Full Terms & Conditions of access and use can be found athttp://www.tandfonline.com/action/journalInformation?journalCode=sgra20

Download by: [Naturhistoriska Riksmuseum] Date: 06 March 2016, At: 23:29

Grana

ISSN: 0017-3134 (Print) 1651-2049 (Online) Journal homepage: http://www.tandfonline.com/loi/sgra20

Sporoderm development in Asimina triloba(Annonaceae)

Nina I. Gabarayeva

To cite this article: Nina I. Gabarayeva (1992) Sporoderm development in Asimina triloba(Annonaceae), Grana, 31:3, 213-222, DOI: 10.1080/00173139209432033

To link to this article: http://dx.doi.org/10.1080/00173139209432033

Published online: 01 Sep 2009.

Submit your article to this journal

Article views: 46

View related articles

Citing articles: 19 View citing articles

Grana 31: 213-222, 1992

Sporoderm development in Asimina triloba (Annonaceae). I. The developmental events before callose dissolution

NINA I. GABARAYEVA

Gabarayeva, N. I. 1992. Sporoderm development in Asinziria triloba (Annonaceae). I. The develop- mental events before callose dissolution. - Gram 31: 213-222, 1992. Odense. August 1992. ISSN

The development of the sporoderm in Asitnitin rriloba before the dissolution of callose proceeds as follow: 1) the initiation and accumulation of prematrix substances, 2) the establishment of the “phantom of cctexine”, 3) the prematrix formation of double origin: microsporal and tapctal, 4) the differentiation of primexine matrix and the beginning of protosporopollenin accumulation on the primordial lamella of the foot layer and in protecturn, 5) the differentiation of procolumellae in primcxine matrix. The sequence of events corresponds to the general scheme of Dickinson, but the peculiar feature is the formation of prematrix prior to appearance of the ordinary primexine matrix.

Nina I . Gabarayeva, Koniarov Botanical liistitiite, Prof. Popova str. 2, SI. Petersbicrg. 197376, Russia.

(hlaiiriscript received 5 February 1991; revised version accepted 10 February 1992)

0017-3131.

Comparatively few works have been dedicated to the in- vestigation of pollen wall development in primitive Angio- sperms (Dahl 8: Rowley 1965 - Degetieria, Meyer 1977 - some Gymnosperms and Angiosperms, Zavada 1984 - Aiistrobnylen iizaciilatn, Waha 1987 - Asitititzn trilobn). These plants are of special interest from the point of view of their outgoing archaic status in the system of Angio- sperms, at least with respect to corresponding views of Takhtajan (1954), Cronqiist (1957) and Thorne (1976).

My \Vork on Asitiiitin triloba was incorporated into a paper, dealing with the question of existence of endexine in primitive Angiosperms (Gabarayeva 1988). Maria Waha (1987) published a paper on sporoderm development in Asitiiiiin triloba. Having analysed the results and conclu- sions in that paper I discovered the author probably had not been able to observe several early stages of primexine matrix development, while the remainder were beautifully illustrated. Naturally, due to the absence of the observation over the whole period of sporoderm development, all the accents have been displaced and some of the author’s con- clusions seem rather paradoxical, e.g. the conclusion that the accumulation of sporopollenin (SP) precursors occur first over the plasmalemma, and then in second place, the primexine matrix appeared between them.

It seems that this paper will be complementary to Waha’s (1987) and may provide clarification of the complex process of “untangling” the general continuous picture of sporo- derm development.

MATERIAL AND METHODS About 140 flower-buds of different developmental stages in Asi- inirza triloba (L.) Dun. cultivated in the Batumi Botanical garden were fixed during two years in 3% glutaraldehyde for 21 h, post- fixed in 2% OsO,, and embedded into Epon. The ultrathin sec- tions were post stained with uranyl acetate and lead citrate, and examined in TEhl TESLA-BS-500. For the determination of chemical composition of slime, surrounding the tetrads during the callose stage the Thitry method (1967) was used. To clear up the chemical composition of tapetal secretions on different stages of development the enzymes lipase, pronase and tripsin were applied (Dashek et al. 1971. & Belitser et al. 19S2).

A bbreviations: PhfC-microsporocyte, TC- tapetal cell, C-callose. S-slime, LS - place of the late slime localization, hi - microspore, PS - peri- plasmie space, FIV - fibrillar wall of tetrad, ST - stripe of pre- matrix of microsporal origin, PT - protectum, Phl. - primexine matrix, P - plasmalemma, PCO - procolumella.

