlled by an underlying system of microtubules radiating from the pore parallel to the microfibrils (Galatis, 1974). Similar observations have been made by Singh and Srivastava (1973) and Palevitz and Hepler (1976). According to the foregoing considerations, the absence of a radial arrangement of wall microfibrils in P-GMC and P-SM can be attributed to the absence of a microtubular system similar to the one found in guard cells. In guard cells, microtubules appear not only to influence the orientation of wall microfibrils, but also to determine the local deposition of material in about the middle of the ventral wall, where the stomatal pore will later develope (Kaufman etal., 1970; Landr6, 1970b; Srivastava and Singh, 1972; Singh and Srivastava, 1973 ; Galatis, 1974). Such microtubular activity is known from numerous other studies (see reviews by Newcomb, 1969; Hepler and Palevitz, 1974). In P-SM and P-GMC the rather symmetrical distribution of microtubules possibly results in the equal deposition of material along the over-all length of anticlinal walls. It seems likely that only the parie-
113 tal cytoplasm abuted on definite areas of the middle of the anticlinal wall resulting from the symmetrical division of the GMC has the ability to function as an organizing center for a unique microtubular system. To conclude the above discussion, it may be suggested that the microtubules have a particular morphogenetic role in the differentiating guard cells. Under conditions used here, this problem cannot be approached directly by studying the action of colchicine on microfibrillar texture of the wall and on the shape of stomata. The colchicine effect on the microtubules is of short duration and takes place only in very young stomata (possessing plasmodesmata). In the slightly abnormal in shape stomata, the microtubules soon reappear and the patterned wall thickening as well as the microfibrillar orientation occurs as in the untreated stomata. Since microtubular control of the microfibril orientation in the wall has not been definitely accepted (see O'Brien, 1972) and since it is possible that colchinine in high concentrations may affect cellular processes unrelated to microtubules (Hart and Sabnis, 1976) the preceding suggestion about the morphogenetic significance of microtubules in guard cells needs further experimental documentation. The single guard cells observed in several plants by Reese (1950), Dehnel (1961) and other investigators have also been observed in Vigna sinensis (data, not shown). In this plant they do not seem to represent differentiated GMC or meristemoids II, but always result from an early degeneration of their partners in young stomata. In these stomata the guard cells are considerably unequal in size and seem to have derived from unsuccessful symmetrical divisions of GMC. Among the specific physiological properties displayed by the guard cells, we could study and verify the ability of starch synthesis from exogenously supplied sugars and the existence of a potassium ion pump between P-SM or P-GMC and their surrounding cells. If the potassium ion pump operating in guard cells results in the turgor change, which causes stomatal opening, it can be assumed that the existence of such a mechanism in the P-GMC and P-SM may imply the mechanism of guard cell movement in these cells too. The failure of this activity to be expressed as a change in cell shape is due to the fact that they lack the proper wall structure and ultrastructure. As a general concluSion of the present paper it can be said that the P-GMC and P-SM of Vigna sinensis are actual!y guard cells in structure and possibly in some functional activities, although they lack their form.
114
References Allaway, W.G., Setterfield, G.: Ultrastructural observations on guard cells of Vicia faba and Allium porrum. Canad. J. Bot. 50, 1405-1413 (1972) Aylor, D.E., Parlange, J-Y., Krikorian, A.D. : Stomatal mechanics. Amer. J. Bot. 60, 163-171 (1973) Brat, L., Weber, F.: Anthoxanthin in den Schliegzellen der Kronblfitter yon Victoria amazonica und Nymphaea zanzibariensis. Phyton 3, 22-28 (1951) Brown, W.V., Johnson, Sr. C.: The fine structure of the grass guard cell. Amer. J. Bot. 49, 110-115 (1962) B/inning, E., Sagromsky, H. : Die Bildung des Spalt6ffnungsmusters in der Blattepidermis. Z. Naturforsch. 3b, 203-.216 (1948) B/inning, E., Biegert, F. : Die Bildung der Spalt6ffnungsinitialen bei AIlium cepa. Z. Bot. 41, 17-39 (1953) Dehnel, G.S. : Abnormal stomatal development in foliage leaves of Begonia aridicaulis. Amer. J. Bot. 48, 129-133 (1961) Fischer, R. A.: Role of potassium in stomatal opening in the leaf of Viciafaba. Plant Physiol. 47, 555-558 (1971) Galatis, B.: Ultrastructural studies on stomatal development. Ph. D. Thesis, University of Athens, Athens 1974 Gavaudan, P. : Sur les tissus a constitution mixte diplo'ide et polyploide d~velopp6s chez les v~g6taux par action de la colchicine. C.R. Soc. Biol. (Paris) 128, 717-719 (1938) Gertz, O. : Studier 6fver klyf6ppningarnas morfologi reed s/irskild hfinsyn till deras patologiska utbildnings former. Acta Univ. Lurid N.F. 15, 3-84 (1919) Guyot, M. : Action de la colchicine sur le d+veloppement des stomates de Viciafaba L. C. R. Soc. Biol. (Paris) 158, 1722-1725 (1964) Guyot, M., Pikusz, A., Humbert, C. : Action de la colchicine sur les stomates de Dianthus caryophyllus L. C. R. Acad. Sci. (Paris) D 266, 1251-1253 (1968) Hart, J.W., Sabnis, D.D.: Colchicine and plant microtubules: a critical evaluation. Curt. Adv. Plant Sci. 8 (8), 1095 1104 (1976) Hepler, P.K., Palcvitz, B.A.: Microtubules and microfilaments. Ann. Rev. Plant Physiol. 25, 309-362 (1974) Humbert, C., Guyot, M. : Modifications ultrastructurales des cellules stomatiques d'Anemia rotundiJblia Schrad. C. R. Acad. Sci. (Paris) D 274, 380-382 (1972) Kaufman, P.B., Petering, L.B., Yocum, C.S., Baic, D. : Ultrastructural studies on stomata development in internodes of Arena sativa. Amer. J. Bot. 57, 33-49 (1970) Kenda, G., Weber, F. : Stomata - Zahl vergr/inter Verbascum Blattaria - Kronblfitter. Osterr. Bot. Z. 97, 503-509 (1950) Konagamitsu, Y., Ono, H. : The period of starch forming ability in guard cell during the shaping of stomata. Sieboldia 2, 131-135 (1959) Kropfitsch, M. : Apfelgas-Wirkung auf Stomatazahl. Protoplasma 40, 256-265 (1951 a) Kropfitsch, M.: UV-Bestrahlung und Stomatazahl. Protoplasma 40, 266-274 (1951 b) Landr6, P. : Premi6res observations sur l'6volution infrastructurale des cellules stomatiques de la Moutarde (Sinapis alba L.) depuis leur mise en place jusqu'/t l'ouverture de l'ostiole. C. R. Acad. Sci, (Paris) D 269, 943-946 (1969a) Landr~, P.: Quelques aspects infrastructuraux des stomates des cotyl6dons de la Moutarde (Sinapis alba L.). C. R. Acad. Sci. (Paris), D 269, 990-992 (1969b) Landr6, P. : Activit6 golgienne en liaison avec celle du plasmalemme dans les cellules stomatiques de la Moutarde (Sinapis alba L.) lors de la formation de l'ostiole. C.R. Acad. Sci. (Paris) D 271, 904-907 (1970)
