Protoplasma 115, 176- 192 (1983) PROTOPLASMA �9 by Springer-Verlag 1983

Microtubules and Their Organizing Centres Guard Cells of Adiantum capillus veneris

B. GALATIS*, P. APOSTOLAKOS, and CHR. KATSAROS

in Differentiating

Institute of General Botany, University of Athens

Received July 19, 1982 Accepted in revised form October 8, 1982

Summary

The cortical cytoplasm of the young guard cells ofAdiantum capillus veneris is locally differentiated. At an early post-telophase stage, numerous microtubules diverge from the cytoplasm occupying the junctions of the midregion of the ventral wall with the periclinal ones, towards the periclinal and ventral wall faces as well as towards the inner cytoplasm. Microtubule-vesicle complexes (MVCs) are detected in these regions. Their appearance is accompanied by the initiation of local wall thickenings in the same areas. Afterwards, more distinct MVCs anchored to the plasmalemma were seen in the cortical cytoplasm of the periclinal walls, close to the growing thickenings, usually at a distance up to 3 ,am from them. Sometimes, they seemed to contain an electron dense substance in which the microtubules were embedded. Cortical microtubules converging from more than one direction terminate at the MVCs. Besides, the microtubule population lining the periclinal walls radiate from the regions where the above cytoplasmic formations are localized. The overlying cellulose microfibrils exhibit the same orientation. The vesicles localized at the MVCs appear to be of dictyosomal origin, very electron dense and react positively to periodic acid-thiocarbohydrazide-silver proteinate (PA-TCH-SP) test. Another population of microtubules fan out from the MVCs, entering deeper into the cytoplasm. They become associated with the nucleus and mitochondria, and traverse the peridictyosomal cytoplasm. In some instances the nucleus formed a protrusion towards an MVC and appeared associated with it via microtubules which radiate from the MVC and flank the nuclear envelope.

The observations favour the hypothesis that prominent microtubule organizing centres (MTOCs) function in the cortical cytoplasm of the midregion of the periclinal walls surrounding the ventral one for a relatively long time. The MVCs and/or their adjacent plasmalemma sites may represent MTOCs or at least they

* Correspondence and Reprints: Institute of General Botany,

specify the cortical cytoplasmic sites where microtubules are nucleated.

Keywords.- Microtubules; Microtubule organizing centres; Adiantum capillus veneris ; Guard ceils.

1. Introduction

The informat ion accumulated so far on s tomata

supports the concept that in Angiosperms the

microtubules are indispensable organdies for guard cell

morphogenesis. They accurately mirror the or ienta t ion

of the radially deposit ing cellulose microfibrils in the

periclinal walls which underl ie the mechanism of

acquisi t ion of the kidney-like shape by the guard cells.

The same organdies also appear implicated in the local

deposi t ion of material on the midregion of the ventral

wall (KAUFMAN e t al. 1970, GALATIS 1974, PALEVITZ

and HEVLEe, 1976, GALATIS 1980, GALATIS and

M ITRAKOS 1980; for a recent review see H EPLER 1981).

Therefore, the identif ication of the cortical MTOCs,

nucleat ing the highly organized micro tubula r systems

in differentiating guard cells will advance our

knowledge on their morphogenesis.

Al though there are reasons to assume that in higher

plant cells cortical microtubules are init iated at specific

sites of the cortical cytoplasm (GUNNING and HARDHAM

1979, 1982, GUNNING 1981) distinct MTO C s have not

been identified yet. The fern A z o l l a is an exception. In

this p lant GUNNING and col. discovered and studied in

detail MTO C s along the cell edges (GuNNIN~ 1980,

B. GALATIS et al. : Microtubules and Their Organizing Centres in Adiantum capillus veneris 177

1979). They were determined as microtubule clusters

possessing variable quantities of an electron dense

matrix as well as vesicular components.

Recently published work has provided evidence favouring the hypothesis that in young Zea mays

_ stomata, prominent MTOCs function in the cortical cytoplasm and/or the plasmalemma of the periclinal walls around the middle of the ventral one. They appear generating the radial microtubules which line the periclinal walls as well as those palisading the ventral wall thickening (GALATIS 1980). MTOCs seem to become activated in the cortical cytoplasm of the mid- region of the periclinal walls of the guard cell mother cells (GMCs) of Zea mays at an advanced interphase stage (GALATIS 1982). PALEVITZ (1981 a, b) identified putative MTOCs in guard cells of Phleumpratense at an advanced stage of their differentiation. In differen- tiating guard cells of Zea mays and Vigna sinensis (GALATIS 1980, GALATIS and MITRAKOS 1980) the above mentioned MTOCs are related to the deposition of the external and internal ventral wall thickenings. Extending our work on stomatal morphogenesis, we investigated the development and organization of the microtubular systems in guard cells of the fern Adiantum capillus veneris, attempting to identify

cortical cytoplasmic sites where MTOCs operate. Among others, the possibility that in guard cells of the above fern, as in root cells of Azolla (GUNNING et al. 1978), the cortical MTOCs could be more discernible than in guard cells of Angiosperms was considered.

