Calcium distribution in fertilized and unfertilized ovules and

Abstract. Potassium antimonate was used to localizeCa2+ in tobacco ovules from 0 to 7 d after anthesis inpollinated and emasculated flowers. Antimonate binds‘‘loosely bound’’ Ca2+ into calcium antimonate; less-soluble forms are unavailable and free calcium usuallyescapes. Ovules are immature at anthesis. Abundantcalcium precipitates in nucellar cells surrounding themicropylar canal. A difference between calcium in the twosynergids emerges at 1 d, which is enhanced in pollinatedflowers. The future receptive synergid accumulates moreprecipitates in the nucleus, cytoplasm and cell walls. Afterfertilization, micropyle precipitates diminish, and theovule is unreceptive to further tube entry. In emasculatedflowers 6 d after anthesis, ovular precipitates essentiallydisappear; however, flowers pollinated at 4–5 d andcollected 2 d later largely restore their prior concentra-tion of precipitates. Ovular precipitates occur initially inthe nucellus, then the embryo sac, and finally the synergidand micropylar filiform apparatus. Possibility, calcium isreleased from the embryo sac, although no structuralevidence of exudate formation was observed. Calciumprecipitates in the ovule correlate with the ability of theovule to be fertilized, suggesting that successful pollentube entry and later development may require calcium ofthe class precipitated by antimonate.

Key words: Antimonate precipitation – Calcium –Embryo sac – Fertilization – Nicotiana (fertilization) –Synergid

Introduction

Calcium is an essential element in biological systems. Insomatic cells, it has been implicated as a coenzyme,

signal ion, charge generator, structural component andsecond messenger depending on its binding and solubil-ity (Bush 1995). In reproduction, pollen tubes purport-edly respond to calcium gradients by elongating towardthe gradient in in-vitro assays (Mascarenhas andMachlis 1962; Reger et al. 1992). Also, the calcium-richenvironment of the synergids (Jensen 1965; Chaubal andReger 1990) may have a special role in terminating theelongation of the pollen tube and triggering its dis-charge. The distribution of calcium in the pistil, ovuleand embryo sac (ES) has therefore been the subject ofconsiderable interest. Calcium in the ovule and ES hasbeen examined using a variety of techniques, includingfreeze-drying and microincineration of sectioned mate-rial (Jensen 1965), energy-dispersive X-ray analysis(EDXA; Chaubal and Reger 1990, 1992a, 1992b) fortotal calcium, chlorotetracycline (Huang and Russell1992b; Tirlapur et al. 1993) for membrane-boundcalcium, and potassium antimonate precipitation(Chaubal and Reger 1993, 1994; He and Yang 1992)for loosely bound calcium (Wick and Hepler 1982) atconcentrations above 10–6 M (Klein et al. 1972), therebyusually excluding free cytosolic calcium. Calcium that isbound to phosphates and calcium carbonates is moretightly bound and therefore is unlabeled by antimonate.

Studies of embryo sacs have indicated that calcium:(i) is extremely abundant in synergids; (ii) accumulatessimilarly in both synergids of some species (e.g., Triticumaestivum, Pennisetum glaucum and Helianthus annuus)and differently in others (e.g., barley) (Chaubal andReger 1990, 1992a, 1993, 1994; He and Yang 1992); (iii)is more abundant in the receptive synergid (e.g., Nicoti-ana tabacum and Petunia hybrida; Huang and Russell1992b; Tirlapur et al. 1993), and (iv) is more abundant inthe micropylar synergid region (although accumulationis chalazal in H. annuus; He and Yang 1992). Thesethemes suggest that calcium within the synergids isrelated to the receipt of the pollen tube, from wheresperm cells are transferred to the egg and central cell.

Prior studies using chlorotetracycline in tobacco tolocalize membrane-bound calcium in the ES (Huang andRussell 1992b; Tirlapur et al. 1993) do not provide

Planta (1997) 202: 93–105

Calcium distribution in fertilized and unfertilized ovulesand embryo sacs of Nicotiana tabacum L.Hui-Qiao Tian1,2, Scott D. Russell1

1Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019, USA2Department of Biology, Wuhan University, Wuhan, China

Received: 14 August 1996 / Accepted: 9 October 1996

Abbreviations: EDXA = energy-dispersive X-ray analysis; ES =embryo Sac; ppt = precipitateCorrespondence to: S.D. Russell; Fax: 1 (405) 325 7619; E-mail:[email protected]

insight on the accumulation or distribution of looselybound or unbound calcium. The current study discussesthe nature of antimonate precipitation, involvement ofpollination in differences between the two synergids andchanges in loosely bound calcium near the time offertilization.

