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Proc. Natl. Acad. Sci. USA Vol. 85, pp. 2594-2597, April 1988 Botany

Exclusion of male mitochondria and during syngamy in barley as a basis for maternal inheritance (embryosac/fertilization/quantitative ultrastructure/ 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 and . 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 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 ; 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 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 duringfusion betweenegg and sperm. The observationof egg of barley. The results indicate that the only one cytoplasmicbody per 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) gen- emasculatedand bagged2 or 3 days before hand erative cell or one spermcell duringthe first or second pollen were performed. were trimmedunder moist condi- , respectively (6, 7); (ii) deletions in 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 during microspore, generative, or 5-min intervals during a 90-min period after . sperm cell maturation(8-11); (iii) organelleexclusion during After 6 hr at room temperaturethe was rinsed in 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 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 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 , 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 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 (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 . 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- 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 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 would be expected in such a body from sperm and egg. Its position is near the level of the sperm which its nucleus had exited. That this vacuolate area is nucleus within the egg cell (Fig. 1). Analysis of complete somewhat smaller than the sperm nucleus may be due to serial ultrathinsections throughthis structureindicates that some invasion of surrounding cytoplasm into the space it contains 59 mitochondria,3 plastids, 7 dictyosomes, and a previously occupied by the nucleus. It is also possible that ratherlarge nonmembrane-boundvacuolate area at the side the sperm nucleus enlarges after entering the egg cell. (v) toward the egg cell. A plasma membraneis present around The location of the cytoplasmicbody within the degenerated much of the cytoplasm, but there are large areas of discon- synergid and next to the egg cell places it in the vicinity of tinuities on the sides away from the egg cell and adjacentto the spermnucleus withinthe egg cell. (vi) Alternativeorigins Downloaded by guest on September 27, 2021 2596 Botany: Mogensen Proc. Natl. Acad. Sci. USA 85 (1988)

FIG.3. Spermnucleus (SN) withinthe egg cell, nearthe egg FIG.5. Cytoplasmicbody (CB) within the chalazalend of the nucleus(EN). L, lipidbody; M, mitochondria.From the embryo sac degeneratedsynergid (DS) showing mitochondria (M), dictyosomes of Fig. 1. (x10,600.) (D), anda vacuole(V). Serialsections show thatthis structureis enucleatedand that the vacuoleextends to, andis largestnear, the sidenext to theegg (E). Fromthe embryosac of Fig. 1. (x 10,600.) for this structureare difficultto explain, since the cytoplasm of both synergids and that of the pollen tube have already PNlBlS^ll-llBBSB^r degenerated. (vii) Plastid inheritance in barley is strictly WMS: ~~~I?l'liill^Sl?X^ _--maternal (2). Assumingthat this interpretationis correct, results of the present investigationstrongly suggest that a mechanismfor major, if not total, exclusion of paternalcytoplasm is oper- ating at the time of gamete fusion in barley. Although data from hybridizationstudies indicate that it may not matter which of the two donor cytoplasmic genomes is eliminated(24, 25), the mechanismproposed here for barley ensures that only maternal cytoplasm is inherited. This mechanismmay be importantduring sexual reproductionfor the nontransmissionof genetically altered male organellar genomes, such as largedeletions in chloroplastDNA known to occur in microsporesof wheat and barley (8, 26). The findingof only a single cytoplasmic body outside the recently fertilizedegg cell could indicate that the sperm cell fusing with the central cell transmits its cytoplasm, which could subsequently be incorporated into the developing . Alternatively, the presence of a single male cytoplasmicbody could mean that, similarto the interpreta- tion for spinach (12), of the two sperms have fused. The occurrenceof at least 59 mitochondriawithin this body lends support to this conclusion, since the mean numberof mitochondriaper sperm cell at anthesis in barley is 31 (11). However, it is very possible that sperm cell mitochondrialproliferation occurs after pollination. This would not be surprisingin light of the postpollinationsperm morphogenesisknown to occur in barley (21). Informationhas not yet been obtainedin barleythat would FIG.4. Portionof the spermnucleus (SN) within the central cell offer insight into the precise mechanism resulting in the (CC),near one polarnucleus (PN). E, egg cell; DS, degenerated exclusion of male cytoplasm during gamete fusion. That synergid.From the embryosac of Fig. 1. (x 10,600.) gamete fusion begins with the coalescence of participant Downloaded by guest on September 27, 2021 Botany: Mogensen Proc. Natl. Acad. Sci. USA 85 (1988) 2597