2. Materials and Methods

Young leaves of Adiantum capillus veneris collected from the field or developed in the laboratory were processed for electron microscope study according to methods which have been previously described (GALATIS 1980). In addition to the above, some leaves were fixed for up to 45 minutes in a 3% glutaratdehyde solution, with or without 0.5% tannic acid. The sections were examined and photographed with a Hitachi HS-8 or a PhiIips 300 electron microscope.

3. Results

3.1. Microtubule Organization During the Initial Stages of Wall Thickening

During an early post-telophase stage, the microtubules reappear in the cortical cytoplasm of all the walls in the guard cells of A. capillus veneris. They are more abundant along the periclinal and ventral walls than along the dorsal ones. Before the deposition of any recognizable wall thickening in guard cells, most of the

microtubules traverse the cortical cytoplasm of the

ventral walls in an anticlinal direction forming dense

palisade arrays along their midregions (Figs. 1, 4, and 5). Some microtubules were also seen at their ends. Although the majority of microtubules are adjacent to the plasmalemma and quite often cross-linked to it, there are many others having the same orientation and running through the cytoplasm at some distance from

the plasmalemma (Figs. 4 and 5; see also Fig. 9). The latter are more numerous in the external and internal paradermal sections.

The microtubules lining the dorsal walls are also anticlinally oriented (Fig. 2), On the contrary, those underlying a limited area of the periclinal .walls around the midregion of the ventral ones do not exhibit a predominant arrangement (Fig. 12). They follow different orientations and seem to terminate at sites containing a large number of electron dense vesicles. The latter form distinct complexes (MVCs) with the microtubules (Fig. 12; see also Fig. 11). However, when examining the overall alignment of microtubules it is clearly seen that those bordering on the rest of the periclinal walls converge on their midregion where the MVCs are localized (Fig. 12). It must be noted here that due to the curvatures of the periclinal walls it was quite difficult to obtain a paradermal section through their cortical cytoplasm including the whole surface of the guard cells. Most of the guard cell sections made were slightly oblique (see Fig. 12 inset). The investigation of post-telophase guard cells in serial transverse sections shortly after microtubule re- installation, confirmed the above observations. Numerous microtubules diverge from 1;he cortical cytoplasm filling the junctions of the midregion of the ventral wall with the periclinal ones (Figs. 3, 7, and 9). They are directed towards the ventral and periclinal walls, while a significant number of them penetrates deeper into the cytoplasm and does not become juxtaposed with the plasmalemma; hereafter, the latter will be termed "cytoplasmic microtubules". As was observed in paradermal sections many vesicles are gathered in these regions forming distinguishable complexes with the microtubules (Figs. 3 and 7). In the same sections it was discerned that some of the microtubules which are not adjacent to the plasma- lemma of the ventral wall terminated at some distance from its junctions with the periclinal ones (Figs. 8 and 9). In these areas the microtubules seemed to end at sites possessing a finely granular electron dense substance and some vesicles (Figs. 8 and 8 inset). Few of the vesicles exhibited angular profiles (Fig. 8 inset).

i78 B. GALAT1S et al.: Microtubules and Their Organizing Centres in Adianlum c'apillu.~ 'eJw;l.~

Although particular attention has been paid, cyto- plasmic sites where microtubules converge were not found along any other lace of the walls or edge made by them in post-telophase guard cells. In A. capillus veneris, as in the fern Polypodium (STEVENS and MARTIN 1978), the formation of the stomatal pore begins internally (Fig. 6). The ventral wall splits along its whole depth before the periclinal ones, which for a considerable time remain intact (Figs. 6 and 9). In the plant examined here the detachment of the ventral wall partners commences immediately after the completion of the cell plate and before the initiation of any detectable thickening (Fig. 6). This happens after microtubule reappearance (Fig. 9). Actually, the separated plasmalemmae are overlain with a thin layer of loosely arranged substance (Fig. 9). They exhibit an undulated appearance. Afterwards, local thickenings are laid down at the external and internal ends of the midregion of the ventral walls at the positions where the MVCs are located and the microtubules converge (Fig. 13; compare with Fig. 6). As in Angiosperm "stomata they appear triangular in transverse sections and lens- shaped in paradermal ones (Figs. 13 and 14). Simultaneously, material starts being uniformly deposited along the whole depth of the ventral walls (Fig. 13). In the latter the cellulose microfibrils are exclusively anticlinally oriented. As guard cell differentiation proceeds, the microtubules obtain a more accurate orientation, showing extensive linking to the plasmalemma. Those of the ventral walls become restricted to their median regions forming more distinct groups compared to the post-telophase guard cells (Fig. 10; compare with Figs. 4 and 5).