Materials and methods

Nicotiana tabacum L. was grown in controlled growth chambers at20–27 °C with 16 h daylength. Flowers were emasculated 0.5 dbefore anthesis and pollinated at anthesis or 4, 5, and 6 d afteranthesis. Ovules were dissected at 1-d intervals to 7 d after anthesis,fixed 4 h at room temperature or 4 °C in 2% glutaraldehyde (v/v) in0.1 M KH2PO4 buffer (pH 7.8) containing freshly added 1%potassium antimonate (K2H2Sb2O7 · 4H2O; from fresh 4% [w/v]stock in double-distilled H2O), washed (five 20 min changes inbuffered 1% antimonate), postfixed in 1% (w/v) buffered OsO4containing 1% antimonate 16 h at 4 °C, washed in buffer (four20 min changes), dehydrated in a graded acetone series andembedded in Spurr’s resin.

At least six ovules from each treatment were sectioned andstained with 2% uranyl acetate (w/v) in 70% methanol (v/v).Precipitates (ppts) were examined using unstained material byEDXA using a Kevex Quantum detector on a JEOL (Tokyo,Japan) JEM-2000FX transmission electron microscope at 40–100 kV using < 30% deadtime and a 12-ls pulse width. Peakdeconvolution software was employed to analyze specimens usingthin-section assumptions. Transmission electron micrographs weretaken using a Zeiss 10a at 80 kV. As additional controls: (i) selectedovules were observed without potassium antimonate treatment,and (ii) sections were incubated in 100 mM EGTA (pH 7.8) for 1 h.

Results

Identification of precipitates. The EDXA spectra ofFormvar (Fig. 1a) and Spurr’s resin (Fig. 1b) werecompared to embedded material; the Cu signal wasgenerated by backscattered electrons striking the grid.Calcium-induced antimonate ppts produced a distinctivecomposite peak near the characteristic energy peaks ofantimony and calcium. Analyses of ppts in nucellar cellwalls (Fig. 1c,e) and the central cell vacuole (Fig. 1d,f)are shown. A significant overlap occurs between diag-nostic antimony (La1 = 3.605 keV, Lb1 = 3.843 keV,Lb2 = 4.100 keV, LIIIab = 4.132 keV) and calcium peaks(Ka1 = 3.691 keV, Ka2 = 3.688 keV, K b1 = 4.012 keV,Kab = 4.038). The composite Ca-Sb (Fig. 1g) was de-convoluted using modeled Sb and Ca contributions untilan acceptable fit was achieved using a v2 test (Fig. 1h;v2 = 2.53). Based on the relative intensities of the SbLa1and the CaKa1 peaks, the calculated atomic percentageof Sb is 66.93% ± 0.58% and Ca is 33.07 ± 0.85%(approximately 2:1).

Ultrastructurally, calcium antimonate ppts variedfrom < 1nm to > 200 nm in diameter. Most could bedistinguished by their contents, density, particulatetexture, and high-contrast margins (Fig. 1e, f). Otherdistinctive inclusions were osmiophilic lipid bodies,which had large Os peaks (Fig. 1i), and starch grains,which had large C and O peaks (Fig. 1j). Osmiophilicbodies were homogeneously gray with indistinct bound-aries (Fig. 1e).

Calcium antimonate ppts (Fig. 2a) were mostlyremoved using the calcium-selective chelator EGTA(Fig. 2b, c). Precipitates not encapsulated in plastic wereremoved, as indicated by stereo imaging.

At anthesis. Embryo sacs are immature (often four-nucleate) at anthesis. Calcium antimonate ppts areabundant in nucellar cells, especially surrounding theES, and are not abundant inside the ES (Fig. 3a). Aftercellularization and early differentiation, ppts becomeabundant in ES cells. Initially, there are no differences inppts between the two immature synergids, which lack afiliform apparatus at this stage. The young egg cellcontains few ppts – these mostly in the nucleus. Thethree antipodal cells have few ppts (Table 1).

Day 1 after anthesis. Synergids form prominent vacuolesat their chalazal ends; the nucleus and cytoplasm aremicropylar (Fig. 3b). Cell walls begin to thicken to forma filiform apparatus. The egg cell forms a conspicuouscentral vacuole and the nucleus becomes chalazal(Fig. 4). The polar nuclei are still located at oppositeends of the central cell.

Pollinated flowers. Precipitates in the two synergidsbegin to differ in pollinated flowers at this stage. Onesynergid acquires more ppts, especially in the nucleus(Fig. 3c). The common synergid cell wall containsnumerous small ppts, which are located throughout thethickening cell wall. In the filiform apparatus, largegranules accumulate in addition to the small pptscharacteristic of the cell wall, especially at tip of thefiliform apparatus (Fig. 3d). In the egg cell, ppt abun-dance decreases but in the polar nuclei, the number ofppts increases.