B. Galatis: Differentiation of Stomatal Meristemoids Landr6, P. : Origine et d~veloppement des ~pidermes cotyl6donaires et foliaires de la Moutarde (Sinapis alba L.). Diff6renciation ultrastructurale des stomates. Ann. Sci. Nat. Bot. 12" S. Tome XIII, 247-322 (1972) Macallum, A.B. : On the distribution of potassium in animal and vegetable cells. J. Physiol. 32, 95-118 (1905) Meidner, H., Mansfield, T.A.: Physiology of Stomata. London: McGraw-Hill (1968) Newcomb, E.H. : Plant microtubules. Ann. Rev. Plant Physiol. 20, 253-288 (1969) O'Brien, T.P. : The cytology of cell-wall formation in some eukaryotic cells. Bot. Rev. 38, 87-118 (1972) Palevitz, B.A., Hepler, P.K. : Cellulose microfibril orientation and cell shaping in developing guard cells of Allium: The role of microtubules and ion accumulation. Planta (Berl.) 132, 71-93 (1976) Pallas, J.E. Jr.: Guard-cell starch retention and accumulation in the dark. Bot. Gaz. 125, 102-107 (1964) Pallas, J.E. Jr., Mollenhauer, H.H. : Electron microscopic evidence for plasmodesmata in dicotyledonous guard cells. Science 175, 1275-1276 (1972a) Pallas, J.E. Jr., Mollenhauer, H.H. : Physiological implications of Vicia faba and Nicotiana tabacum guard-cell ultrastructure. Amer. J. Bot. 59, 504-514 (1972b) Pant, D.D.: On the onogeny of stomata and other homologous structures. Plant Sci. Allahabad Ser. I, 1-24 (1965) Raschke, K.: Stomatal action. Ann. Rev. Plant Physiol. 26, 309-340 (1975) Reese, G. : Beitrgge zur Wirkung des Colchicins bei der Samenbehandlung. Planta (Berl.) 38, 324-376 (1950) Schwendener, S. : Ober Bau und Mechanik der Spalt6ffnungen. Sitzgsber. Berl. Akad. Wiss. Berlin 833-867 (1881) Singh, A.P., Srivastava, L.M.: The fine structure of pea stomata. Protoplasma 76, 61-82 (1973) Srivastava, L.M., Singh, A.P.: Stomatal structure in the corn leaves. J. Ultrastruct. Res. 39, 345-363 (1972) Stebbins, L.G., Jain, S.K. : Developmental studies of cell differentiation in the epidermis of monocotyledons. I. Allium, Rhoeo and Commelina. Develop. Biol. 2, 409~26 (1960) Stebbins, L.G., Shah, S.S. : Developmental studies of cell differentiation in the epidermis of monocotyledons. II. Cytological features of stomatal development in the Gramineae. Develop. Biol. 2, 477-500 (1960) Thomson, W.W., De Journett, R.: Studies on the ultrastructure of the guard cells of Opuntia. Amer. J. Bot. 57, 309-316 (1970) Tonzig, S., Ott-Candela, A. : L'azione della colchicina suflo sviluppo degli apparati stomatici. Nuovo Gior. Bot. Ital. N.S. 53, 535-547 (1946) Weber, F.: Spalt6ffnungsapparat -- Anomalien colchicinierter Tradescantia-Blfitter. Protoplasma 37, 556-565 (1943) Weissenb6ck, K.: Studien an colchizinierten Pflanzen I. Anatomische Untersuchungen. Phyton 1, 282-300 (1949) Whatley, J.M. : The ultrastructure of guard cells of Phaseolus vulgaris. New Phytol. 71, 175-179 (1972) Ziegenspeck, H. : Die Farbenmikrophotographie ein Hilfsmittel zum objektiven Nachweis submikroskopischer Strukturelemente. Die Radiomicellierung und Filierung der SchlieBzellen yon Ophioderma pendulum. Photographic und Wissenschaft 4, 19 22 (1955a) Ziegenspeck, H. : Das Vorkommen yon Fila in radialer Anordnung in den SchlieBzellen. Protoplasma 44, 385-388 (1955b) Ziegler, H., Shmueli, E., Lange, G. : Structure and function of the stomata of Zea mays. I. The development. Cytobiologie 9, 162-168 (1974) Received 20 December 1976; accepted 6 May 1977
Planta 136, 103-114 (1977)
9 by Springer-Verlag 1977
Differentiation of Stomatal Meristemoids and Guard Cell Mother Cells into Guard-like Cells in Hgna sinensis Leaves after Colchicine Treatment An Ultrastructural and Experimental Approach B. Galatis Institute of General Botany, University of Athens, Athens 621, Greece
Abstract. The temporary development of Vigna sinensis seedlings in the presence of colchicine results in the inhibition of stomata generation and the formation of numerous persistent stomatal meristemoids (P-SM) and guard cell mother cells (P-GMC). Before dividing differentially or becoming GMC, the untreated meristemoids undergo a 'preparatory' differentiation, during which a synthesis of new densely ribosomal cytoplasm, an increase of nuclear size, and a detectable proliferation of all the organelles are observed. The same process appears depressed and delayed in treated meristemoids; the cells have usually undergone only part of it when they reach the C mitosis. After the inhibition of their division, the bulged meristemoids II and GMC increase further in size, synthesize new nonribosomal cytoplasm, and start vacuolating slowly. The plastids also increase in size, change in shape, and become able to synthesize large quantities of starch. The cells retain a ribosomal cytoplasm, rough ER membranes, and active dictyosomes for a long time. At the advanced stages of differentiation, the microtubules reappear in the cells even when the plant remains under colchicine treatment. When mature, the P-GMC and P-SM are quite similar to the guard cells and possess considerably thickened periclinal walls, numerous mitochondria, and small vacuoles, while the nucleus, the plastids, and the cytoplasm occupy significant parts of the cell volume. In the epidermis displaying open stomata in light, significant K + quantities are detectable in guard cells and P-GMC or P-SM, while they are almost absent from their surrounding cells. When the stomata close in darkness, K § is accumulated primarily in the subsidiary or typical epidermal cells surrounding these idioblasts and only minimally inside them. Besides, the P-GMC and P-SM, like the Abbreviations: P-GMC=persistent guard cell mother cell; PSM= persistent stomatal meristemoid; ER:endoplasmic reticulum
guard cells, retain the starch for a long time and build up considerable starch quantities from exogenously supplied sugars.
