Sexual Reproduction in Chlamydomonas reinhardtii

Overview of the Life Cycle of Chlamydomonas reinhardtii

Chlamydomonas (Ch.) reinhardtii was first isolated as a green soil alga and is used as a model organism in plants. It retains two flagella subsequently lost by most plants and a chloroplast being functionally equivalent to the chloroplasts of green plants. Its complete genome sequence is available (Merchant et al. 2007) and its life cycle is well characterized. Ch. Reinhardtii has two mating types: mating type plus (mt+) and mating type minus (mt–), which are controlled by a single complex mating type locus (MT+ or MT–) (Ferris et al. 2002). They proliferate asexually when an adequate source of nitrogen exists in the environment. After mitosis in the mitotic cell cycle, the newly formed daughter cells are liberated by the effect of sporangin (a subtilisin-like serine protease) acting on the breakdown of the sporangial cell walls (Matsuda et al. 1995; Kubo et al. 2009). When nitrogen levels fall below a certain threshold, vegetative cells having the MT+ locus differentiate into mt+ gametes and cells having the MT– locus differentiate into mt– gametes. Three hierarchically regulated gene expression programs are generally recognized as a response to nitrogen depletion: a program to adapt to nitrogen starvation, a gamete differentiation program, and a zygote formation program (Abe et al. 2004, 2005; Kubo et al. 2008). Within minutes of being mixed, gametes start to agglutinate. Gametes of opposite mating types pair with each other and fuse to form bi-nucleate quadriflagellated cells (QFCs). The two nuclei in the cell fuse, and a novel set of zygote-specific genes is expressed to form a dormant zygote that is resistant to both freezing and desiccation. When conditions improve, the dormant zygote initiates meiosis and the four recombinant haploid products resume vegetative growth (Fig. 1).

Fig 1

Sexual Adhesion

The sexual adhesion between the gametes is mediated by agglutinin molecules on their flagellar membranes. The plus and minus agglutinins are sex-specifically displayed by nitrogen-starved mt+ and mt– gametes, respectively. These molecules are encoded by different genes and possess complementary adhesive properties. They are both huge monomeric glycoproteins (>1000 kDa; Adair et al. 1983), possessing a large globular head and a fi brous shaft (Goodenough et al. 1985), and are members of the hydroxyproline-rich glycoprotein (HRGP) family (Cooper et al. 1983). Ferris et al. (2005) isolated the SEXUAL AGGLUTINATION1 (Sag1) and SEXUAL ADHESION1 (Sad1) genes encoding plus and minus agglutinins, respectively. Gene expression is restricted to gametes of one mating type.

The presence of the Minus-dominance (Mid) gene localized on the MT– locus suppresses the expression of Sag1 but induces the expression of Sad1. Both deduced proteins are organized into three distinct domains: a head (C-terminal), a shaft, and an N-terminal domain. The plus and minus heads are quite large domains (2006 and 2404 amino acids, respectively) having 12 and 14 putative N-glycosylation sites, respectively. Six of the putative N-glycosylation sites are in similar locations. They are poorly conserved in the amino acid sequence except for two regions of the predicted hydrophobic α-helix. The shafts contain numerous repeats of the PPSPX motif. Head–head interactions, heads–shafts interactions, and antiparallel shaft–shaft interactions may be involved in the sexual adhesion between two specific agglutinins (Ferris et al. 2005).

Signal Transduction after the Interaction of Agglutinins

After the interactions between plus and minus agglutinin molecules on the flagellar membranes, a gamete-specific flagellar adenylyl cyclase is activated via a protein kinase- and kinesin-II-dependent pathway (Saito et al. 1993;

Zhang and Snell 1994; Pan and Snell 2002) and the intracellular cAMP level is elevated nearly tenfold, triggering dramatic alterations in the cell (Pasquale and Goodenough 1987; Saito et al. 1993; Zhang and Snell 1994). The addition of a cell-permeable analog of cAMP, dibutyryl cAMP, can induce most of the cellular changes (Pasquale and Goodenough 1987). The mating-related effects of cAMP elevation include the following. First, flagellar motility is altered and the adhesiveness of the flagellar surface is increased by the translocation of inactive agglutinin molecules from the plasma membrane of the cell body onto the contiguous flagella membrane where the agglutinins become active (Saito et al. 1985; Goodenough 1989; Hunnicutt et al. 1990).

The process is mediated by the kinesin/dynein-mediated intra-flagellar transport system (Snell et al. 2004; Wang et al. 2006; Piao et al. 2009). Second, activated gametes secrete a serine protease (p-lysinase) that converts an extracellularly stored prometalloprotease into an active matrix-degrading enzyme (Buchanan et al. 1989; Snell et al. 1989; Kinoshita et al. 1992), and the cell wall (multilayered glycoproteinaceous extracellular matrix) surrounding each cell is degraded so that the gametes are able to fuse.

Third, mt+ gametes erect an actin-filled microvillus (“fertilization tube”) as a mating structure at the apical ends near the bases of the flagella and the mt– gametes also erect a small, dome-like, actin-free mating structure. The mating structures of both types of gametes display an extracellular coat of material referred to as fringe (Goodenough et al. 1982).

