Proc. Nati. Acad. Sci. USA Vol. 82, pp. 6129-6132, September 1985 Botany Preferential fertilization in Plumbago: Ultrastructural evidence for gamete-level recognition in an angiosperm (cytoplasnic heritable organelles/male gamete/sexual reproduction) SCOTT D. RUSSELL Department of Botany and Microbiology, University of Oklahoma, Norman, OK 73019 Communicated by John G. Torrey, May 13, 1985 ABSTRACT Gametic fusion patterns in the angiosperm can be followed with transmission electron microscopy. The Plumbago zeylanica were determined by using cytoplasmically present report uses the dimorphic distribution of sperm dimorphic sperm cells differing in mitochondrion and plastid plastids to trace the pattern of fertilization in this plant, content and then identifying paternal organelles through their demonstrating that such fusion is preferential. ultrastructural characteristics within the maternal cytoplasm at the time of fertilization. The virtual absence of plastids MATERIALS AND METHODS within the sperm cell that is physically associated with the vegetative nucleus allows paternal plastids to be used to trace Specimen Preparation. Plants of P. zeylanica L. (Palmen- the fate of the two male gametes after fusion. Such paternal garten, Frankfort, FRG) were grown at 18-230C with 16-hr plastids were present in the egg in >94% of the observed cases, days in growth chambers at the University of Oklahoma. indicating the preferential fusion of the plastid-rich, mitochon- Flowers were emasculated prior to anthesis and were artifi- drion-poor sperm cell with the egg. In only one instance did the cially pollinated. Ovaries were collected 81/2 to 9 hr after opposite pattern occur. Since the possibility of this result artificial pollination (following the time table published in ref. occurring as the consequence ofchance in random fusions is <1 7), dissected, and fixed at room temperature in 3% glutaral- in 7000, this represents strong evidence for the presence of a dehyde in 0.067 M phosphate buffer (pH 6.8) for 6-8 hr. final putative recognition event occurring at the gametic level. Tissue was rinsed briefly and fixed in 2% osmium tetroxide in the same buffer, dehydrated in ethanol followed by Double fertilization is a phenomenon characteristic of flow- propylene oxide, and embedded in Spurr's low-viscosity ering plants involving two separate fusion events that incor- resin. Material was stained with uranyl acetate and lead porate both sperm cells into developmentally different lin- citrate by using techniques described elsewhere (7) and eages, both of which are biologically significant. Although observed with a Zeiss 10a transmission electron microscope. numerous pollen tubes compete for the opportunity to fer- Interpretation. To accurately discriminate the fate of the tilize the female gametophyte, if successful, the pollen tube plastid-rich sperm cell, the following served as minimal delivers only two male gametes and both fuse with separate requirements that were met for each included data set: (i) the female reproductive cells. In contrast with animals and female gametophyte had to appear normal and show the non-seed plants, the angiosperms display a remarkable de- presence of male nuclei in both the egg and central cell, gree of conservation of gametic material at late stages of indicating the normal occurrence of double fertilization; (ii) sexual reproduction. Of the two sperm cells, one fuses with two populations of plastids had to be readily identifiable the egg cell to produce the embryo. Nearly simultaneously, within either the fertilized egg, central cell, or both; (iOi) the the second sperm cell fuses with the central cell in a second, ratio of male to female plastids could not exceed 1:5, the separate fusion producing the nutritive endosperm. Whether upper limit of the expected ratio; (iv) at least three paternal the gametic fusion that initiates this process is a random event plastids (as recognized by characteristics given in Table 1 and or one that results in a directed outcome has remained among the text) had to be present in either the egg or central cell, but the most refractory problems in angiosperm embryology (1, not both. The plants used in the study displayed about 95% 2) and one that is addressed in the present study. seed set, with no evidence of apomictic reproduction. Since sperm cells are outwardly similar cells derived from a single mitotic division of the precursor generative cell, the RESULTS male gametes of angiosperms have traditionally been as- identical with neither cell Sperm Structure. The microgametophyte of P. zeylanica is sumed to be essentially (3), pos- tricellular and heterospermic, containing two dimorphic sessing a preferential tendency to fuse with the egg. Evidence sperm cells distinguished by whether or not the cell is contradicting this assumption has recently been reported in physically associated with the vegetative nucleus (4, 5). The Plumbago zeylanica (4, 5), indicating cytoplasmic hetero- sperm cell physically associated with the vegetative nucleus morphism in size, shape, organelle