Key words: Colchicine - Meristemoids -
Stomata
Vigna.
Introduction The ontogeny of the guard cells is the outcome of a complicated process, including: (1) a number of divisions, the first one(s) of which are differential and sometimes obviously asymmetrical and the last of which always results in the symmetrical formation of a pair of guard cells (Bfinning and Sagromsky, 1948 ; Btinning and Biegert, 1953 ; Stebbins and Shah, 1960; Stebbins and Jain, 1960), and (2) a divergent differentiation sequence in the young guard cells expressed structurally by conspicuous protoplasmic changes, the most prominent of which are related to a peculiar microtubule organization, an intense dictyosome activity, and a characteristic plastid differentiation (Landr6, 1969a, b, 1970, 1972; Kaufman et al., 1970; Srivastava and Singh, 1972; Singh and Srivastava, 1973; Calatis, 1974). During the extensive light microscope studies on stomatal ontogeny, a wide range of developmental stomatal anomalies, naturally occurring or experimentally induced, has been illustrated. Naturally occurring P-GMC were observed by Kenda and Weber (1950), Brat and Weber (1951), and Dehnel (1961). Besides, it has been reported that similar cells are formed after different treatments i.e., colchicine (Gavaudan, 1938 ; Weber, 1943 ; Tonzig and Ott-Candela, 1946; Weissenb6ck, 1949; Reese, 1950; Guyot, 1964; Guyot et al., 1968), ultraviolet irradiation (Kropfitsch, 1951a), or ethylene (Kropfitsch, 1951b). The formation of P-GMC in pathological conditions re-
104 lated to galls of fungal or insect etiology has been reported as well (Gertz, 1919), In some o f the preceding studies a great n u m b e r of plastids large in size and containing starch and the thickenings of cell walls have been noted. Both o f these structural characteristics have led to the suggestion that these cells must differentiate toward the direction o f the guard cells. W e b e r (1943) has already questioned whether the P - G M C possess some of the properties o f the guard cells. The present report describes the fine structural differentiation o f meristemoids II and G M C o f Vigna sinensis into guard-like cells under conditions preventing their division. Moreover, presented evidence suggests that these cells possess some o f the functional activities o f the guard cells.
Materials and Methods Six-day-old seedlings of Vigna sinens&,grown in a greenhouse, were further developed for 24, 48, 72, or 96 h in a half-strength Hoagland solution that contained 0.08% colchicine (Sigma). After this treatment the primary leaves were fixed either immediately or after 1, 2, 3, or 14 days of further development in Hoagland solution. Fixation was carried out by immersing small pieces of leaves in 5% glutaraldehyde in 0.025 M phosphate buffer, pH 7, at room temperature, for 2 h. After washing in buffer the samples were postfixed in 1% osmium tetroxide in the same buffer, pH 7 at 4~ C, for 4 h. The specimens were infiltrated in mixtures of Epon 812 or Durcupan ACM (Fluka)/propylene oxide and embedded in the respective resin. Thin sections of material were cut on an LKB Ultrotome III microtome, double-stained with 4% uranyl acetate in 70% ethanol and lead citrate and examined with a Philips 300 electron microscope. Thick sections were cut from the same blocks with an LKB Pyramitome or Ultrotome III microtomes, stained with toluidine blue and photographed with a Zeiss light microscope. K + was localized histochemically in epidermal strips or paradermal sections using Macallum's method (1905) with cobaltinitrite, as has been performed by Fischer (1971).
Observations
Light Microscopy The majority o f the stomata population on the epidermis o f 6-day-old primary leaves o f Vigna sinensis derives f r o m a mesogenous process o f development, which finally forms typical paracytic s t o m a t a (Figs. 1 and 7). This sequence is in brief as follows: A p r o t o d e r m a l cell divides differentially and cuts off the stomatal initial (meristemoid I) and a larger cell that either divides again or differentiates into a typical epidermal cell. The meristemoid I undergoes two successive divisions, the first o f which gives rise to the meristemoid II and the one subsidiary cell, while the
B. Galatis : Differentiation of Stomatal Meristemoids second forms the meristemoid III, which will become the G M C , and the other subsidiary cell. Finally, a symmetrical division taking place in G M C produces the pair of g u a r d cells (Fig. 1). A limited n u m b e r o f s t o m a t a originate f r o m mesoperigenous and perigenous m o d e s of development. The different developmental stages and the completed stomata a p p e a r in a mixed fashion on b o t h epidermides. The terms mesogenous, perigenous and mesoperigenous were proposed by Pant (1965) for the classification of ontogenetic stomatal types. A mesogenous stoma is one in which the guard cell mother cell and all subsidiaries or a single ring-like subsidiary cell arise from the same meristemoid. A mesoperigenous stoma is one in which the surrounding cells are of dual origin, one neighboring cell is mesogenous, and the others perigenous. A perigenous stoma is one in which all neighboring or subsidiary cells derived independently from the guard cell mother cell, which divides only once to form the two guard cells. Examination o f the epidermis o f p r i m a r y leaves, 48-72 h after the onset o f administration o f the drug, revealed that s t o m a t a f o r m a t i o n was noticeably inhibited. M o s t of the developing stomata were blocked at the two- and three-celled stages, which in untreated leaves consist of one or two subsidiary cells, respectively, abuting or surrounding a meristemoid II or I I I or a G M C . A p a r t f r o m these meristemoids II, III, or G M C n o r m a l at the light microscopic l e v e l - n u m e r o u s cells of an idioblastic appearance, larger in size and r o u n d in form, were observed (Fig. 2). The presence of two distinct subsidiary cells confirms that they represent persistent guardcell m o t h e r cells ( P - G M C , Figs. 2, 4 and 5), while the existence of one subsidiary cell and one typical epidermal cell confirms that they must be persistent stomatal meristemoids II (P-SM, Fig. 2). Three different stages of differentiation of G M C into PG M C can be observed in Figures 3-5. Since some mesoperigenous s t o m a t a are f o u n d on the same surface, a small n u m b e r of t h e m m a y represent P - G M C o f mesoperigenous stomata. Nevertheless, the meristemoids II of the mesogenous process are easily recognized because the surrounding cells (one subsidiary and one typical epidermal cell) show p r o m i n e n t differences in size and developmental stage (Fig. 2). The P - G M C and P - S M contain p r o m i n e n t plastids (Figs, 4 and 5) and considerably thickened periclinal walls easily observed in transverse sections (Fig. 6). Conspicuous structural similarities exist between these epidermal idioblasts and the guard cells (cf. Figs. 5 and 6 with Figs. 7 and 8, respectively).
