structural and cytochemical characteristics of the stigma and style

Annals of Botany 78 : 759–764, 1996

Structural and Cytochemical Characteristics of the Stigma and Style in Vitisvinifera L. var. Sangiovese (Vitaceae)

FABRIZIO CIAMPOLINI, CLAUDIA FALERI, DONATELLA DI PIETROand MAURO CRESTI

Dipartimento di Biologia Ambientale, Uni�ersita[ degli Studi di Siena, Via P.A. Mattioli 4, 53100 Siena, Italy

Received: 18 April 1996 Accepted: 1 July 1996

Studies were carried out on structural and cytochemical aspects of the stigma and style of Vitis �inifera. The stigmais of the wet papillate type with a continuous cuticle and pellicle. During the development of the papillae, the cell wallsincrease in thickness and produce a secretion product constituted of lipids that pass through the wall forming theexudate. The style is solid with a central core of transmitting tissue which has conspicuous intercellular spaces thatincrease remarkably from the periphery to the centre where the cuticle is present. The intercellular spaces, where thepollen tubes grow, contain a matrix that includes polysaccharides, pectic substances and scattered areas of lipidicnature. # 1996 Annals of Botany Company

Key words : Cytochemistry, stigma, style, ultrastructure, Vitis �inifera.

INTRODUCTION

The cultivar Sangiovese of the species Vitis �inifera has itsorigin in Tuscany: it is distributed widely in the Senese areaand quantitatively it is the most important component of‘Chianti ’, ‘Brunello ’ and ‘Vino Nobile di Montepulciano’wines.

Vitis �inifera belongs to the group of species with a wetpapillate stigma (Heslop-Harrison and Shivanna, 1977) anda solid style (Hanf, 1936). Several studies have been carriedout on solid-styled systems such as Lycopersicum peru�ianum(Cresti et al., 1976), Malus communis (Cresti, Ciampoliniand Sansavini, 1980), Primula �ulgaris (Heslop-Harrison,Heslop-Harrison and Shivanna, 1981), Olea europaea(Ciampolini, Cresti and Kapil, 1983), Hypericum calycinum(Ciampolini, Shivanna and Cresti, 1988), Nicotiana syl�estris(Kandasamy and Kristen, 1987, 1990), Brugmansia sua�eo-lens (Vennigerholz, 1992; Huda! k, Walles and Vennigerholz,1993), Smirnium perfoliatum (Weber, 1994) and Tibouchinasemidecandra (Ciampolini, Faleri and Cresti, 1995). In thispaper the ultrastructural and histochemical characteristicsof stigma and style in Vitis are described and discussed.

MATERIALS AND METHODS

Plants of Vitis �inifera L. var. Sangiovese growing in theBotanical Garden of the University of Siena, Italy wereused. For optical microscopic studies, the pistils were fixedin 3% glutaraldehyde in 6±6 m cacodylate buffer pH 7±2for 1 h at room temperature, dehydrated in an ethanol seriesand embedded in historesin (LKB). Semithin sections(2–3 µm) were cut with glass knives and used to localizedifferent cytochemical components.

Total proteins were localized with Coomassie BrilliantBlue (Heslop-Harrison et al., 1973), pectinaceous material

with Ruthenium Red (Heslop-Harrison, 1979), total in-soluble polysaccharides with periodic acid-Schiff (PAS)reagent (McGuckin and McKenzie, 1958), lipoidal materialwith Sudan Black B (Pearse, 1961) and tannin material withAzure II}Methylene Blue (O’Brien and Mc Cully, 1981).

For transmission electron microscopy (TEM), the glutar-aldehyde fixed pistils were post-fixed in 1% osmiumtetroxide, dehydrated in an ethanol series and embedded inSpurr’s resin (Spurr, 1969). Sections were cut with a LKBNova ultramicrotome using a diamond knife.

Some sections were stained with uranyl acetate and leadcitrate for 10 min, respectively. Others were stained withphosphotungstic acid (PTA) for detecting pectins (Hayat,1970).

Some sections were collected on gold grids and treatedwith periodic acid-thiocarbohydrazide-silver proteinate(Thie! ry, 1967) to detect polysaccharides. All ultrathinsections were examined with a JEOL JEM 100B electronmicroscope at 80 kV. For scanning electron microscopyfresh stigmata mounted without previous fixation andsputtering were observed with a Philips SEM 501 B scanningelectron microscope at low voltage.

RESULTS

Stigma

The stigma of Vitis �inifera is wet and papillate (Fig. 1) ; thedensely arranged papillae are long, multicellular, uniseriateand uniform in diameter. The papillae are covered with alining of cuticle and pellicle layer as revealed by stainingwith Auramine-O (Fig. 2) and Coomassie Brilliant Blue(Fig. 3). The cuticle and pellicle layer is intact in the distalpart of the papillae but shows discontinuities in the lowerpart. Electron dense homogeneously stained areas repre-senting stigmatic exudate are present both between the

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760 Ciampolini et al.—Characteristics of the Vitis Stigma and Style

F. 1. Scanning electron micrograph of the stigma from a flower before anthesis. ¬70.

F. 2. Fluorescent micrograph of some papillae stained with Auramine O to show the cuticle (arrows). ¬440.

F. 3. Semithin section of papillae stained with Coomassie Brilliant Blue to show the pellicle (arrows). ¬440.

F. 4. Section of the tip cell of a papilla : the cell wall is bipartite—an outer layer made up of loose fibrils and an inner layer of compact fibrils.Note also exudate (Ex) entrapped in cuticle and pellicle (arrows). ¬6000.

