Pollen Development of Rondeletia odorata (Rubiaceae)

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American Journal of Botany 88(1): 14–30. 2001.

POLLEN DEVELOPMENT OF RONDELETIA ODORATA

(RUBIACEAE)1

GAMAL EL-GHAZALY,2,4 SUZY HUYSMANS,3 AND ERIK F. SMETS3

2Palynological Laboratory, Swedish Museum of Natural History, Roslagsvagen 101, S-104 05 Stockholm, Sweden; and3Laboratory of Plant Systematics, Institute of Botany and Microbiology, K.U.Leuven, Kard. Mercierlaan 92, B-3001 Heverlee,

Belgium

Pollen wall ontogeny of Rondeletia odorata was studied with transmission electron microscopy (TEM) and scanning electronmicroscopy (SEM) from tetrad stage until maturity. The ontogenetic sequence of wall development in Rondeletia follows, to someextent, the basic scheme in the angiosperms, i.e., development starts centripetally with the pro-columellae in a plasmalemma surfacecoating (primexine) at the early tetrad stage when the microspores are still enveloped by callose, until intine formation in young pollengrains. The main ontogenetical features of Rondeletia odorata pollen are (1) the very thin irregular foot layer, (2) development of acontinuous layer of radially oriented membranous granular material under the thick endexine, (3) initiation of intine before first mitosiswith characteristic radial plasmalemma invaginations, and (4) a strong stretching force upon engorgement just prior to dehiscence,which leads to reduction in thickness of all wall layers. The possible function of Golgi vesicles in the considerable increase in surfacearea of the plasmalemma at intine initiation is discussed. The endocingulum observed on acetolyzed and sectioned mature grains isexplained ultrastructurally.

Key words: endocingulum; Gentianales; membranous granular layer; pollen wall development; Rondeletia; Rubiaceae; ultrastruc-ture.

The cosmopolitan family Rubiaceae is essentially tropicaland comprises ;11 000 species (Robbrecht, 1988, 1994). It isregarded as a key family for understanding the phylogeny ofthe Gentianales. Palynological interest in Rubiaceae has re-cently resulted in several mostly morphological-systematic pa-pers (e.g., Andersson, 1993; Pire, 1997; Stoffelen, Robbrecht,and Smets, 1997; De Block, 1998).

Rondeletia odorata Jacq. is a small tree with bright orange-reddish flowers native to Cuba and Panama. The genus Ron-deletia Linn. (;250 species) belongs to the tribe Rondeletieaein the subfamily Cinchonoideae, where tribal relationships areblurred by recent fundamental changes in the systematics, e.g.,by works dealing with the delimitation of the Cinchoneae (An-dersson and Persson, 1991), the Isertieae (Bremer and Thulin,1998), and the Rondeletieae (Delprete, 1996). The pollen mor-phology of Rondeletia has been studied previously with lightmicroscopy (LM) and scanning electron microscopy (SEM)(Igersheim, 1993).

Our knowledge of pollen ultrastructure and development issurprisingly poor for such a vast family as Rubiaceae. Trans-mission electron microscope (TEM) images of mature pollenexines have been published for a few genera, mainly in sys-tematic papers (Johansson, 1987; Igersheim and Weber, 1993;Weber and Igersheim, 1994; Endress et al., 1996; Tilney andvan Wyk, 1997). Abadie and Keddam-Malplanche (1975) il-lustrated briefly two rubiaceous species with TEM.

Available data on pollen wall development are even more

1 Manuscript received 22 July 1999; revision accepted 6 April 2000.The authors thank Dirk Korstjens and Dr. Erik Schoeters (Leuven), and

Elisabeth Grafstrom (Stockholm) for technical assistance, and Prof. BjornWalles (Stockholm University) for the use of TEM. The directors of the Bo-tanical Gardens in Meise and Ghent (Belgium), and the Botanical Institute ofStockholm University are acknowledged for permission to collect material.This study was supported by a grant from the Research Council of the K. U.Leuven (OT/97/23), the Fund for Scientific Research-Flanders (FWO, NumberG.0143.95 and 2.0038.91) and by a fellowship to the second author fromWenner Gren Foundations, Sweden.

4 Author for reprint requests (e-mail: [email protected]).

scarce. Andronova (1984) investigated pollen development inseveral species with special attention to the tapetum. In theirseries of light microscopy studies on the floral morphologyand embryology of Pavetta gardeniifolia, von Teichman, Rob-bertse, and van der Merwe (1982) briefly described microspo-rogenesis.

There is only one study of pollen wall development of Ru-biaceae, namely on Mitriostigma axillare, a species with per-manent tetrads (Hansson and El-Ghazaly, in press). As for theorder Gentianales, we know of two similar studies, one onCatharanthus roseus in the Apocynaceae (El-Ghazaly, 1990)and the other on the Asclepiadaceae (Dannenbaum and Schill,1991).

In our work on the development of pollen in Rondeletiaodorata we aimed at documenting the main developmentalfeatures of the exine and intine from tetrad stage until pollenmaturity. Special attention was paid to relate cell organellecontent of the microspores with the ontogenetic sequence ofwall formation. Our data on tapetum and orbicule developmentin the same species will be published later.