Electron Microscopy In untreated leaves the y o u n g meristemoids II and III are small lens-shaped cells similar to each other
B. Galatis: Differentiationof Stomatal Meristemoids in shape, structure, and size. Their recognition as meristemoids II or IlI is based on the presence of one or two subsidiary cells. Furthermore, the 'preparatory' differentiation occurring in a young meristemoid I! resembles closely that of a meristemoid III. In addition, the final differentiation of a meristemoid II into a P-SM is identical to that of a GMC into a P-GMC. To avoid duplication of the presented results, illustrations will be given for the meristemoid III and GMC only. The greatest part of meristemoid II and III cell space is occupied by a nucleus, exhibiting a pronounced nucleolus; yet, they are devoid of vacuoles (Fig. 9). In the densely ribosomal cytoplasm a few undeveloped plastids, mitochondria, rough ER membranes, a few very small microbodies, and rare paramural bodies were observed (Fig. 9). A significant number of ribosomes appear arranged in polyribosoreal complexes. Along the anticlinal walls numerous and densely distributed microtubules were detected (Fig. 17). They are anticlinally oriented and crossbridged to the plasmalemma and to one another with fine links (Fig. 17). The nontreated meristemoids III before becoming G M C - i . e . during the time preceding their symmetrical division-and the meristemoids II before dividing differentially usually undergo a 'preparatory' differentiation. This is expressed structurally by the synthesis of a new ribosomal cytoplasm, the increase of nuclear size and of the number of ER membranes, as well as the proliferation of plastids, mitochondria, and dictyosomes (Fig. 10). Vacuolation does not usually participate in the increase of cell size. The action of colchicine results in the disappearance of subplasmalemmal and spindle microtubules and, after the completion of C mitosis, in the formation of a large, irregular shaped and often lobed nucleus (Figs. 14 and 15). After the inhibition of their division (Fig. 16) the GMC and the meristemoids II, which have completed most of their 'preparatory' differentiation, enter a new differentiation process. The 'preparatory' differentiation in treated meristemoids II and III appears to be depressed and advances slowly in relation to the untreated ones. When these cells reach the onset of C mitosis, they have usually undergone only part of this process and have completed it after the restoration of the nuclear membrane. The nondivided GMC increase further in size and tend to become round in shape (Fig. 11). There is pictorial evidence that this increase represents the synthesis of new nonribosomat cytoplasm and the formation of small vacuoles (Fig. 11). The number of plastids and mitochondria remains more or less constant, while the ER membranes and the dictyosomes appear
105 further increased in number (Fig. 11). The first structural indications of the final differentiation of the meristemoids II and GMC into epidermal idioblasts, which actually commence after the completion of C mitosis, are: the development of plastids that begin increasing in size, changing in shape, and accumulating starch, the development of a vacuole, and the dominant dictyosome activity (Figs. 11 and 14). Among these indications, the divergent plastid differentiation is the most reliable. The cell remains in an active differentiating condition for a long time. This is evidenced from the maintenance of a ribosomal cytoplasm, an extensive rough ER membrane system, and an active Golgi apparatus for a prolonged period of time. The cell in Figure 15 illustrates a persistent meristemoid II o f a mesogenous process (in the inset Figure see the difference in size and developmental stage between the subsidiary cell and the other typical epidermal cell abuted on the meristemoid). The meristemoid final differentiation is identical to that of GMC (see plastid differentiation and wall thickening). On the contrary, only rarely did we observe idioblast cells like the ones described above and devoid of subsidiary cells. It seems likely that they are P-GMC of perigenous type of stomata. The dictyosome activity continuously provides a great number of vesicles and yields a detectable thickening of anticlinal walls and a considerable one of the periclinal walls (Fig. 27). Although dictyosomes were observed at every cell region, a number of them were repeatedly found close to the internal periclinal walls, where numerous mitochondria and ER membranes were placed. These organelle accumulations are possibly related to the cell wall deposition. At more advanced stages of differentiation, patterned pads are deposited at the corners of the internal periclinal walls (Fig. 27). A few plasmodesmata penetrate the anticlinal walls in both meristemoids II and III as well as in GMC (Fig. 10). In exceptional cases, the GMC do not undergo a distinguishable 'preparatory' differentiation and fail to increase in size. Their cytoplasm gains a higher electron opacity and appears densely filled with ribosomes. In such cells the differentiation toward the direction of P-GMC commences and completes regularly (Fig. 14). The plastids start to accumulate starch, while an increase of dictyosome secretory activity yields a pronounced thickening of the cell walls (Fig. 14). It is interesting that in some GMC of minute size, whose failure to develop further leads to their progressive degeneration, differentiation into P-GMC tends to be considerable (Fig. 13). In the 6-day-old primary leaf epidermis a considerable variability in plastid size and complexity exists.
