(Plumbaginaceae). 2. Quantitative cell and organelle dynamics

ORIGINAL ARTICLE

Scott D. Russell Æ Gregory W. Strout

Microgametogenesis in Plumbago zeylanica (Plumbaginaceae).2. Quantitative cell and organelle dynamics of the male reproductivecell lineage

Received: 1 April 2005 / Accepted: 6 July 2005 / Published online: 4 August 2005� Springer-Verlag 2005

Abstract Quantitative cell and organelle dynamics of themale gamete-producing lineage of Plumbago zeylanicawere examined using serial transmission electronmicroscopic reconstruction at five stages of developmentfrom generative cell inception to sperm cell maturity.The founder population of generative cell organellesincludes an average of 3.88 plastids, 54.9 mitochondria,and 3.7 vacuoles. During development the volume of thepollen grain increases from 6,200 lm3 in earlymicrospores to 115,000 lm3 at anthesis, cell volume ofthe male germ lineage decreases more than 67% from362.3 lm3 to 118.4 lm3. By the time the generative cellseparates from the intine, plastid numbers increase by>600%, mitochondria by 250%, and vesicles by 43times. A cellular projection elongates toward andestablishes an association with the vegetative nucleus;this leading edge contains plastids and numerous mito-chondria. When the generative cell completes its sepa-ration from the intine, organellar polarity is reversedand plastids migrate to the opposite pole of the cell.Cytoplasmic microtubules are common in associationwith cellular organelles. Plastids accumulate at the distalend of the cell as a linked mass, apparently adhered bylateral electron dense regions. Before division of thehighly polarized generative cell, plastids decrease innumber by 16%, whereas mitochondria increase by�90% and vacuoles increase by �140% from the priorstage. After mitosis, the resultant sperm cells differ insize and organelle content. The sperm cell associatedwith the vegetative nucleus (Svn) contains 62.7% of thecytoplasm volume, 87% of the mitochondria, 280.4vesicles (79% of those in the generative cell), and 0.6%

of the plastids. At maturity, the Svn mitochondria in-crease by 31% and the cell contains an average of 0.4plastids, 158.9 vesicles, and 0.36 microbodies. The ma-ture unassociated sperm (Sua) contains 39.8 mitochon-dria (up 3.3%), 24.3 plastids (down 31%), 91.1 vesicles(up 54.9%), and 3.18 microbodies. The small number oforganelles initially in the generative cell, followed bytheir rapid multiplication in a shrinking cytoplasmsuggests a highly competitive cytoplasmic environmentthat would tend to eliminate residual organellar heter-ogeneity. Cell and cytoplasmic volumes vary as a con-sequence of fluctuations in the number and size of largevesicles or vacuoles, as well as loss of cytoplasmic vol-ume by (1) formation of ‘‘false cells’’ involving amitoticcytokinesis, (2) ‘‘pinching off’’ of cytoplasm, and (3)dehydration of pollen contents prior to anthesis.

Keywords Male cytoplasmic inheritance Æ Generativecell (angiosperm) Æ Male germ unit ÆMicrogametogenesis Æ Plumbago Æ Sperm cell(angiosperm)

Introduction

Although flowering plant sperm cells are typicallyviewed as being equal and essentially interchangeable,during their formation the two sperm cells within apollen grain often differ in their cytoplasmic organiza-tion and organelle content—a circumstance occurring ina number of species of flowering plants examined to date(Russell 1991; Mogensen 1992; Tian et al. 2001). Themost extreme examples of this phenomenon documentedto date are in the family Plumbaginaceae, where fluo-rescence studies have revealed that one sperm cell of thetwo present in the pollen contains essentially all of theplastid DNA (Corriveau and Coleman 1988; Sodmergenet al. 1995; Saito et al. 2002; Zhang et al. 2003).Plumbago zeylanica—the most intensively studiedmember of the family—displays significant differences insperm cells with respect to cell size, shape, surface area,

S. D. Russell (&)Department of Botany and Microbiology,University of Oklahoma, Norman, OK 73019, USAE-mail: [email protected].: +1-405-3254391Fax: +1-405-3257619

G. W. Strout Æ S. D. RussellSamuel Roberts Noble Electron Microscopy Laboratory,University of Oklahoma, Norman, OK 73019, USA

Sex Plant Reprod (2005) 18: 113–130DOI 10.1007/s00497-005-0005-1

and occurrence of a physical association with the vege-tative nucleus (Russell and Cass 1981). They also differin organelle content and quality (Russell 1984) andplastid DNA (Sodmergen et al. 1995), confirming thepotential for differential organelle transmission. Thesperm cell that is linked with the vegetative nucleus (Svn)contains most of the mitochondria (mean 256.2 per cell)and typically no plastids (mean 0.4), whereas the spermcell that is unassociated with the vegetative nucleus (Sua)contains over 98% of the plastids (mean 24.3) and�87% fewer mitochondria (mean 38.5) (Russell 1984).

Differences in the sperm cells are consistent inpolarity, having been observed in thousands of pollengrains during the course of prior studies and indepen-dent investigations (Sodmergen et al. 1995). Sperm cellorganization and polarity are the basis for preferentialfertilization in which, at gametic fusion, the smaller,plastid-rich Sua fuses with the egg cell in over 95% ofcases observed, transmitting its plastids into the zygote(Russell 1983, 1985). The numerical basis of thesesperm cell differences arise, however, is unclear. Mor-phological changes occurring during this process havebeen described using three-dimensional computer-as-sisted reconstruction (Russell et al. 1996) and directobservation (Sodmergen et al. 1995). The present studyextends these observations to a quantitative level,describing details of organelle differentiation within thesperm cells.

Epifluorescence microscopic studies have demon-strated that generative and sperm cell plastids maycontain ptDNA (Corriveau and Coleman 1988; Sod-mergen et al. 1995; Saito et al. 2002), and thus the fateof paternal cytoplasm has genetic consequences. Thestatus of mitochondrial DNA (mtDNA) is less clear,since mtDNA is normally near the threshold of detec-tion and thus not as easily visualized (Sodmergen et al.1995). These researchers did not detect mtDNA fluo-rescence, but it would not be difficult to screen such cellsusing molecular biology to determine if mtDNA ispresent or absent.

Organelle dynamism throughout the process of malegerm line development in flowering plants has been de-scribed in detail in only one species, tobacco (Yu et al.1989, 1992, 1994; Yu and Russell 1994a, b, c, d). In theorchid Cymbidium goeringii only two stages prior topollen dissemination were examined (Yu and Russell1993), but a similar pattern was evident. In these reports,sperm cells were statistically similar and did not initiallydisplay differences in organellar DNA between the twosperm cells. More detailed studies of tobacco sperm cellsjust prior to fertilization, however, indicated that sig-nificant differences in the two sperm cells emerge late indevelopment, near the time of sperm entry into the ovary(Tian et al. 2001), and these become accentuated in theovary and ovule (Tian et al. 2005).

Studies of male germ cells to date have indicated thatmultiple redundant mechanisms are involved in thereduction of organelle numbers during development.This reduction is mostly, but not completely effective at

preventing paternal plastid expression in the offspring.In tobacco, ultrastructural evidence of occasionalplastids in the sperm cells (Yu and Russell 1994a, b, c, d)and genetic evidence of paternal plastids were numeri-cally nearly identical, indicating paternal ptDNAtransmission and that the usually uniparental maternalinheritance was leaky (Medgyesy et al. 1986). Mostenigmatic was the occurrence of cytoplasmic organelleswithin the sperm nucleus itself, which occurred as a re-sult of constraint of the mitotic field during sperm cellformation in the pollen tube transmitting organelles di-rectly into the fusion nuclei of the zygote and primaryendosperm (Yu and Russell 1992b).

One particularly attractive reason to examinePlumbago is that the fate of its sperm cells is non-random and is easily predicted on the basis of thesperm’s complement of cytoplasmic organelles; theplastid-rich Sua preferentially fertilizes the egg cell toproduce the zygote and embryo and the Svn fertilizesthe central cell to form the endosperm (Russell 1985).The current study delimits further the concept andextent of cytoplasmic heterospermy (Russell 1985,1992) in angiosperms. The establishment of heritableand non-heritable organelles in the generative cell, theirmultiplication and segregation in the generative cellfrom inception to sperm cell formation and maturationin P. zeylanica form an important part of a long-termexamination of preferentiality in flowering plants thatis increasingly the subject of molecular inquiry (Zhanget al. 1998).

Materials and methods

Anthers of P. zeylanica L. (Plumbaginaceae) wereexamined at five different developmental stages (Russellet al. 1996). The condition of the anther was used as anapproximate developmental index to the maturity of thegenerative cells and these were screened using 1–3 lmthick sections to verify stages of generative cell devel-opment. Five major developmental phases were sam-pled: (1) early lenticular generative cell (soon afterformation); (2) ‘‘peeling’’ generative cell during separa-tion of the cell from the intine; (3) ‘‘freed’’ generativecells; (4) immature sperm cells soon after formation; and(5) mature sperm cells at floral anthesis (Table 1).Developmental substages were also present simulta-neously in different cells within a specific anther(Table 2)—a condition that has also been reported inanthers of other plants (Mogensen and Rusche 1985).Material for this study was fixed in phosphate-bufferedglutaraldehyde and osmium tetroxide, dehydrated inethanol, embedded in low viscosity epoxy resin, seriallysectioned at 70–110 nm, and collected on formvar-coated 1·2 mm slot grids according to a techniquemodified from Galey and Nilsson (1966). The malereproductive cells reconstructed at each stage are asfollows: stage I, N=8; stage II, N=8; stage III, N=9;stage IV, N=8 sperm pairs; stage V, N=11 sperm pairs.