RESULTS

The early tetrads surrounded by callose envelope contain microspores with a plasmalemma without any sinuosity. Callose is adjacent to the plasmalemma. A bit later the profile of the plasmalemma becomes slightly sinuous, and at some places plasmalemmasomes appear. Membrane vesicles of different sizes are closely pressed one to another and to the plasmalemma; morphologically the membrane of vesicles does not differ from that of the plasmalemma (Fig. 1A).

Grana 31 (1992)

Dow

nloa

ded

by [

Nat

urhi

stor

iska

Rik

smus

eum

] at

23:

29 0

6 M

arch

201

6

214 N.I. Gnbarayeva

and then - t h e peeling off this layer in the form of a stripe from the plasmalemma (Fig. IB). At this time the cy- toplasm of microspores is rich in free ribosonies (Fig. 1B,C) and tubular SER. The tubules of SER touch with their ends the inner surface of plasmalemma in its distal part, whereas in proximal region they underlay plasma- lemma, touching it along their entire length.

There are few Golgi vesicles at this time. The plastids are of irregular-ovoid shape, with even external membrane and sinuous internal one, the plastid stroma is rather light, with small inclusions. Autolytic processes appear to begin, and the plastids turn first into cytosegresomes and then - into cytosomes. A part of plastid generation are cup-like, with fragments of cytoplasm, captured in their cavities. Plastids of irregular shape with small plastoglobulcs occur. The mitochondria are dedifferentiated so much that they be-

Fig. I. A-F. The initial stages of prematrix formation. (A) hlcm- brane vcsicles are adjacent to plasmalemma. (B) Vesicles or sec- tions of finger-like cvaginations of plasmalemma, containing cyto- plasm; stripes of prcmatrix substance (nrroivheads). (C) Layer of closely pressed ribosome-like particles on plasmalemma. (D, E) Different stages of spreading out of vcsicles (arrows) and finger- like cvaginations of plasmalemma (nrroivhencis). (F) Beginning of cobweb-like pattcrn formation. Bar for A, E, F = 0.3 pm, B-D = 0.5 pm.

1. The iiiitiatiori arid accirrrzirlatiorl of pre1tintri.r siibsfrrrices 011 plasttialeitiriin

Later two different images are observed of the plasma- lemma in different parts of the surface in the same micro- spore or next one in the tetrad: (1) the membrane vesicles seem to be the sections of finger-like plasmalemma cva- ginations containing the fragments of microspore cyto- plasm ( ~ i ~ . IB) , or (2) tliere is a layer closely one to another of ribosome-like particles (Fig. 1C). It seems that later the redistribution of these particle substances, the exfoliation of this layer from the plasma membrane occurs,

Fig. 2. A-C. Formation of slime-containing cavities around te- trads. (A) hleiosis in PhlC and mitosis in tapetal cells, some tapetal cells undergo Iysis, leaving remnants of cytoplasm (nr- roichend). (B,C) Process of autolysis in progress, remnants of cytoplasm gradually turn i n t o slime. Bar = 1 Itm.

Grnrro 3t (1992)

Dow

nloa

ded

by [

Nat

urhi

stor

iska

Rik

smus

eum

] at

23:

29 0

6 M

arch

201

6

Dewloprnerit of sporoderrri iri Asiniirin 215

for the deliver of slime (Fig. 2B, C). To find out the chem- ical composition of the slime, the ThiCry method (1967) was used. The result of this method indicated that the main component of the slime is polysaccharides (Fig. 3A-D).

2. The estnblislirrierit of “phnrirori ccte~~-irie” (cobweb-like prerrintrix) rhroirgh periplnsrriic space

At the next stage the dissolution of the part of callose layer adjacent to microspore plasmalemma proceeds. As a result the electron-transparent layer appears (the periplasmic space) betiyeen the main part of callose and plasmalemma (Fig. 1D,E). Simultaneously the spreading out of mem- branes of vesicles (Fig. ID) and of finger-like evaginations of plasma membrane take place (Fig. 1E). as a conse- quence, their contents are found in the periplasm. The substances filled in with periplasm are, referred to as, the

Fig. 3 . A-D. ThiCry reaction (A, B) and the controls (C, D) on the slime around tetrads. (A) Slime is revealed. (B) The same; excrc- tion of slime between two tapetal cclls is seen (arronhead); callosc is not revealed. (C) Control I : Treatment with H20z instead of HIO,. then thiosc-micarbnsidc and silver proteinate; slime is not revenled. (D) Control I I : treatment with HIO,. then silver pro- tcinatc without preliminary treatment with thioscmicarbazide. Bar = 1 pm.

came scarely recognisable, and are cup-like. The multi- membranous and myelino-like bodies are often observed in microspore cytoplasm, while the nucleus occurs with roundish nucleolus. The bundles of microtubules oriented in different directions are discovered in peripheral cyto- plasm.