MATERIALS AND METHODS

This study is based on fresh flower buds of cultivated Rondeletia odorataJacq. collected in the National Botanic Garden Belgium (specimen number39-2109) on 10 and 24 October 1996, the greenhouses of Ghent University(Belgium) on 2 December 1996, and the Botanical Institute of StockholmUniversity (specimen SU-c-88.24) on 2 June 1997.

The approximate stage of development can be determined by the dimen-sions of the anthers, but light microscopic squashes give a more accurateindication of the developmental stage. For different flower bud sizes, oneanther was crushed on a slide, stained with toluidine blue, and examined withLM. Anthers with microspores in tetrads were ;1.5 mm long, and matureanthers were 62.7 mm. In Rondeletia the early stages of pollen development,and hence the most significant events, proceed very rapidly; this has also beenobserved in Mitriostigma (Rubiaceae; T. Hansson, personal communication,Palynological Laboratory, Stockholm).

TEM/LM—Decapitated anthers were fixed in 2% glutaraldehyde in 0.05

January 2001] 15EL-GHAZALY ET AL.—POLLEN DEVELOPMENT OF RONDELETIA

mol/L Na-cacodylate buffer, pH 7.4 for 624 h and postfixed in 1% OsO4 for1 h. Anthers were blockstained with uranyl acetate for 10 min, dehydrated ina series of acetone and propylene oxide, and embedded in araldite. Ultrathinsections were stained with uranyl acetate and lead citrate. Electron micro-graphs were taken with a Zeiss EM906 electron microscope at 80 kV.

SEM—Fresh decapitated anthers were fixed as for TEM and dehydrated inan acetone series. Anthers were transferred to gelatine capsules filled withacetone 100% and frozen in liquid nitrogen and fractured on a TF-2 chamber.After critical point drying, the anther fragments were fixed on a stub by silverpaint. Pollen in Figs. 28 and 29 was acetolyzed and sectioned using a Ameslab Cryostat freeze microtome. Pollen sections were transferred to a stub.SEM observations were made with a Jeol SM6400 and a Jeol JSM-5800 LVmicroscope.

RESULTS

Rondeletia odorata has tetrasporangiate anthers. The antherwall typically consists of epidermis, endothecium, one, two orrarely three middle layers and tapetum. The tapetum is uni-nucleate and one or two layered. In rare cases we observedtapetal cells with two nuclei. The microspore mother cells(MMCs) are angular in shape with a large nucleus and a dark-staining nucleolus, which often shows one or sometimes twosmall vacuoles. The cell wall of the MMCs and the tapetumappears undulating.

Although the development of the pollen wall is a continuousprocess in Rondeletia, we have organized the descriptions ofdevelopmental events and cytological changes in four stages:tetrads, free microspores, young pollen grains (after mitosis),and pollen grains at dehiscence.

Tetrad stage—After meiosis, tetrads of haploid micro-spores randomly filled the entire space of the anther locules.The tetrads were arranged in tetrahedral and decussate units.Each tetrad was surrounded by a thick, asymmetrical calloseenvelope. The shape of tetrads in the callosic envelope wasmultiangular (Fig. 1). Callose was thickest on tetrads in thecenter of the locules. The thickness of the callosic partitionsbetween the microspore units of a tetrad was variable. Theedges of the callosic layer appeared porous in early tetrads(Figs. 1, 2).

Early tetrads—The plasmalemma of the microspores wasinitially straight and in direct contact with the callose (Fig. 3).The cytoplasm was rich in vacuoles, ribosomes, and Golgivesicles; mitochondria were not observed. Exocytosis was ap-parent through the fusion of vacuoles with the plasmalemma(Fig. 3). Between the callose and the plasmalemma a fibrillarsurface coat developed (Fig. 3) that, upon further develop-ment, increased in thickness. This plasmalemma surface coat(primexine matrix) had a loose, irregular fibrillar texture. Itsthickness was uneven on the circumference of microspores.Pro-orbicules were nested in cup-like depressions of the plas-malemma of tapetum cells (Figs. 2–4).

In slightly older tetrads, rod-shaped electron-dense unitswere radially oriented in the distal part of the plasmalemmasurface coat. These rods formed the columellae (Fig. 5). Some-times more than one developmental stage was observed in thesame anther. This suggests that the early events in microsporedevelopment occur rapidly.

Late tetrads—By end of tetrad stages, all layers of the exinewere obvious (Figs. 6–9). The columellae were not solid and

contained distinct cavities (Figs. 6, 7). The foot layer was thinand the endexine started to develop on a white line centeredlamella (Figs. 7–9). The tectum was thin and interrupted byperforation, and the apertures were distinguished by develop-ment of numerous lamellae and thick granular material (Fig.9).

Free microspores—Upon dissolution of the callosic enve-lope and release of the microspores from the tetrads, theirwalls increased considerably in thickness (Figs. 10–16). Thetapetum appeared hyperactive and some cells seemed to pro-trude between the microspores. Tiny orbicules (;0.20 mm)with a thin continuous coat lined the surface of the tapetalcells, including the inner tangential side, the radial and theouter tangential surfaces (Fig. 10). At the end of this stage thetapetum started to mature, tapetal organelles were found in thelocule (Fig. 16), and initiation of the characteristic endothe-cium thickenings took place. In cases where the septum be-tween the locules was continuous, microspores in one loculeshowed a different stage of development from the other locule.When the septum was interrupted, all microspores in the con-nected locules were synchronized in development.

