Developmental studies of cell differentiation in the epidermis of monocotyledons

DEVELOPMESTAL

Developmental

BIOLOGY,

477-500

2,

Studies of Cell Differentiation Epidermis of Monocotyledons

II. Cytological

Features of Stomata1 in the Gramineae’

G. L. Department

( lg(io)

of Genetics,

STEBBISS Unkersity

Accepted

S. S.

AND

Development

SHAH

of California, June

15,

in the

Dads,

Culifornh

1960

INTRODUCTION

In another paper of this series (Stebbins and Jain, 1960) the senior author has pointed out the potential value of studying developmental processes in epidermal cells of higher plants as a means of unraveling the complex sequence of events that connects the primary biochemical action of genes with their ultimate effects in determining morphological differences. The present contribution describes some features of stomata1 development in Gramineae and presents data that may lead toward an understanding of the physiological processes that determine the morphological changes observed. Species of grasseswere selected as a major object for investigation both because they are easily available and because certain species, such as maize and barley, are well known genetically. The adult stomata1 complex of grassesconsists of two guard cells that are narrow, elongate, and have much thickened walls, flanked by two subsidiary cells, which are of the same length as the guard cells but have walls of normal thickness. This complex was recognized as early as 1784 by Hedwig, and its development has been carefully studied by several authors (Strasburger, 1866; Campbell, 1881; Porterfield, 1937; Flint and Moreland, 1946). Both the adult complex and the developmental sequence are essentially similar in all species of grassesstudied and are found in a great variety of families of monocotyledons, includ1 This G3737.

research

was

supported

by

the 477

National

Science

Foundation,

Grant

No.

478 ing such 1960 ) .

6.

primitive

L.

types

STEBBISS

as the

\lATERIAL

AND

S. S. SHAH

Alismatales

AND

(Stebbins

and

Khush,

METHODS

The species studied were chiefly Hordeum vu&we var, Atlas 46, Sorghum vu&we var. Standard Yellow, using seed obtained from the Agronomy Department, University of California, Davis, Dactylis g’omerntn subsp. lusitanicn, a diploid subspecies which has been grown in this department for several years and came originally from Portugal (Stebbins and Zohary, 1959). More cursory examinations were made of Avena fatua, Zea mays, and Ehrharta erecta. Seedlings were grown at 20-25O in darkness and at an age of 2-3 days were fixed for 24 hours in a modified Carnoy’s fluid: 6 parts absolute alcohol, 3 parts glacial acid, I part chloroform. After fixation the material was thoroughly rinsed and stored in 70% alcohol. Most of the observations were made on the lower (abaxial) surface of the basal region of the first leaf. The best preparations were temporary mounts, stained in acetocarmine diluted to $5 strength in 50% acetic acid. After staining under gentle heat similar to that applied to squash preparations of chromosomes, the acetocarmine was replaced by 50% acetic acid, and the preparation was sealed with a paraffin-beeswax mixture. Such preparations lasted for weeks or even months when kept in the refrigerator. Although adequate data could be obtained from whole mounts of meristematic regions, the best preparations, and the only ones that yielded good photomicrographs, were peels in which the epidermal cell layer was isolated from the remainder of the tissue. This was done by making first a diagonal incision with a sharp knife well above the meristematic region, then gently pulling downward with a finely pointed jeweler’s forceps the flap thus obtained. This operation was not difficult using 3-4-day-old seedlings of barley, in which the epidermal walls are relatively tough, but was much harder to do in Dacqlis, and nearly impossible in Sorghum and Zea, in which the walls of the meristematic epidermal cells appear to be much more delicate. Preparations were made permanent by dehydration in absolute alcohol and xylol and mounting in diaphane or balsam, but this method was difficult and erratic in results owing to the tendency of the thin epidermal peels to become curled or folded during the changing of solutions. The drawings presented were made with a camera lucida and are reproduced at a magnification of 1560 X.

STOMATAL

DEVELOPMENT

IX

479

GRAMINEAE

RESULTS

Although the development of the stomata1 complex in Gramineae has already been carefully described, particularly by Porterfield ( 1937)) our own observations of this sequence of events are presented here as a basis for further analysis. The distribution of the stomata1 complexes in all grass leaves is in vertical rows, within which a complex usually alternates with a single undifferentiated epidermal cell. These rows usually occur on the flanks of the vascular bundles (Fig. 1)) but they

FIG. 1. the psition

Outline sketch of the stomata1

of a cross section of a young rows relative to the vascular

leaf

of Hordcum

showing

bnndles.

are occasionally found directly above or below one of the smaller bundles. In Hordeum and many other genera the rows are normally single, but in Dactylis, Sorghum, and Zen, two or even three stomata1 rows usually occur adjacent to each other. The undifferentiated epidermal cells in the rows adjacent to the stomata1 rows are shorter than those at a greater distance from the stomata1 complexes. The differentiation sequence starts, therefore, with the formation of the stomata1 rows, This occurs in very young leaf primordia, since stomata1 rows are already differentiated on the first seedling leaf of embryos before seed germination. The nature of this earliest differentiation process is not yet known. The rows first become evident through more rapid divisions which produce a large number of small cells. Divisions in the rows on either side of the stomata1 row produce cells that are smaller and more numerous than those in the completely

