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    Immunogold labeling

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    Geitmann1995

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    Immunogold labeling

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    Geitmann 1995

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    Immunogold labeling

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    ‘J Plant Physiol. Vol. 147. pp. 225-235 (1995) It has been shown that during incompatibility reactions the cell wall formation in incompatible pollen tubes is dis Immunogold Localization of Pectin and Callose in Pollen Grains and Pollen Tubes of Brugmansia suaveolens — Implications for the Self-Incompatibility Reaction Anya Gerrmann*, Jan Hupix**, Fevizrras Vennicernio1z, and Bonn Wattes Department of Botany, Stockholm University, $106 91 Scockholm, Sweden Received March 14, 1995 « Accepted May 9, 1995 Summary Pollen of Brugmansia suaveolens was grown in vitro or in vivo after compatible or incompatible polli- nation. Ultathinsections of fixed and embedded specimens were treated with immunogold label for ultrastructural localization of acidic pectins (monoclonal antibody JIM 5), methyl-esterified pectins (monoclonal antibody JIM 7) and callose (anti(1~3)6-glucan antibody) in order to elucidate the dist- bution and intracellular pathway ofthe cell wall constituents. Pectin, present in the inine of the pollen train and in the outer layer ofthe bilayered pollen tube wall, sem to be synthesized in the dictyosomes activated upon germination. Their intracellular transport from the dictyosomes to the pollen tube tip, the site of fusion with the cell wall, occurs in fibrillar or bipartite particles that contain pectins in a highly _methyleserified form, but no callose. In both intine and pollen tube ell wall 2 radial gradient of pectin esterification is observed. Pectins are less esterified with increasing distance from the plasma membrane. Considering the fact chat pectins are secreted in a highly esterified form, the gradient indicates the pres ence of enzymatic activity, causing pectin de-sterficaion during cll wall development. Upon incompa- tible pollination both layers ofthe pollen tube wall undergo thickening, Pectinaceous particles are depo- sited on the inside of the cell wall or seemingly get stuck during migration through the inner callosic layer, Whereas pectinaceous secretory vesicles in compatible pollen tubes show a high grade of eserifica tion, during the incompatibility reaction pectic aggregates of low esterification grade appear in the cyto- plasm. This might indicate the presence of de-esterifying enzymes in the secretory vesicles. Key words: Brugmansia suaveolens, callos, cll wal, immunocytocherisiry JIM 5, JIM 7, pectin, pollen tubs, slFincompattilir. Abbreviations: B. suaveolens ~ Brugmansia suaveolens; ER = endoplasmic reticulum; MAb = monoclo- nal antibody; PASH = periodic acidsilver hexamine; PATAg = periodic acid-thiocarbohydrazidesilver proteinate; SEM = scanning electron microscopy; TEM = transmission electron microscopy. turbed (de Nettancourt et al., 1974; Heslop-Harrison, 1983). ‘Thus, knowledge of cell wall synthesis is essential for under- standing cell wall formation under normal conditions and its dysfunction as a result of selFincompatbility. The pollen tube being a tip growing cell, che major part of the cell wall synthesis occurs atthe apical end of the tube, where precur- * Present address, author for correspondence: Department of En vironmental Biology, University of Siena, Via P.A. Mattioli 4, 53100 Siena aly. ** Present addres: Department of Plant Physiology, Comenius University, Mlynaki dolina B-2, 842 15 Bratislava, Slovakia, © 295 by Gamay Fiche Veg Sogn sor containing vesicles fuse with the plasma membrane, thus contributing both membrane surface and matrix material to the growing tube. In the present work, callosic and pectic epitopes are localized ultrastructurally in the pollen eyto- 226 plasm and cell wall in order to elucidate their respective intra- cellular pathways. Callose and pectins belong to the main components in the plant cell wal. The latter ave been pos: tulated to play an important role in cell wall hydration, fil tering, adhesion between cells, and wall plasticity during growth (Baron-Epel etal, 1988; Jarvis, 1984). Depending on the source they consist’ of homogalacturonan linked to shamnogalacturonan and arabinogalactan sidechains. The carboxyl groups of the sugar units can be methyl-esterified (for reviews see Fincher and Stone, 1981; Carpita and