Early free microspores—The pro-columellae increased inthickness particularly at their distal ends (Figs. 10–12). Onoblique tangential sections through the columellae, they ap-peared as darkly stained circular units with an electron-lu-cent, hollow center (Fig. 10). The pro-tectum was developedwhile additional sporopollenin-like material was depositedbetween the distal portions of the columellae (Figs. 11, 12).The foot layer appeared as a thin, hardly distinguishablelayer at the bases of the columellae (Figs. 11–13). Therewere indications of a white-line-centered lamella, whichseparated the foot layer from the endexine during early de-velopment. The endexine continued to develop on the prox-imal surface of this white line and consisted of several tri-lamellated structures at later stages. On some sections thewhite lines of the endexine appeared to extend into the baseof the columellae (Fig. 11). At the apertural sites, the end-exine dilated into several white-line centered lamellae witha thin coating of electron-dense material, apparently spo-ropollenin. These lamellae were separated from each otherby fibrillar material, i.e., remains of the primexine matrix(Figs. 12, 13). Beneath the endexine, at apertural sites, athick granular body was differentiated (Figs. 13–16). Uponfurther development, this granular material extended intothe interapertural regions forming a continuous granularlayer with radially oriented elements (Figs. 14–16). Ribo-somes, mitochondria, rough endoplasmic reticulum, smallvacuoles, and vesicles were common in the cytoplasm ofmicrospores. Few dictyosomes were observed. Storageproducts were lacking in microscopes at this stage (Fig. 16).A prominent feature of this stage was the abundance of ri-bosomes (Figs. 14, 15) and endoplasmic reticulum (Fig. 15,arrowheads). The layer beneath the endexine consistedmainly of granular material and tubular units similar tothose of the endoplasmic reticulum (Fig. 15). We refer tothis layer as membranous granular layer (MGL). The tubularunits of the MGL had direct continuities with the electron-dense endexine layer (Fig. 15). As maturation continued,the MGL became compressed and differentiated into a mix-ture of granules and membranous components (see Fig. 22).

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Figs. 1–5. Microspores in tetrad stage. 1. Tetrads in a callosic envelope. Note the more porous edges of the callose (arrows) and the remnants of the cellwalls of the MMCs in between the tetrads (SEM). 2. A tetrahedral tetrad of similar developmental stage as in 1. At bottom left, intact tapetal cells with cellwall and extracellular pro-orbicules (arrowheads). 3. Detail of one microspore in callosic envelope (C) with thin plasmalemma surface coat (arrowheads).Cytoplasm around large central nucleus rich in small vacuoles (V), free ribosomes and poorly differentiated mitochondria (M). 4. Detail of a tapetal cell andpro-orbicules (arrowheads). 5. Detail of the plasmalemma surface coat in a slightly late tetrad stage; note radial, darker stained rods (pro-columellae, arrows)


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and protectum elements in distal part of surface coat. Scale bar 5 0.5 mm.Figure Abbreviations: A, apertural region; C, callose; CM, colpus membrane; Ec, endocingulum; G, granular material; GN, generative nucleus; I, intine; L,

lipid droplet; M, mitochondria; MGL, membranous granular layer; N, nucleus; O, orbicules; S, starch; T, tapetum; V, vacuole; VN, vegetative nucleus. Scalebars 5 1 mm, unless stated otherwise.

Late free microspores—As development proceeded, the mi-crospores expanded and increased both in volume and in wallthickness. The columellae were numerous, and radially ori-ented, and the intercolumellar void contained fibrillar material(Fig. 17).

The sporopollenin coat on the white lines in the aperturalregion became thicker. In several sections we observed thatthe plasmalemma receded in the interapertural regions, creat-ing a large pericytoplasmic space under the radially orientedmembranous granular material. We assume that this eventmight indicate the very start of intine development. At the endof this stage the plasmalemma was highly undulated and pro-truded at several sites (Fig. 17). Several vacuoles were prom-inent in the cytoplasm of the microspores.

Young pollen grains—The main developmental event dur-ing this stage was the formation of the intine, the last walllayer. The young pollen grains had punctate exine and threedistinct apertures (Fig. 18). The tapetum appeared to be de-generating, and tapetal organelles and lipid droplets were ob-served in the locule between the pollen grains (Figs. 19, 20).

The intine started to develop on the radial protrusions ofthe plasmalemma as a continuous, evenly thick layer betweenthe apertures (Fig. 19). Under the apertures, however, the in-tine was thicker and bulged out together with part of the pro-toplasm (Fig. 20). The distal part of the intine, close to theradially oriented MGL, had more fibrillar structure and wasmore electron dense than the rest of the intine (Figs. 19, 20).None of the sections with an early intine showed two nuclei.The vegetative nucleus, however, was often excentric, but thismight also be due to vacuolation. We therefore assume thatinitiation of the intine took place prior to first mitosis. Thecytoplasm contained long profiles of rough endoplasmic retic-ulum parallel with the plasmalemma, many mitochondria, vac-uoles of different size, and numerous dictyosomes (Figs. 19,20). The cytoplasm was dense with vesicles that were buddingfrom dictyosomes. These vesicles were directed toward theplasmalemma and fused with it (Figs. 21, 22). The MGL ap-peared slightly compact and irregular in shape (Fig. 22). Thewhite line separating the foot layer from the endexine was stillvisible. The foot layer was readily distinguishable and had thesame stainability as the sexine (Fig. 22). Additional sporopol-lenin accumulated on the exine of young pollen as well as onorbicules. The tectum appeared solid, and microchannels werepresumably obscured by filling material. The columellae ar-cade contained fibrillar material, probably remains of the pri-mexine (Figs. 21, 22).