F. 5. Section of a papilla cell : globular electron-dense material (arrows) seemingly migrates from the wall to the outer zone forming the stigmaticexudate. ¬24000.

F. 6. Portion of a papillar cell showing the electron-dense cytoplasm: note the nucleus (N) and electron-dense deposits of tannin in the vacuoles.¬2600.

F. 7. Semithin transverse-section of the style, just below the stigma, showing the transmitting tissue (TT) with two types of cells. Note raphidescontaining structures in cortical cells (r). ¬110.

F. 8. Longitudinal section of stigma and style. The transmitting tissue is continuous with the multicellular papillae. ¬90.

All figures reproduced here at 75%.


Ciampolini et al.—Characteristics of the Vitis Stigma and Style 761

F. 9. Plasmodesmata (arrows) in the longitudinal and transversal walls of cells of the transmitting tissue. ¬8300.

F. 10. Cross-section of the transmitting tissue of a young style : the cells of the TT are compactly arranged without any intercellular spaces.¬3800.

F. 11. Cross-section of a ripe style : the cells of TT are surrounded by large intercellular spaces filled with secretion product (IS). ¬10000.

F. 12. Semithin transverse-section of a style, near the ovary stained with PAS reagent for total insoluble polysaccharides. Note the prominentintercellular spaces resembling a cavity (arrow). ¬100.

F. 13. Semithin transverse section of a style stained with Ruthenium Red for pectins. ¬160.


pellicle and cuticle and externally of the pellicle (Fig. 4).During the development of the papillae, the cell wallsincrease in thickness and become bipartite. The outer layer

is made up of loosely arranged fibrils whereas the inner layerconsists of compactly arranged fibrils (Figs 4 and 5). Theouter layer is of irregular thickness and sometimes shows



F. 14. TEM micrograph of a cross-section of intercellular substance in the centre of TT stained with Thie! ry’s test. Near the cuticle scarceelectron-dense globules (arrows) are visible. ¬22000.

F. 15. Same as Fig. 13 but stained with PTA for pectins. ¬22000.

F. 16. Cross-section of pollen tubes growing in the intercellular substance of the transmitting tissue (asterisks). ¬8700.


irregular detaching and disruption of the cuticle due to theaccumulation of secretion product below the cuticle (Fig. 4).Both layers react positively to tests for polysaccharides andpectins. The walls of the papillae show electrondensedeposits representing lipid bodies ; they seem to migrate tothe outer zone to form the stigmatic exudate (Fig. 5). The

exudate is electron opaque after post-fixation with OsO%

(Fig. 4) and stains with Sudan Black B (data not shown)indicating the presence of lipids. The major part of the cellcytoplasm is essentially made up of large vacuoles withstrongly electron opaque deposits consisting of tanninindicated by staining with Azure II}Methylene Blue (data


Ciampolini et al.—Characteristics of the Vitis Stigma and Style 763

not shown). The distal part of the papilla shows acytoplasmic profile with a roundish nucleus containing anucleolus (Fig. 6), active dictyosomes, mitochondria, manyRER profiles, ribosomes mostly clustered in polysomes andplastids sometimes containing one or two little starchgrains. The papillar cells are connected to each other bynumerous plasmodesmata.

Style

The style of Vitis �inifera is of the solid type. It comprises anepidermis layer, a parenchymatic tissue and a centraltransmitting tissue (Fig. 7). The epidermis is composed of asingle layer of cells rich in tannins covered by a thick cuticle.Adjacent to the epidermis there are 12–18 layers ofvacuolated cortical cells. In these cells PAS positivestructures containing raphides are present (Figs 7 and 8).These structures (about 8–16 per section) aremore numerousnear the stigma. The volume of the cortical cells is essentiallyoccupied by a large vacuole and the cytoplasm containschloroplasts with a high quantity of starch. Vascular bundlesare present in the parenchymatic tissue, but only in proximityto the ovary. The cylindrical transmitting tissue is located inthe centre of the style and is continuous with the papillae(Fig. 8). These cells appear roundish in transverse sectionsand elongated in longitudinal sections. They are connectedwith each other by plasmodesmata present both in thetransverse and longitudinal walls (Fig. 9). The transmittingtissue shows two types of cells (Figs 7 and 8): one typeshows cytoplasm rich in mitochondria, active dictyosomesformed by five to six cisternae, RER, ribosomes mostlyaggregated in polysomes, plastids containing one or twostarch grains, and lipid droplets. Part of the cytoplasm isoccupied by vacuoles containing strongly electron densedeposits representing tannins. The nucleus with one nu-cleolus is roundish both in cross and in longitudinal sections.Thewall is thick and appears to fray towards the intercellularsubstance (Figs 11, 14 and 15). In the second cell type in thetransmitting tissue (TT) the cytoplasm is occupied byvacuoles containing strongly electron dense material local-ized either against the tonoplast or clustered in the centralpart of the vacuole. This second type of cells is distributedaround the central core delimiting the parenchymatic cellsand occurs occasionally in the central core between the cellsof the first type (Fig. 7). In the young pistil the cells of theTT are arranged compactly without any intercellular spaces(Fig. 10). In the mature pistil the cells of the TT arearranged loosely and surrounded by large intercellularspaces filled with secretion products (Fig. 11). These spacesprogressively increase towards the centre, where a cuticlebelonging to two carpels is joined together. Furthermorethese spaces progressively increase towards the ovary wherethe TT cells seem to form an area filled with a secretion (Fig.12). These spaces representing the intercellular substancereact positively after staining for pectins (Fig. 13) and totalpolysaccharides (Fig. 12) but not at all for proteins. Atultrastructural level the intercellular substance has agranular and fibrillar appearance and it reacts positively toThie! ry’s test (Fig. 14) and to staining with phosphotungsticacid (PTA) (Fig. 15). Near the cuticle are scattered elements

