Autoradiographic Evidence for the Rapid Disintegration of one Chloroplast in the Zygote of the Green Alga Ulva Mutabilis

T. BRATEN 0 0 Electron Microscopical Unit for Biological Sciences, University of Oslo , Blindern, Oslo 3 , Norway This paper gives evidence suggesting that one of the two chloroplasts contributed to the zygote of Ulva mutabilis is rapidly destroyed. Chloroplasts in an advanced stage of disintegration can be seen only 4 min after copulation. Radioactive labelling of gametes prior to copulation shows that the disintegrating chloroplast is supplied through the + (plus) gamete, while the chloroplast from the - (minus) gamete is retained in the zygote. These observations are in agreement with genetic observations from the same alga. - The fate of the chloroplasts in the zygote of green algae is at present a confusing problem. Interest in this problem arises from the fact that it has been demonstrated that non-Mendelian genes are organized into chromosome-like structures, most probably situated in the chloroplast of these plants (Sager & Ramanis, 1970). The gamete of Ulva mutabilis has only one chloroplast. The diploid cells developing from the zygote also have only one chloroplast. Two alternatives therefore exist: either the chloroplast in the diploid cell is a result of the fusion of the 2 chloroplasts; or the chloroplast from one of the gametes is eliminated in the zygote. There is reason to believe that in U. mutabilis the second alternative is the correct one. In a previous paper (Braten, 1971) it was shown that one of the chloroplasts in the young zygote seems to disintegrate. Fjeld (1971) has presented evidence that there is an unequal contribution from the 2 sexes to the zygote of U. mutabilis. A zygote which is the result of copulation between u.v.-irradiated +(plus) gamete and unirradiated (minus) gamete, has a greater chance of survival than a zygote which is the result of the reciprocal experiment. In this paper the fate of the chloroplasts in the zygote is investigated by autoradio MATERIAL AND METHODS The maintenance of algae and the method for obtaining synchronous release of gametes have been described in a previous paper (Braten, 1971). For autoradiographic study the culture medium contained 10 fid NaH14COs per ml of medium. After the initial incubation of the gametophytes in radioactive medium, the release of gametes took place in cold medium. Zygotes were obtained by mixing gametes of the 2 mating types in a drop of culture medium and were fixed for electron microscopy at intervals ranging from 4 min to 19 h after copulation. The fixation, dehydration and embedding procedures were as described before (Braten, 1971). Silvery sections were mounted on Formvar- and carbon-coated grids. For autoradiography Ilford L-4 photographic emulsion was applied as described by Caro & van Tubergen (1962). After 3-4 weeks the grids were developed for 5 min in Microdol developer. Finally, the sections were stained for 2 min in a saturated solution of uranyl acetate in 50 % ethanol and for 1 min in an alkaline lead citrate solution (Venable & Coggeshall, 1965). RESULTS Young zygotes fixed approximately 4 min after copulation proved to be the most useful in this study. At this stage the radioactivity was still confined to the originally labelled chloroplast. At later stages (15 min or older) it was found that the radioactivity had spread all over the cell, making identification of the unlabelled chloroplast difficult. Fortunately, even at the 4-min stage chloroplasts in an advanced stage of disintegration could be found (Fig. 1). When cold (minus) gametes were mixed with radioactively labelled + (plus) gametes the following result was obtained. In sections of 22 zygotes where 2 chloroplasts could be distinguished, 59 grains were found over the disintegrating chloroplast and 10 over the intact chloroplast (Fig. 2). When the copulation was carried out the opposite way (hot gametes x cold + gametes), 119 grains were found over the intact chloroplast and 31 grains over the disintegrating one in sections of 30 zygotes (Figs. 3, 4). Observations of several hundred gametes of the 2 different mating types prior to copulation have failed to show cells with abnormal chloroplasts. In their study of Chlamydomonas Sager & Lane (1969) claimed that chloroplast DNA from the male parent disappears in the zygote. Coincident with the apparent loss of this DNA was also the loss of non-Mendelian genes from the male. In Chlamydomonas reinhardii it has been shown that both chloroplasts persist in the zygote and that they fuse some 5 to 6 h after copulation (Cavalier-Smith, 1970). Serial sectioning of Ulva mutabilis zygotes has failed to show chloroplast fusion. Chloroplasts in different stages of disintegration can be found in the zygotes of U. mutabilis from a few minutes to about one hour after copulation. The fate of the disintegrating chloroplast in its most advanced stage is difficult to follow because after some time labelled material from the cytoplasm and/or the chloroplast spreads to the originally unlabelled part of the zygote. This might mean that chloroplast material which originally stems from the + gamete is redistributed and possibly reutilized in the zygote. Biochemical analysis of developing zygotes ought to clarify this question. Several workers with the light microscope claim to have seen elimination of chloroplasts from one of the gametes in the zygote of algae (Chmielevsky, 1890; Potthof, 1927). Observations also exist from organisms in which the chloroplast is totally lacking in one of the gametes. This is for instance claimed to be the case in the alga Chloroplast disintegration in Ulva Vaucheria (Jinks, 1964). An interesting position in this connexion is occupied by the green alga Ulva lactuca which is said by Levring (1955) to have male gametes lacking chlorophyll and where only the female gamete is capable of developing without fertilization. Clearly these observations need confirmation from studies with the electron microscope, but if they prove to be correct it is tempting to suggest an evolutionary trend in the development of the chloroplast of gametes and zygotes in algae. One end of an evolutionary line could be represented by species in which the chloroplast of one of the sexes is eliminated at a pro-gamete stage. U. mutabilis would represent an intermediate stage in which the + chloroplast is eliminated in the young zygote. Chlamydomonas would represent the next stage in which the 2 chloroplasts fuse, but where the chloroplast DNA from one parent is either lost or inactivated in the zygote. BRATEN, T. (1971). The ultrastructure of fertilization and zygote formation in the green alga Ulva mutabilis Foyn. J. Cell Set. 9, 621-635. CARO, L. G. & VAN TUBERGEN, R. P. (1962). High-resolution autoradiography I. Methods. J.CellBiol. 15, 173-188. CAVALIER-SMITH, T. (1970). Electron microscopic evidence for chloroplast fusion in zygotes of Chlamydomonas reinhardii. Nature, Lond. 228, 333-335. CHMIELEVSKY, V. (1890). Eine Notiz uber das Verhalten der (...truncated)