The next step in the ontogenetic sequence clearly showedthe spindle-shaped generative cell adjacent to the intine andsurrounded with an intine-like wall (Fig. 23). Later in devel-opment the generative cell migrated toward the central vege-tative nucleus. The generative nucleus was characteristicallysurrounded by lipid droplets (Fig. 24) during its associationwith the vegetative nucleus. The intine-like wall around thegenerative nucleus remained intact. The intine became com-pact and appeared more electron dense. In several sites the

intine protruded between the MGL and reached the endexine(Fig. 25). Later on the MGL compressed and appeared morecompact than before (Fig. 25). The cytoplasm was character-ized by abundance of compound starch grains and some lipiddroplets (Figs. 23, 24). The increase in lipid and starch con-tents of the young pollen grains was concurrent with abun-dance of lipid droplets in tapetal cells. Ribosomes, rER, andsmall mitochondria were abundant near the undulating plas-malemma. Dictyosomes and Golgi vesicles, common in earlypollen grains, were absent in this stage.

The volume of the pollen grains progressively increased,resulting in a stretching force on the pollen wall (compareFigs. 19 and 24). The effect of this stretching was more pro-nounced in pollen grains at dehiscence. The intine consider-ably thickened under the apertures. The aperture was coatedwith the fibrillar layer of the intine (Fig. 26) and the colpusmembrane appeared of endexinous nature (Fig. 27).

Pollen grains at dehiscence—The pollen grains were fullyengorged and appeared more or less circular in outline. Maturepollen grains taken from dehisced anthers were covered by aconsiderable amount of lipidic material (positive staining withSudan III), which may be classed as pollenkitt (Fig. 28). Thismaterial was released by the degenerating tapetal cells, whichwere compressed into a tapetal membrane, densely covered byorbicules (Fig. 28).

The pollen wall was extremely stretched due to an increasein volume compared to the previous stage. This stretching af-fects all layers of the wall, i.e., all layers decreased in thick-ness. At the final stage of pollen development the intine wasvery thin. On the equator the apertures were covered withdarkly stained tangentially oriented tubules or lamellationswith a hollow, electron-transparent center (Fig. 29 and inser-tion). The MGL became more widely spaced and appeareddiscontinuous due to outstretching of the sporoderm (Fig. 30).This is obvious on full outlines of mature pollen grains (Fig.31).

Lipid droplets were observed, often in association with theapertures (Fig. 31). The lipid bodies were much more numer-ous than starch grains and they were mostly enfolded in asingle rER strand (Figs. 29, 31). In some sections lipid glob-ules were conspicuously associated with the generative nucle-us/sperm cells. Moreover, we observed numerous dictyo-somes, elongated mitochondria, and a large number of freeribosomes (Fig. 31). The generative cell was enclosed by theintine-like wall (Fig. 31). Several pollen grains showed elon-gated, spindle-shaped sperm cells; thus some grains were tri-nucleate at dehiscence.

Mature pollen grains (LM and SEM)—The mature pollengrains were monads, small [P (polar axis) 16–(17.7)–19 mm,E (equatorial diameter) 18–(19.3)–21 mm], oblate spheroidal(P/E 0.92 on average) in equatorial view and subtriangular inpolar view. The grains were planaperturate generally withthree, rarely four, compound apertures. The short and narrowectocolpi had acute ends. There was a lolongate pore in the


Figs. 6–9. Microspores in late tetrad stage. 6. The callosic envelope (C) starts to disintegrate. The pro-tectum, pro-columellae, foot layer, and endexine aredistinguishable. Note the start of aperture development and presence of several lamellae at aperture sites. 7. Magnified part of the wall showing pro-columellaewith cavities (arrowhead), very thin foot layer, and endexine. The plasmalemma surface coating is obvious between the pro-columellae. 8. Magnified part of amicrospore wall and an aperture, indicating the separation of lamellae towards the aperture and the presence of coarse granular material at aperture site.Arrowhead indicates the remnant of the callosic envelope. 9. Magnified part of the wall to emphasize the thin foot layer (arrowhead) and endexine with whiteline centered lamellae (arrow). Scale bar of Figs. 7–9 5 0.1 mm.


Figs. 10–12. Early free microspores. 10. Overview of several free microspores with large central nucleus (N). Tectum, columellae, foot layer, and endexineare recognizable. In apertural regions granular material is deposited (arrowheads). On oblique sections, at bottom left, columellae in cross section appear hollow(arrows). At bottom right, part of a tapetal cell (T) and extracellular orbicules with thin sporopollenin coat are shown. 11. Sporoderm in cross section not farfrom aperture at the right. Note parallel white-line-centered lamellae (in distal part of the endexine) tending to go up into base of columellae; the foot layer isvery thin and hardly distinguishable. The plasmalemma retracts to give space to loosely arranged granular material under the endexine, initially only in aperturalregions. Scale bar 5 0.5 mm. 12. Sporoderm in apertural region showing dilated endexine with separated white-line-centered lamellae covered with sporopolleninand thick granular body (G) underneath.