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Each series consisted of 400–650 serial sections, with agiven male reproductive cell representing �150–325sections. Sections were stained using 2% uranyl acetatein 50% ethanol and 0.02% lead citrate at high pH forstages I–IV. Sections at stage V were stained using theperiodic acid-thiocarbohydrazide-silver proteinate reac-tion of Thiery (1967). Material was viewed using a Zeiss10a transmission electron microscope operated at 40–60 kV. Electron micrographs were taken either using aplate camera (3-1/4·4¢¢ format) or roll film camera(35 mm format) at 5,000X. Each micrograph was prin-ted at the same magnification, labeled, and numberedconsecutively for each pollen series. Section numberswere used to determine the three-dimensional order ofeach plane. Section thickness was not intentionallyvaried during any given series; consistency was verifiedby section interference color, constancy of ultramicro-tome settings, and electron density. Section thicknesswas independently estimated using three methods: (1) aspherical object (for example, a nucleolus) was recon-structed by measuring its median diameter and dividingby the number of sections (Russell 1984); (2) thickness offolded sections was observed using median folds anddividing by two (Russell 1987); and (3) spheroidalstructures were adjusted until they appeared circular inprofile in completed three-dimensional reconstructions.

Section thickness was required for the placement ofplanes in three-dimensional reconstructions and forquantitative cytology.

Computer-assisted, three-dimensional images weremade using the ‘‘3-D Reconstruction Software’’ de-scribed in Young et al. (1987) on a PC (Russell et al.1996), which was particularly effective in interpretingorganelle distribution and morphology. Three-dimen-sional reconstructions were made by encoding outlinesof cellular features on electron micrographs using astylus and a digitizing tablet to convert images into (x, y)locations. Fiducial points were selected and marked oneach micrograph to determine orientation, which wascalculated using least squares analysis (Young et al.1987). Section number was encoded to designate the zlocation, which was then scaled to section thickness.

Images for stereopairs were photographed using arotation angle of 7� between images. Quantitative datawere evaluated using either the IBM system describedabove (in conjunction with the program MEASURE,provided by Dr. S. Young and VOLUME and PRO-FILE by SDR). The equivalent radius of an object wascalculated by determining the radius of a sphere ofequivalent volume for solid objects (Table 3) or the ra-dius of a circle of equivalent area for profiles (Table 5).The circularity ratio was determined by dividing the

Table 2 Stages and substages indevelopment of the male germunit in microgametogenesis ofP. zeylanica. (After Russellet al. 1996)

Stage Microgametophyte description Diagnostic features

I Lenticular generative cella Early—flattened Thickness ratio >7:1b Mid—rounding Thickness ratio <7:1c Late—cell wall flexure Flexed generative cell wallII ‘Peeling’ generative cella Early—initially lifting of cell 80–100% attachedb Mid—projection formation 50–80% attachedc Late—immersion in cytoplasm/separation >0–50% attachedIII ‘Freed’ generative cella Early—irregular freed cell Cell profile irregularb Mid—spherical freed cell Smooth, immature projectionc Late—elongate freed cell Smooth, mature projectionIV Generative cell division/immature sperma Mitosis/cytokinesis Division figureb Early immature sperm cells Linear, straight axisc Late immature sperm cells Flexed sperm axisV Mature sperm cells

Table 1 Developmental indices used in this study of Plumbago zeylanica (After Russell et al. 1996)

Stage Microgametophytecondition

Calyx length(in mm)

Anther length(in mm)

Whole Gynoecium(in mm)

Other floral characters

Meiosis <2.05 <0.95 <0.90Microspore 2.05 0.95 0.90 Mature calyx glands

I Lenticular generative cell 2.30 1.20 1.10II ‘Peeling’ generative cell 2.75 1.35 1.20 Bract length=corolla

length anther still whiteIII ‘Freed’ generative cell 3.6 1.60 1.75 Anther turning purpleIV Generative cell division 5.5 1.9 3.3 Anther light purpleV Mature sperm cells 12.0 2.1 26.5 Anther deep purple

Indices are approximate and may vary by up to 0.2 mm under different growth conditions

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measured circumference with that of a circle of equiva-lent area.

Results

Attached, lenticular generative cell

A highly asymmetric division of the microspore formstwo dissimilar cells: (1) a vegetative cell that occupiesmuch of the previous microspore and (2) a slender, lens-shaped cell in contact with the intine at the edge of thevegetative cell. The generative cell thus has cell walls ofdistinctly different origins: the outer wall is contiguouswith the intine and is formed prior to mitosis, whereasthe inner cell wall is formed de novo at the end of post-meiotic mitosis through a transiently callose-positive cellwall that is possessed in common with the vegetative cell(Fig. 1; see also Russell et al1996, Figs. 1–3). The aver-age volume of the generative cell is 362 lm3 with anaverage surface area of 401 lm2 (Table 3). The nucleusoccupies an average volume of 150 lm3 and an averagesurface area of 163 lm2 (Table 4), representing nearly50% of the cytoplasm. The distribution of organelleswithin the generative cell is initially peripheral, forming

a dense annulus of cellular organelles around the nucleus(Fig. 1). Organelles are largely excluded from the centralregion by the size of the nucleus and the lenticular shapeof the cell at this stage. The number of plastids at thisstage ranges from two to eight in different cells, with amean of 3.88 plastids (Table 4); the number of mito-chondria ranges from 33 to 81, with a mean of 54.9mitochondria; and the number of vacuoles ranges fromtwo to six, with a mean of 3.75 vacuoles.

Generative cell during separation from the intine

As the lenticular generative cell matures, the cell be-comes thicker (Figs. 2, 3). The average volume of the cellincreases (to 430 lm3, an increase of 18.7%), while theaverage surface area stays essentially the same (387 lm2,a reduction of 3.2%; Table 3). The average volume ofthe nucleus decreases by 13.6% and its surface areadecreases by 17.8% (Table 3), resulting in approxi-mately a 30% increase in the volume of the cytoplasm.The volume of the nucleolus at this stage decreases byover 70% from 32.17 lm3 to 9.44 lm3. The dispositionof organelles changes from annular to internal as thecytoplasm enlarges. Mitochondria and plastids move to

Table 3 Cell volumes, surface areas, and nuclear volumes of the generative and sperm cells of P. zeylanica as determined from three-dimensional reconstruction and volume of the vegetative cell as determined from diameters of living grains

Parameter GC I GC II GC III S I S II

Svn Sua Svn Sua

n= 8 8 9 8 8 11 11CellVolume (lm3)Range 272.4–445.6 292.1–679.8 167.5–271.6 165.6–389.6 137.5–233.2 45.6–116.4 32.8–83.9Mean (± SE) 362.3±24.1 430.0±45.1 216.1±11.6 305.4±27.5 181.4±11.7 69.5±7.5 48.9±4.4Cell surface (lm2)Range 225.0–486.6 270.6–495.4 251.7–422.4 130.0–424.3 101.8–202.3 100.2–209.9 55.7–119.9Mean (± SE) 401.5±29.3 388.6±24.3 314.4±19.8 309.2±33.3 158.8±12.1 147.9±12.0 84.7±5.3Surface area/volume (lm�1) 1.105±1.21 0.904±0.54 1.455±1.71 1.012±1.21 0.875±1.03 2.128±1.60 1.732±1.20Equivalent radius (lm) 4.426 4.682 3.728 4.178 3.512 2.551 2.269NucleusVolume (lm3)Range 112.4–206.8 75.0–148.0 71.0–106.2 62.3–106.4 44.9–73.9 13.8–27.2 9.3–15.4Mean (± SE) 150.4±12.9 130.0±13.9 88.7±3.8 86.6±4.8 62.9±3.3 19.9±1.4 12.2±0.5Surface area (lm2)Range 76.6–240.5 101.6–160.0 66.3–141.8 57.7–96.5 52.6–90.4 21.7–43.2 17.3–23.0Mean (± SE) 162.8±19.5 133.9±6.4 98.9±8.4 83.7±4.3 70.0±4.2 32.4±2.1 20.4±0.6Surface area/volume (lm�1) 1.082±1.51 1.030±0.46 1.115±2.21 0.967±0.90 1.113±1.27 1.628±1.60 1.672±1.20Equivalent radius (lm) 3.299 3.143 2.767 2.745 2.467 1.681 1.428CytoplasmVolume (lm3)Range 132.5–333.2 206.1–481.6 86.4–170.5 103.3–289.7 84.4–159.3 25.5–90.1 21.1–46.6Mean (± SE) 211.9±21.2 300.0±93.7 127.5±9.1 218.7±23.0 118.5±8.9 45.2±6.0 33.4±2.7Vegetative cellCell volume (lm3)Range 56,000–98,000 56,000–134,000 86,400–141,000 75,300–176,000 105,000–186,000Mean (± SE) 72,300±3,540 91,700±3,900 105,900±3,400 125,200±6,450 143,900±5,200Percent of the volume of the pollen grain occupied by generative or sperm cellMean 0.501% 0.439% 0.204% 0.244% 0.145% 0.0483% 0.0340%

Developmental stages: GC I lenticular generative cell. GC II sep-arating generative cell. GC III freed generative cell. S I immaturesperm cell. S II mature sperm cell at floral anthesis. Svn sperm

physically associated with the vegetative nucleus. Sua spermunassociated with the vegetative nucleus. All means are given ±the standard error of the mean

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the internal cytoplasm distal to the intine (Fig. 2),whereas vacuoles appear to multiply and move to thecytoplasm proximal to the intine face (Fig. 3). Falsecells, defined as areas of cytoplasm without a nucleus,are apparently formed by cytokinetic process (Russellet al. 1996), and are aligned with the main cell body,frequently containing numerous, typical cellular organ-elles (Fig. 3), which are presumably lost from thereproductive germ cell lineage. False cells are believed toundergo autolysis as they are not seen at later stages.

