Ultrastructural studies on the development of microspores to ripe pollen (cv. Sangiovese) showed that the diploid microsporocyte undergoes meiosis in order to form a tetrad of microspores sticked together by a callose wall. After callose digestion, 4 independent microspores were released. With successive growth, the single nucleus of each microspore undergoes an asymmetrical division forming the bicellular pollen grain (vegetative cells containing the generative cells). Only after pollination and pollen tube growth the two sperms will be formed. The mature pollen of Sangiovese is tricolpate; the exine is scabrate-verrucate in the equatorial zone and foveolate in the polar region

Ciampoloni, F.

Cresti, M.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis38(4), 141-144(1999) Ultrastructural characteristics ofpollen development in Vitis vinifera L. (cv. Sangiovese) by M. CRESTJ and F. ÜAMPOLJNJ Dipartimento di Biologia Ambi~ntale, Universita degli Studi di Siena, ltalia Summary: Ultrastructural studies on the development ofmicrospores toripepollen (cv. Sangiovese) showed that the diploid microsporocyte undergoes meiosis in order to form a tetrad of microspores sticked Iogether by a callose wall. After callose digestion, 4 independent microspores were released. With successive growth, the single nucleus of each microspore undergoes an asymmetrical division forming the bicellular pollen grain (vegetative cells containing the generative cells). Only after pollination and pollen tube growth the two sperms will be formed. The mature pollen of Sangiovese is tricolpate; the exine is scabrate-verrucate in the equatorial zone and foveolate in the polar region. K e y wo r d s : Vitis vinifera, Sangiovese, pollen grain, microsporogenesis. Introduction The pollen grain is the male gametophyte of the angiosperms with two kinds of structural and functional cells. The male reproductive cells, with little cytoplasm, are either single generative cells (bicellular type) or pairs of sperm cells (tricellular type). The second type of cell is a !arger, non-reproductive vegetative cell, whose cytoplasm encloses the generative cell or the sperm cells in a cell-within-a-cell arrangement ( see CRESTI et al. 1999). The mature pollen grains can also be considered as stor-age cells, which must supply nutrients to the rapidly grow-ing pollen tube (CRESTI et al. 1975). The present paper is directed to study the pollen devel-opment and mature pollen of Vitis vinifera, cv. Sangiovese, to betterunderstand the biology ofthe sexual reproduction of these species. The cv. Sangiovese has its origin in Tus-cany and is widely distributed in this region, quantitatively it is one of the most important components of Chianti, Brunello, Vemaccia and Vino Nobile di Montepulciano wines. A previous account of certain features on pollen, biol-ogy ofreproduction and genetic biodiversity ofSangiovese was given by LOMBARDO et a/. 1978; CASTELLI et a/. 1986; ÜAMPOLINJ et al. 1996; SENSJet al. 1996; VJGNANI et al. 1996. Material and Methods Anthers of Vitis vinifera L. (cv. Sangiovese) were col-lected at different stages of development from plants culti-vated in the experimental fields of the Siena Agricultural High School in Italy. For scanning electron microscopy (SEM), fresh mature pollen grains were mounted with tape on aluminium stubs, coated with gold and then examind with a Philips 50 I B SEM. For Iransmission electron microscopy (TEM), the ma-terial was fixed in 2 % glutaraldehyde (GA) in 0.05 M Ca-codylate buffer, pH 7.4, for 1 hat room temperature, rinsed in the same buffer and post-fixed in 1 % osmium tetroxyde, dehydrated in ethanol and embedded in low viscosity resin. Sections were cut with a diamond knife using a LKB ultratome JJI and stained with uranyl acetate and Iead citrate; observations were made using a JEOL Jem 1008 electron microscope at 80 kV For cytochemical examination, the samples were embed-ded in Historesin (LKB) after fixation in GA. Semithin sec-tions were then stained with Toloudine blue and Periodic acid-schiff (PAS) reaction (O' BRIEN and McCULLY 1981 ), Sudan Black B (RussELL et al. 1976) and Vanillin-HCI {HESLOP-HARRJSON and HEsLOP-HARRISON 1985) and examined with a Zeiss Axiophot light microscope. Results The microspore mother