of high electron density and of homogeneous texture (Figs14 and 15) which react positively to Sudan Black B. In crosssections of pollinated styles pollen tubes can be identified(Fig. 16).

DISCUSSION

According to the classification by Heslop-Harrison andShivanna (1977), the stigma of Vitis �inifera is of the wettype with multicellular uniseriate papillae. The lipidic natureand the method of production of the exudate looks similarto Tibouchina semidecandra (Ciampolini et al., 1995),Nicotiana tabacum (Cresti et al., 1986), Lycopersicumesculentum (Kadej,Wilms and Willemse, 1985),Olea europea(Ciampolini et al., 1983), Citrus limon (Cresti et al., 1982)and Petunia hybrida (Konar and Linskens, 1966a, b ; Kroh,1967). Unlike Petunia and Citrus, where a stigmatoid tissueis identifiable, in Vitis �inifera, the multicellular papillaeare continuous with the transmitting tissue. The exudatethat covers the mature stigma of Vitis totally originatesfrom the stigmatic cells in opposition to Smirnium perfolia-tum (Weber, 1994), where the stigmatic surface coating istotally produced by the stylar transmitting tissue. Themulticellular papillae of Vitis produce exudate in the formof lipid droplets that penetrate through the cell wall ; thedroplets then coalesce forming large deposits causing therupture of the cuticle and pellicle. The cuticle, positive afterstaining with Auramine O, shows the presence of a pelliclepositive to the basophilic dye Coomassie Brilliant Blueindicating its proteinaceus nature previously demonstratedin dried stigmata (Mattsson et al., 1974). Similar toSternbergia lutea (Ciampolini, Shivanna and Cresti 1990)and Hypericum calicinum (Shivanna, Ciampolini and Cresti,1989) the papillae show a thickening of the wall during theripening of the pistil. The same phenomenon occurs also inthe transmitting tissue of the style. In fact in the youngpistil, the cells of the TT are compactly arranged withoutany intercellular space. In the ripe pistil, however, the cellsof the TT are loosely arranged and surrounded by asecretion product and are morphologically similar to thethickened papillae walls. Both react positively to stainingfor polysaccharides and pectins. Furthermore in the stylethere are scattered elements of lipid nature resembling theexudate of the stigma. Unlike other species such asTibouchina and Lycopersicum, stigma and style in Vitisproduce the same type of histochemical secretion and theircells show the same organelle populations including activedictyosomes probably associated with the synthesis andsecretion of polysaccharides and pectins. In Vitis, accordingto Considine and Knox (1979) the multicellular papillae arecontinuous with the TT; stigmatic exudate and stylarintercellular substance look similar in nature and functionand form a continuous medium through which the pollentube grows towards the ovule. The secretion product is richin pectins and other polysaccharides, similar to the secretionof Brugmansia (Huda! k et al., 1993), Tibouchina (Ciampoliniet al., 1995) and such secretion in hollow styles of Lilium(Loewus and Labarca, 1967) and Sternbergia (Ciampolini etal., 1990). Unlike Petunia, Lycopersicum and Malus wherethe intercellular spaces are equally filled throughout the



whole diameter, the intercellular substance in Vitis increasesfrom the cell neighbouring the cortical cells to the centrewhere the cuticle is present. This phenomenon is especiallyprominent near the ovary resulting in a real cavity or canal.The presence of plasmodesmata in the transverse andlongitudinal walls of TT cells recently found also inTibouchina, strengthens the hypothesis that they are involvedin transduction of signals from the ovary and in the controlof pollen tube growth.

ACKNOWLEDGEMENTS

The authors are indebted to Dr Anja Geitmann, forstimulating discussions and reading the manuscript. Thisresearch is supported by grant of MURST (40%).

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structural and cytochemical characteristics of the stigma and style

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