Plastids become more numerous, ranging from 10 to61 plastids per cell, with a mean of 26.7 plastids(Table 4). The total volume occupied by plastids en-larges to 9.77 lm3 (an increase of 46.5%) but each

plastid is smaller in volume (0.363 lm3 per plastid, adecrease of �79% per plastid) with a sectional profile of0.252 lm2 (�70% reduction), suggesting that the in-crease in plastids is largely the result of fragmentation.These organelles gradually aggregate in the region of thegenerative cell nearest to the vegetative nucleus (Fig. 2).Mitochondria also become more numerous, rangingfrom 104 to 275, with a mean of 187.2 mitochondria percell, representing an increase of �240% (Table 4).Mitochondria occupy a total volume of 37.69 lm3, adecrease of 14.8% from the previous stage (Table 6).Both the size and circumference of individual mito-chondrial profiles decrease sharply (Table 5) from theprevious stage (0.2013 lm3 per mitochondrion, a de-

Table 4 Organelle types in the generative and sperm cells of P. zeylanica as determined from three-dimensional reconstruction

Organelle type GC I GC II GC III S I S II

Svn Sua Svn Sua

Sample size 8 10 8 10 10 11 11MitochondriaRange 33–81 104–275 299–425 106–250 19–66 154–311 22–52Mean (± SE) 54.875±5.5 187.2±17.9 354.7±17.3 195.3±13.6 38.5±5.1 256.2±18.0 39.8±3.3PlastidsRange 2–8 10–61 11–40 0–1 15–46 0–2 8–46Mean (± SE) 3.88±0.7 26.9±4.7 22.6±3.0 0.2±0.1 35.3±4.5 0.4±0.2 24.3±3.9VacuolesRange 2–6 85–268 300–513 84–422 37–75 61–314 58–145Mean (± SE) 3.75±0.6 162.8±21.3 388.9±21.2 221.6±30.0 58.8±4.4 158.9±24.5 91.1±9.5MicrobodiesRange 0 0 0 0 0–1 0–2 0–8Mean (± SE) 0.0±0.0 0.0±0.0 0.0±0.0 0.0±0.0 0.2±0.1 0.36±0.2 3.18±1.0

Table 5 Profiles of mitochondria, plastids, and vacuoles of the generative and sperm cells of P. zeylanica


Svn Sua Svn Sua

Mitochondrian = 449 2053 296 486 51 157 7Surface area (lm2) 0.584±0.015 0.284±0.004 0.199±0.004 0.164±0.003 0.240±0.013 0.062±0.002 0.041±0.013Circumference (lm) 3.364±0.051 2.161±0.016 1.848±0.021 1.565±0.013 1.950±0.082 0.953±0.014 0.759±0.101Equivalent radius (lm) 0.431 0.301 0.252 0.228 0.276 0.140 0.114Circularity ratio 1.242 1.144 1.169 1.090 1.123 1.080 1.057Plastidsn = 22 1289 355 3 1567 2 214Surface area (lm2) 0.828±0.121 0.252±0.005 0.205±0.007 0.206±0.013 0.464±0.010 0.268±0.080 0.384±0.013Circumference (lm) 3.884±0.345 2.295±0.029 1.965±0.038 1.639±0.142 2.923±0.038 2.073±0.503 2.576±0.052Equivalent radius (lm) 0.513 0.283 0.255 0.256 0.384 0.292 0.350Circularity ratio 1.204 1.290 1.224 1.019 1.211 1.130 1.173Vacuolesn = 38 826 1750 1263 409 371 192Surface area (lm2) 2.669±0.343 0.434±0.017 0.200±0.003 0.450±0.009 0.312±0.008 0.152±0.009 0.095±0.007Circumference (lm) 5.677±0.460 2.482±0.048 1.750±0.013 2.567±0.030 2.143±0.033 1.427±0.040 1.123±0.036Equivalent radius (lm) 0.922 0.372 0.252 0.378 0.315 0.220 0.173Circularity ratio 1.020 1.063 1.104 1.079 1.082 1.033 1.028



Developmental stages: GC I lenticular generative cell. GC IIseparating generative cell. GC III freed generative cell. S I imma-ture sperm cell. S II mature sperm cell at floral anthesis. Svn sperm


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crease of 75.1%). The larger vacuoles of the previousstage are replaced by numerous, smaller vacuoles. Totalvolume of vacuoles increases from 24.71 to 29.82 lm3

(17.1%; Table 6). The average number of vacuoles

increases by 43 times from the previous stage (from 3.75to 162.8; Table 4), but the average volume decreases by97.2% to 0.183 lm3. Correspondingly, the average sur-face area of an individual vacuole decreases by 84% to

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0.434 lm2, the circumference decreases by 56% to2.48 lm, and the radius of the equivalent circle decreasesby 60% to 0.372 lm (Table 5). Vacuoles observed at this

stage appear to be generated mainly through the frag-mentation of existing larger vacuoles from the previousstage. No larger vacuoles characteristic of the previous

Fig. 1-7 Computer-assisted reconstructions of the generative andsperm cells of P. zeylanica using a PC-based system with theHVEM ‘‘3-D Reconstruction Program". Horizontal stereopairs areviewable with a standard 3-D viewer (Figs. 1, 2, 6); verticalsteropairs (Figs. 4, 7) are viewable with a vertical-splitter 3-Dviewer (‘‘Nesh’’-type viewer). Outline of the cell is indicated inwhite; false cell is outlined in yellow; mitochondria are small,intracellular red objects; plastids are lighter, intracellular greenobjects; vacuoles are outlined in dark blue. Nuclei and nucleoli areshown in Figs. 3–5 only (in gray). Fig. 1 Computer-assistedreconstructions of the generative and sperm cells of P. zeylanicausing a PC-based system with the HVEM ‘‘3-D ReconstructionProgram’’. Horizontal stereopairs are viewable with a standard 3-Dviewer (Figs. 1, 2, 6); vertical steropairs (Figs. 4, 7) are viewablewith a vertical-splitter 3-D viewer (‘‘Nesh’’-type viewer). Outline ofthe cell is indicated in white; false cell is outlined in yellow;mitochondria are small, intracellular red objects; plastids arelighter, intracellular green objects; vacuoles are outlined in darkblue. Nuclei and nucleoli are shown in Figs. 3–5 only (in gray).Stereopair of tightly appressed lenticular generative cell soon aftermicrospore division. Plastids are sparse and dispersed at this stage,and mitochondria are entirely peripheral in distribution. The twoends of the cell are clearly evident at this stage, apparentlyoriginating during microspore division. These tapering ends remainevident throughout development. Fig. 2 Computer-assisted recon-structions of the generative and sperm cells of P. zeylanica using aPC-based system with the HVEM ‘‘3-D Reconstruction Program’’.Horizontal stereopairs are viewable with a standard 3-D viewer(Figs. 1, 2, 6); vertical steropairs (Figs. 4, 7) are viewable with avertical-splitter 3-D viewer (‘‘Nesh’’-type viewer). Outline of thecell is indicated in white; false cell is outlined in yellow;mitochondria are small, intracellular red objects; plastids arelighter, intracellular green objects; vacuoles are outlined in darkblue. Nuclei and nucleoli are shown in Figs. 3–5 only (in gray).Stereopair of a thickening lenticular generative cell at a slightlymore advanced developmental stage that was illustrated in Fig. 8.The cell is now sufficiently thick that organelles occupy almost theentire inner periphery of the cell. Plastids are numerous andgenerally dispersed within the cell, but the cell remains essentiallyunpolarized. Mitochondria are dispersed toward the interior of thecell, and as viewed with the nucleus superimposed (Fig. 3) theirdistribution corresponds closely with the nuclear profile. Taperedcell ends are also visible in this figure. Fig. 3 Computer-assistedreconstructions of the generative and sperm cells of P. zeylanicausing a PC-based system with the HVEM ‘‘3-D ReconstructionProgram’’. Horizontal stereopairs are viewable with a standard 3-Dviewer (Figs. 1, 2, 6); vertical steropairs (Figs. 4, 7) are viewablewith a vertical-splitter 3-D viewer (‘‘Nesh’’-type viewer). Outline ofthe cell is indicated in white; false cell is outlined in yellow;mitochondria are small, intracellular red objects; plastids arelighter, intracellular green objects; vacuoles are outlined in darkblue. Nuclei and nucleoli are shown in Figs. 3–5 only (in gray).Thickening lenticular generative cell viewed in Fig. 2, with additionof nucleus, vesicles, and false cell. Vesicles are distributed towardthe exterior of the pollen grain (toward the convex face of the cell).The false cell of this pollen grain is formed parallel to the taperingends, as is typical, and contained only mitochondria in this grain.Fig. 4 Computer-assisted reconstructions of the generative andsperm cells of P. zeylanica using a PC-based system with theHVEM ‘‘3-D Reconstruction Program’’. Horizontal stereopairs areviewable with a standard 3-D viewer (Figs. 1, 2, 6); verticalsteropairs (Figs. 4, 7) are viewable with a vertical-splitter 3-Dviewer (‘‘Nesh’’-type viewer). Outline of the cell is indicated inwhite; false cell is outlined in yellow; mitochondria are small,intracellular red objects; plastids are lighter, intracellular greenobjects; vacuoles are outlined in dark blue. Nuclei and nucleoli are