Interestingly, the three antipodal cells resemble egg-apparatus cells at this stage (Fig. 5). Two of the

Fig. 1a–d. Energy-dispersive X-ray spectra of thin sections of tobaccoovules. Elements: C, carbon; Ca, calcium; Cl, chlorine; Cr, chromium;Cu, copper; O, oxygen; P, phosphorus; Sb, antimony; Si, silicon. X-axis is energy of emitted X-rays in keV. Y-axis is X-ray counts from 0to full scale (FS), as given for each figure. a Spectrum of Formvar filmmounted on copper grid. FS = 492 counts. b Spectrum of emptySpurr’s resin on Formvar film mounted on copper grid. FS = 794counts. c Spectrum of antimonate ppt in thin section of nucellus cell.FS = 299 counts. d Spectrum of antimonate ppt in thin section ofcentral cell. FS = 1455 counts

Fig. 1e–f. Transmission electron micrographs of antimonate andosmiophilic inclusions in the tobacco ovule. e Nucellus cell containingan osmiophilic area (*) analyzed in i. Antimonate ppts occur in cellwall (CW). × 31 300; bar = 0.2 lm. f Antimonate ppts in the vacuoleof the central cell similar to those illustrated in d, g, h × 39 000;bar = 0.2 lm

Fig. 1g–j. Energy-dispersive X-ray spectra of thin sections of tobaccoovules. Elements: C, carbon; O, oxygen; Os, osmium; Sb, antimony;Si, silicon. X-axis is energy of emitted X-rays in keV. Y-axis is X-raycounts from 0 to full scale (FS) given below. g Composite spectrum ofantimony and calcium of ppt in the central cell with peaks removedand smoothed. FS = 1397 counts. h Model of the composite spectrumaccounting from the contributions of antimony and calcium in pptshown in g. Arrowhead indicates model. FS = 1385 counts. iOsmiophilic inclusion (presumed lipid body) in plastid. FS = 817counts. j Starch grain in plastid. FS = 431 counts

c

94 H.-Q Tian and S.D. Russell: Calcium distribution in tobacco ovules

antipodals display nuclei toward the edge of the ES,resembling synergids, whereas the other cell has anucleus at the opposite pole, similar to an egg cell. Thesynergid-like antipodal cells, however, contain starchgrains (uncommon in synergids) and do not form afiliform apparatus. Some ppts occur in the antipodalcells but no regularity in the distribution of theantimonate granules is evident (Fig. 5). Calcium pptsare abundant in the ES wall, decreasing in frequencytoward the nucellus, except in the micropylar region

(Table 1). Nucellar ppts become less abundant in thecytoplasm and more abundant in the cell wall, increasingalong the micropylar canal (Fig. 3d).

Unpollinated flowers. Ovules from unpollinated flow-ers display differences in synergid structure that are notas well differentiated as in pollinated flowers.

Day 2 after anthesis. Pollinated flowers. Differences inthe two synergids become conspicuous in both fertilizedand unfertilized ovules. The overall electron density of

H.-Q Tian and S.D. Russell: Calcium distribution in tobacco ovules 95

the cytoplasm in unpenetrated synergids is reduced, ERbecomes the most prevalent organelle, and other cyto-plasmic organelles seem neither as abundant nor as wellformed. One synergid is noticeably more labeled withppts than the other, especially in the nucleus and filiformapparatus, and the cytoplasm seems more poorlyorganized (e.g., Fig. 3c, Table 1). Cytoplasmic pptsinside pollen tubes after discharge are less common.Precipitates are less abundant in the apical pollen tubewall at the base of the ES (Fig. 6a), increasing proxi-mally in the micropyle (Fig. 6a,b). Synergids penetratedby pollen tubes contain more abundant calcium pptsthan unpenetrated synergids. In the penetrated synergid,vesicles of pollen tube origin predominate, some con-taining dense ppts, but the synergid cytoplasm isrestricted to the edge of the cell (Fig. 6c). Small pptsare common within the distal pollen tube wall. Theoverall content of calcium ppts remains low with fewlarger ppts in pollen tube vesicles (Fig. 6d).

Unpollinated flowers. At 2 d after anthesis, the ultra-structure of the two synergids appears similar to thepersistent synergid of pollinated flowers, becoming moreelectron-lucent, ER membranes more common andother organelles less abundant and poorly organized.The precociously altered synergid appears to containmore calcium ppts in its nucleus and cytoplasm, and pptsare very abundant in the filiform apparatus (Table 1). Inthe egg cell, ppts remain uncommon; however, polyri-bosomes appear to increase.

