Proc. Natl. Acad. Sci. USA Vol. 85, pp. 2594-2597, April 1988 Botany Exclusion of male mitochondria and plastids during syngamy in barley as a basis for maternal inheritance (embryosac/fertilization/quantitative ultrastructure/sperm cells) H. LLOYDMOGENSEN Departmentof BiologicalSciences, Box 5640, NorthernArizona University, Flagstaff, AZ 86011 Communicatedby Diter von Wettstein,January 4, 1988 ABSTRACT It is known from genetic analysesthat ma- cytoplasmis "stripped"off from the male nucleus duringthe ternal inheritanceof cytoplasmicorganelles is the rule among fertilization process, since plastids and mitochondriaare plants and animals. Although recognizedas one of several present within the sperm cells of wheat and other cereals, possiblemechanisms for strictlymaternal cytoplasmic inheri- yet cytoplasmic inheritanceis strictly maternal. tance, exclusion of sperm cytoplasmat the time of gametic Based upon the presence of what appeared to be male fusion has remained poorly documentedfor the flowering cytoplasmic sheaths just outside recently fertilized eggs of plants. In the present investigation,enucleated, cytoplasmic barley, I reported(13) that exclusion of paternalcytoplasm bodies approximatelythe size of intact, prefusionsperm cells at syngamy likely occurs in this plant; however, documen- have been observedwithin degenerated synergids and adjacent tationin this case consisted only of light microscopicdata. In to recently fertilizedegg cells. A completeseries of ultrathin the present study, serial thick sections and serial ultrathin sections (68 sections) through such a cytoplasmicbody re- sections from reembedded thick sections were used for a vealed59 mitochondria,3 plastids,7 dictyosomes,and a large detailed ultrastructuralanalysis and quantitationof one of vacuole with no limiting membrane.This structureis inter- these enucleated, cytoplasmic bodies located within the preted as the entire male cytoplasmthat was left outsidethe degeneratedsynergid and adjacent to the recently fertilized egg duringfusion betweenegg and sperm. The observationof egg cell of barley. The results indicate that the organelle only one cytoplasmicbody per embryo sac may indicate a complementof this body accounts for that expected within preliminaryfusion between sperm cells or, more likely, the prefusion sperm cells of this plant. existenceof a fundamentallydifferent mechanism of fertiliza- tion betweenthe second sperm and the centralcell. MATERIALS AND METHODS Barleyplants (Hordeumvulgare L.) used for this study were With few exceptions, extranuclearorganellar DNA is inher- either at the Plant ited in and animals In glasshouse-grown Carlsberg Breeding strictly maternally plants (1-5). Station (Hyldagergaard,Denmark), or field-grown at the floweringplants, several mechanismshave been considered United States of of Ar- the inheritance Department Agriculture-University responsible for uniparental of plastids and izona stationin Tucson, Arizona. were mitochondria: exclusion of the from the plantbreeding Spikes (i) organelles gen- emasculatedand bagged2 or 3 days before hand pollinations erative cell or one spermcell duringthe first or second pollen were performed.Ovaries were trimmedunder moist condi- mitosis, respectively (6, 7); (ii) deletions in plastid DNA, tions and initially fixed in 4% glutaraldehydein Sorensen's degeneration of organelles, or elimination of organelle- phosphate buffer (pH 7.4). Collections were made at 3- to containing cytoplasm during microspore, generative, or 5-min intervals during a 90-min period after pollination. sperm cell maturation(8-11); (iii) organelleexclusion during After 6 hr at room temperaturethe tissue was rinsed in gamete fusion (4, 12, 13); (iv) degeneration,lack of replica- phosphatebuffer, postfixed in 2%osmium tetroxide (in same tion, or compartmentalizationinto suspensor cells after buffer) for 2 hr at room temperature, rinsed in distilled zygote formation(14-16). water, dehydratedin an ethanol/acetone series, and embed- Of these possible mechanisms, exclusion of male cyto- ded in Spurr's resin (11, 13). plasm during syngamy has been poorly documented and, Serial thick sections (about 3 gim) were cut with a glass thus, is the most controversial(4, 6, 17). Structuralevidence knife and observed with phase-contrast optics. Selected for such a phenomenonhas been reportedfor cotton (18), in thick sections were then reembedded(19), serially ultrathin which two membrane-bound,enucleated cytoplasmic bodies sectioned (about 75 nm) with a diamondknife, stained with were found within the degenerated synergid shortly after uranylacetate and lead citrate, and observed with transmis- fertilization. Although the investigators recognized that sion electron microscopy. these structuresmay be of