3.2. Microtubule Organization During the Advanced Stages of Wall Thickening up to the Final Opening of the Stomatal Pore

After the establishment of the terminal ventral wall thickenings, more conspicuous MVCs were observed around them, in the cortical cytoplasm of the periclinal walls (Figs. 13, 16, and 23). The vesicles contain a substance of high electron density and are more or less of the same size (Figs. 15 18, 20, and 21). Usually, they are disposed very close to one another and to microtubules which are localized among them (Figs. 16, 20, 23, and 24). Sometimes, the images give the impression that an electron dense material is present among them, in which the microtubules are embedded (Figs. 16 and 20). The space occupied by the MVCs is usually devoid of ribosomes, while endoplasmic reticulum (ER) membranes end at their periphery (Figs. 15 18 and 20). In a few instances only ER membranes entering them were seen (Fig. 16). It is noticeable that at this stage the ER membranes proliferate rapidly and traverse every site of the cytoplasm, becoming the prevailing cytoplasmic element of guard cells (Figs. 13 and 14).

In serial paradermal and transverse sections of guard cells, microtubules of different orientations were seen terminating at these vesicle accumulations (Figs. 11, 15 18, and 20). The terminal regions of microtubules embedded in the MVCs frequently appear more dense than the rest of them (Figs. 20, 21, and 23). Reconstruction of some MVCs from successive sections shows that some microtubules emerging from them are short, a phenomenon which connotes that they might be at an early stage of formation. In some

Abbreviations

CD V coated dictyosome vesicle, O dictyosome, D V dictyosome vesicle, D W dorsal wall, E R endoplasmic reticnlunr, LB lipophilic body, M

mitochondrion, M b microbody, M t microtubule, M VC microtubule-vesicle complex, N nucleus, P plastid, Pl plasmalemma, P W periclinal wall,

S P stomatal pore, V W ventral wall.

Rig. I. Median paradermal section of a post-telophase stoma. The midregion of the ventral walI has split to form the stomatal pore. x 5,500

Fig. 2. The portion of the dorsal wall shown by the arrows in the stoma of Fig. 1. Microtubnles anticlinally oriented traverse the

subplasmalemmal cytoplasm (see arrows), x 45,000

Fig. 3. Portion of a post-telophase s toma in a median transverse section. It is at a stage of ini t ianon of the terminal ventral wall thickenings (large

arrows). Numerous microtubules traverse the cytoplasrn in an anticlinal direction towards the junction of the walls. Many of them are not associated with the plasmalemma. A number of vesicles has been accumulated in the cortical cytoplasm occupying the junction of the periclinal

walls with the ventral one. x 50,000. The inset depicts the stoma, portion of which (see brackets) is illustrated in Fig. 3. x 1,700

Figs. 4 and 5. Higher magnification of the region of the ventral wall limited by the brackets in the s toma of Fig. 1. Well-developed microtubule arrays iine the separated plasmalemmae which exhibit an undulated appearance. Ventral wall deposition has not commenced yet. The arrows

point to microtubules lying at some distance from the plasmalemmae. • 40,000

B. GALATIS et al. : Microtubules and Their Organizing Centres in Adiantum capillus veneris 179

Figs. 1-5

180 B. GALATIS et al. : Microtubules and Their Organizing Centres in AdialTtum capillu.s ~'elteris

instances the MVCs enclosed very short and electron

dense microtubule port ions which did not extend into

the neighbouring cytoplasm (Fig. 16).

The MVCs were always apposed on the p lasmalemma

(Figs. 13, 16, 20, 21, and 23). When they were located

deeper into the cytoplasm, examinat ion o f neigh-

bouring sections confirmed that they reached the

cortical cytoplasm of the periclinal walls and contacted

the p lasmalemma (Figs. 25 and 30; see also Fig. 35).

The position of format ion of the MVCs is very

consistent. They are confined to the cytoplasm adjacent

to the terminal ventral wall thickenings as well as that

apposed on the midregion of the periclinal walls at a

distance 2-3 ~m from them (Figs. 13, 23, and 30). The

careful investigation of the cortical cytoplasm of the

rest o f the periclinal walls as well as that o f the ventral

and dorsal ones in a large number of guard cells did not

reveal the existence of any MVC. As it has already been

noted, microtubules line the whole surface o f the

protoplast.

At the advanced stages of growth of the ventral wall

thickenings and before the final opening of the s tomatal

pore, the regions o f the periclinal walls joined with them

are thinner than the rest of the periclinal walls (Figs. 13

and 30). It implies that these wall regions are under

expansion. This is probably due to the mechanical

stress which is imposed on them in order to disrupt the

periclinal walls over the ventral wall thickenings.

MVCs are localized in the cortical cytoplasm abutt ing

on the thinner periclinal wall regions and their rims

(Figs. 13, 16, and 30).

The inner cytoplasm of the guard cells continues to be

traversed by a significant number of microtubules

(Fig. 22), M a n y of them become spatially associated

with the nucleus, mi tochondr ia and microbodies (Figs.

24, 32 inset, 36, and 37). The latter organelles are

surprisingly numerous in guard cells of A d i a n t u m

capil lus veneris (Fig. 32). Sometimes, these micro-

tubules are arranged in bundles which traverse the

cytoplasm at a distance up to 6 ~.m (Figs. 19 and 38).