Fig. 1. Light micrograph showing a portion of a differentiating lower epidermis taken from an untreated leaf. All the developmental stages of the mesogenous mode of stomatal development are depicted, a One-celled stage; M~: meristemoid I; h Two-celled stage; Mr~ meristemoid II; e Three-celled stage; G M C guard cell mother cell; d Completed stoma Fig. 2. Surface view of a 6-day-old epidermis after 72 h of continuous treatment with colchicine. P-GMC as well as P-SM can be observed Fig. 3. Typical three-celled stage at the onset of treatment Fig. 4. Nondivided G M C at an early stage of differentiation toward P - G M C Fig. 5. Median paradermal section through a developed P-GMC. Note the prominency of the plastids. P plastid Fig. 6. Transverse section through a differentiating P-CMC. Both periclinal walls appear thickened Figs. 7 and 8. Paradermal and transverse sections of differentiated stomata; compare with the P - G M C in Figures 5 and 6 respectively Fig. 9. Meristemoid III at a young stage. ER endoplasmic reticulum; M mitochondrion; N nucleus Fig. 10. Paradermal section through an untreated GMC, before the onset of mitosis. The comparison with the meristemoid III in Figure 9 reveals that a synthesis of ribosomal cytoplasm, an increase of nuclear size, and a proliferation of the organelles have taken place. Pd plasmodesma Fig. 11. Electron micrograph showing a nondivided G M C at an early stage of differentiation toward P-GMC. It is clear that the plastids have started changing, while the dictyosomes are numerous and highly active. Numerous ER membranes are dispersed throughout the cell space and a vacuole has already been formed. D dictyosome; V vacuole Fig. 12. P-GMC at an advanced stage of differentiation; compare with Figures 10 and 1l. The anticlinal walls are further thickened, while the plastids have changed markedly and are almost filled with starch
B. Galatis : Differentiation of Stomatal Meristemoids
107
108
B. Galatis: Differentiation of Stomatal Meristemoids
B. Galatis: Differentiation of Stomatal Meristemoids
109
Fig. 13. GMC exhibiting early signs of degeneration (see mitochondrial structure and density of cytoplasm). However, the divergent plastid differentiation and the dictyosome activity appear commenced (note the wall thickening) Fig. 14. GMC differentiating toward P-GMC, without recognizable preparatory differentiation (cf. the GMC in Fig. 11). Note the irregular shape of the nucleus, the differentiation of plastids (compare with the one indicated by the arrow in subsidiary cell), and the dictyosome secretory activity Fig. 15. Meristemoid II at first stages of differentiation toward P-SM; compare with the GMC in Figures 11 and 14. The inset shows the two-celled stage of the mesogenous stoma from which the meristemoid lI has been taken Fig. 16. GMC at C mitosis. Note the position of the nucleus. C H chromosome Figs. 17 and 18. Paradermal sections including portions of the anticlinal walls of a meristemoid Ill and a differentiating P-GMC, respectively. In both cells the microtubules show the same orientation. The arrows point to cross-bridges that link them with the plasmalemma Hg. 19. Undeveloped plastid taken frolr! an undifferentiated GMC, 24 h after the onset of treatment Fig. 20. Well-developed plastid of a P-GMC after 72 h of continuous treatment. The increase of plastid size, membrane development, and starch loading is profound. G granum; S starch Fig. 21. Mature plastid taken from a P-GMC after 14 days of recovery from the drug. The internal membrane development has proceeded further. Note the dilated appearance of the thylakoids and the change in plastid shape Figs. 22 and 23. Subsidiary and typical epidermal cell plastids 72 h after the onset of treatment; compare with the plastid of P-GMC in Figure 19 Figs. 24 and 25. Guard cell plastids 72 h after the onset of the treatment and 14 days after the removal of the colchicine, respectively. Note the similarity in plastid structure between P-GMC of Figures 19 and 20 and guard cells of Figures 24 and 25, respectively
110 The more undeveloped cells, like the meristemoids, the GMC, and the young subsidiary cells, have smaller and simpler plastids, since these organelles divide before they develop in size and form a new thylakoidal system. Meristemoids II, III, and GMC contain small plastids with a densely stained stroma, bearing ribosome-like particles, a few thylakoids, and some starch grains (Fig. 19). After the inhibition of cell division, the plastids of meristemoids II and GMC embark on a remarkably divergent pathway of differentiation. They begin increasing in size, developing an internal membrane system, and acquiring starch (Figs. 11, 14 and 15). The elaboration of starch begins early in the development of the P-GMC, P-SM, and the plastid. At the fourth day after the onset of treatment, they appear almost filled with starch deposits and possess a relatively developed internal membrane system that forms some grana (Figs. 12 and 20). Their thylakoids may show a dilated or swollen appearance; they commonly contain plastoglobuli and are indistinguishable from the plastids of the guard cells (Fig. 20; cf. Fig. 24). After 14 days recovery of plants from the colchicine, the plastids of P-GMC and P-SM possess a more developed thylakoidal system and appear identical to those of the guard cells (Fig. 21; cf. Fig. 25). Finally, the plastids of P-GMC and P-SM are larger than those of subsidiary and typical epidermal cells, appear equal to those of the guard cells, but remain smaller than those of the mesophyll. In mature epidermis the plastids of subsidiary and typical epidermal cells reach the same level of membrane development. They show a few grana consisting of an unexpectably large number of thylakoids (sometimes more than twenty), while all epidermal plastids show a grana-fret membrane system adequate to classify them as chloroplasts (Figs. 22 and 23). During the entire developmental process, as well as when they become mature, the plastids of P-GMC and P-SM display spatial associations to mitochondria and microbodies. During the recovery from the administration of the drug, the subplasmalemmal microtubules reappear in both P-GMC and P-SM. In paradermal sections, they are anticlinally oriented and more or less symmetrically distributed along their walls (Fig. 18). Conspicuous bridges connect them with the plasmalemma (Fig. 18). Under the conditions of treatment used here, the colchicine interferes definitely with the subplasmalemmal and spindle microtubules in all epidermal cells but does not affect the subplasmalemmal ones of the differentiated guard cells, P-GMC, and P-SM. Moreover, the microtubules reappear in these cells at the advantaged stages of differentiation,
B. Galatis: Differentiationof StomatalMeristemoids even when the plants remain under colchinine treatment. There are two probable explanations for this microtubular behavior: (1) that the microtubules of differentiated P-GMC, P-SM, and guard cells are more resistant to colchicine or (2) that the plasmodesmata disruption, which seems to occur during the differentiation of these cells (see below), prevents the colchicine penetration. The latter explanation seems more plausible. As the cells approach maturity, the cytoplasm becomes progressively poor in ribosomes, while the activities of the ER membranes and the dictyosomes cease (Figs. 12 and 26). Nevertheless, the mitochondria remain numerous-a fact suggesting high energy requirements for the performance of some cell activities (Figs. 26 and 27). In mature P-GMC and P-SM as well as in guard cells, the nucleus, the plastids, and the cytoplasm occupy significant parts of the cell volume (Figs. 12 and 26 ; cf. Fig. 28). The vacuoles show variable development, but their final size remains limited in comparison to those of subsidiary and typical epidermal cells (Figs. 12 and 27). In most of the examined cases, they exhibit a dissected appearance. During the differentiation process the existent plasmodesmata disappear. Thus, at an adult stage, the P-GMC, P-SM as well as the guard cells, appear isolated from the surrounding cells. The similarity of P-GMC (Fig. 26) and P-SM to the guard cells is obvious in both paradermal and transverse sections (cf. Fig. 26 with Fig. 28 and Fig. 27 with Fig. 29).