Molecules Required for the Fusion

Cell fusion is initiated by an adhesive interaction between the mt+ and mt– mating structures, followed by localized membrane fusion. Two proteins are known to be an essential for the membrane fusion reaction. The first is FUS1, which is a single transmembrane protein on the mating structure of the mt+ gamete (Ferris et al. 1996; Misamore et al. 2003). The FUS1 gene is sex-specifically expressed and is located in the MT+ locus (Ferris et al. 1996). FUS1 is an about 95-kDa protein and has domains related to the Ig-like domains of prokaryotic invasins and adhesins. It is essential for the adhesion of the mt+ mating structure to an unidentified receptor on the mating structure of mt– gametes (Misamore et al. 2002, 2003). The fus1-1 mutant undergoes normal flagellar adhesion and gamete activation, and produces an actin-filled fertilization tube in response to cAMP; however, the fus1-1 fertilization tube fails to fuse with the activated minus mating structure and the cells continue to agglutinate for several days. The mt+ fringe is encoded by the FUS1 gene (Misamore et al. 2003). Fertilization tubes on fus1-1 gametes do not contain the fringe (Goodenough et al. 1982).

When mt+ gametes are incubated with the anti-FUS1 antibody, fusions with mt– gametes are blocked. The second protein required for the membrane fusion reaction is GCS1/

HAP2, which is expressed on the surface of the mt– mating structure as a single transmembrane protein (Liu et al. 2008). The gene has also been identified in other algae, protists, and higher plants (Mori et al. 2006; von Besser et al. 2006; Steele and Dana 2009; Wong and Johnson 2010). In Arabidopsis thaliana, the GCS1/HAP2 gene is specifically expressed in sperm cells, and the mutant fails to fuse with both egg and central cells (Mori et al. 2006). In the case of the malaria organism Plasmodium berghei, GCS1/HAP2 is required at a particular step in the membrane fusion reaction between gamete membranes (Liu et al. 2008). The knockout mutant shows male sterility (Hirai et al. 2008). In Ch. Reinhardtii, expression of the GCS1/HAP2 gene is confirmed in both mt+ and mt– gametes but is far stronger in mt– gametes (Mori et al. 2006). GCS1/HAP2 protein localizes at the fusion site of mt– gametes and mt– gcs1/hap2 mutant gametes can form tight perfusion membrane attachments with mt+ gametes, but they fail to fuse (Liu et al. 2008). Both FUS1 and GCS1/HAP2 proteins are degraded rapidly upon fusion, as would be expected to block polygamy (Liu et al. 2010).

Development of the Zygote

The zygote developmental program is triggered by the heterodimerization of two homeoproteins, Gamete specific plus1 (Gsp1) and Gamete specific minus1 (Gsm1), which are contributed by the mt+ gamete and mt– gamete, respectively (Lee et al. 2008). Gsp1 is distantly related to the BELL class homeoproteins and Gsm1 is an ortholog of the KNOTTED1-like homeobox (KNOX) class (Hake et al. 2004; Scofield and Murray 2006). The expression of the GSM1 gene is dependent on the expression of the MINUS DOMINANCE (MID) gene (detailed below) on the MT– locus, while GSP1 expression is inhibited by MID. GSP1 was identified as a gene expressed specifically in mt+ gametes (Kurvari et al. 1998). Ectopic expression of GSP1 in mt– gametes is responsible for the formation of the zygotic cell walls and the expression of several zygote-specific genes (Zhao et al. 2001). When both the GSP1 and the GSM1 genes were ectopically expressed in vegetative cells, a zygote developmental program was activated: they formed zygote-specific cell walls and expressed zygote specific genes, despite being in a nitrogen-supplemented medium. With ectopic expression of both GSP1 and GSM1 in a generated mt+ diploid background, the resulting zygotes undergo normal meiosis (Lee et al. 2008). GSP1 is also important for the uniparental inheritance of chloroplast and mitochondrial DNA. A mutant, biparental31 (bp31), having a deletion of about 60 kb on chromosome 2, including the GSP1 gene, impairs the uniparental inheritance of chloroplast and mitochondrial DNA. The mutant phenotype can be rescued by a cotrans-formation with both the GSP1 and INOSITOL MONOPHOSPHATASELIKE1 (INM1) genes (Nishimura et al. 2012).

Sex Determination in Chlamydomonas reinhardtii

As explained previously, mating types of Ch. Reinhardtii are controlled by a single complex mating type locus (MT+ or MT–) on linkage group VI (Ferris et al. 2002). Heterozygous mt+/mt– diploids, which are occasionally formed after mating, always mate as mt– gametes, indicating that MT– is dominant to MT+ (Harris 1989). The core of the two MT loci encompasses 200–300 kb (Ferris and Goodenough 1994; Ferris et al. 2002, 2010). The MT loci contain highly rearranged DNA sequences, characterized by several large inversions and translocations, which act to suppress the recombination. Some genes are specifically linked to either MT locus. The FUS1 gene on the MT+ locus, and the MT locus region d (MTD1) and MID genes on the MT– locus have been assigned mating type-specific functions in gametogenesis and mating.

Mt+ cells transformed with the MID gene differentiate as mt– gametes and the functional mutant in an mt– background differentiates into an mt+ gamete having all of the molecules required of an mt+ gamete, except for the FUS1 protein (Ferris and Goodenough 1997; Ferris et al. 2002). These results indicate that MID is necessary both to activate mt– gene expression and to prevent mt+ gene expression, allowing the conversion of wild-type mt+ gametes to mt– gametes. MID encodes a RWP-RK family putative transcription factor. Vegetative mt– cells express basal levels of MID. A pulse of upregulated expression (level 1, threefold increase to basal level) occurs at 30 min after nitrogen removal, followed by a return to the basal level at 1 h.

The expression is strongly upregulated (level 2, eightfold) at 4–6 h together with the acquisition of mating competency (Lin and Goodenough 2007).

Knockdown of MTD1 in mt– cells results in a failure to differentiate into gametes of either mating type after nitrogen removal. From the results, Lin and Goodenough (2007) proposed that the first increase of mid (level 1) is sufficient to activate MTD1 transcription and to repress mt+ gamete-specific genes, and that MTD1 expression in turn allows the second increase (level 2) that is necessary to turn on mt– gamete-specific genes.