Figure 5.

aCRY has a positive influence on the germination efficiency, which is supported by blue and red light, and a negative influence on mating ability and maintenance. A, The influence of blue and red light on germination. Blue (465 nm, BL), red (635 nm, RL) and white light (WL, see “Materials and Methods”) were used to illuminate wild-type (WT) zygotes during the germination process. About 50 zygotes were counted for each condition and each replicate (n = 3). Mean values and standard deviations are shown. B, Quantification of soluble aCRY in WT strain CC-125 (mating type plus) and CC-124 (mating type minus), acry_mut strains and corresponding complemented strains. Seventy-five-microgram proteins of each sample were separated by 9% SDS-PAGE and immunoblotted with anti-aCRY antibodies. CC-125 (WT⁺) and CC-124 (WT⁻) were used for comparison and their levels were set to 100%. Unspecified protein bands from the PVDF membrane were used as loading control (LC). The quantifications of aCRY in these strains are based on three independent biological replicates and shown with standard deviations. C, Germination profiles of crosses between WT, acry_mut, and acry_compl strains. Germination of zygotes of several crossing between WT and mutant strains, including WT homozygotes (WT⁺ × WT⁻), heterozygotes (WT⁺ × acry_mut⁻ or acry_mut⁺ × WT⁻), and acry mutant homozygotes (acry_mut⁺ × acry_mut⁻) are shown. In addition, the germination rate of homozygotes of the complemented strains (acry_compl⁺ × acry_compl⁻) is presented. At least three biological replicates were performed. Error bars indicate standard deviations. Student’s t tests were performed (***P < 0.001). D, Quantification of aCRY in total (soluble and membrane-bound) proteins of the different cell types of gametogenesis. The cell types (see “Materials and Methods”) include (1) pG kept in the dark, (2) pG illuminated for 1 h with light resulting in G1 gametes (G1), and (3) G1 gametes that have been exposed to darkness for 1 h resulting in dG1 of WT, acry_mut, and acry_compl mt⁺ strains. Fifty-microgram proteins of each sample were separated by 9% SDS-PAGE and immunoblotted with anti-aCRY antibodies. The expression level of aCRY in pG of WT was used for comparison. Three biological replicates were performed. Error bars indicate standard deviations. E, The influence of aCRY on mating ability and the maintenance of mating ability. Mean values and standard deviations of at least three biological replicates are shown. Student’s t tests were performed (***P < 0.001; **P < 0.01, *P < 0.05). Mating ability assays of WT, acry_mut, and acry_compl strains. pG, G1, and dG1 cells (see D for explanations) were used for the mating ability tests. The mating ability of G1 of WT was set to 100% and used for comparison.