480

G. L.

STEBBIKS

AND

S. S. SHAH

undifferentiated epidermal cells between the rows (Figs. 2 and 3). The sequence of mitoses in the stomata1 rows ends with an asymmetrical division, resulting from the polarization of the cytoplasm, which is denser near the distal ends of all of the cells in this region (Fig. 3). Hence there occurs a polarization-asymmetry sequence, such as those described for Alliun~ and the Commelinaceae (Stebbins and Jain, 1960), which produces distally a small cell, with a smaller, heavily staining nucleus, and proximally a large, more weakly staining cell, the cytoplasm of which quickly becomes vacuolate, as can be seen in living material. The distal cell is the guard mother cell (GMC ). and the proximal one is destined to become an undifferentiated epidermal cell. Soon after it is formed, the GMC enlarges somewhat and its walls become convex adjacent to the epidermal cells in the same row, lvhile remaining flat on the sides opposite the lateral epidermal cells. There then follows a series of asymmetrical mitoses undergone by these lateral epidermul cells, producing two subsidiary cells per GMC (Figs. 4 and S). After subsidiary cell formation is complete the GMC divides transversely to form the two guard cells (Fig. 6), and the whole complex matures (Fig. 7). At maturity, the nuclei of the guard cells are elongate and dumbbell shaped, their middle portion being constricted by the much thickened cell walls, and those of the subsidiary cells have a typical ellipsoid shape (Fig. 8). The asymmetrical divisions to form the subsidiary cells are particularly favorable for studying the asymmetry in chromosomal behavior, since the cells are relatively large and the chromosomes are comparatively well spread out, In the numerous anaphases observed the chromosomes were identical in size and form at the two poles, and no difference could be seen between the two chromosomal groups until the nuclear membrane had formed in early telophase. After this stage, the nucleus which remains in the epidermal cell increases rapidly in size, while that belonging to the subsidiary cell retains essentiallv the same volume. On the other hand, the differential behavior ok the Figs. 4, 5, formation of subsidiaries; Figs. to form the 4-celled stomata1 complex; Fig. x 300; Fig. 3: x 600; Figs. 5, 6: x 1800. at the right. FIG. 9. A portion of a double row of Izktonicn, showing subsidiary cell formation. distal end of the leaf at the right.

6, 7, division of the guard mother cell 8, two mature stomata. Figs. 2, 4. 7, 8: All are oriented with distal end of leaf stomata in Dactylis 1lngnification: x

glomerckn 600. Oriented

~ubsp. with

STOMATAL

DEVELOPMENT

IN GRAMINEAE

481

FIGS. 2-8. Photomicrographs showing stages in development of the stomata1 apparatus in the first seedling leaf of Hordeum vulgare var. Atlas 46. Fig. 2, formation of guard mother cell; Fig. 3, the same, stained by the Feulgen technique;

482

C.

L.

STEBBINS

AND

S. S.

SHAH

chromosomes is sometimes evident, and in this respect the epidermal nucleus retains the telophase coiling of the chromonemata for a longer time than does the subsidiary cell nucleus (Fig. 5). The succession of developmental stages, although proceeding in general from base to apex of the leaf, shows many irregularities in this

la

3

lb

FIG. 10. Diagrams to show the five successive stages in stomata1 development. la, lb, formation of guard mother cell; 2, formation of subsidiaries; 3, subsidiaries completely formed; 4, division of guard mother cell; 5, completed stomata1 complexes.

respect, with earlier stages often appearing distal to later ones. In future work, the disturbance of deveIopmenta1 regularity that can be produced by environmental agents, as well as genotypicahy controlled alterations in the deveIopmenta1 sequence, will be measured by changes in the amount of regularity in the succession of stages, as well as in the relative proportion of the different stages in the stomata1 rows.

STOMATAL

DEVELOPMENT

IN

GRAMINEAE

483

For this reason a graphical representation of the succession of stages in a stomata1 row seemed desirable. This was done by dividing the process of development into five stages, as follows (see diagrams, Fig. 10). (1) differentiation of the GMC from the epidermal cell proximal to it; (2) A, B: differentiation of the two subsidiary cells; (3) “triad” stage, consisting of a GMC and two subsidiary cells, their nuclei in the metabolic condition; (4) division of the GMC; (5) completed stomata1 complex of four cells. Several stomata1 rows have been scored in this fashion, starting with the first (most proximal) complex in the 2A stage. Since the distinction between a fully differentiated GMC (stage 1) and a stage prior to the asymmetrical division which produces the GMC cannot be clearly made in all instances, an earlier, more proximal, beginning of the scoring process was not sufficiently reliable. The scoring was ended when 10-12 consecutive complexes in stage 5 had been observed. Scores for two typical rows are shown in Fig. 11. In the material studied, 155 and 147 complexes, respectively, were scored between the two limits set. The irregularity is most clearly evident at the upper end of the sequence, where the drop from stage 5 to stage 1 is not uncommonly found. Since studies of completely mature stomata1 rows have revealed from 1 to 4% of GMC’s that have failed to differentiate, some of these drops may represent such failure rather than extreme irregularities of timing. This irregularity can be estimated quantitatively by a scoring system that measures the amount of deviation from complete regularity in the succession of stages. An “index of irregularity” was established as follows. The number of complexes at each of the five stages was counted for the row, and an “ideal sequence” was made; this begins with a number of complexes at stage 1 equal to the number counted, followed by the number counted of complexes at each of the later stages in regular succession. Then by comparing the stage of each individual complex in the actual row with that of the complex occupying a corresponding position in the “ideal sequence,” figures of deviation ranging from 0 to 4 were obtained for all the complexes in the row. Their sum, divided by the number of complexes in the row, constitutes the “index of irregularity” for the row. The index values for the rows represented in Fig. 10 are 0.490 and 0.585. The presence of this irregularity is evidence in favor of the hypothesis that polarization is brought about by forces operating within

484

G. L.

STEBBINS

AND

S. S.

SHAH

each individual cell, rather than by any general gresses along the row. Further evidence for this by certain rare abnormalities of development. In sidiary cells were found adjoining two successive

ROW

in5uence which propostulate is provided a few instances, subcells of the stomata1

1.