shown in Figs. 3–5 only (in gray). Stereopair of the back of a‘peeling’ generative cell near initiation of separation. The vegetativenucleus (not shown) was located near the green plastid aggregateand is near the area from where the cell projection will develop.This pole initiates separation of the cell from the intine. Bothmitochondria and plastids are aggregated in a cap-shapeddistribution to the interior of the pollen grain, and vesicles arelocated almost exclusively toward the exterior of the pollen grainnext to the intine. An atypically large false cell is located parallel tothe generative cell with mitochondria, plastids, and a prominentcentral vacuole evident. Polarization is not detected in the falsecells. Fig. 5 Computer-assisted reconstructions of the generativeand sperm cells of P. zeylanica using a PC-based system with theHVEM ‘‘3-D Reconstruction Program’’. Horizontal stereopairs areviewable with a standard 3-D viewer (Figs. 1, 2, 6); verticalsteropairs (Figs. 4, 7) are viewable with a vertical-splitter 3-Dviewer (‘‘Nesh’’-type viewer). Outline of the cell is indicated inwhite; false cell is outlined in yellow; mitochondria are small,intracellular red objects; plastids are lighter, intracellular greenobjects; vacuoles are outlined in dark blue. Nuclei and nucleoli areshown in Figs. 3–5 only (in gray). Another ‘peeling’ generative cellat approx the same developmental stage as in Fig. 4, withvegetative nucleus shown in dark brown profiles in the background.Separation of generative cell from the intine is more advanced,however, distribution of mitochondria and plastids remainspolarized toward the vegetative nucleus, with vesicles occupyingthe region of the cell attached to the intine. Fig. 6 Computer-assisted reconstructions of the generative and sperm cells of P.zeylanica using a PC-based system with the HVEM ‘‘3-DReconstruction Program’’. Horizontal stereopairs are viewablewith a standard 3-D viewer (Figs. 1, 2, 6); vertical steropairs (Figs.4, 7) are viewable with a vertical-splitter 3-D viewer (‘‘Nesh"-typeviewer). Outline of the cell is indicated in white; false cell is outlinedin yellow; mitochondria are small, intracellular red objects; plastidsare lighter, intracellular green objects; vacuoles are outlined in darkblue. Nuclei and nucleoli are shown in Figs. 3–5 only (in gray).Stereopair of newly-formed immature sperm cells, viewable by a‘Nesh’-type vertical-splitting viewer. Crosswall formation results inthe formation of two unequal sperm cells, with strong cytoplasmicdifferences. The distribution of mitochondria and plastids is clearlyshown, with only rare plastids ever noted in the Svn. Plastids remainin a dense aggregate at the tip of the newly formed Sua in abouquet-like distribution as evident previously in the generativecell. The near absence of organelles in the cellular projection istypical at this stage, as is the occurrence of one enucleatedcytoplasmic body; such bodies are frequently located near thedeveloping cellular projection and likely originate from it. Thesperm associated with the vegetative nucleus (Svn) is located at thetop of the picture; the smaller, unassociated and plastid-containingsperm cell is located toward the bottom of the figure. Fig. 7 Com-puter-assisted reconstructions of the generative and sperm cells ofP. zeylanica using a PC-based system with the HVEM ‘‘3-DReconstruction Program’’. Horizontal stereopairs are viewablewith a standard 3-D viewer (Figs. 1, 2, 6); vertical steropairs (Figs.4, 7) are viewable with a vertical-splitter 3-D viewer (‘‘Nesh’’-typeviewer). Outline of the cell is indicated in white; false cell is outlinedin yellow; mitochondria are small, intracellular red objects; plastidsare lighter, intracellular green objects; vacuoles are outlined in darkblue. Nuclei and nucleoli are shown in Figs. 3–5 only (in gray).Stereopair of sperm cells within the pollen grain at anthesis,viewable using a ‘Nesh’-type vertical-splitting viewer. The Sua (tothe left) and Svn (to the right) are at an angle relative to oneanother, and both plastids and mitochondria are more randomlydistributed than at the previous stage. Although the Sua isfrequently flexed toward the vegetative nucleus, it is variablyassociated with the vegetative nucleus and never intimately, as isthe Svn

b

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stage are seen at this or later stages. Vacuoles are dis-tributed in the cytoplasm near the pollen intine, whichrepresents the future area of generative cell separation.

False cells contain all typical cellular organelles withthe exception of the nucleus. Their boundaries are thesame as those of the generative cell except for an addi-tional longitudinal wall shared in common with thenucleated generative cell. False cells are located alongone edge of the lenticular generative cell (Russell et al.1996). The volume of the false cell ranges from 10.5 to54.9 lm3 at this stage. The smallest false cells consist ofslender crescents of cytoplasm, similar to the one indi-cated in yellow in Fig. 3, which contains few organelles.The largest are as large as some nucleated generativecells (206.84 lm3, Table 7), similar to the false cell

illustrated in Fig. 4. The large false cell in Fig. 4 con-tains numerous mitochondria, plastids, and a prominentvacuolar region in the center of its cytoplasm that wouldnormally be occupied by a nucleus. Among three falsecells serially reconstructed at this stage, the average totalvolume of mitochondria was 7.79 lm3 (18.7% of theaverage generative cell); for plastids the total volumewas 12.35 lm3 (126% of the average generative cell),and for vacuoles, 32.5 lm3 (109% of the average gen-erative cell; Table 6). The average number of mito-chondria in the false cell was 37.25 (19.9% of theaverage generative cell), 34.25 plastids (127.3%), and20.75 vacuoles (12.75%).

The cytoplasm of the generative cell becomes highlypolarized as the cell outline becomes more irregular and

Table 6 Volumes and surface areas of mitochondria, plastids, and vacuoles of the generative and sperm cells of P. zeylanica


Svn Sua Svn Sua

Organelles in nucleated MGU cells:n = 6 9 5 9 9 7 7MitochondriaVolume (lm3) 44.26±4.5 37.69±5.5 16.61±3.16 16.75±1.77 2.81±0.27 11.40±2.40 0.25±0.04Surface area (lm2) 256.571±35.2 289.69±38.0 154.26±21.7 159.09±17.3 22.76±2.44 76.56±8.67 0.92±0.110Average volume (lm3) 0.807 0.2013 0.047 0.086 0.073 0.044 0.0063PlastidsVolume (lm3) 6.67±0.12 9.77±0.15 9.91±2.06 0.01±0.001 19.22±2.50 0.06±0.006 14.25±16.3Surface area (lm2) 54.31±5.41 89.42±8.54 94.93±15.84 0.10±0.001 122.31±14.31 0.44±0.049 23.49±25.6Average volume (lm3) 1.72 0.363 0.438 0.02 0.544 0.150 0.586VacuolesVolume (lm3) 24.71±11.93 29.82±14.05 98.90±33.21 127.62±39.88 27.91±9.21 4.50±1.22 1.56±0.43Surface area (lm2) 56.29±28.31 128.55± 59.10 863.44±288.3 727.13±243.5 191.47±54.3 41.53±13.56 24.48±8.45Average volume (lm3) 6.59 0.183 0.254 0.576 0.475 0.028 0.017Organelles in false cells and ECBs:n = 3 4Mitochondria 7.33±3.9 37.25±16.5Volume (lm3) 7.79±10.7Surface area (lm2) 54.20±79.8Plastids 0.00±0.0 34.25±17.2Volume (lm3) 12.35±0.5Surface area (lm2) 129.29±21.3Vacuoles 0.00±0.0 20.75±12.3Volume (lm3) 32.50±4.3Surface area (lm2) 185.43±14.0

Table 7 Cell volumes and surface areas of false cells and ECBs during generative cell development in P. zeylanica as determined fromthree-dimensional reconstruction

Parameter GC I GC II GC III

n= 3 5 2ECB and false cell volume (lm3)Range 10.45–54.93 7.11–206.84 10.13–21.29Mean (± SE) 27.16±13.98 114.02±41.33 15.71±5.58ECB and false cell surface (lm2)Range 1.63–37.643 31.61–354.49 0.75–3.55Mean (± SE) 15.26±11.28 202.48±58.92 2.16±1.40Surface area/volume (lm�1) 0.56±0.81 1.78±1.43 0.14±0.25

Developmental stages: GC I lenticular generative cell. GC II separating generative cell. GC III freed generative cell. All means are given ±the standard error of the mean



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the cell begins to separate from the intine (Fig. 4).Plastids become aggregated in the most deeply immersedpart of the pollen cytoplasm; in most cases, this isnearest the vegetative nucleus. Mitochondria are dis-tributed in the apical cytoplasm surrounding the aggre-gated plastids. Vacuoles are distributed in the cytoplasmof the generative cell nearest the intine (Fig. 4). As thegenerative cell separates from the intine and migratesinto the vegetative cell cytoplasm, the generative cellundergoes a circumferential constriction near itsattachment site on the intine. Ultimately, this causes thegenerative cell to separate from the intine and the cell isenveloped in vegetative cell cytoplasm (Russell et al.