At this time the tapetal cells contain large vacuoles, full of slime; these mucilage depositories are mainly concen- trated in parts of the tapetal cells adjacent to the loculus. Near the vacuoles the stacks of ER are seen. Until the beginning of meiosis in microsporocytes (PMC), some ta- petal cells surrounding PhlC and lying nearly, undergo the process of lysis. leaving cavities with remnants of cyto- plasm, gradually changing to slime. At this time in neigh- bouring tapetal cells. mitosis still occurs (Fig. 2A). It is observed that from the very beginning of tetrad develop- ment there are narrow. irregular shaped cavities around tetrads. which already at the next stage serve as reservoirs

F ; ~ . 4. Cob\veb-likc prematrix in periplnsrnic space and the establishment Of “Phantom Of ectexinc”. (*) UncCrtain pattern of flocculent prcrnatrix substance- (B) Thin. radially oriented strands of a substance arc SCcn (urro1r3)* as \vdl as ;I substance along callose border (nrrolv/ le~t is) . Flocculent substance bcttvcen strands remains (double t r r r o i d i d ~ ) .

Bar = 0.3 pm.

Grana 31 (1992)

Dow

nloa

ded

by [

Nat

urhi

stor

iska

Rik

smus

eum

] at

23:

29 0

6 M

arch

201

6

216 N.I. Gnbnrnyeva

cobweb-like pattern of primexine matrix, the phantom of ectexine, because of its columellate-tectate type: it will become distinct later, after the fully dissolution of callose.

All these changes take place on distal pole of micro- spore, on the proximal surface where the plasmalemma is underlaid with the tubular cisternae of SER, some quantity of prematrix-like substance appears too, but no further changes occur here at this and next two stages.

The cytoplasm of microspores is still rich in ribosomes, ER is seen in form of tubular SER, multimembranous spheres and myelin-like bodies are present, indicating auto- lytic processes. Dictyosomes are rather abundant and pinch off vesicles with fibrillar contents. Plastids and mitochon- dria are dedifferentiated and continue to take part in the autolytic process.

In tapetal cells the slime is secreted from the vacuoles

Fig. 5. Penetration of dark massules of tapetal origin to peri- plasmic space. (A) A massule invades through primary fibrillar en- velope of tetrad. (B,C) A massule with light nimbus penetrates through callose. Prematrix stripe of microspore origin (arrow). Bar = 0.25 pm.

substances of prematrix, since at the next stage a special structure is formed from these substances. This structure is ephemeral, disguished for some time by the imposition of other structures. I have named this structure prematrix of primexine, or “the phantom of ectexine”, as it surprisingly represents a,plan of the future exine.

Early formation of this unusual structure is shown in Fig. 1F. In periplasm, in interval between callose and plasma- lemma the disorganized dissociated substances of prema- trix are seen. No order can yet be traced in its localization. Somewhat later the amassment of these substances is ob- served along the border of the periplasm and callose (Fig. 4A), and then thin, even, radially oriented strands of a substance can be distinguished, while the flocculent mas- sules of all the rest of the prematrix substances remain between the strands (Fig. 4B). This is that very fragile

Fig. 6 . Prematrix establishment of both microsporal and tapetal origin. (A) Penetration of tapetal dark massules to periplasmic space of microspore (arrows). (B) Prematrix stripes of microspore origin (arrowheads; also see Figs. 1B gL Fig. 5C). (C) Continuation of tapetal dark massules penetration to periplasmic space (arrows) and establishment of double-origin prematrix (arroic3hcads - pre- matrix stripes of microspore origin). Bar = 0.3 pm.

Gram 31 (1992)

Dow

nloa

ded

by [

Nat

urhi

stor

iska

Rik

smus

eum

] at

23:

29 0

6 M

arch

201

6

Developriieiit of sporoderrii iri Asiriiiria 217

pass through. It appears possible that there are some lytic enzymes in these massules, such as callase and eellulase. which allow the massules to penetrate through the fibrillar and callose layers of tetrads. On reaching the region of the prematrix, the dark massules occupy some positions there which still seem uncertain (Fig. 6A). The radially oriented strands of cobweb-like pattern are still noticeable, whereas the stripes of prematrix which will be later the basis for the accumulation of protosporopollenin (SP) of the protectum, already take their places in periplasm at some distance from the sinuous plasmalemma (Fig. GB, C).