“I

50

NUMBER FIG.

11.

successive at 3 days’ the text.

lb0

OF

I50

COMPLEXES

Diagram showing the degree of irregularity in the development of stomata1 complexes in two rows of the first seedling Ieaf of barley, fixed age after having been grown in darkness at 20”. Further explanation in

row (Fig. 12). Sometimes this was followed by division of only one of the cells in the row, so that the mature configuration consisted of a pair of subsidiaries flanking a pair of guard cells plus a short undifferentiated epidermal cell. In other instances both cells of the stomata1 row divided transversely, giving rise to “twin stomata” which consisted of two adjacent pairs of guard cells flanked by a single pair of subsidiaries (Fig. 17). These were separated from the normal stomata1 complexes proximal and distal to them by the usual intervening epidermal cells.

STOMATAL

DEVELOPMENT

IN

GFtAMINEAE

485

16

FIGS. 12-16. Drawings showing some unusual abnormalities in the development of the stomata1 complex. Figs. 12, 15, 16 are from Ho&urn; Figs. 33, 14 from Sorghum. Fig. 12. Division of two adjacent guard mother cells, flanked by one pair of subsidiaries, which wilI lead to the formation of “twin stomata,” as in Fig. 17. Fig. 13. Twin “guard mother cells,” one divided and one undivided, with two nuclei in one of the subsidiaries. Fig. 14. An extra division of one of the subsidiary cell nuclei, photographed in Fig. 18. Fig. 15. Formation of two subsidiary cells on the same side of a GMC, as a result of the position of a dividing wal1 opposite the middle of the GMC. Fig. 16. Occurrence of a large subsidiary which covers two GMC’s plus the intervening epidermal ceI1. All figures reproduced x 1560.

486

G. L.

STEBBINS

AND

S. S. SHAH

The best explanation of this abnormality is to assume that in rare instances a protodermal cell in the stomata1 row fails to go through its final division which would produce a GMC and an intervening cell. Such a cell might be capable of both induction and later division to form guard cells, and would constitute the distal of the two “GMC’s” in the twin stomata1 complex. The even more complex abnormality found in Sorghum and illustrated in Fig. 13 could be explained by assuming that the same sequence of events could be followed by transverse division of the subsidiary cell. Division of a subsidiary cell located in the normal position was found once in both Hordeum and Sorghum, and is illustrated in Figs. 14,18, and 19. Regularities

and Abnormalities

in Subsidiary

Cell Formation

The presence of an inductive force emanating from the GMC, which brings about division of the nuclei of lateral epidermal cells to form subsidiary cells, is indicated by the following lines of evidence. 1. At the time when these divisions are occurring, no other epidermal cells are dividing. In the leaf meristem, mitoses can be seen in the parenchyma cells of the subepidermal layer, but even these are absent at the time when lateral epidermal cells are dividing for subsidiary cell formation in stomata1 complexes of the developing awn of the lemma. 2. Before division, the epidermal nucleus occupies a position close to the GMC. Later its mitotic spindle, although oriented at various angles, always has one pole directed toward the GMC. 3. The number of divisions carried out by an epidermal cell corresponds exactly to the number of GMC’s to which it is laterally adjacent. In Hordeum and Dactylis, for instance, the longer of the lateral epidermal cells have a length equal to four or six times that of the cells in the stomata1 row, and so are adjacent to two or three GMC’s (Figs. 4, 7, 9). Their nuclei, therefore, divide two or three times, to produce as many subsidiary cells, while the nuclei of the shorter lateral epidermal cells divide only once. On the other hand the nuclei of the epidermal cells that alternate between GMC’s in the stomata1 row, which will be termed intervening cells, usually do rot divide at all in Hordeum, in which the stomata1 rows are ordinarily single. In Dactylis, Sorghum, and Zea two stomata1 rows normally occur side by side, with the intervening cell of one row lying beside

STOMATAL

DEVJSLOi’M%NT

IN

GIIAMINEAE

487

the GMC of the adjacent row (Fig. 9). These intervening cells regularly divide to provide subsidiary cells for the GMCs adjacent to them. In the occasional examples of double stomatal rows found in Hordeurn, the intervening cells also divide. 4. Occasionally the wall dividing two lateral epidermal cells emerges from the middle of the lateral wall of a GMC, so that the two halves of the GMC are adjacent to different lateral epidermal cells (Figs. 15 and 19). In this case, both epidermal cells divide, and the mature complex has three subsidiaries, two on one side and one on the other. This abnormality was seen repeatedly in l-2% of the complexes of Hordeum and has also been noted in mature complexes of Scirpus and Juncus. Another abnormality suggests that GMC’s adjacent to the same lateral epidermal cell may compete with each other in attracting its nucleus for the formation of a subsidiary. Figure 16 shows a single example noted in which a giant subsidiary covers three cells, two GMC’s and the intervening cell. This would develop into the type of mature complex shown in Fig. 20, which also was seen very rarely. It would be most easily explained by assuming that the attractive forces of the two GMC’s were so nearly equal that the nucleus of the lateral cell divided while occupying a position exactly intermediate between the two GMC’s. Evidence that consecutive GMC’s differ in the intensity of the inductive force which they exert at any one time is provided by observations of pairs of GMC’s which are flanked on eith& side by a single pair of long laterals, as in Fig. 22A. In most examples of this type observed in Hordeum the lateral nuclei divided successively opposite the same GMC, so that one of the two GMC’s acquired both subsidiaries before the second acquired any, In 123 of the examples the first GMC to acquire subsidiaries was the distal one, while in the remaining 75 examples the proximal GMC first acquired subsidiaries. Since the over-all progression of stomata1 differentiation is basipetal, the acropetal differentiation of a particular pair of complexes in the lastmentioned examples is opposite to the general trend and is further evidence that the differentiation of GMC’s is largely an autonomous function of individual cells. Further studies of the succession of subsidiary cell formation suggest that the competence of the lateral epidermal cell to respond to the inductive stimulus of the GMC depends partly upon the size of the