Experimental Data It is well known that the guard cells have an inherent ability to accumulate starch and retain it for a long time (Konagamitsu and Ono, 1959; Pallas, 1964). To examine whether the exogenously supplied sugars are available for starch production in P-GMC and P-SM, like in guard cells (Konagamitsu and Ono, 1959; Pallas, 1964), leaf paradermal sections were floated on a 0.03 M solution of sucrose or 0.02 M glucose in dark for some days. After 24-48 h of incubation the P-GMC and P-SM appeared completely filled with starch, a phenomenon that is common in guard cells and that becomes prominent after staining with Lugol's iodine (Fig. 31). P-GMC, P-SM, and stomata of sections floated on sugar solution obviously bear more starch than those of sections floated on water (Fig. 30; cf. Fig. 31). However, starch acquisition occurs only in the regions of the sections that carry adherent subepidermal tissue, a fact also underlined by Pallas (1964). In cultured epidermal peels it was observed that the P-GMC and the P-SM retain the elaborated starch and remain viable during a long starvation period,
B. Galatis: Differentiation of Stomatal Meristemoids
111
Fig. 26. Mature P - G M C characterized by prominent plastids and n u m e r o u s mitochondria. The inset is a low power electron micrograph of the P - G M C in Figure 26 including its subsidiary cells Fig. 27. Late stages of differentiation of a P - G M C in transverse section. The considerable thickening of the periclinal walls and the deposition of pads at the corners of the internal wall are clearly seen (arrows) Fig. 28. Differentiated stoma in a median paradermal section; compare with Figure 26 Fig. 29. Median tranverse section of a mature stoma; compare with the P - G M C illustrated in Figure 27
even when the surrounding tissue seems dead. It has been suggested by some investigators that an accumulation of potassium in guard cells is immediately involved in the mechanism by which turgor pressure of guard cells is increased, causing stomatal opening (see review by Raschke, 1975).
With a commonly applied histochemical method (Macallum, 1905), it was observed that in epidermis of illuminated leaves that had substantially opened stomata, significant potassium accumulations were localized in guard cells, P-GMC, and P-SM. At this stage, little or no potassium was detected in the sur-
112
B. Galatis: Differentiation of Stomatal Meristemoids Discussion
Figs. 30 and 31. Portions of freehand epidermal sections, floated on water (Fig. 30) and on a solution containing 0.02 M glucose (Fig. 31). Each of them includes one stoma and one P-GMC. After Lugol's iodine staining, the guard cells and the P-GMC of sections incubated on sugar solution are loaded with starch Figs. 32 and 33. Epidermal sections containing one stoma and one P-GMC each, taken from leaves that maintain open stomata in light and closed ones in dark, respectively. In Figure 32 the potassium accumulations are detected in the guard cells of the open stoma and in the P-GMC. In Figure 33 the reaction product is seen in the subsidiary cells surrounding the closed stoma and the closelylying P-GMC
rounding cells (Fig. 32). When epidermal peels or freehand sections of leaves, maintained in dark for 3 h, were stained with cobaltinitrite, the potassium appeared concentrated in subsidiary cells surrounding P-GMC, P-SM, and guard cells of closed stomata (Fig. 33). Large potassium quantities were also seen accumulated in typical epidermal and mesophyll cells, making the definition of the phenomenon more difficult. The foregoing indications strengthen further the similarity of P - G M C and P-SM to guard cells, which will be discussed in detail.
The guard cells differ from the other epidermal cells in form, anatomical characteristics, cytoplasmic elements, vacuolar properties, as well as 'in metabolism, particularly as it is affected by light' (Meidner and Mansfield, 1968). These unique structural features and physiological properties make guard cell differentiation quite interesting. The preceding observations deal with the fine structure of differentiating and mature idioblast epidermal cells experimentally induced from guard cell precursors. Of particular interest was the extent to which these cells acquire the structure and some of the unique functional activities of the guard cells. Light microscopic studies have established that the guard cells exhibit a characteristic shape, highly variable thickenings of cell walls, a radial orientation of wall microfibrils (' Radiomicellat': Ziegenspeck, 1955a, b), and prominent plastids. The mechanism of stomatal movement is based on the differential wall thickening of the guard cells and particularly on the microfibrillar orientation in the walls in relation to the ones of their surrounding cells (Schwendener, 1881 ; Ziegenspeck, 1955a, b; Aylor et al., 1973). F r o m the fine structural studies published so far, some generalizations concerning the ultrastructure of guard cells can be made. In most of the examined cases, the mature guard cells are characterized by: (1) plastids with poor (in monocotyledons) or slightly developed (in dicotyledons) grana-fret membrane systems almost filled with starch grains, (2) the rarity or absence of plasmodesmata, and (3) many mitochondria and vacuoles variable in number, size, and appearance (Brown and Johnson, 1962; Landr~ 1969a, b, 1970, 1972; Thomson and De Journett, 1970; Kaufman et al., 1970; Allaway and Setterfield, 1972; Whatley, 1972; Pallas and Mollenhauer, 1972a, b; Humbert and Guyot, 1972; Srivastava and Singh, 1972; Singh and Srivastava, 1973 ; Galatis, 1974; Ziegler et al., 1974). The structural features of the mature guard cells correspond to those of the mature P-SM and P-GMC, except for the shape, the radial orientation of the wall microfibrils, and the deposition of a local thickening on a part of the anticlinal wall. The highly specialized structure and physiological behavior of guard cells is the manifestation of a peculiar differentiation exhibiting an intense dictyosome and E R membrane activity, a divergent differentiation of plastids, and particularly a unique organization of microtubules. The appearance and existence of the latter keep pace with the patterned thickening of the cell wall (Kaufman et al., 1970 ; Landr6, 1970 b; Srivastava