In the microspore cytoplasm, there are as many ribo- somes as earlier, the tubular SER fills the cytoplasm and is in contact with the plasmalemma (Fig. GC). Dictyosomes remain active; mitochondria attain the extreme degree of dedifferentiation and often appear to have no contents. The majority of plastids are cup-like and it appears that the autolytic processes in the captured cytoplasm are very ac-

Fig. 7. Differentiation of primexine matrix and initiation of protec- turn, primordial laniella and some procolumellae. (A, B) Protec- tum appears on the surface of primexine matrix between the places. occupied earlier by dark massules from tapetum (nrroivs). Also primordial lamella emerges (nrrowAeads). (C) In this place protecturn appcars betwen the upper surface of primexine matrix and lo\ver surface of prematrix stripe. Note the process of primor- dial lamella gcncration (nrroichends) and formation (arrows). Ttvo procolumellae appear also. Bar for A. B = 0.3 [im, C = 0.25 pm.

beyond the tapetum to the cavities, originated earlier from the degradated tapetal cells and which now are adjacent to tetrads.

3. Secreriori of prmia~rix siibsraiices by toperiini

At the next stage the secretion of dark massules of a sub- stance by the tapetum begins. These massules invade through the primary fibrillar envelope of tetrads (Fig. 5A) and penetrate to callose layer (Fig. 5B) and further - to the pcriplasms space ( ~ i ~ ~ . 5 ~ ; 6A). The e~ectron-transparent nimbus is formed around each massule (Figs. 5A-C; 6A-C). which, perhaps shows the dissolution of Small quantities of substances in those layers which the massules

Fig- 8. Distal re&ion with protecturn between prematrix and pri- mexine matrix. (B) Proximal region with a single sinuous lamella. (C) Transition region between the two poles. (D) Differentiation of procolumellae in prirncxine matrix (nrroir/Ieads), dark massules of tapetal origin (arroit’s). Bar for A. B = 0.25 [im, C = 0.2 prn, D = 0.5 iim.

Graria 31 (1992)

Dow

nloa

ded

by [

Nat

urhi

stor

iska

Rik

smus

eum

] at

23:

29 0

6 M

arch

201

6

218 N.I. Gobnroyeva

stance. As earlier, a lot of tubular SER cisternae are in contact with plasmalemma.

5. Differentiation of premexirie rrintrix orid the iriitintiori of protectirrn ond primorclinl loriielln of the foot layer

The synthesis and secretion of prematrix substances from microspore cytoplasm to periplasm lead to their accumu- lation, and after the redistribution of substances, the struc- ture of the prematrix changes, acquiring the features of more o r less classical fibrillar primexine matrix. The homo- geneous electron-opaque substance - the precursors of SP - start to accumulate, and as a result, the protectum appears (Fig. 7A, B). In those places which had been occupied by dark massules from tapetum at previous stages, the pre- cursors of SP never accumulate, and occur only befwecri the massules on the surface of matrix (Fig. 7A) or under the surface of stripes of prematrix (Figs. 7B, C; SA).

The formation of membranes begins a t the same time over the plasmalemma under the primexitie matrix (Fig. 7A, C), the process of membrane generation occurs step by step on the surface of the plasmalemma, and the fragments of new-formed membrane are at first indistinct and re- semble shadows (Fig. 7C - arrows). The same process goes on the proximal pole of microspore, where the typical matrix is absent. The proximal membranes are very sinuous (Fig. SB). The transition from distal (on the left) to proxi- mal (on the right) regions of microspore surface is shown on Fig. SC. As the activity of the dictyosomes increases. the mitochondria undergo redifferentiation, step by step ac- quiring cristae. The plastids are cup-like as earlier and the process of autolysis inside them comes to an end: the rem- Fig. Y. (A) Altcrnativc localization of pro-colurnellae and dark

rnassuks from tapeturn (nrrolrs). (B, c) Sliding m d Erazing SCC-

tions of microspore surface. Note the alternation of dark rnassulcs (nrroirs) and cross-scctions of procolurncllae (arroivhends). Bar = (1.3 urn.

nants of ER cisternae and other organelles are seen in their invaginations. The tetrads begin to loose their callosic en- velope. In tapetum the narrow slime-containing channels

r - - - - - - .. . . . .-

between neighbour cells are formed, which open directly to the locullis.

tive..This state of the plastids is retained during the follow- ing three stages.

6. Differeritintiori of procoluniellne iri pritriexitie rrintrix In tapetum where the stacked RER is prevailed, the synthesis. accumulation and secretion of slime to loculus continuc; besides slime, dark massules surrounded by a light nimbus are released into the loculus.

When most of the protectum is already formed. under its plates (in sections - under stripes) the procolumellae begin to appear (Figs. 7C; SD 6: 9A). Sometimes the base of a procolumella it is possible to observe some membrane in the form of a loop (Fig. 7C).