1996). A thin layer of cytoplasm remains basally ap-pressed to the intine (Fig. 5). This layer of remnantcytoplasm contains few, if any major organelles andcannot be detected soon after the separation of thegenerative cell from the intine. The plastids becometightly aggregated as the generative cell migrates into thevegetative cell (Fig. 5).

Microtubule-organelle associations

Since longitudinally oriented cortical and subcorticalmicrotubules are common within the cytoplasm of the

Fig. 8-11 Transmissionelectron micrographs (TEM) ofgenerative cells of P. zeylanicaFig. 8 Cell periphery (cp) of anobliquely-sectioned ‘lifting’generative cell, displaying theparallel distribution of corticalmicrotubules (unlabeledarrowheads). Vesicles arefrequently seen in the cellperiphery in chemically fixedmaterial. Stained withPA-TCH-SP reaction ·15,250Fig. 9 Association ofmitochondrion (m), corticalmicrotubules (unlabeledarrowheads), and the plasmamembrane at the edge of the cellperiphery (cp) ·77,100Fig. 10 Stereopair of basalportion of the cellularprojection of a ‘freed’generative cell. Corticalmicrotubules (arrowheads) areevident up to the obliquely-sectioned terminus of the cell.Organelles, includingmitochondria (m) and plastids(not shown), are located nearthe microtubular network. VCvegetative cytoplasm ·44,500Fig. 11 Median longitudinalsection of ‘freed’ generative cell,illustrating the polarizeddistribution of organelles,including the mitochondria (m),plastids (p), vesicles (v), andnucleus (GN). The generativenucleus is displaced toward thedistal end of the cell, frequentlyindented by the plastidaggregate. gb Golgi bodies·7,900

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generative cell, few organelles are distant from amicrotubule; many organelles, however, seem to beassociated by means of short (�25 nm) processes thatappear to connect the boundary of the organelle withone or more microtubules. These associated organellesappear to include plastids, mitochondria, vesicles, thenucleus, and even relatively large vacuoles. As in Fig. 9,associations with organelles may involve numerousmicrotubules. Associations are most frequent just beforegenerative cell division, when microtubules are mostnumerous. After generative cell division, the number ofmicrotubules is markedly decreased.

Freed, elongating generative cell

As the generative cell becomes immersed in the vegetativecell, its cell surface appears progressively more regular,and the main body of the cell becomes largely ellipsoidal.The cellular projection at the end of the cell closest to thevegetative nucleus elongates rapidly and is consistentlyphysically associated with the vegetative nucleus at thisstage (Fig. 11; see also Russell et al. 1996, Figs. 15, 34,40). The average volume of the cell is 216.1 lm3 at thisstage (a decrease of 50.3%) with an average surface areaof 314.4 lm2 (a decrease of 19.1%; Table 3). The averagevolume of the nucleus decreases by 31.8%, the averagesurface area decreases by 26.1% (Table 3), and the nu-cleus occupies a larger proportion (41%) of the volumeof the cell. The nucleolus expands slightly from a volumeof 9.44 to 10.9 lm3 (15.5% increase).

As the generative cell separates from the intine, thedistribution of organelles reverses. Plastids aggregate onthe side of the cell opposite to the vegetative nucleus andmost of the vacuoles move to the proximal side of thegenerative cell. Mitochondria occupy some of theremaining space in the cytoplasm. Although plastidsoccupy a slightly larger volume (9.91 lm3, up 1.4%),their area in profile continues to decrease (down 18.7%)as well as their total numbers (22.6 plastids per cell,decreasing 16%). The average volume of each plastidincreases by 20.7% to 0.438 lm3 and plastids becomemore complex in shape (Table. 3, 5, and 6). Mitochon-dria nearly double from the previous stage, increasing by89.5% to 354.7 mitochondria per cell, whereas the totalvolume of mitochondria decreases by 56% to 16.6 lm3.The average volume of a mitochondrion decreases byover 76% to 0.047 lm3, and the mean profile area de-creases to 0.199 lm. Vacuoles increase in number by108% to 389.9 vacuoles per cell and their total volumealso increases dramatically (up 230%) to 98.9 lm3.Individual vesicles, however, decrease by >38% inmean volume to 0.254 lm3, and their profiles in sectiondecrease in area and circumference (Table 5). Vesiclesoccupy �25% of the volume of the cell, with the hya-loplasm reduced by a corresponding amount. The highcontent of vacuoles at this stage causes the cytoplasm toappear highly vesiculated (Fig. 11; see also Russell et al.1996, Fig. 15).

Associated with the surface of the vegetative nucleusare enucleated cytoplasmic bodies (ECBs) that appear toseparate from the generative cell at many developmentalstages. These ECBs consist of small, spherical membrane-bound bodies with characteristic boundaries that includeplasma membranes of both the generative and vegetativecell. They are often observed in locally abundant aggre-gates. The generation ofECBs seems to be restricted to thetip of the cytoplasmic projection. Frequently, bodies oc-cur that contain fragments of membrane particles andcytoplasmic remnants, but rarely recognizable organelles.Such bodies have been termed vesicle-containing bodies(VCBs) (Yu et al. 1992) and appear to be degenerationproducts of ECBs. The mean total volume of ECB/VCBsis 15.7 lm3 (a decrease of 86% from the false cells of theprevious stage) and the mean total surface is 2.2 lm2 (adecrease of 99%). The surface area to volume ratio ofECB/VCBs is 0.14 lm�1, representing the lowest suchratio occurring in ECB/VCBs and false cells (Table 7).Because of this small size, their disappearance from thecytoplasm is difficult to document.

Organelle apportionment

Axially oriented microtubules are located principally ina single layer immediately beneath the plasma mem-brane (Figs. 8, 10). Associated with these are numerousmitochondria and plastids (Fig. 9), which appear to belinked by small fibers of �25 nm in length (see Russelland Mislan 1986, Fig. 13); these appear to link micro-tubules with individual microtubules and with eachother. These associations appear widespread among thecortically distributed organelles (Figs. 9,10,11, 14, 15).

Plastids are distributed in a tight bouquet-like aggre-gate at the proximal end of the cell (as in the Sua inFig. 11; also in Russell et al. 1996, Figs. 40, 41). Thenucleus is displaced proximally toward the plastidaggregate and appears strongly appressed to the nuclearenvelope, sometimes creating a cup-like indentation inthe profile of the nucleus (Figs. 11, 13). Plastid profilesfrequently have an electron dense substance located be-tween them, apparently linking the plastids. This mate-rial is stainable using the PA-TCH-SP (Fig. 12) and issensitive to periodic acid oxidation suggesting at leastpartially polysaccharidic content. According to seriallyreconstructed images, adhesions often form betweendifferent plastids and may thereby aggregate them at thisstage. Mitochondria remain independent, but are fre-quently located in small, local aggregates as viewed usingcomputer-assisted reconstruction (Figs. 4, 5). These localaggregates may possibly reflect groups of organelles ofcommon descent (e.g., Russell et al. 1996, Fig. 40).

Generative cell division and immature sperm cells

Immediately prior to mitotic division, the generativecell elongates until it extends nearly from the edge of

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the pollen grain to the center of the vegetative cell(e.g., Russell et al. 1996, Fig. 16). The generative nu-cleus is displaced toward the proximal end of the cellat this stage and is located at the future site ofcytokinesis. No evidence of a pre-prophase band wasobserved. Cytoplasmic organelles, which are stronglypolarized in distribution in the freed generative cell,become permanently allocated to their respectivesperm cells when cytokinesis occurs, and the organelledistribution present in the polarized generative cell isreflected in the composition of the resulting spermcells.

After generative cell division, the combined volumeof the resulting sperm cells is 486.8 lm3 (an increase of125% from the generative cell) with a combined surfacearea of 468.0 lm2 (an increase of 48.9%). The meannuclear volume of the combined cells increases by 68.5%to 149.5 lm3 and the mean nuclear surface area in-creases by 53% to 153.7 lm2 (Table 3). The volume ofthe cytoplasm increases by 164%. The Svn (sperm cellassociated with the vegetative nucleus) accounts for62.7% of the combined volume of the two sperm cellsand 66.1% of the surface area. The volume of the Svnnucleus is also larger than the Sua (57.8%) as is surface

Fig. 12-13 TEM of the ‘freed’generative cell of P. zeylanicaFig. 12 Plastids in ‘freed’generative cell appear linked byPA-TCH-SP positive plaques(unlabeled arrowheads) formingorganellar aggregates at thedistal end of the cell. ·31,000Fig. 13 Cross-section of theindented generative nucleus(GN) at the ‘freed’ cell stage andnearby plastids (p). Othercellular organelles are present atthe periphery of the nucleus,including mitochondria (m),and Golgi bodies (gb) ·12,700

Fig. 14 Stereopair of TEM ofan oblique section of the distalend of ‘freed’ generative cellillustrating the location ofcortical microtubules (unlabeledarrowheads) and theirassociation with nearbymitochondrion (m) and plastids(p). The lobed nature of thegenerative cell is indicated byarrows near the cell periphery(cp) ·25,700

Fig. 15 Stereopair of TEM ofplastids (p) associated with amicrotubule (arrowhead)·215,000

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area (54.5%). A nucleolus is present in immature spermcells during interphase, but the combined volume ofsperm nucleoli decreases by 70%—from 10.93 lm3 inthe generative cell to 2.16 lm3 for the Svn and 1.05 lm3

for the Sua.The total volume of plastids in the sperm cells almost

doubles (an increase of 94%) to 19.23 lm3, and thenumber of plastids in the male reproductive cells in-creases from a mean of 22.6 to 35.5 plastids (an increaseof 56%). A mean total of 35.3 plastids are located in theSua (99.4% of the total). The volume of individualplastids increases by 24.2% to 0.544 lm3 in the Sua andthe average profile increases by 126% to 0.464 lm2,while decreasing dramatically in the Svn, where at mostone was seen. Interestingly, the profiles of plastids re-mained nearly the same size in the Svn from inception tomaturity (at stage GC-III: 0.205±0.007, Svn-I:0.206±0.013, Svn-II 0.268±0.080; Table 5). ObservedSvn contained at most one plastid with a mean volume of0.02 lm3, representing among the smallest of the gen-erative cell plastids, which have an average volume of0.438 lm3. This single plastid represents less than 0.9%of those present in the generative cell and 0.6% of thosepresent in the two combined sperm cells.