Gi- beaut, 1993). In order to localize pectins in pollen grains and tubes, we used the monoclonal antibodies JIM 5 and JIM 7 (Vandenbosh et al., 1989; Knox et al., 1990). Immunodot- blot analyses had shown that JIM 5, which is specific for an acidic pectin epitope, recognizes pectin with up to 50% este rifiction whereas JIM 7, which is largely unreactive with ‘un-sterified pectin, reacts with pectin, having a degre of ex terifcation ranging from 38% to 90% (Knox et al, 1990). ‘An anti-1,3-glucan antibody was used for localization of callose ‘The scopes of the present work are i) the ultrastructural lo- calization of pectins and callose in the cytoplasm and cell ‘wall of pollen grain and pollen tube, i) the characterization of the intracellular pathway of the pectin precursors in ger inating pollen, and ii) the possible implications ofthese re sults for the processes involved in the selfincompatibility reaction in pollen tubes. Material and Methods Plant material ‘Two clones of Bragmansia suaveolens (white red), exhibiting self incompatibility and’ crosrcomputbility (Preise and Preise, 1991), were grown under long day conditions in the green house at the Department of Botany, Stockholm University. Day temper sure was 18°C and night temperature 15°C. Pollen grains were collected I week before anthesis and shorly after anthesis. For in Avan Garmaanas, Jan Hopi, Feuaras Vewntcennot2, and Byonx WALLes ico studies white flowers were pollinated with pollen fom red flowers for compatible pollination and with pollen from white flowers for incompatible pollination. After 48h the compatible pole len tubes had reached the middle part ofthe style (length between 220 and 270 mm). Pieces from the upper and middle part of the style ‘were cut and prepared for electron microscopy. For oxo studies, pollen grins were sown on 2 thin layer of culture medium spread ‘on coverslips after Raudashoski et al, 1987). Elecron Microscopy Immarure and mature polen, pollinated styles were fixed in 25% glutaraldehyde in 05M phosphate buffer, pH72, for Sh tom femperatue. The materia was thoroughly washed in butler and postted in osmium tetroxide (1 in phosphate bute) for 2h room temperature. The specimens were dehydrated in ethanol and embedded in LR.White resin ard grade for immunological Studies or dehydrated in acetone and embedded in Spurs epoxy re Sin, Urathin sections were eut with 3 Sorvall ultramicrotome MT 520 and coed on formar cated gold (lor PATAg treatment) tickal (or immunocytochemisy) or copper grids. Specimens were Slbyerved in 4 Zeiss EMIOA clecron miconcope operated 3 CORY. Micrographs were taken with plat camera and electron micro. scope hn (Kodak) Immenocyocheristry Ueathin sections of specimens embeded in LR-White were in cubated in buffer containing 10mM TrisHHCl, pH 7.5, 150mM NaCl, 05% bovine serum albumin, and 0.08% Tween 20 for 20min for preabsorption of unspecific binding. For immunolabel- ling the monoclonal antibodies JIM 5 and JIM were diluted 13100 in butter, followed by several washes in buffer and incubation in goatantirabbit IgG coupled with 10nm colloidal old (BioCell or ‘Amersham) for 45min (dition 1:50 in buffer). The anti(3)2- tlocan antibody (Biomupplies, Australia) was used in a dilution of 1:100 in buffer, and the secondary antibody. was 2 goavanti- ‘mouse IgG coupled with 10mm colloidal gold (Amersham Interns- tional ple, UK, dilution 1:20 in buffer). The sections were washed, etd aod sained wih ray] amate (2%) and led crate (ey old). Fig. 1 SEM micrograph of a pollen grain germinated on the stigma. The pollen tube ha ibilar surface and shows alight swelling at che ddstal end. Noce the wrate structure ofthe pollen grain exine. bar ~ 10 im, Figs.2-5: TEM micrographs of pollen grains embedded in Spur's epoxy resin. If not noted otherwise the sections are posestained with vuranyl acetate and lead cits, Fig. 2: Now germinated pollen gran treated with PATA for label of polysaccharides. The cytoplasm is rch in mitochondria, plastids with clectron dense starch grains and small vacuoles. No bipartite vesicles are found in the cytoplasm before germination. The cell wall ofthe pollen grain consists of the eleczron translucent intine (), andthe electron opaque ezine, consisting of eine (n) and sexne (3). The arrow {ndieates the equatorial zone where intine and nexine layers alternate. bar = 2m, Fig 3 Diceyosomes in germinated pollen grain. Note she similar texture of the dictyosome derived vesicles (arrows) and the contents ofthe fibrillar and biparive particles in Figs 4 and 5. bar = 0.2m. Fig 4 Meridional section through the aperture of a