Alternative localization of procolumellae, supporting the plates of protectum and of the cavities occupied by the dark

4. The cstablishnierit of prcitintrix of both rnicrosporcil nrid tnpetol origin

The substances in the periplasm have a dual origin and consequently different distribution. The dark massules coming from the tapetum (Fig. 6C, arrows), progress to the intervals between the prematrix stripes of microsporal ori- gin (arrowheads) and remain there; the remainder of pre- matrix substances begin to be redistributed in periplasm (Fig. 6C). Besides, in the cytoplasm, the number of Golgi vesicles considerably increases; they contain a fibrillar sub-

tapetal niassules is clearly observed by comparing sections oriented perpendicularly to the surface of the plasma- lemma (Figs. SD; 9A) and sections of microspore surface. oriented tangentially to the surface of the microspore (Fig. 9B. C). These micrographs show very clearly that in places where the dark massules of tapetal origin have settled, proto SP never accumulates. Later the substances of the massules will be destroyed, and the luminae of mature

Dow

nloa

ded

by [

Nat

urhi

stor

iska

Rik

smus

eum

] at

23:

29 0

6 M

arch

201

6

Developtnetit of spororlertii in Asirt~ir~n 219

Fig. IO. Cytochcmical trcatmcnt of sections with 0.01% solution of trypsin for 3 h (B) and for 21 h (C). In both cases dark massulcs disappcar (mroii*s). Scc (A) for control. Bar for A = 0.3 pm. B, C = 1 pm.

sporoderm result from these processes in A . trilobo. I t is important to emphasize the hexagonal pattern of primexine matrix (Figs. 8D; 9C).

To define the approximate chemical composition of the dark massules, sections were treated with 0.01% solution of trypsin for 3 h and 21 h to test the possibility for the presence of proteins. The disappearance of the dark mas- sules after treatment with trypsin suggests that those mas- sules consist partly of proteins (Fig. 1OA-C).

Before the dissolution of callose in the distal region, the primexine consists of a protectum, procolumellae and 1-2 membranes over the plasmalemma. The first membrane (external one) will be a basis for the foot layer. In the proximal region. the changes involve 2-3 sinuous mem- branes. The number of ribosomes in the cytoplasm de- creases, the tubular SER occupies considerable part of the

cell volume, and active dictyosomes are found. Some plas- tids retain the cup-like form, the rest undergo redifferentia- tion: their stroma becomes more electron-opaque. and plastoglobulae appear. The mitochondria acquire the typ- ical form with ovoid cristae.

The tapetal cells continue to secrete slime to the loculus. preserving their initial localization along the peripheral part of the loculus, but they already begin to loose the radial walls.

During this time in the tapetum development no activity connected with the formation of pro-orbicules has been observed. The envelope of tapetal cells, consisting of fri- ably distributed fibrils, becomes gradually destroyed simul- taneously with the dissolution of callosic envelopes of te- trads. Numerous cisternae of E R , polysomes and vacuoles with slime are observed in tapetal cytoplasm.

DISCUSSION

The ciirol origiti of prcrtintri.r

First, outgrowths of plasmalemma occur between callose and plasmalemma of the very young tetrad, which are usually called plasmalemmasomes (Marchant 6: Robards 1965). This stage is quite common in early pollen wall development (Dunbar 1973; Rowley et al. 1973; Dickinson 1982), and also occurs in primitive Angiosperms (Gaba- rayeva 1987, 1990n, 1991). However, what is unusual for the primitive Angiosperms at such an early stage. is the mode of delivery of substances for preexine structures. Almost simultaneously over the plasmalemma we can ob- serve outgrowths with cytoplasmic content, the different stages of spreading out of these vesicles and the distribution of the contents of these vesicles on the plasmalemma. Simi- lar images of pinching off the vesicles and spreading their membranes out were observed in hlicliclin fiisctrtn (Gaba- rayeva 1986), but at a later stage - during the building of endexine lamellae at the beginning of the post-tetrad pe- riod. The presence of a different kind of spreading profiles over the plasma membrane is indicated in many species during the process of pollen ndl development (Rowley 1962, 1961; Rowley 6r Dunbar 1967); a role in exine pat- tern determination is ascribed to the outgrowths of plasma membrane (Dickinson 19S2).

The transfer of a part of cytoplasm together with ribo- some-like particles beyond the plasmalemma is the initial “constructing material”, which forms later the prematrix of primexine, or “exine phantom”. It is noteworthy that Ro- land’s (1973) review has shown that according to the data of several authors, some spherical granules from the peri- pheral cytoplasm can occur beyond plasmalemma; these granules are considered to be enzymic complexes.