488

G. L.

STEtBBINS

AND

S. S. SHAH

FIGS. 17-21; 23-25. Photomicrographs showing abnormalities in stomata1 development. All are from Hordeurn except for Fig. 18, which is from Sorghum. Fig. 17. Mature “twin stomata.” Figs. 18 and 19. Abnormalities of subsidiary formation, comparable to the drawings (Figs. 14 and 15). Fig. 20. Two stomata and an intervening cell, covered by a pair of giant subsidiaries. Fig. 21. Abnormal stomata1 complex with one subsidiary missing. Fig. 23. Normal transverse division and abnormal longitudinal division (at left) of a GMC, associated with abnormal elongation of the cell. Figs. 24 and 25. Immature and mature complexes resulting from the abnormal type of division illustrated in Fig. 23. Figs. 17, 20, 21: x 800; Figs. 18, 19: x 1800; Figs. 23-25: x 600.

STOMATAL

DEVELOPMENT

22-A.

da 000 00 +!3%

IN

489

GRAMINEAE

I”“.0 PD I (o)o(o)cq3) (cj0”‘(0)0 (0) I07 BLL.

22-B.

22-E.

FIG. 22. Diagrams showing the relationships between guard mother cells and lateral epidermal cells which lead to regular succession in subsidiary cell formation. A. Two GMC’s flanked by a pair of long laterals directly opposite to each other. B. Two GMC’s flanked on one side by a long lateral and on the other by two short laterals. C. Three GMC’s, flanked by long laterals which are not opposite each other, and by short laterals. Double arrows indicate directions in which the nucleus of the lateral epidermal cell nearly always divides first to produce a subsidiary cell; double arrows consisting of one continuous and one broken line indicate less strong tendencies. D and E. Surface view and cross section of part of a double stomata1 row in Dactylis, showing the positions of the types of cells described. For explanation of abbreviations, see text.

lateral cell. Critical evidence was obtained in Hordeurn from examples in which a GMC was flanked on one side by a long lateral and on the other by two short laterals, as in Fig. 22B. In 133 examples of this type observed, the first subsidiary was formed by the long lateral in 132 cases, and by the short lateral in only 1 case. Even more critical evidence in favor of the assumption that large laterals form subsidiaries more readily than small ones was provided by studying the more

490

G. L.

STEBBINS

AND

S. S. SHAH

complex configurations found in Dactyl& Here the stomata1 rows are usually double, so that each G-MC is flanked on one side by a lateral and on the other by an intervening epidermal cell (Figs. 9 and 22E). The lateral cells on the side of the double row which faces the bundle are smaller than those on the “outer” side away from the bundle, partly because they are usually somewhat narrower, and also because the epidermal cells in the vicinity of a bundle are more flattened than those between bundles, as can be determined both from cross sections and by focusing downward with the fine adjustment screw in the usual whole mounts. The order of size of the various types of epidermal cells which can flank a GMC is, therefore, as follows, from largest to RELATION BETWEEN FORMATION IN LATERAL

TABLE I CELL LENGTH AND PRECEDENCE IN SUBSIDIARY CELL EPIDERMAL CELIS OF Dactylis glomerata SUBSP. lusitanica Number of examples where first subsidiary is formed by:

Types of eella opposite each othw

Outer long oil (second Bundle side bll (second Outer short Bundle side

(oil)-bundle side intervening subsidiary)-bi long (bZl)-outer intervening subsidiary)-& (osZ)-bundle side intervening short (bsl)--outer intervening

(bi) (oi) (bi) (oi)

Longer cell

Shorter cell

Total

144 32 68 6 62 39

1

1 3 2 27 -

145 38 69 9 64 66 -

40

391

351

6

smallest: (1) long lateral on side away from bundle (otl); (2) long lateral nearest bundle (bll); (3) short lateral away from bundle (osl); ‘(4) short lateral nearest bundle (bsl); (5) interstitial away from bundle ( oi) ; (6) interstitial nearest bundle (bi) . Table 1 gives the figures for 391 GMC’s scored on one leaf of Dactylis glomerata subsp. lusitanica. When an outer long lateral ( oZZ) was opposite an interstitial cell of the row nearest to the bundle (bi), it formed the first subsidiary in all but one of the 145 examples observed. Furthermore, in 32 of 38 examples observed, the nucleus of the outer long lateral divided a second time before the second of the two GMC’s adjacent to it had acquired a subsidiary from the interstitial cell (bi) on the other side. The behavior of long laterals on the bundle side (bll) was similar, although fewer examples were available. In 68