germinated pollen after PATAg treatment. The particles seemingly moving towards the tule show a granular or fibrillar eructure(arows and insert). bar = 1m. Fig 5: Transverse section ofthe apical end of a pollen tube filed with bipartite particles. The particles are partly surrounded by 3 mem brane (arrows), ocasionally they coalesce (arrowhead) or fuse with the cll wall (bold arrow). bar = 05 um. 228 Periodic acid thicerbohydrazideslverproteinate (PATA) tin for polyaccheides Uathinections were collected on costed gold grids. Incubation ‘was performed on drops ofthe respective solutions placed in cera ‘mic mold following the protocol of Thidry (1967) Seanning electron microscopy Pollen grains fixed and dehydrated as above were critical point ried in Balzers CPD 020 entcal point deyer and sputtered with gold in a Polaron E5100 sputter. Specimens were observed ina Ste- Feoscann 260 scanning electron microscope (Cambridge Instr ‘ments) operated at IOKV. Buorscence Microscopy For observation ofthe calloe distribution in pollen tubes grown, in civ, pollinated stigmas were slit longitudinally. The haves were ‘mounted with the transmitting tissue facing up ina drop of decolo- rized aniline blue on a microscope slide. Specimens were observed ina Fluorescence Microscope (Zest) with « Bp 365/11 fe. Pictu- res were taken with a data back camera (Yashiks) on T1-X Pan film (Kodak). Hoxration and Germination After pollination of the stigma the pollen remains shrunk for ca, 40min before assuming the spherical shape character istic forthe hydrated grain. On the other hand, pollen sown con agar plates hydrates immediately. The B. suaveolens pol: Jen grain is spherical in the hydrated state and its sexine is distinctly meridional striate. ‘The grain has a diameter of 35-37 pm, Shortly before germination it assumes an oblate spheroidal shape with a major axis of 29-32um and a minor axis of 33~35 4m (Fig, 1). The cell wall of the B. suaveolens pollen grain consists of 2 1.5~1.6)1m thick exine and a 0-4— (.46um thick intine (Fig, 2). Electron dense material occurs between the baculae of the exine. The equatorial zone of the Ania German, Jan Huh, Faurzrmas Vinanceniorz, and BySan Wastes {grain is characterized by alternating exine and intine layers (Fig. 2). One hour after pollination or sowing, respectively, the first pollen tubes emerge from one of the apertures of the three colporate pollen grains in both the in vivo and in vitro systems (Fig. 1). On the ultrastructural level germination is companied by structural changes of the cytoplasm. The ER that is aggregated in huge stacks in the mature grain is mostly replaced by single ER profiles upon germination. Dictyosomes show increased activity indicated by the in- creased number of vesicles surrounding them (Fig. 3) A par ticular type of inclusion appears. These inclusions are ves clelike and have a diameter of 0.2-0.4 1m. They are either of loose fbr or gran structure (Fg 4) oF conan ¢ core of densely granulated or fibrillar” appearance sur- rounded by an electron translucent halo (Fig. 5). Some of these particles are completely or partially surrounded by a membrane. The fibrils in these particles are stained after the PATAg reaction (Fig. 4). The particles appear first close to the aperture and move into the pollen tube upon germina. tion (Fig, 4). They will be referred to as fibrillar or bipartite particles, respectively. ‘The pollen tube ‘The pollen tube of B suaveolens is regularly cylindric with the exception of the most distal part, which shows swelling (Fig. 1). Ithas a diameter of 7~ 11 um and shows a polar dis tribution of the organelles along the longitudinal axis allow ing a differentiation into zones (according to Crest et al, 1977). The pollen tube wall appears bilayered. The outer layers 1090 200:m hic and has lie strrare with strong electron scattering ability after PATAg staining (Fig. 4). The inner layer is 30 to 501m thick and considerably less clectron dense than the outer layer, also after PATAg stain- ing Ic is absent a che pollen tube tp ‘The most apical part of the pollen tube, the growth zone (after Crest eta, 1977), contains 2 high number of inclu sions similar to the bipartite particles observed in the germ Figs.6~ 11: TEM micrographs of pollen grains embedded in LR-White and treated with immunocytochemistry Post-stain with uranyl ace- tate and lead citcate 6 lnc of the pollen grain bled with JIM for aide pins. Labi conspicuous more dense towards the outer portion ofthe inne. Note thatthe intine material alternating Wi sine, « ~ sexine, bar = 05m, fexine material close to the