cells (MMC) at the prophase-leptotene stage (Fig. I) show a polyedric form, a roundish nucleus with only one nucleolus and numerous organelles, e.g. mitochondria, plastids, dictyosomes and rough endo-plasmic reticulum (RER) profiles. Tapetum and microspore mother cell walls are pectocellulosic in nature. At this stage the plasmodesms between tapetum·and MMC are closed, meanwhile some plasmodesms between MMC and MMC give origin to cytomictic channels (Fig. 2). The deposition of callose between the primary cellulose wall and the plasmalemma of each individual microspore occurs at the end of the simultaneus cytokinese leading to tetrad formation. Each tetrad is enclosed in the callosic wall of the MMC from which it is derived and the single microspores arealso separated from the callosic walls. The Correspondence to: Prof. M. CRESTI, Dipartimento di Biologia Ambientale, Via P. A. Mattioli 4, 1-53100 Siena, ltaly. Fax: + 39-577-232 860. E-mail: cresti@unisi.it 142 M. CRF.STI and F. CIAMPOLINI Figs. 1-4: Mierespore molher cells (MMC) at the prophase-lep-totene stage; tapeturn (T); x 4200 ( l ). Cytomictic channel between two microspores (arrows); x 19200 (2). Tetrad showing rounded nucleus (N) and plastids (P) containing starch grains; x 6000 (3). Portion of a young microspore at the Ietrad stage showing primexine deposition (arrow). Note the electrondense cytoplasm with active dictyosomes (D); x 38000 (4). tetrads, which are roundish in shape, contain a single nu-cleolus which is also roundish. The nuclear membrane con-tains numerous pores and the plastids reveal one or more starch grains (Fig. 3). During the deposition ofthe primexine (Fig. 4) the cytoplasm continues to be electrondense; the dictyosomes, forrned by elongated cistemae, produce nu-merous vesicles, and the ribosomes appear to be aggregated in polysemes. After the dissolution ofthe callosic wall, the microspores are released; they grow in volume and become roundish. The roundish shaped nucleus contains one or two !arge nucleoli. In the cytoplasm there are amyloplasts containing numerous starch grains (Fig. 5). During this pe-riod there is a concomitant rise in width and complexity of the exine (sexine and nexine I increase: nexine 2 is laid down) and the cytoplasm begins to fill with vacuols (Fig. 6). The microspore nucleus, which remains in a centrat po-sition during the tetrad stage, migrates to the cell periphery and is positioned near the plasma membrane (Fig. 6). At the cortical position, the spore nucleus undergoes a mitotic di-vision resulting in a bicellular pollen grain with a smaller generative cell (GC) within the cytoplasm of a !arger vegeta-tive cell (VC) (Fig. 7). Vacuoles forrned during the free microspore period are reabsorbed and the vegetative nu-cleus (VN) migrates to the center ofthe cell. Initially the GC Figs. 5-7: Portion of a free microspore showing amyloplasts (A) with numerous starch grains; mitochondria (M); x 16000 (5). Free microspore with the exine (E) and nexine I (Ne) increased in thick-ness; x 5600 (6). Bicelled microspore showing a portion of the centrat vegetative nucleus (VN) and generative cell (GC-dotted line) appressed to the plasma membrane; x 16000 (7). is appressed to the plasma membrane (Fig. 7). Before shed-ding the GC is located in the central portion ofthe VC closely connected to the VN. Maturegrain E x t e r n a l m o r p h o I o g y : Sangiovese pollen is tricolpate (Fig. 8), the exine is scabrate-verrucate in the equa-torial zone and foveolate in the polar zone (Fig. 9) (F AEGRI and !VERSEN 1964; CIAMPOUNJ and CRESTI 1981 ). Inter n a I m o r p h o l o g y : The GC migrates into the VC assuming a spindie shape (Fig. 1 0). The GC is separated from the cytoplasm ofVC by an in<i.ented wall-like structure; in cross sections it is roundish whilst in longitudinal sec-tians it has the shape of a crescent moon; it is localized in the middle of the grain near or closely associated with the VN forrning the Male Germ Unit (MGU). The nucleus fills most ofthe cell and has electrondense masses of chromatin. The cytoplasm of the GC contains many ribosomes, dictyosomes, mitochondria and some small cistemae. A re-markable feature of the GC is the !arge quantity of small electrondense roundish bodies localized at the two