Day 3 after anthesis. Pollinated flowers. The first divisionof the endosperm is frequently completed at this stage.The disorganization of the persistent synergid becomes

conspicuous; its large vacuole disappears, and it isreplaced by numerous small vacuoles, ER, and smallvesicles containing numerous calcium ppts. Abundantppts occur in the micropylar end of the ES wall (Fig. 6e).Precipitates are less common in fertilized egg cells(Fig. 6f ) than in surrounding endosperm and synergids(Fig. 7a). Antipodal cells have begun to degenerate.

Unpollinated flowers. Synergids remain intact(Fig. 7b,c) and continue to differ in ppt distribution(Table 1). The antipodal cells also remain intact and mayenlarge.

Day 4 after anthesis. Pollinated flowers. Cell wallsaround the zygote and persistent synergid are completedwithin 2 d of fertilization and the large vacuolesdisappear, replaced by abundant organelles. Abundantppts occur in the endosperm, ES and adjacent nucellarcell walls. Nucellar starch grains are less frequent.

Unpollinated flowers. Precipitates are still conspicu-ous in the filiform apparatus and in the synergidcommon wall (Fig. 7d), but otherwise synergid ultra-structure is essentially unchanged. Precipitates in ES and

Fig. 2a–c. Transmission electron micrographs of antimonate-labeledcytoplasm before and after extraction using EGTA. × 29 300;bar = 0.5 lm. a Electron-opaque ppts range in size from ca. 1 to250 nm in this 100-nm-thick section of the central cell and adjacentvacuole. b, c Stereopair of same section as a, after EGTA extraction.Larger ppts (>100 nm) are removed. Smaller ppts are not removedunless they are present on the surface of the section, as demonstratedusing stereo viewer. Precipitates entirely enclosed in the embeddingresin are unavailable to the chelator. Osmiophilic bodies (arrowheads)are not removed

Fig. 3a–d. Transmission electron micrographs of antimonate-labeledtobacco ovules from anthesis to 1 d after anthesis. Abbreviations usedin Figs. 3–8: A, antipodal; CC, central cell; CW, cell wall; E, egg cell;EN, endosperm; ES, embryo sac; FA, filiform apparatus; N, nucellus;n, nucleus; PT, pollen tube; S, synergid; Z, zygote. a At anthesis, theES is frequently immature (four-nucleate). Cell walls around the EScontain dense ppts near the nucellus. Large granules are also evidentin the ES. × 1800; bar = 3 lm. b One day post-pollination. Mature eggapparatus is evident with completely formed synergids and egg cellsharing boundaries with the central cell. Calcium antimonate ppts aresomewhat more abundant in ES than in surrounding nucellus. × 1800;bar = 3 lm. c Larger magnification of synergids in b. Differences incalcium antimonate ppts are evident between the nuclei; the nucleus inthe upper-left accumulated numerous small ppts, whereas the othernucleus accumulated fewer but larger ppts. Comparison with ES afterfertilization suggests that the latter will become the persistent synergid.× 4100; bar = 2 lm. d One day post-pollination. In some ES, thefiliform apparatus of one synergid shows a greater accumulation ofppts. At this stage, a gradient in ppts is evident in the cell walls of thenucellus; this concentration diminishes with distance from themicropylar canal (arrowheads). × 5200; bar = 2 lm

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nucellus cell walls decrease in abundance (Table 1).Starch grains occur infrequently in nucellar cells at thistime and those in the egg and central cell decrease in sizeand abundance.

Days 5, 6, and 7 after anthesis. Pollinated flowers. Thefirst division of the zygote is completed at this stage andfew ppts are seen at the onset of embryogenesis. Thesuspensor contains numerous mitochondria and nearlyhalf of its cell walls contain wall ingrowths.

Unpollinated flowers. At 5 d after emasculation, EScells remain intact; but the number of ppts diminishesdrastically and becomes highly variable. In the synerg-ids, ppts remain evident, especially in the filiformapparatus, although in lesser abundance (Fig. 8a). At6 and 7 d after emasculation, few ppts persist in theovule and these are in the filiform apparatus (Fig. 8b,Table 1).

Emasculated flowers with delayed pollination. Flowerspollinated 4 d after emasculation set about 2500 seeds(the normal amount), whereas flowers pollinated at 5 dproduce distinctly smaller fruits and only about 1000seeds, and those pollinated at 6 d set no fruit and showno signs of post-fertilization development. Unpollinatedflowers normally drop after 8 d. The stigma and styleappear to support appreciable pollen tube growth at thistime, but ovules are not fertilized.

Flowers pollinated 4 d after anthesis and grown for2 d more display ppts in nucellus cells and in themicropylar wall; some ppts occur in the filiform appa-ratus and ES wall (Fig. 8c, Table 1). In some ESs, one ofthe synergids appears predisposed to degenerate (asbefore); the synergid cell wall frequently thickensnoticeably.