aperture is beled intensely (arrows). ~ intine, n ~ ne- Fig.7:Intne of the pollen grain labelled with JIM 7 for methylesterifed pectins. The intne is labelled densely throughout, wheres the ex ines almost devoid of any gold gains. bar = 0.5 um. Fig. 8: Transverse section ofa pollen tube grown in vito labelled with JIM 7 for methyl esterified pectns. The cell wall shows intense label throughout the outer layer (ol) In the eytoplasm dictyosomes and vesicles ae abelled (arrows). ~ inner layer. bar = 0.5 )4m, Figs 9 Serial transverse sections of one pollen tube grown in vivo labelled with JIM 7 (Fig. 9), JIM 5 (Fig 10) and the anc-(-3}#glucan told (gl). The oer var layer) ofthe olen tabled ens fer cc pets IM) and wih dea ime Stans the nue for mayne pin ya or cnc coc the nator el eal Gh Rr ‘ment wih he pectin psc abode the inn laer shows only few gold gan svayeaoxined ah fleas roe) The asm shows no label for eallose epitopes, whereas the cytoplasmic inclusions are labelled intensely for mthylesterified pectins, Only a few ofthe inclusions ae labeled for acidic pectins (arowhead), bars = 05m Immunogld Localization of Pectin and Callosein Pollen Grains and Pollen Tubes 229 20 nating pollen grain. Occasionally they coalesce and particles localized at the periphery seem to fuse with the cell wall (Fig. 5). Most of these fusions with the cell wall occur in the tip region where the fibrillar layer of the cell wal is directly adjacent to the plasma membrane. The material in the bipar tite particle shows a striking resemblance tothe structure of the outer layer of the cell wal. In the organelle zone, adjacent to the growth zone, there ate bipartite particles present as well, but less numerous ‘There are numerous fibrillar particles present, especially in the vicinity of dietyosomes, thus indicating their putative origin (Fig. 4). A third type of vesicles is possibly derived from dicrysosomes as well. They are smaller ($0100 nm) and contain material of varying electron density. Immunolocalization Electronmicrographs of immunogold treated ultrathinsec tions of pollen grains show that label with JIM 7 is localized throughout the intine Fig. 7), whereas after teatment with JIM 5 gold grains are mostly confined to the outer part of ‘the intine (Fig. 6). Depending on the developmental state of the pollen grain the intensity of label for pectins in the cy- toplasm varies. In pollen grains sampled before anthesis only few gold grains are observed. Occasionally, diryosome vest cles are labeled with JIM 5 and JIM 7 (not shown). During ‘maturation and germination, the number of fibrillar and bi partite particles increases. These are labelled intensely with JIM 7 (Figs, 8 and 9) whereas Iabel with JIM 5 was less in- tense (Fig, 10), indicating the high grade of methyl-esterifica tion of the pectins in these particles. In the pollen tube ine tense label is present in the outer layer of the cell wall after treatment with the pectin specific antibodies. Epitopes lx belled by JIM 5 are distributed evenly throughout the outer layer (Fig. 10), whereas methylesterified pectins (labeled by JIM 7, Fig 9) are concentrated atthe inner part of the outer layer in tubes grown in vivo. On the other hand, in tubes grown in vitro the entire outer cell wall layer is labelled with JIM? (Fig 8). In the cytoplasm JIM 7 labels all three types of Figs. 12-17: Pollen tubes after incompatible pollination Avia Gurraane, Jan Huis, Frnzras Visoacennotz, and Bion WALLES vesicles (fibrillar, bipartite and small) intensely whereas label with JIM 5 is rare. Acidic pectins are mostly present in some larger inclusions resembling vacuoles. Dictysomes are le: belied at their periphery and surrounding vesicles with JIM 7 for methylesterfied pectins (Fig, 8). Label with the anticallose antibody shows that the inner layer of the pollen tube wall is labelled intensely, whereas the outer layer is not marked. Neither the bipartite particles nor the other eytoplasmic inclusions are marked (Fig. 1), On control ‘sections, obtained by omitting incubation ‘with the first antibody, no label was found (not shown). Morphology of pollen tubes after the gametophtic self incompatibility reaction For the study of the seltincompatibilty reaction stigmas ‘of white flowers were self pollinated. Samples were taken at 6,8, 10, 12, 24 and 48h after pollination from the upper part of the stigma. The first visible changes of tube morphology ae huge stacks of circular rough ER that appear 8h after pollination. Beginning 10h after pollination the inner callo- sic cell wall layer normally having a diameter of 30nm starts