extremi-ties ofthe cell (Fig. IO).lt is on the basis ofSudan B black and Vanillin-Hel staining and its ultrastructural appearance that this substance is considered as tannin. Ultrastructural characteristics of pollen 143 Figs. 8-10: Mature pollen grain at scanning electron microscope; x 4000 (8). Portion of an enlarged pollen grain in the polar and equatorial zone; x 8000 (9). Maturepollengrain showing the gen-erative cell and the vegetative nucleus: Note the electrondense rounded bodies localized at the extremity of the generative cell; X 8000 (10). The nucleus of the VC is !arge, irregularly shaped, the chromatin is less electrondense than the generative nucleus. Many stacked RER cistemae can be seen in the cytoplasm, especially between GC and VN. The cytoplasm contains numerous mitochondria usually located in the peripheral zone while the ribosomes are distributed all over the cytoplasm or gathered in irregular masses. In the cytoplasm there are roundish weakly electrondense bodies (Fig. 8) that could represent the proplastids that are seen later on in the pollen tube forming lamellae and starch grains. Many inclusions, the nature of which is rather difficult to interpret, are fre-quently encountered inside vacuoles; they should be con-sidered small autophagic vacuoles containing residual prod-ucts. Discussion The ultrastructural features of Sangiovese pollen dur-ing its development does not seem to have any particulari-ties in comparison with other bicellular pollen grains that have been described to date (reviews: MASCARENHAs 1989, CREsn and TIEZZI 1992). The process ofmicrogametogenesis in Vitis vinifera can be briefly summarized in two different steps. First, within the anther locule, a diploid microsporocyte undergoes meiosis in order to originate a tetrad of microspores which are held together by a thick callose wall. Successively the callose is digested by appropriate enzymes and 4 independent microspores are released. With the further growth of each microspore, the single nucleus migrates to the peripheral portion ofthe cell where it undergoes an asymmetrical divi-sion (first haploid pollen mitosis). The resulting bicellular pollen grain consists ofa small GC and a !arge VC. Inside the anthers the Vitis pollen development stops at this stage. Secondly, after anthesis and pollen shedding, the GC is destinated to divide once more (second haploid pollen divi-sion) in order to produce the two sperm cells involved in double fertilization. In general, in the last few decades particular attention has been paid to the main steps: meiosis, first and second haploid pollen division. Thesestages have been extensively investigated in different species focussing the attention on the composition ofthe organelles, and cytoskeletal elements and their association with the VN forming the MGU (ANGOLD 1968; HESLOr-HARRISON 1968; SANGERand JACKSON 1971 a, b; RussELL and CAss 1981 ; CRESTI et al. 1984; V AN WENT 1984; DuMASet al. 1985; RussELL et al. 1990; seealso CRESTI et al. 1999). On the contrary only a few reports have focussed on the differentiation and development of the VC within the pollen (GIMENEZ-MARTIN et al. 1970; SANGER and JACKSON 1971 c; CHARZYNSKA et al. 1989; KuANG and MusGRAVE 1996) even though there have been many reports ofpollen germi-nation and tube growth (review: CAI et al. 1997). The organelies ofthe VC generally provide evidence of high-Ievel metabolism of sugar, Iipids or proteins (CRESTI et al. 1991) used after pollen activation for the tube e\onga-tion and transport ofmale gametes along the style and ovary. In Vitis the vegetative Storage substances are mainly pro-teins and Iipids, the presence ofsugar seems rather limited. In any case the VN and VC play a more active roJe in the development of pollen than the GC. However, the VC is a cell with a programmed dead because its role ends after fertiliza-tion and it does not undergo further mitotTc division. The molecular basis ofthe VC fate has not yet been darified but recently T ANAKA et a l.' 1998, with the immunofluorescence method using specific antisera raised against two histones of Lilium longiflorum, gave an interesting explanation. They suggested that a preferential decrease in the Ievel ofhistone (H I) in pollen nuclei is essential for