Flowers pollinated 5 d after anthesis and grown for2 d more, display ppts in the filiform apparatus and ESwall, but still ppts are more abundant than in unpolli-nated controls. The synergid containing more ppts mayappear to undergo pre-degenerative changes; mitochon-dria become more conspicuous in the egg than in thecentral cell, with plastids more conspicuous in thecentral cell. In some ESs only a few ppts occur in thefiliform apparatus and ES wall (Fig. 8d, Table 1).

Discussion

Nature of potassium antimonate precipitation. Potassiumantimonate has long been known to trigger distinctiveprecipitation in the presence of Ca2+ (review: Wick andHepler 1982). The Sb-rich ppts have sharp borders, arelargely electron opaque and highly variable in size. Noppts meeting these criteria were found to lack Sb usingEDXA, so they could easily be discriminated from otherinclusions. While EGTA removed Sb-rich ppts, pptsenclosed in the embedding material, which EGTA didnot penetrate, were not removed. Otherwise, ppts werehighly insoluble.

The exact identity of the ppts has been controversial;however, EDXA suggested a simple 1:2 ratio of Ca:Sb,which is consistent with formation of calcium antimon-ate (CaH2Sb2O7) and EGTA removal of exposed ppts.No evidence of significant Na, K or Mg peaks wasnoted, perhaps in part because of the higher solubility oftheir corresponding cations.

Precipitates form using calcium concentrations as lowas 10–6 M (Klein et al. 1972) in 2% potassium antimon-ate, suggesting that calcium antimonate has a lowsolubility constant. Less-soluble forms of calcium, forexample, calcium phosphates or carbonates do not

Fig. 4. Transmission electron micrograph of antimonate-labeledtobacco ovule 1 d after pollination. Mature egg and central cellcontain few ppts near the egg nucleus. Abbreviations as in legend forFig. 3a. × 7500; bar = 2 lm

Fig. 5. Transmission electron micrograph of antimonate-labeledtobacco ovule showing all three antipodals 1 d after pollination.The left and middle antipodals have nuclei toward the chalaza,whereas the right antipodal has a nucleus (not shown) nearer thecentral cell. All have conspicuous starch grains (arrowheads).Abbreviations as in legend for Fig. 3a. × 2800; bar = 3 lm



appear to release their Ca2+ at all and are therefore notdetected using this method. Free cytosolic calcium maybe at such a low concentration (usually <1 lM) that it isprobably undetected (Bush 1995). Calcium available forbinding by antimonate is termed ‘‘loosely bound’’ sinceonly Ca2+ present in more-soluble compounds willrelease cationic calcium for antimonate precipitation.Although the biological significance of this pool of Ca2+

is not well known, it may represent a dynamic storemade available through changes in hormone levels andpH; the Ca2+ may be bound to a variety of proteins,lipids and carbohydrates. Calcium ions associated withallosteric compounds may be nearly as dynamic as thefree cytosolic pool, responding to near and/or distantsignals with changes in availability (Bush 1995). Anti-monate-labeled ppts share common solubility character-istics, but the ppts visualized in different cellularcompartments may have very different physiologicalfunctions.

Calcium distribution during development. Calcium anti-monate ppts in developing tobacco ovules are dynamic.Although calcium ppts are located in specific sites in theovule, these appear to change during development, aresensitive to pollination, and appear to reflect a redistri-bution of loosely bound Ca2+. The pattern of pptsreflects local accumulations and suggests the net direc-tion of Ca2+ flow (Table 1).

At anthesis (day 0), ppts are predominantly found innucellar cells and are less common in the free-nuclearES. During maturation, ppts accumulate in the ES(day 1) and are depleted in the nucellus. Precipitates thenaccumulate in the cell walls of the micropylar nucellus,synergids and central cell. The ppts become increasinglyabundant despite an increase in volume, thicker cell

walls and greater cytoplasmic volume, suggesting mas-sive uptake of calcium into the ovule. Maturation of theES occurs before fertilization (days 1 and 2), accompa-nied by significant calcium accumulations in the ovule,ES, and surrounding the micropylar canal. The massiveaccumulations of calcium in the wall of elongatingpollen tubes suggests that this calcium is available fortube growth; ppts are less abundant both proximally anddistally. The role of calcium accumulation in the ovule ispotentially related to the chemotropic attraction ofpollen tubes, which in many plants grow toward calciumgradients (Mascarenhas and Machlis 1962; Reger et al.1992). Calcium accumulating in the micropyle maycreate a tightly focused gradient from the micropyle,which may form an attractive enough signal forelongating pollen tubes to veer 90° to enter the ovule.High calcium concentrations following fertilization maybe triggered by cell death of the synergid and tube afterdischarge.