Extensive microtubule overlappings were observed in

the above bundles (Figs. 19 and 38). Quite often,

microtubules traverse the peridictyosomal cytoplasm

(Fig. 29). Examinat ion o f a large number o f guard cells

has evidenced that most o f the above microtubules

approach the cortical cytoplasm of the periclinal walls

which surrounds the ventral wall thickening (Fig. 22).

Microtubules lining the periclinal walls and others

entering deep into the cytoplasm were seen fanning out

f rom the MVCs (Figs. 15-18). It is wor thy of note that

numerous mi tochondr ia and microbodies are placed

close to the terminal ventral wall thickenings as well as

in the cytoplasm intervening between the nuclei and the

ventral walls (Figs. 31 and 32). The lipophilic bodies

which are abundant in the young guard cells are also

preferably posit ioned in these cortical cytoplasmic

regions (Fig. 30). The above observations emphasize

the intense polarization of the guard cells.

A phenomenon which deserves some attention, is the

shaping of the guard cell nucleus which is illustrated in

Fig. 33. In transverse sections it exhibits a projection

towards an M V C lying close to the junct ion region of

the periclinal wall with the ventral wall thickening

(Figs. 33 and 35). The MVC contains microtubules

overlapping with each other (Fig. 34). A detailed study

of these deformed nuclei in a number of guard cells has

shown that some microtubules emerging f rom the

MVCs flank the nuclear envelope encompassing this

projection. In Figs. 36, 37, and 38 one can observe an

MVC from which microtubules radiate towards the

nucleus. A perinuclear microtubule bundle converges

on the same cytoplasmic site (Fig. 38).

Fig. 6. Median transverse section of a young stoma at a stage of differentiation more or less similar to that of the stoma shown in Fig. I. N ore the internal initiation of the stomatal pore. The periclinal walls remain intact, x 5,500

Fig. 7. The junction region of the periclinal walls with the separating ventral ones of the guard cells illustrated in Fig. 6, in another section. N ore the accumulation of the vesicles and their associations with the microtubules. The ventral wall thickening has not been initiated yet. Microbodies are preferentially placed in this region, x 43,000

Fig. 8. Portion of a very young stoma. A microtubule bundle is shown by the arrows. The microtubules of the bundle seem to terminate at some distance from the junction of the ventral with the periclinal wall. x 31,000. The inset shows the cytoplasmic region where the microtubule bundle shown in Fig. 8 terminates. It contains some electron dense material as well as vesicles, some of which exhibit angular profiles (arrows). x 36,000

Fig. 9. The lower portion of the ventral wall of the stoma shown in Fig. 6 in another section. Note that many microtubules (arrows) traverse the cytoplasn~_. Some of them are adjacent to the plasmalemma, some others at some distance from it. Microtubules from the ventral walls and others from the perictinal ones converge on the junction of the periclinal walls with the ventral ones. Note the position of microbodies in this area. The same region in another section is shown in Fig. 7. Some loose material externally covers the plasmalemma limiting the stomatal pore (transparent arrow), x 39,000

B. GALATIS et al. : Microtubules and Their Organizing Centres in Adiantum capillus veneris 181

Figs. 6 9

182 B. GALATIS et al. : Microtubules and Their Organizing Centres in Adiamum capi[lus veJleris

As far as the origin of the vesicles participating in the

MVCs is concerned, the observations suggest that they are pinched off by the dictyosomes. They resemble

vesicles accumulated in the immediate vicinity of the

dictyosomes (Figs. 28 and 29; compare with Figs. 20,

25, and 30). In addition, after the application of

periodic acid-thiocarbohydrazide-silver proteinate

(PA-TCH-SP) test, the MVC vesicles become im-

pregnated with Ag deposits (Figs. 26 and 27).

The dictyosomes in differentiating guard cells of A. capillus veneris are numerous and exhibit an intense

secretory activity. They are associated with numerous

smooth and a significant number of coated vesicles (Figs. 28 and 29). The smooth vesicle population

consists of vesicles significantly differing from one

another in size and electron density (Figs. 28 and 29).

Among them those which are the most electron dense

are detected in the MVCs. It is important that most of the dictyosomes in the differentiating guard cells of the

fern examined in this paper occupy positions in the cytoplasmic regions neighbouring to the midregion of

the periclinal walls. Some of them are detected in the

cytoplasm intervening between the nucleus and the

ventral wall and only a few in the rest of the cytoplasm (Fig. 13). The rich in dictyosomes cytoplasmic areas are

those which are traversed by "cytoplasmic micro-

tubules". At the final stages of guard cell differentiation in which

the opening of the stomatal pore has been completed by the local disruption of the periclinal walls over the pore

and the guard cells have gained their kidney-like shape,

the MVCs become gradually rare and indistinct. On the

contrary, the microtubule arrangement under the periclinal walls remains typically radial. The overlying

cellulose microfibrils radiate away from the pore. The above observations confirm ZIE~ENSP~CK'S (1941)

conclusions that the periclinal walls of the fern stomata

are radially micellated. The thinner junction regions of

the periclinal walls with the terminal ventral wall thickenings become thicker than the rest of the

periclinal walls. This is apparent after the opening of

the stomatal pore.