content, and physical is designated Syn, and the sperm cell not associated with the association with the vegetative nucleus. Although details of vegetative nucleus is designated Sua. The two sperm cells the fusion of gametes are concealed by nucellar cell layers differ in size, shape, nuclear dimensions, and organelle surrounding the egg, ultrastructural studies have revealed content. The S, is smaller, has a smaller nucleus, and that sperm cytoplasmic organelles in Plumbago are transmit- contains numerous plastids (average, 24.2 plastids) and few ted (6, 7) and can be identified by ultrastructural differences mitochondria (average, 39.8; ref. 5). In contrast, the larger in the fertilized female gametophyte (7). Differences present Svn infrequently contains plastids (average, 0.45) but contains in organelle content within the sperm cells can therefore be numerous mitochondria (average, 256; ref. 5) and an exten- employed as an ultrastructurally observable probe, which sive cellular projection that is physically associated with the The publication costs of this article were defrayed in part by page charge Abbreviations: Sw,,, sperm cell physically associated with the vege- payment. This article must therefore be hereby marked "advertisement" tative nucleus; Sua, sperm cell not associated with the vegetative in accordance with 18 U.S.C. §1734 solely to indicate this fact. nucleus; TEM, transmission electron micrograph. 6129 Downloaded by guest on September 30, 2021 6130 Botany: Russell Proc. Natl. Acad. Sci. USA 82 (1985) Table 1. Differences in organellar morphology between plastids Table 2. Frequency of plastids present in the Sv,, based on a of sperm origin and those of the megagametophyte Poisson distribution of absolute organelle counts of 11 sperm Characteristic Sperm plastid Egg plastid cells (5) Size Up to 1.5',m Up to 2.5 ,um No. of plastids Frequency Average sec- 0 0.6347 tional area 0.518 ,tm 0.893 g±m 1 0.2885 Shape Ellipsoidal, elongate Pleiomorphic 2 0.0658 Stroma Denser than cytoplasm Equally electron dense 3 9.9351 x 1O-3 Lamellae Inflated Constricted 4 1.1290 x 1O-3 Plastoglobuli Aggregated Randomly distributed 5 or more 1.1095 x 1o-4 vegetative nucleus. These differences in organelle content ovules used, with a male nucleus observed either approach- alone are significant enough to distinguish between the two ing or in the process of fusing with the nucleus of the female sperm cell types, with P < 0.001 for both mitochondrial and reproductive cell that it entered. In the remaining ovules, (i) plastid content (5). the required number of plastids was not observed, (ii) plastid Cytoplasmic Transmission. Mitochondria and plastids of populations could not be distinguished, (iii) reproductive sperm origin can both be identified within the female abnormalities were observed, or (iv) fixation was inadequate. gametophyte following fertilization (6). Differences in plastid The region where gametic fusion occurs was often evident structure being greater, these were used as the primary in the egg/zygote and central cell/endosperm (Fig. 3) located criteria for determining sperm cell fusion patterns in the near the terminal aperture of the pollen tube (6, 7) within present study. Sperm plastids may be distinguished from 20-30 ,um of the summit of the egg (8). Sperm mitochondria maternally originating plastids by the characteristics enumer- can sometimes be identified by size and structural differ- ated in Table 1 (see also ref. 7) and illustrated in Fig. 1. Since ences, as described in a previous paper (7). Near the site of one sperm lacks the presence and cell essentially plastids, unfused, bodies of sperm cyto- location of a number of sperm plastids in female cytoplasm gametic fusion, enucleated should be sufficient to identify which sperm cell fused with plasmic material were frequently seen between the zygote the egg. However, the infrequent presence of plastids in the and endosperm (Figs. 3 and 4). These enucleated bodies opposite sperm cell (Svn) implies that a certain number of (Figs. 3 and 4) arise in part through severance from the paternal plastids must be identified before this judgment can nucleated portion of the sperm cell during its expulsion from be made. Therefore, it is necessary to calculate the expected the pollen tube (7) or possibly through diminution of the cell, frequency of sperm plastids in the S, (Table 2), which was as has been suggested in barley (9). Such bodies contain only accomplished by using data generated from a previous study mitochondria of dimensions similar to those located in the (5). As the presence of three or more paternal plastids in the sperm cell (Fig. 4) and were not observed to contain plastids. cytoplasm of either the zygote or endosperm should accu- These observations indicate a probable origin of enucleated rately establish the fate of the plastid-rich Sua in all but 1% of cytoplasmic bodies from the Svn. A previous study indicates the cases (Table 2), this was selected as a requirement for that such bodies can remain between the egg and central cell each included data set.