The other source of substances, which are secreted at this time to periplasm, is evidently the tubular SER. My suppo- sition is that at the initial stages of sporoderm development tubular S E R synthesizes and secretes into prematris

Dow

nloa

ded

by [

Nat

urhi

stor

iska

Rik

smus

eum

] at

23:

29 0

6 M

arch

201

6

220 N.I . Gabnrnyeva

whether receptors for SP precursors, or enzymes for their polymerization.

In the periplasmic space the formation of fragile cobweb- like pattern occurred, that predicted the structure of the future colurnellate-tectate exine. Such a phenomenon - the formation of prefigure of the future sporoderm layer - has been already observed in the study of the fern sporoderm in Arietiiiapliyllitinis (Surova 1985). At this stage the distribu- tion of the receptors for SP precursors in the periplasmic space probably occurs, that may determine the pattern of future exine. I believe that this process is mainly autoorga- nizing and proceeds as a result of physical and chemical characters of colloidal solutions in thin layer (Gabarayeva 1 990 b) .

As noted earlier, almost simultaneously with the forma- tion of the cobweb-like ephemeral pattern, dark massules of substances from tapetum begin to appear. They go through the primary fibrillar envelope of tetrads and oc- cupy distinct places between the stripes of prematrix. It is probable that such a precise localization of both prematrix stripes and the dark massules from tapetum is connected with the previous short cobweb-like stage, which in some way determines their alternative and mutually excluding localization. This stage approximately corresponds to that, which marked as the initial stage by Waha 1987 (Fig. 1D). Moreover, I suppose that the prematrix elements of tapetal origin (dark massules) were identified by Waha (1987) as SP precursors. My data have demonstrated that these dark massules prevent the accumulation of the SP precursors around them and thus lead to the formation of the cavities in the tectum of the mature sporoderm. Remnants of dark massules are seen in luminae.

Pritiiexirie tiiatrix atid pritnexirie: which is priniary?

It is noteworthy that the bulk of primexine matrix, which appears as a result of Golgi activity, can be observed almost simultaneously with protectum. However, there were some stages of prematrix before the appearance of the usual primexine matrix. Beside there were definite elements in the prematrix of both microspore and tapetal origin which had already predetermined where SP precursors d o accu- mulate and where they d o not.

The cytoplasm of microspores a t this stage is packed with the tubular SER, and the continuous contact of these tu- bules with plasmalemma implies that these organelles are responsible for the synthesis of SP precursors, which occur beyond the plasma membrane, accumulate on the recep- tors of matrix, o r on polymerisation-promoting surfaces (Dickinson 1976).

The principle of negative-positive correlation between the space pattern of primexine matrix and of primexine itself (Heslop-Harrison 1968; Dickinson 1976; Dickinson & Potter 1976) has been fully implemented in the case of sporoderm development in Asitriirin rriloba. The only modification of this general principle is that not all parts of

primexine matrix are of microspore origin; a certain part of it, namely those massules of osmiophillous substance, are of tapetal origin. Waha (1987), reported the primary ap- pearance of protoSP and the secondary appearance of pri- mexine matrix. My data have shown that all elements of prematrix and some elements of matrix emerge before the appearance of protoSP.

It is interesting to compare the processes in developing microspores of Poiticiatin gilliesii (Skvarla & Rowley 1987) with those in Asitiiitin triloba. The authors have noted that the developed primexine matrix appears simultaneously with the initiation of probaculae. But, first, this event is preceded by the emergence of the receptors of the future tectum on plasmalemma: “Placoid receptors for the exine template are widely spaced on the plasma membrane”. (Skvarla & Rowley 1987). Second, the appearance of these receptors is preceded by some changes on surface of mi- crospore i.e. the formation of invaginations of plasma- lemma (cavations) and stacks of parallel fibrillae in other places of the microspore surface. The estimation of these events depends oq point of view. I consider all the pro- cesses, described by Skvarla and Rowley (1987) t o be the necessary preparatory steps for the appearance of typical matrix. I have observed a very similar picture in Asittiitin triloba, where some developmental phases of preparatory structures before the initiation of the orthodox matric oc- cur. I have called these preparatory structures “prematrix”. It is of no consequence that these preliminary stages differ from those of Poitzciatia. The other thing is important: the emergence of primexine matrix before primexine, whether it is or is not preceded ‘by some phases of prematrix whether it is formed long before the initiation of primexine or appears not long before.