4. Discussion

4.1. Organization of Microtubules- Localization

of Cortical MTOCs

The microtubule organization m guard cells of A.

capillus veneris is precisely patterned and constitutes the first structural manifestation of their divergent

differentiation. It closely resembles that of Allium cepa

(PALEWTZ and HEELER 1976) and Vigna sinensis (GALATIS and MITRAKOS 1980) as well as that of young

guard cells of Zea mays (GALATIS 1980). The presented

results show that: 1. the deposition of radial microfibrils in the periclinal walls in presence of an

attending radial microtubule system in the underlying cytoplasm characterizes the s tomata of a fern, 2. very

distinct microtubule arrays line anticlinally the

thickening midregions of the ventral walls, and 3. anticlinally oriented microtubules line the dorsal

walls equidistantly distributed along them. Considering

the above it seems that the mechanism of morpho-

genesis of guard cells of A. capillus veneris is similar to that of kidney-shaped guard cells of Angiosperms.

Regarding the development of the microtubule systems

in guard cells, the observations made in this article support earlier conclusions reached during the study of

shaping guard cells of Zea mays (GALATIS 1980; see Introduction). In A. capillus veneris guard cells, the

cortical cytoplasm adjacent to the periclinal walls

around the midregion of the ventral ones is structurally and functionally differentiated from the rest of the

cortical cytoplasm. This specification is manifested immediately after the completion of cytokinesis, by the

Fig. 10. Midregion of the ventral walls of a stoma at a more advanced stage of differentiation, as'it appears in a paradermal section.The ventral walls have been thickened (compare with Figs. 4 and 5). Note the numerous microtubules which are adjacent to the plasmalemma arranged one to three units deep into the cytoplasm. Other microtubules lie at some distance froln the plasmalemma forming bundles (see arrows), x 40~000

Fig. 11. Paradermal section through the cortical cytoplasm of a periclinal wall close to an established ventral wall thickening. Note the convergence of microtubules (arrows) on the site shown by the arrow-heads, x 45,000

Fig. 12. Slightly oblique section through the cortical cytoplasm of the periclinal walls of a post-telophase stoma, before the initiation of the ventral wall thickening. Note the accumulation of numerous electron dense vesicles in the region where the terminal ventral wall thickening will emerge. The vesicles form distinct complexes with the microtubules. In this region the microtubules do not exhibit a predominant orientation. However, those lining the rest of the periclinal walls converge on this region (arrows). They appear radiating from the midregion of the periclinal walls towards the dorsal ones. Note again the localization of two microbodies close to the cytoplasmic region where the MVCs appear, x 43,000. The inset shows the stoma, portion of which is illustrated in Fig. 12. x 2,200

B. GALATIS et al. : Microtubules and Their Organizing Centres in Adiantum capillus veneris 183

Figs. 10 12

184 B. GALATIS et: al. : Microtubules and Their Organizing Centres in Adiantum capillus ve*leris

Fig. 13. Transverse section of a stoma at a relatively advanced stage of differentiation. The periclinal walls have not been disrupted yeL to

complete the stomatal pore. The triangular external and internal ventral wall thickenings are distinct (arrows). The junction region of the

periclinal walls with the ventral wall thickenings are thinner than the rest of periclinal walls (see arrow-heads). MVCs are localized in those

regions. The preferential gathering of dictyosomes and lipophilic bodies at the cortical cytoplasm close to the ventral wall thickenings is obvious.

The ER membranes have greatly proliferated, x 8,500

Fig. 14. Paradermal view of a terminal ventral wall thickening. Note the prominence of the ER membranes, x 13,000

Figs. 15-18. Junction region of a periclinal wall with a ventral wall thickening (see Fig. 16 inset) in four successive sections. An M VC is localized in

the adjacent cytoplasm. Many microtubules radiate from this structure entering deep into the cytoplasm. Each of the microtubules nos. Z 4 can be

followed in two of these sections. It is evident that they terminate at the MVC. The terminal portions of nos. 1, 2, and 4 are seen in Fig. 16 and no. 3 in Fig. 17. The transparent arrows in Figs. 15 and 16 indicate overlapping microtubules portions longitudinally sectioned. The arrow in Fig. 18

points to a cross-sectioned overlapping microtubule portion, and in Fig. 16 to a microtubule segment enclosed in the MVC. It seems likely that an electron dense matrix is localized among the vesicles (Fig. 16). ER membranes terminate at the MVC periphery (Figs. 15, 17, and 18). The arrow-

head in Fig. 16 shows an ER portion entering this structure, x 43,000. The inset in Fig. 16 shows the s toma from which the periclinal wall region

illustrated in the above Figures has been taken. The arrow points to the MVC site. x 1,600

