Ectopic expression of a Chlamydomonas mt+-specific homeodomain protein in mt- gametes initiates zygote development without gamete fusion

Abstract

The molecular mechanisms that activate expression of zygote genes after fertilization are obscure. In animals, receptor-ligand interactions during sperm-egg membrane fusion as well as delivery of putative regulatory molecules by the sperm into the egg cytoplasm are proposed to activate zygote development and subsequent transcription of zygote genes. The mechanisms of activation of zygote development in higher plants also are mysterious, in part because of the difficulty of isolating female gametes of higher plants. In the unicellular, biflagellated green alga Chlamydomonas, the early steps in zygote development are much more accessible to investigation. Within minutes after mating type plus (mt+) and mating type minus (mt-) gametes fuse, expression of several zygote-specific transcripts is induced independently of protein synthesis. Here, we show that ectopic expression in mt- gametes of an mt+ gamete-specific, homeodomain protein, GSP1, induces a zygote-like phenotype and activates expression of zygote genes. One of the genes, zsp2, expressed in these "haploid zygotes" encodes a zygote cell surface adhesion molecule that promotes formation of multicellular aggregates. In total, expression of six out of seven zygote genes examined was induced by ectopic expression of GSP1. Our experiments show that in addition to contributing their genomes to the zygote cytoplasm, gametes also deliver proteins that can activate gene transcription.

Figure 1

Diagrammatic representation of the Chlamydomonas life cycle. Gametes are formed when vegetative mt+ and mt− cells undergo gametogenesis induced by nitrogen deprivation (middle left). When gametes of opposite mating type (or sex) are mixed together, they adhere to each other via mating type specific adhesion molecules (agglutinins) on their flagella to form large collections of agglutinating cells (upper center). Flagellar adhesion induces gamete activation that leads to release of cell walls, erection of mating structures at the apical ends of the gamete cell bodies (upper right), and several other cellular and biochemical changes that prepare the gametes for cell fusion. All of the events associated with gamete activation can be induced in gametes of a single mating type by incubating them in dibutyryl cAMP (Pan and Snell 2000b). The tips of the mating structures fuse to establish cytoplasmic continuity, and soon thereafter the two gametes merge completely to become a zygote (lower right). Zygote formation can be extremely rapid, and within 5–10 min after gametes are mixed together, 70%–90% of the cells fuse. Immediately after cell fusion, the zygote developmental pathway is initiated and at 2–4 h after fusion, the zygotes form large aggregates of tightly adherent cells (lower center) as a consequence of synthesis of zygote-specific extracellular matrix molecules and cell body adhesion molecules. At about the same time, the mt− chloroplast DNA is degraded. After an obligatory several days in the dark and upon return to nutrient-rich medium, meiosis and germination occur, and each zygote forms four progeny, two mt+ and two mt− vegetative cells (lower right). Vegetative cells undergo mitotic divisions until the nitrogen is depleted from their medium and the cycle begins again. Diagram modified and adapted from Figure 1 of Pan and Snell 2000b.

Figure 2

Ectopic expression of HSV–GSP1 in mt− gametes. (A) Anti-HSV antibody screening of HSV–gsp1 transformed vegetative cells. Clones 16, 23, 56, and 105 out of 120 vegetatively growing mt− nit+ clones expressed a 120 kD HSV-reactive antigen detected by immunoblotting. (B) HSV–gsp1 transformed vegetative cells and gametes produced HSV–GSP1 protein that reacted with anti-GSP1 antibody. Immunoblotting showed that clone 105 cells expressed HSV–GSP1, which was reactive with both anti-GSP1 (lower panel) and anti-HSV (upper panel) antibodies, when grown as vegetative cells and gametes; endogenous GSP1 was expressed only by mt+ gametes. Clone 104 mt− cells, which were nit+ but did not express HSV–GSP1, were unreactive with both antibodies as vegetative cells and gametes.

Figure 3

HSV–gsp1 transformed mt− gametes exhibit a zygote-like phenotype. (A) Wild-type zygotes and the mt− transformants formed macroscopic aggregates visible to the unaided eye. The two left panels show photographs of 125-mL Erlenmeyer flasks containing wild-type mt+ and mt− gametes, which distributed themselves homogeneously in the medium. Wild-type zygotes and clone 105 gametes formed extensive aggregates as seen in the left two panels. (B) Microscopic, dark-field, pseudocolor images of wild-type and mt− transformants. Cells in the mt+ and mt− gamete cultures existed as individuals (left two panels), whereas cells in the cultures of wild-type zygotes and in cultures of the HSV–GSP1 transformed clone 105 gametes (two right panels) formed large, multicellular aggregates of closely adherent cells. Gametes are about 10 μm in diameter.