Takahashi (1989), working on exine development in Caesalpitiia japotiica describes special “radial structures’’ which occupy the invaginating regions of plasmalemma at the initial stages of the tetrad period. Later, when protec- turn and procolumellae are already established, these aste- risk-like structures remain in the gaps of protectum, in the places of future luminae. A t the same time Takahashi (1989) affirms that primexine matrix is formed in coincide with the probacules, without any reason disregarding his own observations on “radial structures”, which precede the appearance of the typical primexine matrix. The sequence of events during the primexine establishment in Caesalpitiia japotiica seems t o analogous one in Asituitin triloba. The radial structures (Caesalpitiia) and the dark massules (Asi- triitin) are most probably the structures of prematrix. Both preventing the emergency of ectexine structures in the places of their location i.e. the places of the future lumina.

The role of tapetiitn

One of the major issues demonstrated in the present paper concerns the permeability of callose, raised in connection with the passage of dark massules of substances from tape-

Grana 31 (1992)

Dow

nloa

ded

by [

Nat

urhi

stor

iska

Rik

smus

eum

] at

23:

29 0

6 M

arch

201

6

Dciduprrrcrr! of sporoderrrr iri Asirnirin 22 I

tapetuni contain sonic protcins, probably - callase en- zyme. Their 1ocaliz;ition in preniatrix is predetermined earlier, at the stage of the cobweb-like pattern, and they, in their t u r n , determine the placcs in tlie spo- roderm where sporopollenin does not accumulate (the places of luminae). Tlie usual fibrillar matrix of primexine develops on the ground of prcmatrix. Protcctum is formed almost siniul- tancously, because its basis with corresponding recep- tors had already appeared in prcmatrix. llrocolumcllac of exine are seen somewhat later, in the end of callosc period. Simultaneously the membranc- like lamellac appear which are the ground for the foot layer and later for cndexine. ‘Ihpetuni during the callose period is of cellular sccrc- tory type. I t excretes large quantity of slime and pre- serves its location but does not form any orbicules. As seen froni the Thicry reaction, the excreted sliriic has a polysaccharide nature. The primcxine does not precede the appearance of pri- mexine matrix. The contribution of tapctuni to the formation of spo- rodcrm in the callose period is very considerable.

turn to microspore prcniatrix. These data suggest that some unusual mode of uptake through the callose envelope is likely to exist. This took place by means of borderline dissolution of small quantities of callose around massules and step-by-step sliding of these massules towards the nii- crospore. lt is natural to suppose that in dark massules sonic quantity of the enzyme callose is present, which tvas “captured” by niassulcs from tapeturn. Certainly, it might be useful to determine tlie chemical composition of these dark niassulcs of tapetal origin. hly supposition is that these substances arc of glycoprotein nature, and the cy- tochemical enzymatic test undertaken with trypsin suggest that they do contain some proteins. I cannot agree with \Vaha. (19S7). who commented: “At this stage little or no contribution to sporoderm development conies froni tape- t u ni “.

Tlie tapctuni in Asiririiro rrilohn is of unusual type. I t behaves ;is cellular secretory one, but only t i l l the moment of the dissolution of the tetrad callose envelopes. Then. it behaves as i f i t was a plasmodium, surrounding the tetriids and penetrating betwen the niicrospores. At the initial stages dark massulcs are formed i n tlicm, intended for the iriscrtion into primexine matrix and determination to sonic extent the exine pattern. hloreover, mass of slime is se- creted i n tapeturn. The synthesis of slime by the cisternae of KER and its excretion from tapeturn to loculus has been also observed in Micheliufirscnfn (Gabaraycva 1956), but i n this case i t occurs aftcr thc dissolution of callose, and is not s o intense. The explanation of an abundant excretion of slime in Asirriirrn rrilubn, in an early phase of sporoderm development could be (1) the tetrads i n Asirrrirrn friloha do not separate into niicrospores often callosc dissolution (2) each tetrad is enclosed into separate logettc of tapeturn. ‘the consequence of these points are: niicrosporcs in h i - nririn triloho arc more isolated froni the influence of tape- tiini t h a n microspores in species with free microspore pe- riod. Hence, i t must be some mechanism which \vould facilitate the penetration of different substances. for exani- ple nutritive ones and the prccursors of SP, to microspores. 1 consider that the hypersecretion of the slime is that very necccssary mechanism. The slime translocates some sub- stances from tapctuni cells to microsporcs.

CONCLUSIONS

1. Some initial stages of sporoderm development in h i - rrrirrn rriloha rcsult in formation of the prcmatrix of exine. The niost important features of this period are a) a brief stage of cobweb-like pattern (the phantom of ectexinc), represents a period when the main process of exinc pattern determination occurs b) some substances of tapctal origin in the form of dark rnassulcs take part in building of exine prcmatrix. They are able to pcne- tratc through callosc envelope of tetrads. The enzp:ltic test undertaken with t r y p i n , shows dark niassules from

2.

3.

4.

5.

6.