STOMATAL

DEVELOPMENT

IN

GRAMINEAE

491

out of 69 examples observed, a long lateral nearest the bunde (bll) formed its first subsidiary before the outer intervening cell opposite to it. Only 9 examples were observed of the second subsidiary of a long lateral on the bundle side opposite an outer intervening cell; here the figures were 6 to 3 in favor of the long lateral. In respect to short laterals the differences between the outer and the bundle side of the stomata1 row was the most striking; the outer short lateral (osl) was ahead of the intervening cell on the bundle side (bi) in 62 out of 64 examples, and the bundle side short lateral (bsE) was ahead of the outer intervening cell (oi) in only 39 out of 66 examples. These data indicate that the greater the size difference between the two laterals adjacent to a particular GMC, the greater is the chance that the larger lateral will be the first to form a subsidiary. Similar observations made on Avena fatua indicate that the same relationship holds also in this species. Another regularity in the formation of subsidiaries occurs when a GMC lies between the distal and the proximal ends of two long laterals (Fig. 2%). In this case the first subsidiary is usually formed by the lateral with its distal end opposite to the GMC. For Hordeum the figures obtained were 52 examples of distal preceding proximal, and 10 of the reverse. The comparable figures for Avenu fatua are 30 to 6. The assumption that the precedence of a subsidiary over the one opposite to it is actually due to a greater competence to react to the stimulus of the GMC is strengthened by data from the distribution of abnormalities in stomata1development produced in certain experiments that will be described in greater detail elsewhere. Following the suggestion of Dr. Daniel Mazia, e-day-old seedlings of Atlas barley were immersed for periods of l-4 hours in solutions of mercaptoethanol, a chemical agent that, because of its production of chemically active SW groups, interferes with the formation of the mitotic spindle. One effect of this treatment was to produce a large number of mature stomata with one of the subsidiaries missing (Fig. 21). In mature leaves of seedlings which had undergone this treatment, 34 abnormal stomata1 complexes of this type were scored in which one of the flanking lateral cells was of the long, and the other of the short, type; in 23 examples the missing guard cell was next to the short lateral and in only 11, opposite the long lateral. In eight of the latter examples, the missing subsidiary would have been formed at the proximal end of a long lateral, and since a subsidiary had been formed at the distal end of the

492

G. L.

STEBBINS

AND

S. S. SHAH

same lateral, these examples probably represent failure of its nucleus to divide twice. In the normal developing epidermis of Ho&urn, the second division of the nucleus of a long lateral often contributes a subsidiary after the short lateral opposite it; hence these 8 examples cannot be considered critical. More significant were the data on missing subsidiaries in examples in which the distal and proximal ends of long laterals were on opposite sides of the same GMC, as in Fig. 22C. In 22 examples of this type, the missing subsidiary was opposite the proximal end in 21 cases and the distal end in only 1 example. Another type of abnormality, illustrated in Figs. 23-25 may help to explain why the mitotic spindle of the GMC division to form the guard cells is transverse, and therefore at right angles to all the other divisions that take place in the leaf at this stage, except for the induced divisions that form the subsidiary cells. Very rarely in normal leaves the GMC division was longitudinal, and hence at right angles to its normal orientation. This condition was much more frequent in seedlings that had been subjected to mercaptoethanol treatment according to a method which will be described elsewhere. As a result of this change, the two guard cells become placed in a proximal-distal position, and the mature stomata1 complex, although possessing a full complement of cells, is nonfunctional because of the aberrant shape of the guard cells (Fig. 25). Although most of the guard cells lying in this position had well-differentiated walls as in Fig. 25, sometimes their walls failed to develop and were as thin as those of the subsidiary cells. Figures 23 and 24 show that when this abnormality occurs the cells showing it are distinctly more elongated than the neighboring GMC’s which divide in the usual fashion. This difference was consistent in all observed examples of this type of abnormality and suggests that a relationship exists between the orientation of the mitotic spindle and the shape of the GMC. The fact that the GMC normally divides at right angles to the plane of division of the surrounding cells is apparently associated with its nearly isodiametric shape. Differences

between

Hordeum

and Sorghum

Since the genera Hordeum and Sorghum are almost at opposite ends of the classification system of the grass family, differences between them in the developmental features of the stomata1 complexes may reflect the amount of variation that may be expected in the family as

STOMATAL

DEVELOPMENT

IN

493

GRAMINEAE

a whole. These are of two general types: differences in the arrangement and shape of the cells, and those in the appearance and behavior of the nuclei. In respect to cell arrangement, one difference has already been noted: in Hordeum the stomata1 rows are usually single, whereas in Sorghum they are usually paired. Since Dactylis, which belongs with Hordeum in the subfamily Festucoideae, resembles Sorghum in having is probably not of major paired stomata1 rows, this characteristic taxonomic significance. A second difference lies in the relative length of the cells in the stomata1 row as compared to those in the adjacent rows. One measure of this difference is the proportion of lateral epidermal cells that lie adjacent to two GMC’s, and consequently form two subsidiaries. This was found to be 40% in Hordeum and only 3.5% in Sorghum (Table 2). A contingency table analysis for chi square TABLE 2 OF LATERAL EPIDERMAL

FREQUENCY Type of cell Short Long

(forming (forming

1 subsidiary) 2 subsidiaries)

CELL

Hordeurn

Sorghum

106 70 (39.8%)

249 23 (3.5%) 272

176 a X2 = 64.23;

significant

at 0.1%

TYPEP

level.

based upon these data gave a highly significant result. A third difference in cell arrangement concerns the number of epidermal cells intervening between two complexes in a stomata1 row, as scored at stage 5 (cell division complete). In Hordeum, this number is almost always 1, with intervals occupied by two epidermal cells representing only 6.3% of the total (Table 3). In Sorghum the number of intervals TABLE 3 OF ONE- AND TWO-CELLED STOMATAL COMPLEXEP

FREQUENCY

Type of interval With With

one intervening two intervening

epidermal epidermal

significant

BETWEEN

Hord~um

cell cells

949 64 (6.37~) 1013

a X2 = 19.11;

INTERVAIA

at 1%

level.