Figure 4

The zygote-like phenotype was detected only in HSV–gsp1 transformed mt− cells. Photographs of cell cultures in 24-well plates show that only the HSV–gsp1 transformed mt− gametes (clones 16, 23, 56, and 105) and wild-type zygotes displayed the zygote-like phenotype. Nontransformed mt− parental cells (B215) and mt+ parental cells (G1), HSV–GSP1 transformed mt− vegetative cells and HSV–GSP1 transformed mt+ vegetative cells and gametes did not form aggregates and were homogeneously distributed in the wells.

Figure 5

Expression of the zygote-specific extracellular matrix adhesion molecule, zsp2, is restricted to zygotes and HSV–gsp1 mt− transformed gametes. (A) zsp2 expression in wild-type zygotes does not require protein synthesis. Northern blotting indicated that zsp2 transcripts were not expressed in unmixed mt+ (mt+ G) or mt− (mt− G) gametes. Within 5 min after wild-type mt+ and mt− gametes were mixed together to induce zygote formation, the zsp2 transcript appeared and continued to be present until at least 40 min after zygote formation began; expression was not inhibited if the cells were mixed together in the presence of the protein-synthesis inhibitor cycloheximide (10 μg/mL). cblp was used as a loading control. (B) HSV–gsp1 transformation induced expression of zsp2 transcripts, but only in mt− gametes. HSV–gsp1 transformed clones 16, 23, 56, and 105 expressed zsp2 transcripts if they had undergone gametogenesis (G), but failed to express zsp2 transcripts as vegetative cells (V). Transformed cells that were nit+, but did not express HSV–GSP1 (clones 16 and 104), also did not express zsp2 as vegetative cells or gametes. Neither the mt− parental cells (B215, G) nor the mt+ parental cells (G1, G) expressed zsp2, nor did HSV–gsp1 transformed mt+ gametes (HSV–GSP1, G). Wild-type zygotes at 1 h after mixing expressed substantial amounts of zsp2 transcripts (wt, 1 h, Z). (C) ZSP2 protein is expressed only in zygotes and HSV–GSP1–expressing mt− gametes. Vegetative cells (V) and gametes (G) of clones 104 and 105, wild-type mt+ gametes, and wild-type zygotes were analyzed for the presence of ZSP2 by use of immunoblotting. The arrowhead on the right indicates the ZSP2 antigen. The band above ZSP2 was present in all of the lanes except the zygote one, and presumably represents a cross-reactive, constitutively expressed cell-wall molecule, which would have been released by zygotes and discarded when the samples were harvested by centrifugation in preparation for SDS-PAGE and immunoblotting.

Figure 6

Northern blotting analysis of transcripts for ezy1 and Class V genes. Transcripts for ezy1 were not induced in HSV–gsp1 transformed mt− gametes vegetative cells (V) or gametes (G). Transcripts for the late-appearing, zygote-specific Class V molecule, which were detected in 2-h zygotes but not 30-min zygotes, were also detected in the HSV–gsp1 transformed mt− gametes (G) but not vegetative cells (V). cblp was used as a loading control.

Figure 7

Regulation of transcripts for zys1A, but not ezy1, by gamete activation. (A) HSV–GSP1 undergoes posttranslational modification during gamete activation. Immunoblotting with an anti-HSV antibody showed that HSV–GSP1 underwent posttranslational modification during gamete activation induced either by adhesion with nonfusing mt+ imp1 gametes or during zygote formation after mixing with wild-type mt+ gametes. Because clone 104 is an mt− gamete not expressing HSV–GSP1, it did not react with the anti-HSV antibody. In addition, samples containing only wild-type mt+ gametes or only imp1 mt+ gametes did not react with the anti-HSV antibody, even though these cells contain endogenous GSP1 (data not shown). (B) Transcript levels for zys1A are up-regulated during gamete activation. Northern blotting showed that transcripts for the zygote-specific molecule zys1A were up-regulated several fold during activation of HSV–gsp1 transformed gametes that had been mixed with imp1 mt+ gametes, whereas levels of zsp2 and Class VIII transcripts were unaffected by gamete activation. Wild-type 30-min and 2-h zygotes, clone 104 gametes, and clone 104 and 105 vegetative cells are shown as controls. The total RNA loaded in each lane was identical, but only one half of the RNA in the four right-hand lanes is from mt− cells.