thickening, reaching a diameter of up to 400~600nm after 24h (Fig. 12), Callose depositions are particularly conspicu- ous towards the tip of the pollen tube (Fig. 13). The ealosic plugs separating the active cytoplasm from the inactive distal part of the tube are more irregularly distributed in incompa- tible tubes and have a longer and more massive shape (not shown). The outer pollen tube layer i subject to thickening, too. Its diameter increases from 100 to 200nm to up to 400nm after 24h, In addition, large numbers of bipartite particles are deposited at the inside ofthe cell wall without being integrated into it Figs. 14 and 15) or forming crescent shaped inclusions in the inner callosic inner wall (Figs. 16 and 17), Parallel to the thickening of the cell wall the eyto- plasm shows increasing signs of degeneration. The appearance of vacuoles with fibrillar contenes strongly labelled with JIM 5 for acidic aids is remarkable (Fig. 17). ig. 12: Transverse secion of an incompatible pollen tube showing thickening ofthe inner translucent ayer (i). Treatment with the anti(13}glucan antibody shows intense label ofthe inner layer but no label of the outer layer (ol) or the eytoplasmic inclusions. bar = thm, Fig 13: Fluorescence micrograph of incompatible pollen tube stained with declorized aniline blue for label of calloe. Especially the ip re ton (aro) shows swelling and iregular deposits of callose. bar = SOum. Fig. 4: Transverse section of Spurr embedded pollen tube showing deposition of bipartite particles atthe inside of the cell wall. bar = 5m, Fig. 15: Transverse section of LR-White embedded pollen tube treated with JIM? for methylestrified pectns, The gold grains indicate the pectinaceous nature ofthe material deposited at the inside of the cell wall upon incompatible pollination. bar = 0.5 jm. Fig. 16: Transverse section of Spurr embedded pollen tube showing crescent shaped fibrillar inclusions inthe thickened inner cll wall ayer (arrow). bar = OSum. Fig. 17: Transverse section of LR-White embedded pollen tube treated with JIM 5 fr acidic petins. The crescent inchisions inthe inner eel wall yer (arrows) and many inclusions (asterisk) in the degenerating cytoplasm ae labeled bar = 0S um. Immunogold Localization of Pectin and Callose in Pollen Grains and Pollen Tubes 231 232 Discussion The cell wall ‘The ulkrastructure of the pollen tube cell wall has been subject to many investigations. For most of the examined species a bipartite structure of the cell wall was reported. With the help of histochemical or biochemical methods and X-ray diffraction analyses several groups were able to charac- terize the composition of the cell wall layers of pollen tubes (lor a review see Harris etal, 1984; Steer and Steer, 1989). ‘The outer fibrillar layer ofthe tube cell wall consists mainly of pectin, hemicellulose and cellulose, whereas the inner layer contains mainly callose and proteins (Heslop-Harrison, 1987). [As in Nicotine alata (Meikle etal, 1991) the inner layer of, the B suaveolens pollen tube cell walls intensely labelled for callose with the respective antibody, but i is not marked by PATAg. Since eallose isa 8-1,3-glucan, it has no free vi slyeol groups that could be labelled by the PATAg reaction (Rougier eta, 1973). On the other hand, che outer layer ap- pears strongly electron scattering after PATAg treatment, in- dlicating its high content of polysaccharides other than eal: lose. The outer layer has a fibrillar appearance and is marked by antibodies specific for pectns, a result consistent with the results of labelling for acL-arabinofuranosyl residues in Nico- tiana alata pollen (Anderson et al, 1987). In B. suaveolens, acidic pectins labelled by JIM 5 seem to be abundant throughout the entire outer wal layer whereas methyLeste re ests abel by YM 7, speared moreconcmed ‘on the inner part of the outer layer of in vivo grown pollen tubes. This labeling pattern is in accordance with the results ‘obtained after immunofluorescence and immunogold label ‘of Nicotiana tabscum pollen tubes with the MAbs JIM 5 and JIM? (Liet al, 1994; Geitmann etal, 1995). Inthe intine of “Arabidopsis thaliana pollen, label with JIM 5 appears more dense towards the outside and towards the plasma mem- brane than in the middle parc (Van Aelst and Van Went, 1992). In the B. surveolens pollen, however, there is a radial gradient of methylesterifiation of the pectins in the intine, demonstrated by the increasing label density towards the ou ter part after teatment with the same antibody. A radial grax dient of esterification is also present in the pollen cube