Structure and Function of the Primary Zygote Wall of Chlamydomonas Monoica

The Chlamydomonas zygospore is formed after gametic cell fusion by a progression of ultrastructural and physiological changes, including the synthesis of a protective and elaborately sculptured secondary wall (Figure 1). Assembly of the secondary wall occurs within the confines of a transient primary zygote wall, synthesized immediately after gamete fusion, and assembled beneath the residual gamete walls. Upon completion of secondary wall assembly, the primary zygote wall is shed from the maturing zygospore (Figure 2). This primary zygote wall, composed primarily of a β-1,3 glucan (callose), may be important in the regulation of secondary zygospore wall formation.A temperature-sensitive mutant strain of C. monoica, zym30, has been isolated that shows marked abnormalities in both primary and secondary wall ultrastructure. The degree of wall formation in the mutant strain is inversely proportional to temperature. At 25° C most zygotes fail to synthesize either the primary or the secondary wall. At 15° C both primary and secondary wall assembly occurs to some extent and as many as 50% of the zygotes develop a “pseudo-wildtype” phenotype (Figure 3). Although release of the primary wall rarely occurs in the zym30 strain (even at permissive temperatures), those walls that are released appear thinner than the walls shed from wildtype zygospores, as viewed by phase contrast microscopy. Fluorescence images incorporating aniline blue, a fluorochrome specific for β-1,3 glucans, show decreased fluorescence suggesting reduced callose synthesis (Figure 4).Several permanent, ultrastructurally distinct layers comprise the secondary wall, the assembly of which may be controlled, in part, by the primary wall. One or more of these secondary wall layers contain the highly durable, autofluorescent biopolymer, sporopollenin. Autofluorescence of zym30 zygospores produced at the permissive temperature is enhanced relative to wildtype zygospores. This correlates with ultrastructural changes in the primary wall and overproduction and/or misassembly of the surface secondary wall layer (Figure 5). Work is in progress to determine whether the environmental resistance, and/or viability of the zym30 “pseudowildtype” zygospores are reduced relative to wildtype zygospores.

The Chlamydomonas Zygospore: Mutant Strains of Chlamydomonas monoica Blocked in Zygospore Morphogenesis Comprise 46 Complementation Groups

Chlamydomonas monoica undergoes homothallic sexual reproduction in response to nitrogen starvation. Mating pairs are established in clonal culture via flagellar agglutination and fuse by way of activated mating structures to form the quadriflagellate zygote. The zygote further matures into a dormant diploid zygospore through a series of events that we collectively refer to as zygosporulation. Mutants that arrest development prior to the completion of zygosporulation have been obtained through the use of a variety of mutagens, including ultraviolet irradiation, 5-fluorodeoxyuridine, ethyl methanesulfonate, and methyl methanesulfonate. Complementation analysis indicates that the present mutant collection includes alleles affecting 46 distinct zygote-specific functions. The frequency with which alleles at previously defined loci have been recovered in the most recent mutant searches suggests that as many as 30 additional zygote-specific loci may still remain to be identified. Nevertheless, the present collection should provide a powerful base for ultrastructural, biochemical, and molecular analysis of zygospore morphogenesis and dormancy in Chlamydomonas.