Figs. 13–16. Free microspores. 13. Detail of microspore wall in apertural region (aperture to the right); tops of columellae are stained darkest and areconnected to form the tectum. The white lines in the endexine are only visible in the distal portion; the foot layer is extremely thin. Close to the apertures, theendexine consists of several white-line-centred lamellae that are coated with sporopollenin (arrows) and separated by fibrillar material. Underneath this structuredeposition of the granular material (G) takes place. Scale bar 5 0.25 mm. 14. Slightly later in development than in Fig. 13. The granular material is now acontinuous layer (MGL) under the endexine. Scale bar 5 0.5 mm. 15. Slightly oblique section showing condensation of the granular material into radiallyoriented elements. Note tubules in the peripheral cytoplasm, perpendicular on plasmalemma (arrowheads), which continue through the exine up to the columellararcade (arrows). Scale bar 5 0.5 mm. 16. Overview of full microspore showing a continuous membranous granular layer under the endexine.


Figs. 17–20. Initiation of intine. 17. Late free microspore with excentric vegetative nucleus (N) and well-developed exine, including continuous granularlayer under endexine; plasmalemma is undulating (arrowheads), indicating start of intine formation. Note fibrillar deposits between columellae. 18. SEM ofpollen grains (in same stage as Fig. 19). Exine punctation and apertures are well developed. Note imprints of microspores in tapetum (T). 19. Slightly later indevelopment than in Fig. 17. Vegetative nucleus (VN) with prominent nucleolus and start of generative cell (GN). Initiation of intine on distal face of theundulating plasmalemma (arrowheads). Maturing tapetal cells (T) with orbicules in upper right corner (arrows). Note tapetal organelles and lipid droplets inlocule between the young pollen grains. 20. Detail of aperture in same stage as Fig. 19. Note highly undulating plasmalemma producing membrane fragments(arrowheads). Active cytoplasm with rER-strands, mitochondria, small vacuoles, and many vesicles. At the left, remnants of maturing secretory tapetal cellswith many lipid droplets (L) in contact with the exine.


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0.5 mm. 22. Same developmental stage as Fig. 21, but cross section of inter-apertural region. Early intine with structural differences distal from undulatingplasmalemma; membranous granular layer under solid endexine is more com-pressed; foot layer is easily distinguishable; active cytoplasm is rich in mi-tochondria, rER, vesicles, and free ribosomes.

Figs. 21–22. Maturation of intine. 21. Detail of early intine (I) at aperture;plasmalemma protrusions are particularly visible; cytoplasm is bulging out ataperture and covered by darkly staining fibrillar material. Note structural dif-ferences within early intine. Dictyosome vesicles are abundant in hyperactivecytoplasm and fusing with undulating plasmalemma (arrows). Scale bar 5

center of each colpus; protruding onci were not observed inunacetolyzed grains. The sexine was punctate with a smoothtectum (Figs. 32–35). The perforations were variable in shapeand size. The columellae were considerably longer in the me-socolpia giving rise to the subtriangular amb (Fig. 32). Theinside ornamentation of acetolyzed grains was uniformly gran-ular except for the endocingulum, which is ;2 mm wide. Thesurface structure of the endocingulum was more coarse withrod-like elements (Figs. 32, 33). The MGL probably corre-sponds with the inner granular ornamentation that is observedon acetolyzed and fractured grains (Figs. 32, 33).

DISCUSSION

The discussion will focus on the characteristic features ofthe development of Rondeletia pollen and attempt to relatecytological events with the ontogenetic sequence of sporodermformation.

Pattern initiation of early exine—During the callose periodthe exine is initiated as rods extending from the plasmalemma.These rods are exine units that become columellae as well aspart of the tectum and foot layer. Oblique sections of the earlyexine show that the tectum consists of the distal portions ofclose-packed exine units. The pro-columellae are tubular instructure, and they subsequently develop into mature wall el-ements by the accumulation of sporopollenin. We assume thatunits of pre-exine are continuous with structures in the cyto-plasm, which are cytoskeletal.

In early tetrad stages of Rondeletia microspores, when thetectum becomes evident, the exine units show a honeycombpattern resulting from close packing and interdigitation of theunits. Dickinson (1970) showed the occurrence of such a hon-eycomb arrangement in Lilium. El-Ghazaly and Jensen (1985and 1986) presented a similar pattern in Triticum in early stag-es of microspore development. Wodehouse (1935) pointed outthat a reticulate pattern is a common theme in nature and isformed where even shrinkage occurs within a uniform matrix.