Fig. 19. Microtubule bundle running through the cytoplasm in an anticlinal direction towards an MVC adjacent to the periclinal wall (not

included in this Figure). The arrows show microtubule overlappings, x 48,000

B. GALATIS et al. : Microtubules and Their Organizing Centres in Adiantum capillus veneris 185

Figs. 15 19

186 B. GALATIS et al. : Microtubules and Their Organizing Centres in Adiantum capil/us ~e~mri.s

local function of prominent MTOCs, nucleating the radial microtubules underneath the periclinal walls and possibly those of the ventral and dorsal ones. The major findings leading to this conclusion are the facts that (a) the reappearing microtubules lining the periclinal walls as well as those invading inner cytoplasmic regions of the guard cells are focused on the above limited areas of the cortical cytoplasm, and (b) the MVCs are localized there. These structures arrest the attention of the observer. There are no reasons to believe that the microtubules are nucleated in the rest of the cortical cytoplasm or in different sites of the inner cytoplasm and later grow and converge on the midregion of the

periclinal walls.

In root cells of Azolla the cortical MTOCs constantly function along the cell edges (GuNNiNG et al. 1978). In

differentiating guard cells of Zea mays (GALATIS 1980) and Vigna sinensis (GALATIS and MITRAKOS 1980) examined so far as well as of A. capillus veneris

examined here their localization seems to be somewhat different. Very prominent MTOCs are selectively activated at the onset of the guard cell differentiation at particular and limited regions of the cortical cytoplasm adjacent to the periclinal walls. This difference probably reflects the peculiarity of guard cell morphogenesis and correlates with the highly patterned organization of microtubular systems in guard cells. The prolonged function of the MTOCs in the above cells does not seem meaningless. Their persistence for some time might secure the longevity of the microtubules and the maintenance of the pattern of their organization in guard ceils for the time for which they are needed. It must be noted here that we are largely ignorant of the means by which the activation of the MTOCs takes place, and of the factors involved in this process. In Zea

mays stomata, some inheritance of the MTOCs functioning in their cortical cytoplasm seems to occur. The MTOCs operating in the cortical cytoplasm of the middle of the periclinal walls in the young guard cells appear to be activated in GMCs at an advanced interphase stage (GALATIS 1982). It remains to be investigated whether this phenomenon characterizes also the stomata ofA. capillus veneris. The existence of a type of inheritance of the cytoplasmic regions where MTOCs function has been repeatedly stressed by GUNNING and col. (see GVNMNG and HARDHAM 1979). Finally, the possibility of activation of MTOCs by the local wall expansion and the concomitant deformation of the underlying plasmalemma and cortical cytoplasm must be considered. Major MTOCs function at the thin junction regions of the periclinal walls with the terminal ventral wall thickenings.

4.2. Possible Function o f MVCs and/or Adjacent

Plasmalemma Sites as M T O C s

Although very preferable associations between micro- tubules and dictyosome vesicles, as well as microtubule focal points where vesicles are collected, have been seen in differentiating guard cells of the Angiosperm Zea

mays (GALATIS 1980), MVCs like the ones observed in the fern investigated here were not found. Obviously, the accumulation of the vesicles cannot be considered to be accidental or as vesicle gatherings like those occurring commonly in the vicinity of the active dictyosomes. It reflects particularly intimate micro- tubule-vesicle associations or interactions between MTOCs or another related factor and the vesicles. Many times the microtubule-vesicle intimacy is so close that the MVCs can be considered to be integrated cytoplasmic formations. Their uniqueness is also

Figs. 20 and 21. MVCs apposed on the periclinal walls of guard cells at an advanced stage of their differentiation, preceding the completion of the

opening of the stomatal pore. In Fig. 20 the microtubules shown by the arrows terminate at the MVC and overlap each other (arrow-head). In

both MVCs the ends of microtubules are denser than the rest of them. Note the high electron density of the vesicles, x 48,000, x 50,000

Fig. 22. Junction region of the periclinal wall with the terminal ventral wall thickening. Microtubules radiate f rom this region towards the inner

cytoplasm (arrows). Numerous dictyosome vesicles are accumulated at this region, x 45,000

Fig. 23. MVC apposed on the ventral wall thickening at the time of breaking of the external periclinal wall. • 40,000

Fig. 24. Microtubule-mitochondrion juxtaposition. An MVC can be observed close to the mitochondrion. The arrows point to microtubules.

x 50,000

Fig. 25. MVC entering deeper into the cytoplasm. The arrow shows two short electron dense microtubule segments surrounded by vesicles.

x 43,000

Figs. 26 and 27. MVCs as appear after the application of the PA-TCH-SP test. The vesicles as well as the cell walls have reacted intensely.

x 60,000, x 32,000

B. GALATIS et al. : Microtubules and Their Organizing Centres in Adiantum capillus veneris 187

Figs. 20 27

188 B. GALATIS etal.: Microtubules and Their Organizing Centres in 4diaHtum capillus venetia

underlined by the fact that they are residents of a

limited portion of the cortical cytoplasm of the guard

cells.