the development of the male gametophyte cell through large-scale expression of genes that include pollen-specific genes which results in pollen germination and growth oft)le pollen tube. One interesting feature present in the GC cytoplasm of Vitis are dark deposits (tannins) located at the two poles of the cell. Tannin deposits occur very widely among the higher plants (LEDBETTER and PORTER 1970). No generat rule seems to govem the distribution of tannins: extracellularly deposited along the primary walls, dispersed in the cyto-plasm or concentrated in the vacuoles. The functions ofthese deposits are different. Some hypotheses have been based on their antioxidant activity and still others on their capacity to discourage predators (see LEDBETTER and PoRTER 1970). In Vitis the function of GC tannin deposits, in addition to the above mentioned functions could be storage of nutrients 144 M. CREsn and F. CIAMPOLINI for the carbohydrate metabolism necessary for GC division and sperm cell formation after pollination. References ANGOLD, R. E.; 1968: The fonnation of generative cell in the pollen grain of Endymion non-scriptus (L.). J. Cell Sei. 3, 573-57~. CAI, G.; Mosl'ATELLI, A.; CRESTI, M.; 1997: Cytoskeletal organization and pollen tube growth. Trends Plant Sei. 2, 86-91. CHARZYNSKA, M.; Mt.:RGJA, M.; CRESTJ, M.; 1989: Ultrastructure of the vegetative eell of Brassica napus pollen with particular refer-ence to microbodies. Protoplasma 152, 22-28. CASTELLI , S.; PISANJ, P. L.; RINAWtLLI, E.; 1986: Research on pollen morphology of some grapevinc clones. Riv. Ortotlorofrutticult. ltal. 69, 287-297. CIAMPOLINI, F. ; CRESTI, M.; 1981: Atlante dei Principali Pollini Allergenici Presenti in ltalia. Ed. Univ. Siena. - -; FALERJ, C.; Dr PIETRO, D.; CRtSTI, M.; 1996: Structural and cyto-chemical characteristics of the stigma and style in Vitis vinifera L. var. Sangiovese (Vitaceae). Ann. Bot. 78, 759-764. CRESTI, M.; PACINI, E.; SARFAITI, G.; SIM<>Nnou, C.; 1975: Ultrastructural features and storage funetion a n Lycopersicum peruviamim pol-len. In: L. MuLCAHY (Ed.): Garnele Competition in Plants and Animals. North Holland Pub!. Co., Amsterdam. - -; ÜAMPOLJNI, F. ; KAPIL, R. N.; 1984: Generative eells of some angiospenn with particular emphasis on their microtubules. J. Submicrose. Cytol. 16, 317-326. - -; BLACKMORE, S.; VAN WENT, J. L.; 199 1: Atlas of Sexual Reproduction in Flowering Plants. Springer-Verlag, Berlin, Heidelberg, New York . · - -; TiEZZI, A.; 1992: Sexual Plant Reproduction. 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Protoplasma 107, 85-107. - -; CRESTI, M.; DUMAS, C.; 1990: Recent progress an spenn characteri-zation in tlowering plants. Physiol. Plant. 80, 669-676. SANGER, J. M.; JACKSON, W T.; 1971 a: Fine structure study of pollen development in Haemathus Katherinae Baker. I . Formation of vegetative and generative cells. J. Cell Sei. 8, 289-30 I. - -; - - ; 1971 b: Fine structure study of pol ten development in Haemathus Katherinae Baker. 2. Microtubules and elongation of the generative cells. J. Cell Sei. 8, 303-315. - - ; 1971 c: Fine structure study of pollen development in Haemathus Katherinae Baker. 3. Changes in organelles during development of vegetative cells. J. Cell Sei. 8, 317-329. SF.NSI, E.; VJGNANI, R. ; RHooE, W; BIRICOLTI, S.; 1996: Characterization of genetic biodiversity with Vitis vinifera L. Sangiovese e Colorino genotypes by AFLP and ISTR DNA marker technology. Vitis 35, 183-188. T ANAKA, I.; ÜNo, K. ; FuKUDA, T.; 1998: The developmental fate of angiosperm pollen is assoeiated with preferential decrease in the Ievel of histone H I in the vegetative nucleus. Planta 206, 561-569. VAN WENT, J. L.; 1984: Unequal distribution of plastids during genera-tive eell formati on in Impatiens. Theor. Appl. Genet. 68, 305-309 . VJ< ;"ANI, R.; BowERS, J. E.; MEREDITH, C.; 1996: Microsatellite DNA polymorphism analysis of clones of Vitis vinifera Sangiovese. Sei. Hort. 65, 163-169. Received May 18, 1999 

Ultrastructural characteristics of pollen development in Vitis vinifera L. (cv. Sangiovese)

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Ultrastructural characteristics of pollen development in Vitis vinifera L. (cv. Sangiovese)

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