Wall layers that deserve special attention—Columellae—The columellae in Rondeletia pollen are the first wall com-ponent to be synthesized in the fibrillar plasmalemma coat inearly tetrad stage. In oblique (tangential) sections from theearly free microspore stage, the columellae appear circularwith an electron-lucent center. Thus in three dimensions theyare hollow cylindrical and radial supportive elements. Later indevelopment the centers become obscured by material of thesame electron density as sporopollenin. Nowicke, Bittner, andSkvarla (1986) used plasma-ashing on many species of Paeon-ia and found that rod-shaped substructures were evident inpollen that had not been observed before ashing. Blackmoreand Claugher (1984, 1987) used fast atom bombardment instudying the exines of Fagus and Scorzonera and found thatthe exines were composed of hollow tubes. Blackmore (1990)in a developmental study of pollen of Echinops found thatexine processes appeared to be hollow during early stages. In


Figs. 23–27. Young pollen grains. 23. Pollen grain after first mitosis showing generative cell adjacent to intine and surrounded by intine-like wall (arrow-heads); remnants of vacuole (V) in cytoplasm, and initiation of storage products, lipids (L) and compound starch grains (S). 24. Later in development, thegenerative cell (GN) associates with vegetative nucleus (VN) and is characteristically surrounded by lipid droplets; starch grains and lipid droplets are abundantin cytoplasm. 25. Slightly oblique cross section of sporoderm in interapertural region. Granular material compressed into thin layer under solid endexine; whiteline separating endexine from foot layer remains visible (arrowhead); mature intine (I) with a distal fibrillar sublayer and a proximal part with the characteristicradial membrane fragments. The distal part of the intine protrudes into the granular material, touching the endexine (arrow). 26. Cross section of sporoderm ataperture. The proximal part of the intine (I) is considerably thicker under the aperture. 27. Cross section of sporoderm at aperture, showing thick intine (I),colpus membrane (CM) is shown to be of endexinous nature.


Figs. 28–31. Mature pollen grains at dehiscence. 28. Pollen grains in locule covered with lipidic droplets (L) of tapetal origin; tapetal cells completelydegenerated, orbicules (O) lying on endothecium (SEM). Scale bar 5 10 mm. 29. Cross section through aperture. Cytoplasm under aperture with many freeribosomes and lipid droplets (L) characteristically surrounded by a single strand of rER, starch granules (S) less abundant than before; note the thin stretchedintine (I) even under the aperture. The pore is covered with tubular endexine components with a low contrast core (arrows). Insertion shows detail of theendexine low contrast cores in cross section (small arrows). 30. Detail of sporoderm in interapertural region; all wall layers stretched and reduced in thickness,especially the membranous granular layer (MGL) and the intine (I). Scale bar 5 0.5 mm. 31. Pollen grain prior to anther dehiscence with lipid droplets on itssurface (L); storage products in cytoplasm are mainly lipids with fewer starch grains; the cytoplasm appears grey because of the abundance of free ribosomesand dictyosomes. The generative nucleus (GN) is located towards the periphery of the vegetative cell.


Figs. 32–35. Endocingulum at inner surface of grains. 32. Inside view of half of mature pollen grain showing endocingulum (Ec) on equator (SEM). 33.Detail of Fig. 32. Structure of endocingulum is coarse and rod-like in contrast to granular nexine (SEM). 34. The same area as in Fig. 33, as seen in TEM;near aperture (A) membranous granular layer and endexine are lacking, and fasciated columellae are in direct contact with intine (I). 35. Three dehydratedgrains showing that endocingulum is not a particular area for folding in the process of harmomegathy.

Echinodorus, El-Ghazaly and Rowley (1999) also observedprobaculae with an unstaining, hollow-appearing core zone.

Foot layer—The foot layer was hardly visible in early stag-es. It became more pronounced and had a similar stainabilityas the sexine, i.e., darker than endexine, from the vacuolatestage on. In Rondeletia the junction between the endexine andthe foot layer appears as a white line. Similar structure wasobserved in other species and described as ‘‘junction plane’’

(Xi and Wang, 1989; Rowley and Rowley, 1996) or ‘‘com-missural line’’ (Simpson, 1983).

Endexine—Formation of the endexine clearly involveswhite-line-centered lamellae. The ‘‘lamellations’’ can be rod-lets, but there are several reports of actually sheet-like lamel-lations (e.g., Stone, Sellars, and Kress, 1979). Rowley (1996)suggested that a tubular organization with reversible lateralcross-linking offers versatility for growth and internal trans-


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Figs. 36–43. Schematic drawings of main stages of pollen wall development in Rondeletia odorata. The corresponding TEM micrographs are mentioned ifthey are included in the present paper; otherwise we refer to figures that show a similar developmental stage. 36. Tetrad stage. Initiation of pro-columellae andpro-tectum in primexine (;Fig. 5). Figs. 37–41. Free microspore stage. 37. Formation of endexine mediated by white lines which dilate in apertural region tothe left; foot layer hardly discernible. 38. Initiation of membranous granular layer proximal to endexine; significant sporopollenin polymerization in sexinecompared to previous stage (;Fig. 11). 39. Membranous granular material forms continuous layer, and tubular connections with peripheral cytoplasm appear;endexine becomes more solid and compressed (;Fig. 14). 40. Slightly later in development. Membranous granular material shows a radial orientation. Tubularstructures from peripheral cytoplasm extend through the exine up into the columellar arcade (;Fig. 15). 41. Start of intine development on plasmalemma withnumerous radial extensions (;Fig. 19). 42. Young pollen grain with fully developed sporoderm, note the structural difference in intine (;Figs. 21, 22). 43.Mature pollen. Wall layers are stretched upon engorgement prior to dehiscence (;Fig. 30).

port. Researches should consider the possibility of extensivelateral cross-linking of the rodlets or tubules into sheets. Atthe apertures endexine components separate from each other(most conspicuously in free microspore stage) contrary to thesolid appearing endexine in interapertural regions. In these ap-ertural regions the endexine shows lamellations with white-line centers in longitudinal profile and tubular componentswith a low-contrast core in cross sections. In Poinciana (Le-guminosae) Rowley and Skvarla (1987) also showed that end-exine components in apertural regions were tubular with alow-contrast central core. In interapertural regions, wherewhite-lines in the endexine are commonly believed to repre-sent lamellae, an alternate interpretation of their morphologymight be fascicles of the tubular form.