Regarding the functional significance of the MVCs

and/or the adjacent portions of the plasmalemma there

are many indications favourably arguing that 1. they

represent MTOCs or at least they specify cortical

cytoplasmic sites functioning as MTOCs, and 2. they

directly or indirectly promote the local wall thickenings

at the junctions of the midregion of the periclinal walls

with the ventral one.

The first hypothesis is advocated from the following

observations: 1. Microtubules terminate at the MVCs

which sometimes appear to contain an electron dense

matrix in which the microtubule ends are embedded.

The latter microtubule portions are more electron

dense than the rest of the microtubules. 2. Microtubules

running through the cortical cytoplasm of the periclinal

walls as well as others entering into inner cytoplasmic

regions are focused on these structures. 3. Very short

microtubules terminate at the MVCs or are completely

enclosed in them. 4. They resemble in someway the

MTOCs described by GUNNING etal . (1978). 5. The

appearance of MVCs coincides with the initiation of the

microtubule organization in guard cells. In our opinion, the local deformation of the guard cell

nucleus in A. capillus veneris strongly suggests the

fnnction of proximal MTOCs which seem to be the

MVCs. Numerous microtubules radiate from the latter

towards the nucleus. They also give rise to microtubule

bundles. As far as we know, there is enough literature

showing that the shape of the nucleus changes in cases

in which MTOCs operate close to it, Microtubules

assembled by the latter are involved in this process.

Similar nuclear shapings have been described in

Closterium (PICKETT-HEAPS and FOWKE 1970), in

Marchantia (GALATlS and APOSTOLAKOS 1977, FOWKE

and PICKETT-HEAPS 1978), in Ditrichum pallidurn (BRowN and LEMMON 1980), and in GMCs of Zea mays (GALATlS 1982), Presumably, the above-mentioned

nuclear deformations can be used as a reliable criterion

for the recognition of the MTOCs.

The survey of the current literature on the cytology of

both animal and plant cells casts no doubts on the

generalization that the most consistent pictorial mark

of the cytoplasmic regions functioning as MTOCs is the

presence of a granular electron opaque material. This

may be either diffuse or may exhibit some form (for

reviews see PICKETT-H EAPS 1969, ROBERTS 1974, HEPLER

and PALEVITZ 1974, DUSTIN 1978, TUCKER 1979). A

similar matrix seems also to be present in some MVCs.

The above mentioned facts argue against an essential

involvement of dictyosome vesicles in microtubule

nucleation, i.e., eliminating the Ca'-~ from their

microenvirons. However, it must be remembered here

that the increased staining of dictyosome vesicles by

OsFeCN when they fuse with the expanding cell plate

may reflect the increase of the vesicular calcimn

(HEELER 1982; see also WICK and HeeLER 1980). The

calcium is presumably bound to acid polysaccharides

(HEELER 1982). The ability of dictyosome membranes

to sequestrate Ca, - has been pointed out by B ELITSER et

al. (1982). The above lines of information could explain

the high electron density of the vesicles of the MVCs. If

the dictyosome vesicles in MVCs sequestrate Ca' 4 the

possibility that they are implicated in microtubule

assembly should be considered.

4.3. MVCs and Local Wall Thickening

The promotion of local wall deposition by the MVCs is

also plausible. Gatherings of dictyosomes as well as of

their vesicles, containing polysaccharidic substances,

Fig. 28. Portion of the cytoplasm of a guard cell close to the ventral wall, containing two dictyosomes. They produce vesicles of different size and electron density, x 50,000

Fig. 29. Dictyosomal area lying close to the midregion of the perictinal wall. This area is traversed by microtubules (arrows). Note also the intense staining of membranous elements produced by the dictyosomes where coated vesicles are localized. The arrow-head indicates a microtubule linked by a cross-bridge with a dictyosomal membranous element, x 45,000

Fig. 30. Lower magnification of a guard cell portion including the external ventral wall thickening. At the thinner junction region of the periclinal wall with the ventral wall thickening an MVC is organized. Note the close accumulation of tee tipophilic bodies. The arrows point to microtubules, x 23,000

Fig. 31. Median paradermaI section of a stoma, including the midregion of the ventral walls. The majority of mitochondrial population of the guard cells is preferentially gathered in these areas, x 12,000

Fig. 32. External ventral wall thickenings. Note the preferential localization of microbodies, x 13,000. The inset shows a microbody flanked by a microtubule, x 48,000

B. GALATIS et al. : Microtubules and Their Organizing Centres in Adiantum capillus veneris 189

Figs. 28-32

190 B. GALATIS et al.: Microtubules and Their Organizing Centres in 4diantum capi/lu~ ve~zeri.s

are found at positions where intense local wall thickenings develop. As in guard cells of Zea mays and Vigna sinensis (GALATtS 1980, GALATIS and MITRAKOS 1980), in the fern examined in this paper the terminal ventral wall thickenings rise at cortical cytoplasmic areas where prominent MTOCs seem to function. A similar positional relationship between local wall formation and MTOCs has been recorded in differentiating xylem elements of Azolla (GUNNING