present and the difficulty of distinguishing between pro- plastids and poorly differentiatedmitochondria (11, 22, 23). Putative proplastids have been reported within the sperm cells of barley (13). I thankProfessor Diter von Wettstein,Carlsberg Laboratory, and Dr. R. T. Ramage, University of Arizona, and their staffs for growing the plants for this study and for performingthe hand pollinations.Expert technical assistance by Vincent Wagner,Max- ine Rusche, Peggy Pollak, and CarlRay is gratefullyacknowledged. This work was supportedby the NationalScience Foundationunder GrantDCB-8501995 and by the OrganizedResearch Fund, Northern Arizona University. 1. Gillham, N. W. (1978) Organelle Heredity (Raven, New York). 2. Kirk, J. T. O. & Tilney-Bassett,R. A. E. (1978) The Plastids: TheirChemistry, Structure, Growth, and Inheritance(Elsevier Biomed, Amsterdam). 3. Conde, M. F., Pring, D. R. & Levings, C. S., III (1979) J. Hered. 70, 2-4. 4. Hagemann,R. & Shroder,M. B. (1984) in Proceedings of the Eighth InternationalSymposium on Sexual Reproductionin Plants, , , eds. Willemse, M. T. M. & van Went, J. L. (Podoc, Wageningen, The Netherlands), pp. 53-55. 5. Gyllensten,U., Wharton,D. & Wilson, A. C. (1985)J. Hered. 76, 321-324. 6. Hagemann,R. (1983) in Fertilization and Embryogenesisin OvulatedPlants, ed. Erdeleska,0. (VEDA, Bratislava,Czech- oslovakia), pp. 97-99. 7. Russell, S. D. (1985) Proc. Natl. Acad. Sci. USA 82, 6129- 6132. 8. Day, A. & Ellis, T. H. N. (1985) Curr. Genet. 9, 671-678. FIG. 6. Same cytoplasmic body (CB) as in Fig. 5, sectioned 9. Clauhs, R. P. & Grun, P. (1977)Am. J. Bot. 64, 377-383. -1.4 jtm from the plane of Fig. 5, showinga plastid(P), mitochon- 10. Vaughn, K. C. (1981)J. Hered. 72, 335-337. dria (M), and a dictyosome (D). E, egg; DS, degeneratedsynergid. 11. Mogensen, H. L. & Rusche, M. L. (1985) Protoplasma 128, Note wall precursorvesicles (PV), which are of pollen tube origin. 1-13. From the embryo sac of Fig. 1. (x 10,600.) 12. Wilms, H. J. (1981)Acta Bot. Neerl. 30, 101-122. 13. Mogensen,H. L. (1982)Carlsberg Res. Commun.47, 313-354. plasma membraneshas long been known and is well docu- 14. Tilney-Bassett,R. A. E. (1976)Heredity 37, 95-107. mented in plants and animals (27-30). The initial phases of 15. Russell, S. D. (1980) Science 210, 200-201. fusion in the case of barley need not differ from the usual 16. Kuroiwa, T. & Nakamura, S. (1987) Molecular Biology of situation where membrane fusion and the formation of Mitochondriaand Plastids (Cold Spring Harbor Lab., Cold SpringHarbor, NY), p. 62 (abstr.). cytoplasmic bridges occurs between the two cells. It is 17. Russell, S. D. (1986)in The Chondriome,eds. Mantell, S. H., conceivable that once an opening is created the sperm Chapman,G. P. & Street, P. F. S. [Longman (Harlow), Es- nucleus alone may have a propensity toward relocation, , U.K.], pp. 69-116. which would result in the spermcytoplasm being left behind 18. Jensen, W. A. & Fisher, D. B. (1968)Planta 78, 158-183. within its original membrane.Subsequent closing of 19. Mogensen, H. L. (1971)J. Ariz. Acad. Sci. 6, 249-250. plasma 20. D. D. & W. A. Am. J. Bot. 62-70. the at the of fusion could be by Cass, Jensen, (1970) 57, opening point accomplished 21. Mogensen, H. L. & Wagner, V. T. (1987) Protoplasma 138, membranereformation involving the numerousvesicles pres- 161-172. ent within the egg and sperm cell or, perhaps, by a mem- 22. Cass, D. D. (1973) Can. J. Bot. 51, 601-605. brane constriction process. Mechanisms similar to those 23. Cass, D. D. & Karas, I. (1975) Can. J. Bot. 53, 1051-1062. proposed here have been described by Jensen and Fisher 24. Shiller,B., Herrmann,R. G. & Melchers, G. (1982)Mol. Gen. (18) and Wilms (12). Genet. 186, 453-459. The finding of three plastids within the cytoplasmicbody 25. Ziegler, M. L. & Davidson, R. L. (1981) Som. Cell Genet. 7, the of 73-88. of the present study lends support to conclusion A. & T. H. N. Cell 359-368. contain 26. Day, Ellis, (1984) 39, Hagemannand Shr6der(4) that all cereals probably 27. Friedman,I. (1962)Science 136, 711-712. plastidsin their generativeand spermcells. Failureto report 28. Colwin, H. L. & Colwin, A. L. (1963)J. Cell Biol. 19, 501-518. plastids in the generative and sperm cells of barley studied 29. Tyler, A. (1965)Am. Nat. 99, 309-334. previously is probably due to the low number of plastids 30. Russell, S. D. (1983)Am. J. Bot. 70, 416-434. Downloaded by guest on September 27, 2021