Our results show that ER units at the periphery of the cy-toplasm extend radially toward the developing wall and formthe membranous part of the MGL beneath the endexine. Wehave also observed these units further extended to the end-exine and even to the arcade between columellae. Because ourobservations were consistent in several sections and the ERunits were quite obvious, we resist the idea that ER might havea role in transfer of material between microspores or pollengrains and tapetal cells.

Membranous granular layer (MGL)—Echlin and Godwin(1969) described in Helleborus that after formation of the end-exine on lamellae, deposition of sporopollenin appeared assmall granules that gradually coalesce. A similar phenomenonwas observed in Rondeletia where membranous tubular unitsintermingled with granular material accumulated on the prox-imal side of the endexine. A similar layer was observed inNelumbo (Kreunen and Osborn, 1999), Nymphaea (Gabaray-eva and El-Ghazaly, 1997), and Catharanthus (El-Ghazaly,1990). The question of whether this layer is mainly developedfrom extensions of the ER or whether it belongs to the end-exine, e.g., endexine II, or a layer not homologous with theendexine remains open. It is definitely not part of the intinesince its granular part can resist acetolysis. The granular or-namentation of the inside of mature acetolyzed grains corre-sponds most likely to this granular material. A histochemicalstudy of this layer in different species will provide useful in-formation on the chemical nature of this layer. Such a studyis in progress by the authors of this paper.

Intine—The intine, like other primary plant cell walls, de-velops generally after mitosis (Robards, 1970) and thus in pol-len grain stage. In Rondeletia, however, intine formation mightbe initiated before mitosis as was also observed by Heslop-Harrison (1968) in Lilium and Silene, and in Dioscorea du-metorum (P. Schols, personal communication, Laboratory ofPlant Systematics, K. U. Leuven). Although the exact timingof intine initiation might be doubtful, the mode of deposition

is not. The Golgi vesicles are clearly deposited onto the plas-malemma and are spilled out by exocytosis into the periplas-mic space to form the intine, which is found pressed againstthe exine. This is common in other families and has been ob-served by Echlin and Godwin (1969) in Helleborus foetidus,in other Ranunculaceae by Roland (1971), in Platanus bySuarez-Cervera and Seoane-Camba (1986), etc. In Rondeletiathe irregular, sometimes branched ingrowths of the plasmalem-ma into the developing intine are very pronounced in late freemicrospore stage and immediately after first mitosis (Figs. 17,19–22).

Apertures—We did not observe parallel endoplasmic retic-ulum profiles in future aperture sites as was reported for Lilium(Dickinson, 1970), Helleborus (Echlin and Godwin, 1968) andSecuridaca (Coetzee and Robbertse, 1985). From early tetradstage, however, the plasmalemma surface coat is much thinneron three equally distributed areas believed to be the destinedapertures. The intine is characteristically thicker under the ap-ertures before engorgement of the grains. Prior to dehiscencethe intine becomes very much stretched and of equal thicknessall around the profile of the grain (see below). In mature pollengrains we showed that the colpus membrane is of endexinousnature (Fig. 27) and that in cross sections the pore is coveredwith tubular endexine components with a low-contrast core(Fig. 29).

Cytology—The dynamics of the cytoplasm components dur-ing pollen ontogeny is astonishing. Cell organelles are formedaccording to the demands of the developmental processes.Next to this variation in time there is also variation in space.Comparison of sections of different angles in the same devel-opmental stage showed that the cell organelles are not evenlydistributed in the cytosol. The peripheral cytoplasm at the latevacuolate stage is dense with Golgi vesicles and dictyosomes,and the vesicles fuse with the plasmalemma (Figs. 21, 22). Asstated above, the distal face of the intine is formed by materialtransported in Golgi vesicles that through exocytosis is re-leased into the periplasmic space. We believe that there is alsomembrane flow from the dictyosomes to the plasmalemma.During the exocytosis process the membranes of the vesiclesare incorporated into the plasmalemma, increasing consider-ably its surface area. This membrane flow explains how theplasmalemma retains its surface area during formation of theradial membrane fragments in the proximal part of the intine.Membrane flow and differentiation from the ER through theGolgi apparatus to the plasmalemma have been long known(e.g., Morre et al., 1971; Gabarayeva, 1987; see also reviewsby Steer, 1991 and Sitte, 1998).

