Gene Regulatory Networks for the Haploid-to-Diploid Transition of Chlamydomonas reinhardtii

Abstract

The sexual cycle of the unicellular Chlamydomonas reinhardtii culminates in the formation of diploid zygotes that differentiate into dormant spores that eventually undergo meiosis. Mating between gametes induces rapid cell wall shedding via the enzyme g-lysin; cell fusion is followed by heterodimerization of sex-specific homeobox transcription factors, GSM1 and GSP1, and initiation of zygote-specific gene expression. To investigate the genetic underpinnings of the zygote developmental pathway, we performed comparative transcriptome analysis of both pre- and post-fertilization samples. We identified 253 transcripts specifically enriched in early zygotes, 82% of which were not up-regulated in gsp1 null zygotes. We also found that the GSM1/GSP1 heterodimer negatively regulates the vegetative wall program at the posttranscriptional level, enabling prompt transition from vegetative wall to zygotic wall assembly. Annotation of the g-lysin-induced and early zygote genes reveals distinct vegetative and zygotic wall programs, supported by concerted up-regulation of genes encoding cell wall-modifying enzymes and proteins involved in nucleotide-sugar metabolism. The haploid-to-diploid transition in Chlamydomonas is masterfully controlled by the GSM1/GSP1 heterodimer, translating fertilization and gamete coalescence into a bona fide differentiation program. The fertilization-triggered integration of genes required to make related, but structurally and functionally distinct organelles-the vegetative versus zygote cell wall-presents a likely scenario for the evolution of complex developmental gene regulatory networks.

Figure 1.

A gene-regulatory network model of sexual development in Chlamydomonas reinhardtii consisting of at least six regulatory programs. Gray bars depict the duration of each program. The upper bar shows physiological/cellular events providing cues for the programs. Arrows denote the consequence of specific events/factors as induction or repression. Known molecular players are shown as boxes, blue for plus-specific and red for minus-specific. NS/GAM, N-starvation induced/gametogenesis program; PG/MG, plus- and minus-specific gamete programs; MR, mating reaction-induced program (dibutyryl-cAMP-inducible); gL, g-lysin-induced program; FD/HB, fusion-dependent/homeobox transcription factor-dependent program.

Figure 2.

Identification of coregulated genes in the early zygote transcriptome by hierarchical clustering analysis. Relative expression patterns among 13 conditions are shown for selected gene clusters representing the early zygote-specific clusters (C33, C43, C50, and C10) and the g-lysin-induced clusters (C44 and C24). Box plots show distribution of percentage expression values per condition, calculated relative to the sum of FPKM values in all 13 conditions per gene. Boxes show the median and the 25th and 75th percentile. Whiskers indicate the 10th and 90th percentiles. Details pertaining to the remaining clusters are found in Supplemental Tables S4 and S5.

Figure 3.

GSM1/GSP1-dependent activation of EZ-core genes. A, Relative abundance of transcripts in early stage zygotes to minus gametes was analyzed using qRT-PCR. Error bars: sd of three biological replicates. g+: plus gametes; g−: minus gametes; Z.5, Z1, and Z2 represent 0.5 h, 1 h, and 2 h after mixing gametes. Black bar, CC-621 (mt−) X CC-125 (mt+); Gray bar: CC-621 (mt−) X bp31 (mt+). B, Promoter-luciferase reporter assay of two EZ-core promoters. Fold-difference was calculated from cumulative luciferase-based promoter activity during the early zygote development relative to samples without mating. Error bars, sd of three biological replicates.

Figure 4.

Differential activation of g-lysin-induced genes during the early zygote development. A, Relative abundance of transcripts in early stage zygotes to minus gametes was analyzed using qRT-PCR. Error bars, sd of three biological replicates. g+: plus gametes; g−: minus gametes; Z.5, Z1, and Z2 represent 0.5 h, 1 h, and 2 h after mixing gametes. Black bar, CC-621 (mt−) X CC-125 (mt+); Gray bar, CC-621 (mt−) X bp31 (mt+). B, Promoter-luciferase reporter assay of two EZ-core promoters. Fold-difference was calculated from cumulative luciferase-based promoter activity during the early zygote development relative to samples without mating. Error bars, sd of three biological replicates.

Figure 5.

Distribution of annotated EZ-core/gL+EZ/gL-EZ genes in functional categories related to cellular differentiation (excluding the unknowns). The categories listed in the legend represent, in order, the categories in the pie charts starting from midnight and proceeding clockwise. Cell wall-related categories are marked by thick outlines. Left, EZ-core (n = 204); middle, gL+EZ (C44) (n = 129); right, gL-EZ (C24) (n = 113). Number of genes in the top two categories are shown in pie charts. Annotation details are found in Supplemental Tables S18, S19, and S20.

Figure 6.

Identification of the MAW (membrane-anchored cell wall protein) family encoding HRGPs with a C-terminal hydrophobic domain. A, Domain structure of MAW family proteins in Chlamydomonas. B, Bayesian phylogeny of MAW domains among Volvocacean MAW proteins. Predicted GPI-anchored MAW proteins are found in clades I and II. C, Cys-rich MAW domain consensus sequence. Domain sequence alignment is found in Supplemental Figure S6. D, Differential expression of MAW family genes in Chlamydomonas based on average FPKM values among nine conditions. Upper graph, EZ-specific genes. Lower graph, Non-EZ-specific genes. Due to very low expression, CreMAW5 and CreMAW11 are not included in the graph.

Figure 7.

Predicted flow of cytosolic hexoses into nucleotide-sugar metabolism in support of glycosyl transferases up-regulated during the early zygote development. Cytosolic hexose pool is supplied by gluconeogenesis and export from the chloroplast. Arrows show directional or bidirectional conversions. Red arrows, predicted sugar flow changes in early zygotes, resulting in a large increase in available nucleotide-sugars both in the cytosol and in Golgi via NST-family transporters. Blue arrows, predicted sugar flow increase into GDP-d-Man and GDP-l-Gal in the cytosol. Red metabolites, forms usable by glycosyl transferases. Shaded symbols represent genes. Brown, early zygote-specific or up-regulated (EZ or gL+EZ). Green, g-lysin induced (gL-EZ). Gray, found in other clusters. Details of these genes are found in Supplemental Tables S24 and S25. Putative nucleotide-sugar transporters in ER/Golgi are found in six classes (B-I excluding C and D classes known to be localized to the plasma membrane; Supplemental Table S26). Frc, Fructose; Man, Mannose; Glc, Glucose; Gal, Galactose; Rhm, Rhamnose; GlcA, Glucuronic acid; GalA, Galacturonic acid; Xyl, Xylose; Arap, Arabinose-pyranose; Araf, Arabinose-furanose.