Mitochondria undergo a modest increase in totalvolume (up 17.8%) from the generative cell throughsperm cell formation (Table 6), with the mean volume ofindividual Svn mitochondria increasing by 83% to0.086 lm3 per mitochondrion, while those of the Suaincrease by 55% to 0.073 lm3. Mean mitochondrialprofiles decrease by 17.6% in the Svn and increase 20.6%in the Sua. The combined number of mitochondria de-creases by 34.1% from the high count of 354.7 per cell inthe ‘freed’ generative cell to 233.8 mitochondria per cell.The Sua contains 38.5 mitochondria, a mere 11% of

those present in the generative cell and 16.7% of thosepresent in the two sperm cells.

Vacuoles decrease by 27.9% (combined total) withthe Svn receiving most (79%) of the vacuoles (Table 4).Their average volume, however, increases sharply inboth the sperm cells (to 0.576 lm3 per vacuole in the Svnand 0.475 lm3 in the Sua, compared to 0.254 lm3 in theprior generative cell stage, an increase of 127% and87%, respectively, Table 6). The equivalent sectionalradius increases by 50.0% in the Svn and 25.0% in theSua. Microbodies are first observed at this stage, occur-ring only in the Sua and not in the Svn (Table 4).

Transition of sperm cells to maturity

During the transition of sperm cells to maturity, thecombined volume of the two cells decreases to less thanone quarter of previous volume (118.4 lm3, down75.7%), with the Svn decreasing by 77.2% and the Suadecreasing by 73.0%. The cell surface of the sperm cellsalso decreases (Svn decreasing to 47.8% and Suadecreasing 53.3% of their previous surface area), and thesurface area to volume ratio doubles (Svn up 110% andSua up 97.9%). Sperm nuclei also decrease dramaticallyin volume (Svn decreasing by 77%, Sua by 80.6%) andsurface area (Svn decreasing by 61.3%, Sua by 70.9%).The volume of the nucleolus also decreases in bothsperm cells. In the Svn, the volume of the nucleolus de-creases by 42.9% to 1.23 lm3, and in the Sua by 49.4%to 0.53 lm3.

Plastids become less aggregated in the Sua at anthesisand are distributed equally at both poles of the cell(Russell 1984). The plastids inside the Sua decrease by31.2% in number and to 24.3 and 30.4% in total volumeto 24.7 lm3, respectively. The volume of individualplastids increases by 7.7% in the Sua to 0.586 lm3 andby 7.5 times in the Svn to 0.150 lm3. Plastid profile areain the Sua decreases by 11.9% and circumference by25.9% in the mature sperm. Apparently, plastids de-crease in size, but not complexity. Plastids in the Svnshow only minor changes in sectional profile and cir-cumference.

Mitochondria increase in number in the Svn from195.3 to 256.2 per cell (up 31.2%). Although theirnumber increases, the total mitochondrial volume de-creases by 31.9% and the average surface area of aprofile decreases by 62.2% (Table. 4,5, 6). In the Sua,mitochondria remain essentially as abundant, but con-tain less than one-tenth of their previous volume (a de-crease of 91.1% to 0.0063 lm3) and one-sixth of theirprevious average profile area (a decrease of 82.9%). Incomparison, a mitochondrion in the Sua occupies only14.3% of the volume of a mitochondrion in the Svn,leading to questions about whether they are large en-ough to contain significant quantities of genetic mate-rial.

The degree of vacuolization decreases significantly inboth the sperm cells during maturation. In the Svn,

Fig. 16 Stereological comparison of male germ unit cells duringdevelopment in lm3 during development. Developmental stages:GC I lenticular generative cell. GC II separating generative cell. GCIII freed generative cell. SI immature sperm cell. S II mature spermcell at floral anthesis. Svn sperm physically associated with thevegetative nucleus. Sua sperm unassociated with the vegetativenucleus

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vacuoles decrease by 28.3% in number, 96.5% in totalvolume, and 95.1% in the mean volume of individualvacuoles. The Sua displays an increase of 54.9% in thenumber of vacuoles, but similar decreases in total vol-ume of vacuoles (a decrease of 94.4%) and the volume ofindividual vacuoles (decreased by 96.4%).

Microbodies become more abundant in the maturesperm cells, and their presence correlates strongly withthe presence of plastids. The Sua contains an average of3.18 microbodies per cell, whereas the Svn contains 0.36microbodies per cell—in greatest abundance in theinfrequent Svn cells in which plastids occur.

The sperm cells tend to diverge at a 60–100� angle atthis stage, typically with the Svn bending near theboundary between the two sperms (Russell 1984) in aregion that is often flattened. The Sua therefore ap-proaches the vegetative nucleus more closely, althoughonly rarely it is in near contact with the vegetative nu-cleus. The cytoplasmic density of the sperms increasessignificantly, presumably as a function of decreasedcellular volume during pollen dehydration. At this latestage, uranyl acetate and lead citrate provide little elec-tron contrast, unlike surrounding cells which continue tostain as they did at prior stages (Russell and Cass 1981).

Discussion

Generative cell formation and cytoplasmic diminution

The division of the microspore results in a vegetative cellthat occupies 99.68% of the cytoplasm, and a generativecell that receives an average of only 0.32% of the priormicrospore cytoplasm. During development, the volumeof the vegetative cell lineage increases by almost 20times, from 6,200±250 lm3 in early microspores, to39,000±1,300 lm3 at the time of generative cellformation, and 115,000±6,800 lm3 at anthesis. Simul-taneously, the reproductive lineage decreases in volumeby 67.3%, from 362.3±24.1 lm3 at inception to118.4±11.9 lm3 at pollen release. The decrease in malereproductive cells is largely constant, except during theseparation of the generative cell from the intine and innewly formed sperm cells, when a transitory increase involume occurs. The trend in volume decrease is oppositeto that in the female germ lineage (Huang and Russell1993). At pollen release, the male germ cells togetheroccupy a volume of 0.082% of the pollen grain. The Svnoccupies 0.048% of the pollen grain and the Sua occupies0.034% of the total volume (Russell 1984).

Numerous overlapping mechanisms in addition todehydration of pollen grains appear to contribute todecreases in the volume of the sperm cells. Enucleatedcytoplasmic bodies (ECBs), vesicle-containing bodies(VCBs), and false cells from the male reproductivelineage may all be factors in the loss of generative cellvolume to the vegetative cell, either directly throughcoalescence or indirectly through lysis. Similar trends

have been described in other plants. In barley (Mogen-sen and Rusche 1985), flattened areas of cytoplasmappeared to be pinched off from the sperm cells duringtheir maturation. In tobacco (Yu et al. 1992, Yu andRussell 1993), significantly large ECBs and VCBs appearto shed from both generative and sperm cells duringpollen maturation and descent in the pollen tube. In thegenerative cell of the orchid Cymbidium, in which noheritable organelles appear to be present, VCBs areproduced during generative cell maturation prior topollinium release (Yu and Russell 1993). Both the gen-erative and sperm cells contain large ECBs and VCBs.

The formation of false cells has been reported only inPlumbago to date (Russell et al. 1996). These appear tooriginate through amitotic cytokinesis, as supported bytheir structural relationship with the generative cell andthe presence of a thinner wall between the generative andfalse cells. The formation of these false cells can result ina loss of up to 40% of the cytoplasm, but does notappear to occur in every cell, nor is it easily recognizable.Because of the topographic complexity of pollen grainsand generative cells, false cell detection requires serialreconstruction; the only reliable criteria include the ab-sence of a nucleus and the presence of cellular closure,which are difficult criteria to establish from non-seriallycollected sections.

The formation of ECBs and VCBs during spermdescent in the pollen tube appears to continue duringdevelopment, along with the vacuolization of the spermcells, which results in a further decrease in the concen-tration of cytoplasmic organelles in the sperm cells priorto fertilization, as noted up to the arrival of sperm cellsin the synergid in Populus, as well as sperm cells in theprogamic stage of other plants (Russell et al. 1990,Russell 1992).