In some plants, synergids appear to directly secrete asubstance observable using transmission electron mi-croscopy (Chaubal and Reger 1992b; Willemse et al.1995), which may represent the active secretion of achemotropically substance, such as calcium pectate(Reger et al. 1992). Although there is no evidence forthe release of such a visible discharge in tobacco, such adischarge of calcium may not be visible using thesemethods. Also, the pattern of ppts suggests that calciumaccumulates in the micropylar nucellus and filiformapparatus, continuing to accumulate for two daysthereafter at both sites. It can be inferred that mostcalcium at this stage enters the ES through the nucellusand the central cell, which has the largest surface area incommon with the ES. Since this calcium appears toaccumulate in localized regions within the ovule, pre-sumably, it is transported against a gradient to thesesites. Inside the filiform apparatus, the distribution ofppts is essentially uniform at first, but at day 2, thehighest concentration of ppts is located at the micro-pylar end of the filiform apparatus, decreasing inconcentration chalazally.

The calcium gradient decreases appreciably afterfertilization, with much of the calcium apparentlyaggregating within the pollen tube and in wall deposits.Precipitates in the micropyle also decrease. This wouldhave the effect of decreasing the calcium gradient andpresumably making the ovule a less attractive target forpenetration. Post-fertilization release of calcium fromthe penetrated synergid (Chaubal and Reger 1992b,1993) may represent a widely expressed mechanism forsuppressing the entry of multiple pollen tubes in ovules,although it is not completely effective (Chaubal andReger 1993). After fertilization, intense calcium gradi-ents dissipate in the ES, ppts decrease in the synergidsand filiform apparatus, and ppts increase in adjacentnucellar cells. The concentration of loosely boundcalcium in the egg cell remains low before and afterfertilization, suggesting that Ca2+ is highly restricted andlargely in cellular organelles. Presumably, the highcalcium is related most closely to pollen tube arrival,

Fig. 6a–d. Transmission electron micrographs of antimonate-labeledtobacco ovules with fertilized ESs 2 d after pollination. Abbreviationsas in legend for Fig. 3a. a Distal pollen tube terminates near aperture(at top of figure). Few ppts occur at the periphery of pollen tube wall,although some ppts are evident in the filiform apparatus and in othercell walls of the nucellus. × 3300; bar = 2 lm. b Proximal pollen tubewall located within micropylar canal contain abundant ppts whichalso extend into surrounding nucellar cell walls. × 8300; bar = 2 lm.c Synergid penetrated by a pollen tube. Terminal aperture is indicatedby arrowheads in the degenerating synergid. Persistent synergidcontains fewer ppts and in the nucleus these tend to be larger in size(cf. right nucleus in c). × 4700; bar = 2 lm. d Contents of degeneratedsynergid are rich in pollen tube contents, some of which displaydensely packed aggregates of ppts. Near the synergid hook region,proliferated cell wall contains numerous ppts and adjacent cell wall ofthe nucellus displays local accumulations of ppts. × 6000; bar = 2 lmFig. 6e–f. Transmission electron micrographs of antimonate-labeledtobacco ovules 3 d after pollination. Abbreviations as in legend fore Degenerated synergid next to nucellus contains large particles thatintergradate to smaller granules in the cell wall; surrounding nucellarcell walls contain smaller and sparser ppts. × 15 000; bar = 1 lm.f Degenerated synergid showing various distributions of ppts amongits contents. Arrowhead indicates dense layer of ppts surrounding avesicle of presumed pollen origin. Adjacent zygote with starchgranules has fewer ppts. × 7500; bar = 2 lm

b



discharge and gamete transfer, rather than later stages inembryogenesis.

Synergid position and determination of receptivity. InHelianthus annuus, the receptive synergid is closer tothe funiculus (Yan et al. 1991) and contains more ppts(He and Yang 1992) than the other synergid. Apparentdifferences between the two synergids in H. annuuscould reflect the closer proximity of the receptive synergidto the vascular strand, but in tobacco, synergid degen-eration seems to be random (Huang and Russell 1992b;present study). The ovules of tobacco (100 lm in length)may be too small to maintain a calcium gradient sufficientto form a consistent pattern of synergid degeneration,whereas in the larger uniovulate ovules of sunflower(often 1 mm) this may not be a problem.