1981). However, whether the above positional coincidence between MTOCs and local walI emergence is functional or whether in the same regions other factor(s) (e.g., microfilaments) operate remains to be elucidated. An observation directly related to but not answering the above questions is the following: In "persistent GMCs" of Zea mays, which are formed after the inhibition of the symmetrical division of GMC by colchicine treatment, very conspicuous local wall thickenings are deposited in the midregion of their periclinal walls. These wall pads emerge at the positions where in young guard cells the formation of the terminal ventral wall thickenings take place. MTOCs seem to become activated in these cortical areas of advanced interphase GMCs. This local wall growth proceeds in the absence of microtubules (GALATIS

1982). Eventually, some information related to microtubules or their MTOCs or other relative polarity factor(s) is retained in these sites, promoting the deposition of wall material. The preferential concentration of dictyosome vesicles in areas in which MTOCs operate is not surprising. During the lateral expansion of the celt plate in the late cytokinetic cells, phragmoplast MTOCs are activated at its margins where numerous dictyosome vesicles are accumulated (INOU1; 1964, ALLEN and BOWEN 1966,

INOUE and SArO 1967, HEPLER and NEWCOMB 1967, HEPLER and JACKSON 1968, GUNNING et at. 1978). In guard cells the preferential gathering of vesicles at specific cortical sites, may reflect the directed (unidirectional?) movement of dictyosome vesicles parallel to microtubules towards their MTOCs or particular regions of microtubules, or towards another" factor related to them.

4.4. Functional Significance o f "Cytoplasmic Micro-

tubules" - Conclusions

The existence in differentiating guard cells of A. capillus

veneris of a well developed and rather organized "cytoplasmic microtubule" population is very remark- able, among others because these microtubules are nucleated by cortical MTOCs. Considering the presented results it is tempting to suggest that the "cytoplasmic microtubules" play an important role as "spatial organizers", maintaining the dictyosomes and other organelles, in the cytoplasm close to the rising thickenings. Such an activity has been attributed to cytoplasmic microtubules of animal cells (for a review see TUCKEa 1979). A "cytoplasmic microtubule" population has also been observed in guard cells of Zea mays (GALATIS 1980), but it is not so developed as in A. capillus veneris.

The findings of the present work seem to provide some insights into the complexities of the mechanism controlling the microtubule organization in the guard cells. The cortical cytoplasm underlying the midregion of the periclinal walls close to the ventral ones appears to be a very dynamic site~ It contains the MTOCs nucleating the highly organized microtubular system of the guard cells which seems to function as a prepattern of their morphogenesis. The outstanding observation

Fig. 33. Transverse section of a guard cell through a median plane. The nucleus exhibits an angular profile towards the cortical cytoplasm

neighbouring on the external ventral wall thickening. • 5,000

Fig. 34. MVC located at the cytoplasm close to the ventral wall thickening of the guard cell illustrated in Fig. 33, where the nucleus projects. Note

the electron density of the vesicles. Three electron dense microtubules overlapping with each other are enclosed. The arrow points to a

microtubule which is directed towards the MVC. Some of the vesicles are angular (transparent arrows), x 63,000

Fig. 35. Portion of the guard cell limited by the rectangular in Fig. 33. Note the position of the MVC in relation to the projecting nucleus and the

microtubule directed towards the nucleus, x 36,000

Figs. 36 and 37. Successive sections of an MVC, external section of which is shown in Fig. 38. It is clearly seen that numerous microtubules

(arrows) which are directed towards the nuclear envelope are focused on them. x 50,000

Fig. 38. Microtubule bundle traversing the perinuclear cytoplasm. This bundle as well as other microtubules converge on an MVC cytoplasmic

site (arrow-head) located close to the nucleus. The arrows indicate microtubule overlappings, x 45,000

Fig. 39. A guard cell, portion of which is shown in Fig. 38. The position of the microtubule bundle is indicated by the arrow, while that of the

MVC by an arrow-head, x 1,900

B. GALATIS et al. : Microtubules and Their Organizing Centres in Adiantum capillus veneris 191

Figs. 33-39

192 B. GALATIS et al. : Microtubules and Their Organizing Centres in Adiantum capillus veneri~

of this work is that of the MVCs, formations which probably, together with the adjacent plasmalemma, represent actual MTOCs. In differentiating guard cells of the fern A. capillus veneris the MTOCs or the sites where they function are more distinct compared to those recognized in the guard cells of the Angiosperm Zea mays (GALATIS 1980). As a conclusion, it must be said that despite the above mentioned points, additional work is needed in order to substantiate our knowledge on the localization of the cortical MTOCs, a phenomenon of determinative role in cell morpho- genesis and closely related to cell polarity.

Acknowledgements

This work was supported by a grant from the National Hellenic

Research Foundation (No 25/1982).

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