B. Galatis: Differentiationof Stomatal Meristemoids and Singh, 1972; Singh and Srivastava, 1973; Galatis, 1974). All the cytoplasmic activities occurring in differentiating guard cells are observed in P-GMC and P-SM, except for the organization of microtubules, which is similar to that of other meristemoids. In Vigna sinensis the young guard cells undergo an additional early structural differentiation, during which they synthesize new ribosomal cytoplasmic complement, while its organelles appear to be proliferating (Galatis, 1974). It is interesting that the 'preparatory' as well as the final differentiation of meristemoids II and GMC into P-SM and P-GMC resembles processes of differentiation occurring in guard cells. However, the foregoing observations clearly suggest that the 'preparatory' differentiation, i.e., the cell growth, is not necessary for the operation of differentiation of GMC and meristemoids II into P-GMC and P-SM; it seems likely that it is related to its successful completion and to the final size attained by these idioblasts. The larger GMC and meristemoids II become larger P-GMC and P-SM. The interesting question that arises now is why the P-GMC and P-SM do not take the form of the guard cell, while they gain most of the guard cell characteristics. It is well known that the development of an irregular cell shape is closely related to the microfibrillar wall texture. In the case of stomata the kidney shape of the guard cells is possibly determined by the radial orientation of the microfibrils from the rim of the stomatal pore toward the dorsal walls (Palevitz and Hepler, 1976). In Vigna sinensis this patterned microfibrillar deposition seems to be controlled by an underlying system of microtubules radiating from the pore parallel to the microfibrils (Galatis, 1974). Similar observations have been made by Singh and Srivastava (1973) and Palevitz and Hepler (1976). According to the foregoing considerations, the absence of a radial arrangement of wall microfibrils in P-GMC and P-SM can be attributed to the absence of a microtubular system similar to the one found in guard cells. In guard cells, microtubules appear not only to influence the orientation of wall microfibrils, but also to determine the local deposition of material in about the middle of the ventral wall, where the stomatal pore will later develope (Kaufman etal., 1970; Landr6, 1970b; Srivastava and Singh, 1972; Singh and Srivastava, 1973 ; Galatis, 1974). Such microtubular activity is known from numerous other studies (see reviews by Newcomb, 1969; Hepler and Palevitz, 1974). In P-SM and P-GMC the rather symmetrical distribution of microtubules possibly results in the equal deposition of material along the over-all length of anticlinal walls. It seems likely that only the parie-
113 tal cytoplasm abuted on definite areas of the middle of the anticlinal wall resulting from the symmetrical division of the GMC has the ability to function as an organizing center for a unique microtubular system. To conclude the above discussion, it may be suggested that the microtubules have a particular morphogenetic role in the differentiating guard cells. Under conditions used here, this problem cannot be approached directly by studying the action of colchicine on microfibrillar texture of the wall and on the shape of stomata. The colchicine effect on the microtubules is of short duration and takes place only in very young stomata (possessing plasmodesmata). In the slightly abnormal in shape stomata, the microtubules soon reappear and the patterned wall thickening as well as the microfibrillar orientation occurs as in the untreated stomata. Since microtubular control of the microfibril orientation in the wall has not been definitely accepted (see O'Brien, 1972) and since it is possible that colchinine in high concentrations may affect cellular processes unrelated to microtubules (Hart and Sabnis, 1976) the preceding suggestion about the morphogenetic significance of microtubules in guard cells needs further experimental documentation. The single guard cells observed in several plants by Reese (1950), Dehnel (1961) and other investigators have also been observed in Vigna sinensis (data, not shown). In this plant they do not seem to represent differentiated GMC or meristemoids II, but always result from an early degeneration of their partners in young stomata. In these stomata the guard cells are considerably unequal in size and seem to have derived from unsuccessful symmetrical divisions of GMC. Among the specific physiological properties displayed by the guard cells, we could study and verify the ability of starch synthesis from exogenously supplied sugars and the existence of a potassium ion pump between P-SM or P-GMC and their surrounding cells. If the potassium ion pump operating in guard cells results in the turgor change, which causes stomatal opening, it can be assumed that the existence of such a mechanism in the P-GMC and P-SM may imply the mechanism of guard cell movement in these cells too. The failure of this activity to be expressed as a change in cell shape is due to the fact that they lack the proper wall structure and ultrastructure. As a general concluSion of the present paper it can be said that the P-GMC and P-SM of Vigna sinensis are actual!y guard cells in structure and possibly in some functional activities, although they lack their form.
114