With the exception of false cell formation, which ap-pears to be restricted to the generative cell during itsattachment to the intine, the other cytoplasmic diminu-tion trends appear to extend throughout development.Cytoplasmic diminution mechanisms appear to operatethroughout development in Plumbago, as was also thecase in tobacco (Yu et al. 1992, Yu and Russell 1994a, c).This diminution of male cytoplasm apparently eliminatesorganelles in some Apiaceae (Weber 1988) and Orchid-aceae (Cocucci and Jensen 1969, Yu and Russell 1993);however, in the majority of flowering plant sperm cells,heritable organelles remain present (Russell 1991). Thatflowering plants may display similar trends in cytoplas-mic reduction suggests that genetic control is exerted onmale cytoplasmic expression; however, there is also anemerging evidence that the rate of cytoplasmic decreasemay also be influenced by factors external to the plant,including environmental effects as noted by Yu andRussell (1994d). The extent to which dehydration affectsdiminution of cell volume appears to be minor; thereduction of cytoplasmic volume in male reproductivecells is disproportionately greater than that occurring inthe vegetative cell. An enigmatic change, which maynonetheless relate to the hydration status of the male

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reproductive cells is that the mature sperm cells are notstained well by uranyl acetate and lead citrate (cells areessentially homogeneously gray), despite that surround-ing cells and less mature cells stain normally.

Quantification of surface area and volume changes

Figure 16 illustrates the quantitative cytological changesthat occur in the male germ cells from their inception toanthesis in Plumbago using direct morphometric calcu-lations from three-dimensional reconstruction. Accord-ing to these observations, the nuclear fraction of themale reproductive cells is greatest at generative cellinception and sperm cell maturation (Fig. 16), althoughin absolute terms, there is nearly a consistent decrease involume. Interestingly, of the two sperm cells, the volumefraction of the nucleus is larger in the sperm cell destinedto fertilize the egg (Sua) both at or near inception and atanthesis, although the actual volumes of the sperm nu-clei remain similar. Interestingly, ESTs of the Sua alsoappear to be more abundant and diverse than those ofthe Svn suggesting that transcription may be more activein the Sua (Gou et al., unpublished data; data at http://www.genome.ou.edu/plumbago.html and posted onGenBank). The relatively higher nuclear volume of theSua is due to a reduced volume fraction occupied byhyaloplasm (which is relatively twice as large as that inthe Svn, Fig. 16). Nucleoli occupy the greatest volumes(relative and absolute) at generative cell inception anddecrease rapidly thereafter, never again regaining thesame volume fraction.

Organelles with electron-lucent contents are frequentin the male reproductive cells, but it is not evident whe-ther these bodies, which are approximately the size ofmitochondria (0.2 lm), should be regarded as smallvacuoles or relatively large vesicles. Nonetheless, theoverall volume of vesicles and vacuoles in the generativecell increases. Despite the low number of vacuoles ini-tially present in the microspore before division, decreasesin the size of vacuoles are more than offset by increases inthe numbers of vesicles and vacuoles; at the end of thisstage the relative volume of vesicles and vacuoles mayhave risen by over 25%. As a consequence of cytoplasmicpolarization of the generative, most of the vesicles areinherited by the Svn. By the time of anthesis, vesiclesoccupy the smallest volume fraction of the cell in the Svnand especially, the Sua (Table 6, Fig. 16).

The loss of volume and volume fraction duringmaturation is statistically significant and greater in theSvn than in the Sua, reflecting a 77.2% decrease in the Svncompared with a 73.0% decrease in the Sua. Interest-ingly, polyubiquitin transcription is differentially up-regulated in the Svn compared to the Sua, as well (Singhet al. 2002). Perhaps, increased expression of poly-ubiquitin in the Svn could enhance breakdown of pro-teins in that cell contributing to increased volume loss,as well as enhancing differences in gene expressionbetween the two sperm cells.

Changes in the quantity and qualityof heritable cytoplasmic organelles

In both plastids and mitochondria of male reproductivecells, there are stages in which these organelle popula-tions are less abundant than those formed in normalsomatic cells. At generative cell inception, only two tosix plastids were observed in the cytoplasm of the newlyformed cell; this number increases during developmentto an average of 24.3 plastids in the Sua at anthesis.Mitochondria are more numerous, with 33 to 81 mito-chondria in the newly formed generative cell, and theindividual organelle volumes are initially similar to thosein the vegetative cell (Table 6). The volume of individualmitochondria, however, decreases by over 94% from theearly generative cell to the late, ‘freed’ cell, at firstoccupying nearly 12% of the volume of the generativecell, and subsequently decreasing to less than 3% at thenext stage. At an average mitochondrial volume of0.047 lm3 (Table 6), the internal region available forstoring the entire mitochondria genome may be injeopardy. The division of mitochondria may thus belargely unlinked with organelle replication (Bendich andGauriloff 1984).

The overall quantity of heritable organelles suggeststhat during generative cell development, both mito-chondria and plastids are reduced to low levels ofoccurrence, but they are later restored to prior levels.Although restrictions of organelle number and volumemay be an incidental consequence of generative cellformation, these reductions are likely to restrict anyvariability that might occur in the organellar genomeand would thus restrict the cytoplasmic heterogeneityof paternal organelles arriving in the future zygote andendosperm. Since minority genomes are the mostlikely endangered by reductions in organelle content(Birky 1983), these would be likely to lose throughsorting out and once lost, would not be capable ofbeing restored in the lineage. Presumably, as thephysical size of organelles decreases, fewer organellescontain multiple, or even complete single copies oftheir own genome (Bendich and Gauriloff 1984). Thiswould also add to a reduction of the possible influencethat a minority or altered genome could exert in thesperm cells or their post-fusion derivatives. Except forthe reduction of size, organelles appear structurallysimilar to those in surrounding cells and seem to re-main functional. Sodmergen et al. (1995), and Corri-veau and Coleman (1988) established that the plastidscontain conspicuous DNA.

Origin of the sperm-vegetative nucleus association

The association of the male germ cells with the vegeta-tive nucleus may begin as early as generative cellinception. When the generative cell is first formed, itdisplays a small beak at the tip of the cell near thevegetative nucleus (Russell et al. 1996). At first the

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generative cell appears otherwise unpolarized, but theplastids soon accumulate at the end of the generative celllocated nearest the vegetative nucleus. This end, subse-quently, is usually the one that initiates the separation ofthe generative cell from the intine. Separation progressesfrom the beak of the generative cell to its opposite endby means of an intensifying constriction around thecircumference of the generative cell next to the intine(Figs. 11, 12; Russell et al. 1996). Upon separation fromthe intine, the cytoplasmic polarity within the generativecell rapidly reverses and plastids aggregate at the endopposite to the vegetative nucleus. The beak of thegenerative cell elongates at this stage and becomes morehighly associated with the vegetative nucleus, to a tighteror looser degree in different pollen grains. By the time ofgenerative cell separation, all pollen grains seem to dis-play a tighter association with the vegetative nucleus,which is maintained throughout later development anduntil the time of sperm discharge from the pollen tube.

Role of microtubules in cell morphogenesis

Microtubules in the generative cells of Plumbago occurin two populations: a sparse population of randomlyoriented internal cytoplasmic microtubules and a densersystem of longitudinally aligned cortical microtubuleslocated in parallel arrays. Internal cytoplasmic micro-tubules are sometimes observed near the generative nu-cleus and in association with cytoplasmic organelles, buttend not to be directly associated with the nuclearenvelope. Microtubules are most frequently preserved inthe current preparations in the late, elongated generativecells and were less frequently preserved at other stages.The cortical system of microtubules in the late genera-tive cell appears to be centered near an apparently singlefocal point located at the distal end of the generative cellwith respect to the vegetative nucleus, which may serveas a microtubule-organizing center. This region fre-quently extends near to the intine, and is locatedopposite the cellular projection, in association withessentially all of the plastids. Microtubules in the corti-cal system are also frequently associated with cytoplas-mic organelles, with apparent fibrillar materialtraversing this distance. The association of the presumedmicrotubule-organizing center with the generative celltip and the aggregated plastids suggests that microtu-bules may play a crucial role with regard to both gen-erative cell morphogenesis and polarization of cellularorganelles.

Evidence for microtubule involvement in theestablishment and maintenance of generative cell shapehas been presented in an early work on Haemanthususing electron microscopy and drug studies (Sangerand Jackson 1971a, b). In their study, such microtu-bule-disrupting drugs as colchicine and isopropylN-phenylcarbamate resulted in the elimination ofmicrotubules in the typically spindle-shaped generativecell. In the absence of microtubules, there was a partial

reversion to a nearly spherical shape, but it was pos-sible that the cell boundary retained sufficient rigiditythat complete reversion did not occur. Removal ofisopropyl N-phenylcarbamate in that study resulted inthe reestablishment of microtubules, but without aresumption of the prior spindle shape. As noted innumerous immunofluorescence and EM studies,microtubules in the generative cell typically organizeinto dense cortical arrays (e.g., Cresti et al. 1984;Palevitz and Tiezzi 1992). Microtubules may be orga-nized into dense bundles, particularly in bicellularpollen, or may be organized as individual fibers, but ineither case are commonly found in association with theplasma membrane. In the tricellular pollen of Plum-bago, most of these fibers are separate and not locatedin bundles.