Sorghum

193 34 (15.Oy&) 227

494

G. L.

STEBBINS

AND

S. S.

SHAH

containing two epidermal cells is 15%, a difference that is highly significant according to the contingency table test for chi square. The best explanation for this difIerence is that in Sorghum a significantly higher number of cells in the stomata1 row fail to divide after the cytoplasm has become polarized, and so do not carry through the differentiation into a GMC and an intervening epidermal cell. In respect to the complexes themselves, Hordeurn, which represents the festucoid type, differs from Sorghum and Zea, representing the panicoid type, in the shape of the subsidiary cells. As has been pointed out by numerous authors (Avdulov, 1931; Stebbins, 1956), the festucoid type of stomata1 complex is oblong in outline, due to the elongateellipsoid subsidiary cells, whereas the panicoid type is lozenge-shaped, since the subsidiary cells are broader relative to their length and are nearly triangular in shape. This difference in adult subsidiary cells in due chiefly to the greater amount of cell elongation found in Hordeunr and other festucoid genera. Immediately after their formation, the subsidiary cells are somewhat shorter and broader in Sorghum and Zen than in Hordeum, Dactylis, or Avena. Furthermore, the intervening epidermal cells are broader relative to the GMC in the panicoid genera, while in the festucoid genera the GMC is nearlv as broad as the intervening cells. Hence the room for lateral expansion is greater in Sorghum and Zea than in the other genera. In respect to the nuclei themselves, the differences other than those present in all the metabolic nuclei of these two genera were hard to define. In most preparations, the difference in staining capacity between the darkly colored GMC and subsidiary cell nuclei and the larger, paler nuclei of the ordinary epidermal cells appeared to be more marked in Sorghum and Zea than in Hordeurn. This effect was particularly noticeable before the formation of subsidiary cells. In addition, the epidermal nuclei of Zen and Sorghum appeared to be more variable in size, although we could not be certain whether or not this was a fixation artifact. None of these differences could be defined with precision in the material and with the techniques available to us. DISCUSSION

1. Evidence

on the Nature of the Polarization-Asymmetry

Sequence

Stebbins and Jain (1960) p ointed out that the three critical stages in the differentiation of the stomata1 complex of monocotyledons are

STOMATAL

DEVELOPMENT

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495

the differentiation of the GMC from the intervening epidermal cell, the formation of subsidiaries through the inductive action of the GMC, and the transverse division of the GMC to form the two guard cells. In grasses, a fourth critical stage precedes the other three: the initial differentiation of the cells in the stomata1 row. Except for the final GMC division, all these stages are characterized by the sequence of events designated by Stebbins and Jain as the polarization-asymmetry sequence. This consists of the polarization of the cytoplasm within the cell, the mitotic division of the nucleus across the cytoplasmic gradient formed, and the consequent differentiation of the daughter cells because of differential nuclear-cytoplasmic interactions. The key to an understanding of this sequence will be provided chiefly by an explanation of the nature of the cytoplasmic polarization by which it begins. The present study provides only a few clues toward the solution of this difficult problem, but these are worth enumerating here as a guide for future research. First, polarization is apparently not governed entirely by any general influence that affects the entire tissue or even groups of similar cells uniformly, but is rather a property of individual cells. This is evident from the irregularity of GMC differentiation and subsidiary induction within the stomata1 rows. Although these processes proceed generally in a basipetal direction in the developing leaf, the irregularities of this progression are very great, and stomata1 complexes in the final stage of guard cell formation may lie immediately adjacent to complexes in which subsidiary formation has not even begun. This suggests to the authors that an explanation of polarization would lie not only in studying the action of substances such as hormones, which enter the tissue from elsewhere and might be expected to have a general effect, but more particularly in obtaining a better understanding of gene-cytoin the individual cells themselves during the plasmic interactions process of differentiation. This might be done through studies of DNA, RNA, and protein metabolism, both via differential staining and the behavior of radioactive tracers. Helpful information might also be obtained by preparations designed to show the activity of mitochondria and the distribution of various enzymes, as has been done by Avers (1958) and Avers and Grimm (1959)) and by learning the nature and distribution of the endoplasmic reticulum and the microsomes, using the electron microscope (see Whaley et al., 1959). A second fact that emerges from the present study is that the polarization-asymmetry sequences taking place in the grass epidermis all

496

G. L.

STEBBINS

AND

S. S. SHAH

occur at a period of transition between two developmental stages. The differentiation of stomata1 rows apparently occurs in the young primordia at the time when the neighboring procambial cells are in active division and when the epidermal cells distal to them are beginning to cease division and commence the maturation process. It is thus the stage when the initial formation of leaf primordia and the delimitation of tissues within them is nearing completion and when the over-all meristematic activity of the primordium is being replaced by the basal intercalary meristem that forms all the leaf blade except for its tip. The differentiation of the GMC, on the other hand, takes place when the surrounding epidermal cells are just completing their final division, i.e., at the transition from meristematic condition to that of cell maturation, in respect to the epidermis. Preliminary studies show that the polarizaion-asymmetry sequences that produce epidermal hairs and the short siliceous and suberous cells found in some regions of the epidermis occur in the same region. In the root, the polarization-asymmetry sequences that give rise to root hairs are likewise produced at the zone of transition between the meristematic region and the region of maturation (Avers and Grimm, 1959). This suggests that cytoplasmic polarization may be a by-product of alterations in cell physiology that mark the transition from one developmental stage to another. 2. Evidence