References Allaway, W.G., Setterfield, G.: Ultrastructural observations on guard cells of Vicia faba and Allium porrum. Canad. J. Bot. 50, 1405-1413 (1972) Aylor, D.E., Parlange, J-Y., Krikorian, A.D. : Stomatal mechanics. Amer. J. Bot. 60, 163-171 (1973) Brat, L., Weber, F.: Anthoxanthin in den Schliegzellen der Kronblfitter yon Victoria amazonica und Nymphaea zanzibariensis. Phyton 3, 22-28 (1951) Brown, W.V., Johnson, Sr. C.: The fine structure of the grass guard cell. Amer. J. Bot. 49, 110-115 (1962) B/inning, E., Sagromsky, H. : Die Bildung des Spalt6ffnungsmusters in der Blattepidermis. Z. Naturforsch. 3b, 203-.216 (1948) B/inning, E., Biegert, F. : Die Bildung der Spalt6ffnungsinitialen bei AIlium cepa. Z. Bot. 41, 17-39 (1953) Dehnel, G.S. : Abnormal stomatal development in foliage leaves of Begonia aridicaulis. Amer. J. Bot. 48, 129-133 (1961) Fischer, R. A.: Role of potassium in stomatal opening in the leaf of Viciafaba. Plant Physiol. 47, 555-558 (1971) Galatis, B.: Ultrastructural studies on stomatal development. Ph. D. Thesis, University of Athens, Athens 1974 Gavaudan, P. : Sur les tissus a constitution mixte diplo'ide et polyploide d~velopp6s chez les v~g6taux par action de la colchicine. C.R. Soc. Biol. (Paris) 128, 717-719 (1938) Gertz, O. : Studier 6fver klyf6ppningarnas morfologi reed s/irskild hfinsyn till deras patologiska utbildnings former. Acta Univ. Lurid N.F. 15, 3-84 (1919) Guyot, M. : Action de la colchicine sur le d+veloppement des stomates de Viciafaba L. C. R. Soc. Biol. (Paris) 158, 1722-1725 (1964) Guyot, M., Pikusz, A., Humbert, C. : Action de la colchicine sur les stomates de Dianthus caryophyllus L. C. R. Acad. Sci. (Paris) D 266, 1251-1253 (1968) Hart, J.W., Sabnis, D.D.: Colchicine and plant microtubules: a critical evaluation. Curt. Adv. Plant Sci. 8 (8), 1095 1104 (1976) Hepler, P.K., Palcvitz, B.A.: Microtubules and microfilaments. Ann. Rev. Plant Physiol. 25, 309-362 (1974) Humbert, C., Guyot, M. : Modifications ultrastructurales des cellules stomatiques d'Anemia rotundiJblia Schrad. C. R. Acad. Sci. (Paris) D 274, 380-382 (1972) Kaufman, P.B., Petering, L.B., Yocum, C.S., Baic, D. : Ultrastructural studies on stomata development in internodes of Arena sativa. Amer. J. Bot. 57, 33-49 (1970) Kenda, G., Weber, F. : Stomata - Zahl vergr/inter Verbascum Blattaria - Kronblfitter. Osterr. Bot. Z. 97, 503-509 (1950) Konagamitsu, Y., Ono, H. : The period of starch forming ability in guard cell during the shaping of stomata. Sieboldia 2, 131-135 (1959) Kropfitsch, M. : Apfelgas-Wirkung auf Stomatazahl. Protoplasma 40, 256-265 (1951 a) Kropfitsch, M.: UV-Bestrahlung und Stomatazahl. Protoplasma 40, 266-274 (1951 b) Landr6, P. : Premi6res observations sur l'6volution infrastructurale des cellules stomatiques de la Moutarde (Sinapis alba L.) depuis leur mise en place jusqu'/t l'ouverture de l'ostiole. C. R. Acad. Sci, (Paris) D 269, 943-946 (1969a) Landr~, P.: Quelques aspects infrastructuraux des stomates des cotyl6dons de la Moutarde (Sinapis alba L.). C. R. Acad. Sci. (Paris), D 269, 990-992 (1969b) Landr6, P. : Activit6 golgienne en liaison avec celle du plasmalemme dans les cellules stomatiques de la Moutarde (Sinapis alba L.) lors de la formation de l'ostiole. C.R. Acad. Sci. (Paris) D 271, 904-907 (1970)
B. Galatis: Differentiation of Stomatal Meristemoids Landr6, P. : Origine et d~veloppement des ~pidermes cotyl6donaires et foliaires de la Moutarde (Sinapis alba L.). Diff6renciation ultrastructurale des stomates. Ann. Sci. Nat. Bot. 12" S. Tome XIII, 247-322 (1972) Macallum, A.B. : On the distribution of potassium in animal and vegetable cells. J. Physiol. 32, 95-118 (1905) Meidner, H., Mansfield, T.A.: Physiology of Stomata. London: McGraw-Hill (1968) Newcomb, E.H. : Plant microtubules. Ann. Rev. Plant Physiol. 20, 253-288 (1969) O'Brien, T.P. : The cytology of cell-wall formation in some eukaryotic cells. Bot. Rev. 38, 87-118 (1972) Palevitz, B.A., Hepler, P.K. : Cellulose microfibril orientation and cell shaping in developing guard cells of Allium: The role of microtubules and ion accumulation. Planta (Berl.) 132, 71-93 (1976) Pallas, J.E. Jr.: Guard-cell starch retention and accumulation in the dark. Bot. Gaz. 125, 102-107 (1964) Pallas, J.E. Jr., Mollenhauer, H.H. : Electron microscopic evidence for plasmodesmata in dicotyledonous guard cells. Science 175, 1275-1276 (1972a) Pallas, J.E. Jr., Mollenhauer, H.H. : Physiological implications of Vicia faba and Nicotiana tabacum guard-cell ultrastructure. Amer. J. Bot. 59, 504-514 (1972b) Pant, D.D.: On the onogeny of stomata and other homologous structures. Plant Sci. Allahabad Ser. I, 1-24 (1965) Raschke, K.: Stomatal action. Ann. Rev. Plant Physiol. 26, 309-340 (1975) Reese, G. : Beitrgge zur Wirkung des Colchicins bei der Samenbehandlung. Planta (Berl.) 38, 324-376 (1950) Schwendener, S. : Ober Bau und Mechanik der Spalt6ffnungen. Sitzgsber. Berl. Akad. Wiss. Berlin 833-867 (1881) Singh, A.P., Srivastava, L.M.: The fine structure of pea stomata. Protoplasma 76, 61-82 (1973) Srivastava, L.M., Singh, A.P.: Stomatal structure in the corn leaves. J. Ultrastruct. Res. 39, 345-363 (1972) Stebbins, L.G., Jain, S.K. : Developmental studies of cell differentiation in the epidermis of monocotyledons. I. Allium, Rhoeo and Commelina. Develop. Biol. 2, 409~26 (1960) Stebbins, L.G., Shah, S.S. : Developmental studies of cell differentiation in the epidermis of monocotyledons. II. Cytological features of stomatal development in the Gramineae. Develop. Biol. 2, 477-500 (1960) Thomson, W.W., De Journett, R.: Studies on the ultrastructure of the guard cells of Opuntia. Amer. J. Bot. 57, 309-316 (1970) Tonzig, S., Ott-Candela, A. : L'azione della colchicina suflo sviluppo degli apparati stomatici. Nuovo Gior. Bot. Ital. N.S. 53, 535-547 (1946) Weber, F.: Spalt6ffnungsapparat -- Anomalien colchicinierter Tradescantia-Blfitter. Protoplasma 37, 556-565 (1943) Weissenb6ck, K.: Studien an colchizinierten Pflanzen I. Anatomische Untersuchungen. Phyton 1, 282-300 (1949) Whatley, J.M. : The ultrastructure of guard cells of Phaseolus vulgaris. New Phytol. 71, 175-179 (1972) Ziegenspeck, H. : Die Farbenmikrophotographie ein Hilfsmittel zum objektiven Nachweis submikroskopischer Strukturelemente. Die Radiomicellierung und Filierung der SchlieBzellen yon Ophioderma pendulum. Photographic und Wissenschaft 4, 19 22 (1955a) Ziegenspeck, H. : Das Vorkommen yon Fila in radialer Anordnung in den SchlieBzellen. Protoplasma 44, 385-388 (1955b) Ziegler, H., Shmueli, E., Lange, G. : Structure and function of the stomata of Zea mays. I. The development. Cytobiologie 9, 162-168 (1974) Received 20 December 1976; accepted 6 May 1977