In Rondeletia, the vegetative cell of the maturing pollengrain synthesizes the two intracellular lipid structures found inpollen: oil bodies and intracellular membranes (Fig. 24). In



mature pollen grains of Rondeletia, both starch grains and lipidbodies are found as storage products. Compound starch grainsappear first, i.e., at the late free microspore stage. After thefirst pollen mitosis, oil body accumulation begins and starchcontents of the cytoplasm decrease. At anther dehiscence lipidsare the main storage product. The lipid body accumulation inRondeletia is preceded by a transient accumulation of starchas demonstrated in many other plant species (e.g., Reznickova,1978; Wetzel and Jensen, 1992; Clement et al., 1994; Hess,1995).

The ER is considered to be a site of lipid body formationin both animal (Zaczek and Keenan, 1990; Bozhkov and Dlu-bovskaya, 1992) and plant cells (Grabski, de Feijter, andSchindler, 1993; Lacey and Hills, 1996). In late free micro-spore stages and young pollen grains, we observed a clearproliferation of an extensive network of ER membranes. TheseER units are generally associated with maturation and expan-sion of the cytoplasm of the pollen vegetative cell (Jensen,Fisher, and Ashton, 1968; Charzynska, Murgia, and Cresti,1989). An extensive network of ER during the young pollengrain stage was also observed in Ledebouria (Hess, 1995) andin Brassica (Piffanelli, Ross, and Murphy, 1998). In maturepollen grains of Rondeletia, the storage oil bodies are char-acteristically enfolded in a single rER strand. Similar pocketsof ER have been described in the pollen of Gossypium hir-sutum (Jensen, Fisher, and Ashton, 1968), Impatiens walleri-ana (Fisher, Jensen, and Ashton, 1968), and Tradescantia re-flexa (Noguchi, 1990). It has been suggested that the extensivenetwork of ER membranes might protect the oil bodies fromcoalescencing during de- and rehydration (e.g., Piffanelli,Ross, and Murphy, 1997). Secondly, the intracellular mem-brane system can also provide lipid precursors for the increaseof surface area of the plasmalemma which follows germinationand pollen tube growth (Piffanelli, Ross, and Murphy, 1997).In addition to the prominant ER, there are numerous vesicles,apparently developed from the dictyosomes, that are layingbeneath the surface of the plasmalemma. These membranousbodies undoubtedly contribute to the dramatic increase in sur-face area of the plasmalemma during maturation of the micro-spores and formation of the pollen grains (Piffanelli, Ross, andMurphy, 1998).

We observed several grains with three nuclei at maturity;thus the second mitosis of the generative nucleus takes placebefore release of the pollen grains. This observation contra-dicts Wunderlich (1971) and Puff (1994) who stated that pol-len of Rondeletia is bicellular when shed.

Volume increase at dehiscence—The pollen wall of Ron-deletia becomes abnormally stretched just before dehiscence.This stretching affects the spacing between columellae and thethickness of all distinguished layers of the wall. The effectwas particularly obvious on the radially oriented granular ma-terial. This material appeared irregular in shape and in its dis-tribution at pollen maturity. The intine was no longer thick inapertural regions at dehiscence as is generally the case.

Endocingulum—On the inside of mature grains, a broadendocingulum occurs with an irregular rod-like surface struc-ture in contrast to the granular ornamentation of the remainderof the nexine. The ultrastructural explanation of this structureis clear from Fig. 34. Next to the aperture there is a zone freeof radially oriented granular material, endexine, and foot layer.The columellae are branched and interconnected in this area.

To us, the inside view on the endocingulum shows the prox-imal ends of the columellae. The most plausible functionalinterpretation of the endocingulum is a role in harmomegathic(accommodate variations in the volume of the cytoplasm) pro-cesses but dehydrated grains observed in SEM contradict thishypothesis (Fig. 35).

Summary of major ontogenetic events—The main onto-genetic events in pollen wall development of Rondeletia odor-ata are presented schematically in Figs. 36–43. Our drawingsare mainly based on TEM micrographs included in this workor on unpublished material. Our present study shows someinteresting features that can be related to the dynamic pro-cesses of pollen development. We show ER cisternae extend-ing between plasmalemma protrusions, and material of themembranous granular layer beneath the endexine (Fig. 15).The ER cisternae are also observed within the arcades betweencolumellae (Figs. 15, 22). Our interpretation is that the earlycolumellae with hollow cylindrical center, as well as other lay-ers of the exine, may function as a dynamic transfer systembetween the cytoplasm of developing microspores and the ta-petum. Heslop-Harrison (1963) showed the presence of cister-nae of the endoplasmic reticulum under the plasmalemma inconnection with the conduits of columellae. Similar observa-tions were reported by Skvarla and Larson (1966) as part oftheir ontogenetic study of Zea mays. The interpretation thenwas that there might be a connection between sites of colu-mellar initiation and ER cisternae. To understand the originsof this communication or transport system it would be helpfulto study and understand the processes involved in the locali-zation of initiation sites of exine layers on the plasmalemma.

The development of a very thin foot layer and relativelythick endexine is one of the characteristic features of pollenof Rondeletia. Another feature is the membranous granularlayer initiated at early free microspore stage. The membranouspart of this layer consists of ER units extending from the cy-toplasm, which are clearly presented in our study. Further-more, we have observed several cytological structures that arepronounced in Rondeletia: first, the abundant occurrence ofGolgi vesicles and their demonstrated role in initiating the in-tine before mitosis, and second, the numerous lipid dropletssurrounding the generative nucleus.

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Pollen Development of Rondeletia odorata (Rubiaceae)

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