Synergid degeneration and pollination. Synergid degen-eration has been defined by a variety of criteria andappears to vary by species (reviewed in Huang andRussell 1992a). According to fluorescein diacetate as-sessment for viability, synergid degeneration in tobaccodoes not occur until the pollen tube arrives in the ovule(Huang and Russell 1992b); however, changes in theability of the two synergids to bind chlorotetracyclinehave been observed (Tirlapur et al. 1993) and indicatethat differences in membrane-bound calcium occur in thetwo synergids. In the current study, pollen-dependentchanges in the quantity and distribution of ppts begin at1 d after anthesis – essentially the first time thatsynergids can be regarded as mature. Ultrastructuralchanges in the synergid cytoplasm also occur almost assoon as the cells are mature. Mogensen and Suthar(1979) noted ultrastructural differences in tobaccosynergids as early as 18 h after pollination. Emergingsynergid differences are somewhat less conspicuous inunpollinated flowers (present study), suggesting thatsome aspects of synergid degeneration are prepro-

grammed and part of normal development. Synergidchanges are accelerated by pollen tube growth in thegynoecium, but final degeneration apparently does notoccur until pollen tube arrival in the ovule (Huang andRussell 1992b). The receptive synergid in tobaccoappears to be decided well in advance of arrival, andthe selection of which synergid degenerates is accelerat-ed, but not determined by pollination and pollen tubegrowth.

Pollination and calcium distribution. Pollination-en-hanced changes in calcium distribution were modestduring the first 3 d of the experiment (Table 1), but withES maturation closely followed by fertilization, pollina-tion effects are difficult to separate from maturation.Correlated with this is also starch accumulation.

Experiments using delayed pollination provide fur-ther evidence for pollination-induced changes in ovules.Normally, day 5 is the last day that a flower may bepollinated and successfully set seed. Flowers pollinatedlater abscise on day 8. Fertilization typically occurs twodays after pollination, but this is extended to 3 d forflowers pollinated on day 4 or 5. Calcium begins todecline on day 4, decreasing dramatically on days 4, 5,and 6. On day 7, sparse ppts remain in the synergidcommon wall and no deposits occur in unpollinatedovules (Table 1). Material pollinated on day 4 andgrown for 2 d after pollination displays a retention ofppts compared with emasculated ovules on day 6.Substantial ppt increases occur in the ES wall afterday 5. Specimens pollinated on day 5 and collected onthe second day after pollination display significantincreases in ovule and ES calcium compared withemasculated flowers of the same age (Table 1). Coincid-ing with delayed pollination is also the re-accumulationof starch.

Pollination-enhanced changes in emasculated ovulessupport the presence of communication pathways fromthe pollen tube (in the style) to the ovule, demonstratingthat a pollination-related signal elicits changes in theovule and ES. To date, this may be the clearest evidenceof the ‘‘cross-talk’’ phenomenon proposed by Jensenet al. (1985).

Effectiveness of this response decreases only slightlyup to 5 d after anthesis. During this period, ovules arecapable of setting seed, albeit in declining numbers nearthe end of receptivity. Ovule receptivity seems correlatedwith calcium ppts. If pollination is delayed, calciumdistributions after pollination increase until they resem-ble those present at the height of receptivity, suggestingan important role for calcium in pollen tube receipt inthe ES. Such a relatively simple signal as Ca2+ avail-ability may be rapidly reversed or dissipated uponfertilization and could potentially prevent multiplepollen tubes from penetrating an ovule.

We gratefully acknowledge Greg Strout for technical assistance,research support from US Department of Agriculture grants 91-37304-6471 and 95-37304-2361, and use of the Samuel RobertsNoble Electron Microscopy Laboratory.

Fig. 7a–d. Transmission electron micrographs of antimonate-labeledtobacco ovules 3 d (a–c) or 4 d (d) after anthesis in pollinated (a) andunpollinated ovules (b–c). Abbreviations as in legend for Fig. 3a.a Fertilized ES showing perinuclear region of zygote, nucleus ofendosperm, and adjacent degenerated synergid. Zygote containsfewer, larger ppts, whereas endosperm contains more-numerous andfiner ppts. In the degenerated synergid ppts are distributed throughoutthe cytoplasm and are small and extremely abundant. × 6000; bar = 2lm. b Unfertilized ES showing fine ppts in unfused polar nuclei incentral cell and two intact synergids with generally dispersed ppts incytoplasm. Higher concentration of ppts occurs in nucleus andcytoplasm of right synergid. × 1800; bar = 3 lm. c Higher-magnification view of two intact synergids from another ES. Notedifferences in concentration and distribution of ppts, which areparticularly abundant in nucleus of synergid on left. Precipitates arenot especially abundant in the filiform apparatus at this stage and aremore abundant at the micropyle. × 3700; bar = 2 lm. d Precipitatescontinue to accumulate in chalazal face of filiform apparatus.Abundance and size of ppts varies from larger granules in chalazalFA to finer granules in micropylar wall. × 7500; bar = 2 lm

b



References

Bush DS (1995) Calcium regulation in plant cells and its role insignaling. Annu Rev Plant Physiol Plant Mol Biol 46: 95–122

Chaubal R, Reger BJ (1990) Relatively high calcium is localized insynergid cells of wheat ovaries. Sex Plant Reprod 3: 98–102