ACKNOWLEDGEhlENTS I am grateful to Dr. J. Rowlcy for stiniulnting discussions and n l s n for thc partly revision of the manuscript.

REFERENCES

Grana 31 (1992)

Dow

nloa

ded

by [

Nat

urhi

stor

iska

Rik

smus

eum

] at

23:

29 0

6 M

arch

201

6

222 N . I . G n b n r q w n

riod: the primcxinc formation in connection with cytoplasmic org;inclle activity. - Dot. Zh. (Leningrad) 72: 1310-1317.

Gabaraycva, N. I . 1955. The significance o f ontogenetic studies for clarifying the structure of mature sporodcrm and phylogeny on example o f some hlagnoliaccae and Annonaceae. - In: Palynol- ogy in the U.S.S.R. (cd. A. F. Chlonova), pp. 48-52. - Nauka, Novosibirsk.

Gabarayeva, N. 1. 1990a. Ultrastrutural study of main dcvclop- mental stages of sporoderm and parallel cytoplasmic organelle changes in Njrriplinen coeritlea microspore. - Theses 4th Rc- public Conf. Electron hiicrosc. Kishinev 1990. pp. 4W9. - Acad. Sci. hloldov. SSR, Kishinev.

Gabaraycva. N. I . 1990b. The hypothctieal trends in exine struc- ture determination. - Bot Zh. (Leningrad) 75: 13531362.

Gabaraycva. N. I . 1991. Patterns of development in primitive angiosperm pollcn. - In: Pollen and spores. Patterns of divcrsifi- cation. (ed. S. nlackmore S: S. 11. Barnes), pp. 257-265. - Clarcndon Press, Oxford.

lleslop-Ilarrison, J. 1965. Wall development within one micro- spore tetrad of Lili irrri longifloriivi. - Can. J. Bot. 46: 1185- 1192.

hlarchant, R. 6r Robards, A. \V. 1965. hlembrane system asso- ciated with the plasmalemma of plant cells. - Ann. Uot. 32: 457471.

hlcycr, N. R. 1377. Comparative morphological investigations of sporodcrm ultrastructure and dcvclopnicnt in Gymnosperms and primitive Angiosperms. - D.Sc. Thesis, Univ., hioscow.

Roland, J. C. 1973. The relationship between the plasma nicni- branc and plant cell wall. - Int. Rev. Cytol. 36: 45-92.

Ro\vley, J. R. 1962. Nonhornogenous sporopollenin in micro- spores of I’on ori)iiin. - Gram Palynol. 3: 3 1 9 .

Rowlcy, J. R. 1964. Formation of the pore in pollcn of Pon uwiiin. - In: Pollen physiology and fertilisation (cd. 11. E Linskens), pp. 59-69. - N. Holland Publ. Co., Amsterdam.

Ro\vlcy, J. R., Dahl, A. 0. S: Skvarla, J. J. 1973. Localization of ATP:isc xtivity in pollen grains. - Norw. J. Dot. 20: 31-50.

Rowley, J .R. S: Dunbar. A. 1967. Sources of membranes for cxine forniation. - Svcn. Bot. Tidskr. 61: 49-64.

Skvarla, J. J. it Rotvlcy. J. R. 1957. Ontogcny of pollen in Poiri- cinrin (Legurninoscae). 1. Development of exine template. - Rev. Palacobot. I’alynol. 50: 313-331.

Surova, T. D. 1955. hlorphology and dcvclopmcnt of spores in Schizncaccac - Kaulf. - Thesis diss., Leningrad.

Takahashi, hl. 19x9. Pattern determination of the cxinc in Coesol- piriin jnporiica (Leguminosae: Cacsalpinioidcae). - Ani. J. Bot.

Takhtajnn, A. 1951. The origin of angiosperms plants. - Sov. Nauka, hloscow.

ThiCry, J.-P. 1967. hlisc en 6vidcncc dcs polysaccharidcs sur coupes fines en microscopic Clectroniquc. - J. hlicrosc. 6: 957- 101s.

Thorne, R. F. 1976. A phylogcnctic classification of the Angiospcr- mae. - Evol. Uiol. 9: 35-106.

\Vatla, hi. 1957. Sporodcrm development of pollen tetrads in Asi/?ii/in frilohrr (Annonaceae). - Pollen Spores 29: 31-44.

Zavadn, hi. 1984. Pollen wall dcvclopment of Aitsfrobailcja rnacirlufa. - Bot Gaz. (Chicago) 145: 11-21.

76: 1615-1626.

Grutra 31 (1992)

Dow

nloa

ded

by [

Nat

urhi

stor

iska

Rik

smus

eum

] at

23:

29 0

6 M

arch

201

6