spermorigin, it is not possible to Ultrastructuralinformation on the embryo sac containing assess what proportion of the sperm cytoplasm may be the enucleated, cytoplasmic body of the present study was representedby these bodies-i.e., no quantitativedata were obtainedfrom 4 reembeddedthick sections. The cytoplasmic given and only one mitochondrionwithin the presumedmale structurewas present within 3 thick sections and 68 ultrathin cytoplasm was illustrated. Wilms (12) proposed a similar sections. The drawingof Fig. 1 is a composite based upon exclusion mechanism for the male cytoplasm of spinach; projectionsof photographicnegatives taken from the 4 thick however, his only documentationwas that of a binucleate sections. cell (interpreted as fused sperm cells), containing sparse located within the intercellular between cytoplasm, space RESULTS the egg and central cell. Hagemann(6) and Hagemannand Shr6der (4) propose for several grass species that the male After their discharge from the pollen tube, the two sperm cells become positioned near the chalazal end of the degen- The publication costs of this article were defrayed in part by page charge eratedsynergid (13, 20). Often, the sperms are found beyond payment. This article must therefore be hereby marked "advertisement" the degeneratedsynergid cytoplasm and within the intercel- in accordance with 18 U.S.C. ?1734 solely to indicate this fact. lular space between the egg and central cell (Fig. 2; ref. 13). 2594 Downloaded by guest on September 27, 2021 Botany: Mogensen Proc. Natl. Acad. Sci. USA 85 (1988) 2595 FIG.2. Intact,prefusion sperm cell (SC) containing mitochondria (M)and a nucleus(SN), located between the egg (E) and central cell (CC).DS, degeneratedsynergid; SV, spermvacuole. (x 10,600.) FIG.1. Compositedrawing based upon four serial thick sections fromthe sameembryo sac showingan early postsyngamy stage. The the Two mem- pollentube (Pt) has enteredthe degeneratedsynergid (DS) and degenerated synergid cytoplasm. plasma dischargedthe two spermcells. One spermnucleus (unlabeled branes are evident at a few places along the side where the arrowhead)is within the egg cell (E), near the egg nucleus. The other cytoplasmic structureis adjacent to the egg (Fig. 5). At no spermnucleus (unlabeled arrowhead) is withinthe central cell (CC) point is there an openingbetween the cytoplasmic body and nearthe polarnuclei (PN), whichhave begun to fuse. Withinthe the egg cell. The cytoplasmic body measures 6.7 gm x 3.8 degeneratedsynergid and adjacent to the egg cell is a cytoplasmic ,tm; the vacuolate area is 3.7 /xm x 3.5 ,tm. The sperm body (CB),which is interpretedto be spermcytoplasm that was nucleus within the cell of this sac is excludedat the timeof fusionbetween egg embryo approxi- eggand sperm. FA, filiform 7.0 x 4.0 Intact cells within apparatus;I, innerintegument; N, nucellus;dashed lines outline mately ,tm ,tm. prefusionsperm vacuoles. barleyembryo sacs measurefrom 6.0 ,xmto 8.0 /m in length and from 3.5 /m to 5.9 ,im in width. Althoughthe sperms are connected to each other withinthe pollen tube (21), they have not been observed to be in DISCUSSION contact within the embryo sac (present study; ref. 13). Several embryo sacs show what appears at the light Several factors support the interpretationthat the enuclea- microscope level to be an enucleated, cytoplasmicbody just ted, cytoplasmicbody of the present study representssperm outside the recently fertilized egg (present study; ref. 13). cell cytoplasmleft behindafter the male nucleus entered the One such early postsyngamystage is diagramedin Fig. 1; the egg. (i) Many of these structureshave been observed at the embryo sac on which this drawingwas based is the same as lightmicroscope level withinrecently fertilizedembryo sacs; that from which the following data were obtained.Note that none has been seen prior to fertilization. (ii) The size and one male nucleus is present withinthe egg cell (Figs. 1 and 3) shape of the cytoplasmic body are roughly similarto that of and the other is within the central cell (Figs. 1 and 4). intact sperm cells within the embryo sac. The slightly Neither sperm nucleus has yet reached the female nucleus smallersize of the cytoplasmicbody is not unexpected, since with which it was destined to fuse (Figs. 1, 3, and 4). the nucleus is no longer present. (iii) The cytoplasmic Located within the degeneratingsynergid and at the edge of structure contains organelles known to be present within the egg slightly chalazal to midlevel is an enucleated cyto- barley spermcells (11, 13, 22, 23). (iv) The cytoplasmicbody plasmic body (Figs. 1, 5, and 6), which is interpretedto be contains a nonmembrane-bound,vacuolated area devoid of sperm cytoplasm excluded at the time of fusion between organelles, which