wal. Here, however, it is reflected by the decreasing label with JIM ? towards the ouside, The inn thatthe rade of pes tin esterification is higher in the inner, presumably younger pars of the pollen cell walls consistent with observations in ther plant tissues. In general, the composition of pectic polymers seems to vary during the initial stages of cell wall development (Northcote, 1978). For this distribution pat- tern of methyl-sterifcation ether a modification of the pec synthesized and secreted by the endomembrane sys tem, or gradual enzymatic deesterification ofthe older parts of the cell wall could be responsible. Liners and van Cutsem (1992) support the latter hypothesis since synthesis and se- ‘eretion of pectins is shown to occur in highly esterified form in suspension-cultured carrot cells. This i also the casein B susveolens pollen tubes, indicated by the intense label with JIM 7 and rare label with JIM 5 of the secretory vesicles. In ‘general, during cell wall maturation pectin methyl esterases Awa Germano, Jas Hui, Feuaras Viswrceanot2, and ByOnx WALLES cause de-sterfiction of pectins (Jarvis, 1984; Stachelin et al., 1991; Lynch and Stachelin, 1992). These enzymes are ci- ther present in the wall or provided by external sources. The fact that methyl-esterified pectin are more abundant in the outer cell wall of pollen tubes grown in vitro than of those grown in vito, and the finding thatthe lateer show a decreas- ing label density towards the outer surface ofthe wall, cates that the stylar matrix contributes to the de-sterifica tion of the pectins in the pollen tube wall. In fac, especially older styles of B. suaveolens show a high content of methyles- terase activity (Vennigerholz, unpublished results). On the ‘other hand, the appearance of acidic peetin cotaining vacu colar inclusions in incompatible pollen tubes might indicate that pectin methyl esterases are present in the secretory Ves cles. They might start to act on the pectins upon prolonged presence of secretory material in the cytoplasm caused by the incompatibility reaction, ‘A comparison of the cell walls of pollen grain and pollen tube shows strong similarity in texture and labeling pattern of the itine of the pollen grain and the outer cell wall layer of the pollen tube. Both are labelled intensely for pectins, whereas the exine of the pollen grain seems devoid of pec. tins. This provides further evidence for the assumption that the pollen tube wall is formed in continuation of the intine (Heslop Harrison, 1987). ‘The pectin pathway Peetins are a major matrix component in the plant cl wall. In pollen tubes they are mainly present in the outer layer of the bilayered cell wall. Pectins are formed of homo- galacturonan, shamnogalacturonan I and Il, oligosaccharide side chains and arabinogalactan. The degree and pattern of ‘the methylsterifcation of the homogalacturonan domains has influence on the physial state of the cell wall matrix. ‘The presence of calcium causes dimerization of acidic pec tins to form a relatively rigid gel, whereas methylesterified pects are rather water soluble. The subcellular routes of polysaccharide synthesis have been subject to-many im ‘ations. Delmer and Stone (1988) summarize in their review that membrane-associated glycosyltransferases are_synthe- sized in the rough ER and transported to the Golgi appar tus, where they catalyze the assembly of polysaccharides. ‘The necessary monosaccharides are provided by nucleotide sugar translocators. First polymerization steps might be per formed in the ER; however, immunogold label for pectins indicated thac the assembly of the pectin backbone up to 2 hhigh degree of polymerization takes place in the cis and me- dial cisternae of the dictyosomes (for a review see Levy and Stachelin, 1992). Bowles and Northcote (1976) showed that pectin is present in the membrane system as very lage poly- ‘mers. Nearly simultaneous to the backbone assembly, meth: yhesterfcation of the carboxyl groups by pectin methyl transferases takes place (Moore and Stachelin, 1988; Stache lin et al, 1991; Zhang and Stachelin, 1992; Liners et al, 1994). Alter immunogold treatment ‘with pectin specific antibodies, label was found preferentially on the bulging ‘margins and surrounding vesicles of the dictyosomes. How: ever, it is unclear whether pectins have to cross the entire diceyosome stack for complete addition of the oligosaccha- Immunogold Localization of Pectin and Callose in Pollen Grains and Pollen Tubes ride side chains (Zhang and Stachelin, 1992), or if pectin transporting vesicles might leave the dictyosome stack