Chaubal R, Reger BJ (1992a) Calcium in the synergids and otherregions of pearl millet ovaries. Sex Plant Reprod 5: 34–46

Chaubal R, Reger BJ (1992b) The dynamics of calcium distributionin the synergid cells of wheat after pollination. Sex PlantReprod 5: 206–213

Chaubal R, Reger BJ (1993) Prepollination degeneration in maturesynergids of pearl millet: an examination using antimonatefixation to localize calcium. Sex Plant Reprod 6: 225–238

Chaubal R, Reger BJ (1994) Dynamics of antimonate-precipitatedcalcium and degeneration in unpollinated pearl millet synergidsafter maturity. Sex Plant Reprod 7: 122–134

He CP, Yang HY (1992) Ultracytochemical localization of calciumin the embryo sac of sunflower. Chin J Bot 4: 99–106

Huang BQ, Russell SD (1992a) Female germ unit: organization,reconstruction and isolation. Int Rev Cytol 140: 233–293

Huang BQ, Russell SD (1992b) Synergid degeneration in Nicotiana:a quantitative, fluorochromatic and chlorotetracycline study.Sex Plant Reprod 5: 151–155

Jensen WA (1965) The ultrastructure and histochemistry of thesynergids of cotton. Am J Bot 52: 238–256

Jensen WA, Ashton ME, Beasley CA (1985) Pollen tube-embryosac interaction in cotton. In: Mulcahy DL, Ottoviano E (eds)Pollen: biology and implications for plant breeding. ElsevierBiomedical Press, New York, pp 67–72

Klein RL, Yen SS, Thureson-Klein A (1972) Critique on the K-pyroantimonate method for semi-quantitative estimation ofcations in conjunction with electron microscopy. J HistochemCytochem 20: 65–78

Mascarenhas JP, Machlis L (1962) Chemotropic response ofAntirrhinum majus pollen to calcium. Nature 196: 292–293

Mogensen HL, Suthar HK (1979) Ultrastructure of the eggapparatus of Nicotiana tabacum (Solanaceae) before and afterfertilization. Bot Gaz 140: 168–179

Reger BJ, Chaubal R, Pressey R (1992) Chemotropic responses bypearl millet pollen tubes. Sex Plant Reprod 5: 47–56

Tirlapur UK, Van Went JL, Cresti M (1993) Visualization ofmembrane calcium and calmodulin in embryo sacs in situ andisolated from Petunia hybrida L and Nicotiana tabacum L. AnnBot 71: 161–167

Wick SM, Hepler PK (1982) Selective localization of intracellularCa2+ with potassium antimonate. J Histochem Cytochem 30:1190–1204

Willemse MTM, Plyushch TA, Reinders MC (1995) In vitromicropylar penetration of the pollen tube in the ovule ofGasteria verrucosa (Mill) H Duval and Lilium longiflorumThunb: conditions, attraction and application. Plant Sci 108:201–208

Yan H, Yang HY, Jensen WA (1991) Ultrastructure of themicropyle and its relationship to pollen tube growth andsynergid degeneration in sunflower. Sex Plant Reprod 4: 166–175

Fig. 8a–d. Transmission electron micrographs of antimonate-labeledtobacco ovules at 5 or 6 d after anthesis, with or without delayedpollination. Abbreviations as in legend for Fig. 3a. a Five days afteremasculation. Precipitates are abundant but evenly dispersed in thetwo synergids, whereas the filiform apparatus displays a highly polardistribution with large micropylar ppts. Precipitates continue todiminish in nucellus cells. × 7500; bar = 2 lm. b Six days afteremasculation. Precipitates continue to diminish in size and abundancein synergid cytoplasm and filiform apparatus. Cell walls of filiformapparatus apparently thicken through the addition of an electron-dense material. Synergid common wall (arrowheads) also thickens andbecomes more irregular, with accretion of electron-translucentmaterial. × 6000; bar = 2 lm. c Four days after emasculation, 2 dafter pollination. Precipitates in synergids and nucellus remain present,but not abundant in cytoplasm. Precipitates are common, but nothighly polarized in filiform apparatus. Although the synergid commonwall thickens, electron-dense material is absent near edges of filiformapparatus. Elaborations of synergid cell wall contain large ppts.Nucellus contains more-abundant ppts than in unpollinated materialof same stage (cf. Fig. 8b). × 3700; bar = 2 lm. d Five days afteremasculation, 2 d after pollination. Precipitates are still common atmicropylar end of filiform apparatus, which appears enlargedcompared to previous stages. More-highly vesiculated cytoplasm ofsynergid on left contains greater abundance of ppts. Nuclei appearirregular in shape at this stage and are impoverished in ppts. Nucellarcells still contain some ppts. × 4700; bar = 2 lm

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Calcium distribution in fertilized and unfertilized ovules and