on the Nature

of Induction

The data presented in this paper indicate that the induction of subsidiary cell formation by the influence of the GMC’s depends upon both the stimulus of the GMC and the competence of the lateral epiderma1 cells to respond to this stimulus. The time when the stimulus of the GMC becomes effective varies greatly, even between GMC’s in close proximity to each other and resembling each other closely in size and general appearance. It appears, therefore, to be controlled by physiological forces that are not readily apparent upon cytological study. The suggestion of Biinning (1948, 1957) that induction depends upon a substance which emanates from the GMC, is the most plausible one, although as yet it is idle to speculate on what this substance might be. If such a substance exists, we must assume that each GMC produces it individually at varying rates. The competence of the lateral epidermal cells to respond to this stimulus depends in a regular fashion upon two factors: the size of the

STOMATAL

DEVELOPMENT

IS

GRAMIXEAE

497

cell and the position of the adjoining GMC, whether at its distal or proximal end. It seems unlikely that cell size per se is the determining factor; rather some factor associated with cell size is more likely. The larger cells have relatively large vacuoles, and preliminary experimeuts with plasmolysis, using solutions of mannitol varying between 10 and 15W, indicate that the large epidermal cells between the stomata1 rows are plasmolyzed much more easily than either the lateral epidermal cells or the GMC’s themselves, which are the most resistant to plasmolysis. This suggests that a gradient of decreasing osmotic pressure extends outward from the GMC and that the larger is the adjoining epidermal cell, the steeper may he the gradient. This gradient is due probably to a lower concentration of solutes in the lateral epidermal cells; hence the diffusion outward of mitosis-inducing substances might be expected to be more rapid if the osmotic gradient were steeper. The reduction of osmotic pressure with the increased cell size found in tetraploids has been reported by Becker ( 1931) in mosses, Schlijsser (1936) in the tomato, and Hesse (1938) in Petunia. The tendency for the distal ends of long epidermal cells to form subsidiaries before the proximal ends of the long cells opposite them must be due to other causes, since there is no evidence from plasmolvsis to indicate a distal osmotic gradient within the cells, and none wcLld be expected, On the other hand, there is some evidence to indicate that a tendency for distal polarization exists in all the epidermal cells at the time of subsidiary cell formation. The cells in the stomata1 row are always polarized distally at the time of GMC differentiation, and in the upper leaf sheaths and lemmas of Hordeurn, Avenu, and BTOI)ICI.S, as well as in the leaf epidermis of some speices of Agropyron and other grasses, distal polarization of all epidermal cells is evident from the fact that they undergo polarization-asymmetry sequences for the formation of the siliceous-suberous pairs of short cells. At this time, the nuclei of these cells lie at their distal ends. For this reason, it is likely that the distal polarization force is exerted on the nuclei of the lateral epidermal cells and reinforces the attraction of the GMC adjoining their distal ends. That an attraction exists between the GMC and the nuclei of the lateral epidermal ce1l.s is evident from the fact that in living tissues the epidermal cell which is dividing to form a subsidiarv is always closely appressed to the wall immediately . adjacent to the GMC.

498

3. Application

G. L.

STEBBIXS

AA-II

S. S. SHAH

of These Rest&s to Studies of Meristenzatic

Actidty

The number of GMC’s at each of the five stages of development represented in Fig. 11 and scored in determining the index of irregularitv provides an accurate measure of the amount of intercalary me&tern existing in any grass leaf at various stages of its development. Hence, it can he used to determine whether a correlation exists between either the amount or duration of meristematic activity and the final size of the leaf. Since leaf size and number are directly correlated with the vigor and productivity of a plant, the measure of meristematic activity suggested here may provide a valuable tool for studying the developmental basis of differences in vigor and productivity, and perhaps of making predictions about these valuable economic properties on the basis of examinations of seedlings. Preliminary observations indicate that the amount of meristem varies in the same plant according to conditions of growth, and particularly according to age. In Dactylis, seedling leaves have many times fewer developing complexes in a row than leaves of old clones. In Nortlcum the third and fourth leaves of a shoot. which are the largest, have somewhat more meristcm than the first and second, but this difference is much less marked than in Dnct!glis. If different genotypes are compared, using comparable leaves and growth stages under similar conditions of the environment, genetically controlled diffcrcnces can also be seen. Thus the number of developing complexes in a row is much less in the wild species Hordetlnl nmrinum than in the cultivated H. uzl~gclre. Unpublished observations of G. S. Khush indicate that two wild species of Secnle, the annual S. qluestre and the perennial S. montmwn, both have less meristem than the cultivated species, S. cerecr~e. A svstematic investigation of these differences is planned. The index of irregularity should provide a measure of the constancy of the growth processes in the plants concerned. Students of animal development (1Varburton, 1955; Waddington, 1958) have pointed out that the regularity and orderliness of the adult phenotype mav hc attained bv a somewhat irregular pattern of growth, termed by IVaddington “background noise,” with compensatory adjustment for thesr irregularities in later stages of development. With the present technique, one could determine whether greater homeostasis, as discussed by Lerner ( 1954). is acquired by a greater over-all regularity in the

STOMATAL

DEVELOPMEXT

IN

499

GHAMISEAE

growth processes or by more complete compensation for the same amount of initial irregularity. An investigation of this topic is also planned for the future. SUMMARY