The occurrence of motor proteins in associationwith cytoskeletal elements has received considerableinterest as a mechanism for the translocation anddeployment of organelles in animals and plants (Vale2003). In plants, there are abundant and functionallydivergent motor proteins compared to animals (Reddy2001)—as many as 17 myosins and 61 kinesins in theArabidopsis genome alone (Lee and Liu 2004). Myosinrepresents a motor protein that is functionally associ-ated with microfibrils and results in directional move-ment to the plus end (except myosin VI), and despitethe diversity only a few of the animal subclasses arerepresented in plants. Kinesins are most conventionallyplus-directed motor microtubule-associated proteins;however, plants have a much wider diversity judgingfrom characterizations of the Arabidopsis genome,including 21 minus-directed actin microfilament-asso-ciated motor proteins, another 21 minus-directedmicrotubule-associated kinesins, and a number of lessrepresented divergent kinesins (Lee and Liu 2004). Theclose association of organelles with microtubules,particularly in the elongated, ‘freed’ generative cell ofPlumbago suggests that organelles in this system arepositioned by the action of cytoskeleton-associatedmotor proteins. Although actin and actin-directedESTs have been identified in both sperm cell types atanthesis in P. zeylanica (see http://bomi.ou.edu/russell/,http://www.genome.ou.edu/plumbago.html and Gen-Bank XP Gou et al., unpublished data), microfibrillarsystems have not been evident in generative or spermcells (Palevitz and Liu 1992), nor have myosin ESTsbeen found in anthesis sperm cells. Microtubules areabundant in generative cells but are not represented inESTs of sperm cells after anthesis. Homologs of twokinesins, however, are evident: one EST of the Svn hashomology with a kinesin light chain-like protein ofArabidopsis (At3g27960) and one product of the Suahas homology with kinesin-like protein Tbk5, which isa minus end-directed microtubule-associated kinesin(At3g16060) predicted to be a soluble, mitochondrion-associated motor protein (summary at http://genom-ics.msu.edu/cgi-bin/plant_specific/locus_search.cgi?lo-cus=At3g16060.1). In Plumbago, it is presumed that

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the interphase microtubular system of the generativecell is unidirectional and further that the region nearthe projection serves as a microtubule organizingcenter. This would anchor the minus end of microtu-bules and provide cytoskeletal polarity. Thus, theoccurrence of a gene product similar to kinesin-likeprotein Tbk5 is consistent with the polarized distri-bution of mitochondria. The polarization of plastidsby another kinesin, which would logically be a plus-end directed microtubule-associated motor protein,was not characterized in the anthesis Sua ESTs, but theprocess appears to be completed at a prior stage. Thepolarized distribution of plastids appears to be com-pleted in the generative cell prior to mitosis, so geneproducts involved in migration may not persist in post-anthesis sperm cells. In maturing sperm cells, plastidsare redistributed in the Sua, but remain associated withthe crosswall in the Svn, suggesting that such processesmay remain dynamic during development.

In the case of neuronal axons, which are among thebest-described systems of filament-associated organellemovement, there appears to be a reestablishment ofpolarity of the axon cytoplasm after discharge (Vale2003). Unless associated with microtubules, smallvesicles and mitochondria in the axon exhibit saltatorymovement. In the current study, filamentous structureswere observed between organelles and associatedmicrotubules with a length of 25.14±0.62 nm. Thisobserved length exceeds that of microtubule-associatedmotor proteins alone, reported by Vale (2003), whichare 14–20 nm, but may be consistent with the lengthof a linked motor protein. Axonal organelle transportin neurons represents an extreme degree of cytoplas-mic activity; however, slower migration of organellesto a single pole, such as during generative cell devel-opment, could also be accomplished by a similarmechanism involving cytoskeleton-motor protein-organelle linkages.

Organelle inheritance problems

When cytokinesis occurs in the generative cell, a new cellwall is formed at the site of the metaphase plate. Gen-erative cell formation in tricellular pollen plants is lar-gely similar to that described in somatic cells, but a pre-prophase band is not formed during the prior interphase(Burgess 1970; Palevitz and Tiezzi 1992). From incep-tion, the crosswall between the sperm cells displays in-tense aniline blue-induced fluorescence, indicative ofhigh callose content (Russell et al. 1996). The eccentricposition of the nucleus in the generative cell prior todivision results in two dissimilar sperm cells after wallformation. The generative nucleus tends to be displacedaway from the vegetative nucleus resulting in the Svnbeing the larger cell and the Sua being the smaller cell.The asymmetrical distribution of organelles that exists inthe generative cell prior to mitosis is made permanentwhen cytokinesis occurs.

Organelles within the immature sperm cells are dis-tributed in essentially the same pattern as observedwithin the generative cell; plastids are aggregated at theend of the Sua opposite from the common wall andmitochondria, and small vacuoles are prevalentthroughout the cytoplasm of the Svn. Mitochondria andsmall vacuoles are sparsely present within the Sua nearthe crosswall with the Svn, but not on the opposite sideof the cell. Upon maturity, plastids, mitochondria, andvesicles become randomly distributed within the Sua.The plastids lose their lateral adhesions at this time andare no longer associated with cortical microtubules. Theonly exceptions to this random distribution of organellesoccur in plastids in the Svn, where plastids remainadjacent to the crosswall next to the Sua, and that heri-table organelles remain absent in the narrower confinesof the Svn cellular projection. In cellular projections,there is apparently sufficient space for heritable organ-elles, but they are simply not found in the narrow pro-jections.

At the inception of the generative cell, there are fewheritable organelles in the cytoplasm, an average of 3.88plastids and 54.9 mitochondria. The volume of the malegerm lineage cells is initially 362.3 lm3 but decreases toa combined volume of 118.4 lm3 in the sperm cells atanthesis. Although the volume of the pollen grain in-creases by �500%, from �30,000 lm3 to �150,000 lm3,the generative cell, at its largest volume, is still less than0.5% of the volume of the pollen and loses nearly 67%of its cellular volume. The male germ cell lineage be-comes progressively smaller until the Svn occupies lessthan one-tenth as much as it did originally, with a rel-ative volume of 0.0483%, and the Sua occupies a smallerrelative volume of just 0.0340% (Table 3). Meanwhile,the number of organelles increases dramatically duringdevelopment in the male germ cell lineage. Concurrently,the numerical density of organelles becomes more con-centrated, as organelles become more numerous andsmaller.

Given the small number of organelles in the newlyformed generative cell, it is attractive to suggest thatthe equivalent of an organellar founder effect mayoccur in male germ unit cells, where heterogeneity inthe heredity of a particular organelle class may beeasily lost. Initially, sorting out of organelles duringthe development of the plant (inclusive of all eventsfrom the zygote to meiotic cytokinesis) may eliminateminority genes. Then after meiotic cytokinesis, thesevere restriction of organelle numbers during gener-ative cell formation contributes to a decrease in po-tential heterogeneity as well, virtually assuringorganellar genetic homogeneity. The number of malegerm lineage organelles may represent as few as <1%of those typically found in an average somatic cell.The size of organelles is also drastically reduced aswell. In the case of mitochondria, their volume in theSua is 0.25 lm3 versus 11.40 lm3 in the Svn and in thegenerative cell they are 44.26 lm3. This severe decreasein mitochondrial volume raises the suggestion that

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they may carry few mitochondrial genes into the eggcell during double fertilization, and that possibly notall of the profiles carry genetic material.

In the Svn the opposite pattern occurs, as plastids aredrastically reduced in volume, from 0.807 lm3 in theinitial generative cell to 0.150 lm3 in the Svn, comparedwith 0.586 lm3 in the Sua. The reduction of plastids inthe Svn suggests that although they usually have noplastids and anyhow even if plastids were present, theyare dwarfed in size as well as number in the few Svn cellsthat contain them.

Overlapping mechanisms of (1) founder popula-tions, (2) cytoplasmic diminution, and (3) reductionsin organelle volume serve to reduce any potentialchimeric differences that may arise during developmentthrough retention of organelle heterogeneity or sub-sequent mutation. There is strong structural evidencethat a single restricted founder population of heritableorganelle genes is transmitted to the paternal gameticlineage. If paternal organelles are transmitted into thefemale gametes as a rare occurrence and if theorganelles are small enough to prevent meaningfulquantity, the quality of paternal genes in some orga-nelle classes may be disadvantaged and even elimi-nated. In the case of Plumbago zeylanica, organelletransmission of heritable paternal plastids into thecentral cell occurs through the Svn and converselytransmission of heritable paternal mitochondria occursin the egg cell through the Sua. Given volume andorganellar constitution of these cells, the likelihood ofheterogeneous organelle inheritance seems remote.Thus, in this plant there is a preferential reduction ofheritable organelles.

Although the current data represent results obtainedin a cytoplasmically heterospermic plant, similarreductions in the volume of the male cytoplasmiclineage suggest that the numerical trends and finding inPlumbago are present in many flowering plants,including such model plants as Arabidopsis, and isdocumented in barley (Mogensen and Rusche 1985),corn (Rusche 1988), Cymbidium (Yu and Russell 1993),and tobacco (Yu et al. 1992, 1994). In this and allother sperm cells, it is likely that there is a generalreduction in organelle volume and numbers, even incases where biparental or uniparental paternal plastidinheritance is observed.

Acknowledgements We thank James Campbell, Susan Gray, KellyHolzer, Margaret Kania, Robert Krenek, Tim Mislan, LoriO’Brien, Rahmona Thompson, and Kurosh Valanejad for excellenttechnical assistance. Software for computer-assisted reconstruc-tions (as described in Young et al. 1987) was obtained fromDr. J. C. Kinnamon, (Department of Biology, University of Den-ver, Denver, CO, USA). An expanded format of this program (forobjects with more than 2,000 profiles) was provided by Dr. StephenYoung (Department of Psychology, University of California, SanDiego, USA), for which we are grateful. This research was sup-ported in part by the National Science Foundation grant DCB-8409151. We gratefully acknowledge the use of Samuel RobertsNoble Electron Microscopy Laboratory of the University ofOklahoma.

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(Plumbaginaceae). 2. Quantitative cell and organelle dynamics