The Chlamydomonas hatching enzyme, sporangin, is expressed in specific phases of the cell cycle and is localized to the flagella of daughter cells within the sporangial cell wall.

The timely breakdown of the extracellular matrix by proteolytic enzymes is essential for development, morphogenesis and cell proliferation in plant and animal cells. Sporangin of the unicellular green alga Chlamydomonas reinhardtii that mediates breakdown of the sporangial cell wall to liberate the daughter cells after cell division is characterized as a subtilase-like serine protease. The sporangin gene is specifically transcribed during S/M phase in a synchronized vegetative cell cycle. In immunoblot analyses using a polyclonal antibody raised against the sporangin polypeptide, the enzyme is synthesized after mitotic cell division and accumulated in the daughter cells before hatching. Immunofluorescence analyses showed that sporangin is localized to the flagella of the daughter cells within the sporangial cell wall, and released into the culture medium. The data suggest that sporangin is released from flagella concurrently with the digestion of sporangial cell wall, and then the daughter cells are hatched from the sporangia in the Chlamydomonas vegetative cell cycle.

Characterization of novel genes induced by sexual adhesion and gamete fusion and of their transcriptional regulation in Chlamydomonas reinhardtii.

When mating type plus and minus gametes of Chlamydomonas are mixed, they agglutinate with each other via their flagella, fuse, then initiate the zygote formation program which includes synthesis of the zygote cell wall, fusion of nuclei and chloroplasts, and the digestion of chloroplast DNA from the minus parent. The mRNAs from gamete and zygote cells was isolated and hybridized to cDNA-macroarray filters both to identify new genes expressed during the mating reaction and the early zygote formation process and to analyze the gene expression programs that underlie these sexual processes. Twenty-one novel genes were identified in this screen, designated as EZY (early zygote expressed) genes. The EZY genes included genes encoding proteins whose function is unknown, and genes encoding proteins that appear to be involved in processes such as cell wall synthesis, gene expression, intracellular trafficking or secretion, and vesicular transport in zygotic cells. All of the EZY genes were strongly induced within 1 h during the mating process, including early zygote formation. The transcriptional characteristics of EZY genes were analyzed by using the fusion-defective mutant fus mt(+). Among the EZY genes, 12 genes were not activated in fusion-defective conditions, suggesting that cell fusion is required for their expression. The remaining nine that were transcribed in fusion-defective fus matings were also inducible by cell wall removal in either vegetative or gametic cells, indicating that these genes were induced only indirectly by the cAMP signaling pathway initiated by flagellar agglutination as a result of mating-induced cell wall loss.

The regulatory networks of gene expression during the sexual differentiation of Chlamydomonas reinhardtii, as analyzed by mutants for gametogenesis.

Cells of Chlamydomonas reinhardtii differentiate into gametes under conditions of nitrogen (N) starvation, expressing the genes for the N-adaptation program and the gamete program. To investigate the regulatory networks of transcription among the N-starvation-inducible genes, we examined the gene expression in dif mutants, affecting gametic differentiation. In a conditional mutant, dif2, the cells remained 'vegetative' at the restrictive temperature, and the induction of 20 out of 21 genes related to the two programs was impaired. They were expressed soon after transfer of the cells to the permissive temperature, in parallel with the acquisition of mating ability. In an unconditional mutant, dif3, the cells could not differentiate into gametes at all, but the induction of only four genes (FUS1, NSG3, NSG6 and NSG7) related to the gamete program was impaired. The results suggest that Dif3 regulates putative N-starvation signal transduction pathways downstream of a master regulator, Dif2. We also examined a light-dependent laboratory strain that was unable to become gametes in the dark. The 'pre-gametes' placed in the dark, however, could induce normally all of the 21 genes, suggesting that light is required for the gametic differentiation at the translational and/or post-translational levels.

Genealogical relationships among laboratory strains of Chlamydomonas reinhardtii as inferred from matrix metalloprotease genes.

Almost all research on Chlamydomonas reinhardtii has utilized wild-type strains of three principal stock lines (Sager, Cambridge, Ebersold-Levine), which traditionally has been assumed to be descendants of a single zygote isolated by Smith. We previously noticed that there are several sequence differences in a single-copy gene of gametolysin, mmp1, between the mt+ and mt- strains employed. To further examine the polymorphisms among the three lines, we obtained 18 representative strains of all three descendant lines of Smith's isolate, nine strains recently isolated from the wild and one strain (CC-1373 mt+) of C. smithii, a strain from Smith's collection interfertile with these C. reinhardtii strains; and we compared the mmp1-3'UTR by RFLP and sequencing analyses. Sequence divergences were found between the mt+ and mt- strains of both the Sager and Cambridge lines, but not between the two mating-type strains of the Ebersold-Levine line. We also examined the polymorphisms, using the 3'UTRs of two other mmp genes and the introns of ypt4 and fus1. Based on the results, we conclude that it is genetically impossible for all the current C. reinhardtii lines to be the immediate descendants of a single zygote.