The development of stomata in the grass leaf is divided into five stages: (1) formation of the guard mother cell (GMC); (2) formation of subsidiary cells through mitoses induced by the GMC in lateral epidermal cells; (3) completed triad of cells consisting of the GMC and two subsidiaries: (4) d’lvision of the GMC to form the guard cells; (5) differentiating complex of four cells. When these stages are scored successively from the bottom of a stomata1 row distally, the succession of stages is in general in the order listed, but many irregularities occur, and a quantitative index of irregularity is suggested. The irregularities within a row as well as certain described abnormalities indicate that both the cytoplasmic polarization with leads to G;CIC differentiation and the inductive force which the GMC exerts in subsidiary cell formation are governed principally by gene-cytoplasmic interactions within the GMC itself rather than by general forces acting uniformly throughout the tissue. Four lines of evidence indicate that subsidiary cell formation results from induction by the GMC; the most convincing of these is the formation of two subsidiaries on one side of the GMC in complexes having the dividing wall between two lateral cells opposite a GMC. Other evidence indicates that the competence of the lateral epidermal cell to respond to this stimulus depends upon the size of the cell and its position relative to the GMC. A long, lateral epidermal cell forms a subsidiary more readily at its distal than at its proximal end. The process of induction is associated with regular changes in the size of the GMC. Although the general succession of events is the same in all species of grasses studied, characteristic differences in detail exist between distantly related genera, such as Hordeum on the one hand and Sorghum as well as Zen on the other. Some physicochemical forces that may be controlling these processes are discussed. REFERENCES AVDULOV, N. P. ( 1931). Karyo-systematische Untersuchungen men. Bull. Appl. Botany Suppl. 44, 423 pp. AVERS, C. J. (1958). Histochemical localization of enzyme epidermis of Phleum pratense, Am. ]. Botany 45, 609-613.

der activity

Familie

Grami-

in the

root

500

C.

L.

STEBHISS

ASD

S. S. SHAH

C. J., and G~arar, R. B. ( 1939). Coqarative cnzynle differentiation in grass roots. I. Acid phosphatasc. Am. J. Botcuu/ 46, 19%19:3. B~cxm, G. ( 1931 ) Experinxmtelle Analyse der Genom-und Plasmonwirkung bei \looscn. III. Osniotischer Wert lietcroploider Pflanz~~n. Z. Induktil;e A/xtclmtt~utqs-II. Vererbutrglehre 60, 17-38. BtiXSlh’C, E. ( 1948 ). “Entwicklungs-und Bewegungsphyhiologie dcr Pflanze.” Springer, Berlin. B~~SSINC, E. ( 1957). Polarit%t und inlquale Tcilun 8 des pflanzlichen Protoplasten. Z’,otol’lastnologicL ( Vi~mu) 8 (9,)) 146. C.IXIPBELL, 1). II. ( 1881 ) . On the development of the stomata of Tradescantia and Indian corn. Am. Notrrrnlist 15, 761-766. FLI\ I-, L. II., and ~IORELANI~, C. F. ( 1946). A study of stonutn in sugarcm~. Am. J. Botq 33, 80-82. HE~SE, 1%. ( 1938). Verglcichendc Untcrsuchungen an diploiden und tetrnploiden l’vtunien. Z. Jttdrtktiw Abstcrtnmtotgs-cc. Vererbrrt&.&re 75. l-2:3. LIMSEH. I. 11. ( 1954 ). “Genetic Homcostasis.” \\‘iley, New York. P~TEHFIELI~, W. Xl. ( 1957 ). Histogcncsis in the bamboo, with special referuxe to the epidermis. Bull. Torrey Botcttt. Club 64, 421-432. Sc:HLijbsm. I,. A. ( 1936). Bcfruchtungsschwirrigkciten bei Autopolyploitlcm md ihrc iiberwindung. Ziichter 8, 295-301, STEBBINS. G. L. (1956). Cytogenetics and evolution of the grass fannilv. Am. J. Botom/ 43, 890-905. ~TEBBIN~, G. L., and JAW, S. K. ( 1960). Dcvelopmrntal studies of cell differentiation in the epidermis of Inono~otyletlo~~s. 1. Allirrm, Hhoeo, ud Cotttttwlittit. .%cc~lop Bid. 2, 409-426. STEBBINS, G. L., am1 KHUSH, G. S. (1960). V, ariation in the organization of the stomata1 complex in the leaf epidermis of monocotylrdons and its bearing on their phylogeny. Am. J. Botcmy in press. STEBBIXS, G. L., and ZOHAHY, 1). ( 1959). Cytogenetic and evolutionary stndies in thct genus DocttJli.y. I. The morphology, distribution, and interrelationships of the diploid subspe~ics. C’rtio. Calif. (Berkdey ) Pttbls., Rotcttty 31, l-40. STW.SBUHGER, E. ( 1866). Ein Beitrag zur Entwicklungsgeschichtc der Spnlttiffnunp. T&t&. wiss. B&m. 5, 297-342. WADDINGTON, C. HI. (1958). “The strategy of the Genes.” Allen and Unwin, London FVarmu~~ox, F. E. ( 1955). Feedback in tlcvclopmcnt and its evolutionary signifiCR~CC. Am. Noturdist 98, 129-140. WH.AI.EY, \lJ. J,, MOLLENNAUER, H. H., and KEPHAHT, E. (19,59). The endoplasmic reticulum and the Golgi structures in maize root cells. J. Biophp. Biocizettt. C!/tol. 5, 501-506. Avms,