al- ready from the medial cisternae (Moore etal, 1991). In any case, dictyosome derived vesicles transport the pectins to- wards the plasma membrane, probably with the help of the cytoskeletal system (Delmer and Stone, 1988). During this transport final synthesis steps, such as methybesterifcation, seem to take place (Vian and Roland, 1991; Fincher and Stone, 1981). In pollen tubes these transport vesicles seem to be represented by the bipartite or fibrillar inclusions. Bipar- tite particles and inclusions of similar appearance have been observed in many kinds of pollen grains and tubes. In most cases they appeared in the growing pollen tube, in some cases only after incompatible pollination. In grasses they oc- cur already in ungerminated pollen and are referred to as pppartcles (Heslop-Flarrison, 1979; Heslop-Harrison. and HeslopHarrison, 1982). These authors assume that they contain pectic substances, whereas callase was postulated to be a component of the vesicle contents by Crest and Tiezzi (1990), Crest ctl. (1977), Crest etal. (1979) and Ciampolini tal. (1982), who observed the fusion of fibrillar particles with the plasmalemma in Lycopersicon perwvianum and Pru nus avium, respectively. They concluded that the vesicles contain callosc precursors for the synthesis ofthe inner cell wall layer. However, the authors do not give any evidence for the calosie nature of the particles. The only indication ‘would be their high glucose content (Harris etal, 1884). Exx amination of particles with similar ultrastructural appear ance in other species showed that in these cases their content is indeed not callosc but apparently consists of other poly- saccharides. Cellulose might be present (Engels, 19748), but pectins seem to represent the main component (Rosen etal. 1964; Rosen and Gawlik, 1966; Dashek and Rosen, 1966; van der Woude et al 1971; Crestiet al, 1985; Anderson et ala 1987). Further arguments for the role of the particles as cell wall precursors were provided by Engels (19742), who chemically analysed the contents of isolated particles from Petunia pollen tubes. The results indicated a relatively high protein content, which was suggested to reflect the presence of enzymes responsible for polymerization and methylation ofthe pectic contents In B suaveolens pollen tubes, particles are observed that re- semble very much the mature and immature inclusions de- seribed by van der Woude et al (1971). The fibrillar, less electron dense inclusions appear further away from the api- cal end, whereas the bipartite particles fill the apical tip of the tube. Intermediate states between the two kinds are ob- served, thus indicating that they represent the same particle in different stages of development. Both were labelled with JIM 7, indicating the pectic contents. The rare presence of lie bel after treatment with JIM 5 indicates the high grade of es- teriication of the pectias. None of the particles is labelled by the anticallose antibody, showing the absence of this polysaccharide in the fibrillar and bipartite particles of surveolens. The mature particles fuse with the cell wal, here by obviously contributing material to the formation of the outer cell wall. Fusions occur in the very tip, where the cal- losic inner layer is absent, a5 well as in more distal areas where a thin layer of callose is present. It scems as if in the lamer case the pectic material has to cross the inner callosie 233 wall layer first, before being able to fuse with the outer layer. Evidence for this migration process isthe labelling for pectins of fibrillar inclusions in the inner cell wall, especially conspicuous in the thickened inner layer of incompatible pollen tubes, These inclusions probably represent material fn its way to the outer cell wal. Incompatibility reaction During the gametophytic seltincompatbilty reaction, pollen tube growth is arrested in the styl, in some eases al: ready on the stigma. Numerous investigations on different species showed that the main symptoms observed in the in- compatible pollen tube are a reduced growth rate, the ap- pearance of circular ER (CER) in the cytoplasm, thickening of the pollen tube wall, and the deposition of callose in form of irregular and numerous callosic plugs and a thickened in ner tube wall layer (Linskens and Fsser, 1957; Van der Pluijm and Linskens, 1966; Tupy, 195%; de Nettancourt et al, 1974; Crestiet aly 1979: Herrero and Dickinson, 1981; ‘Anderson et a, 1987; Samaha et al, 1989; Singh and Kao, 1992; Franklin-Tong and Franklin, 1992; fora review see Pa:

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