Fig. 1.

Heat and cold tolerance are oppositely regulated by EMB-4. A) Schematic illustration of cold tolerance and heat tolerance assays. In the cold tolerance assay, worms were cultivated at a constant temperature from egg to adult at 15°C or 20°C or 25°C, and then they were incubated at 2°C for 48 h, after which the survival rate was calculated the next day. In the heat tolerance assay also, worms were cultivated at a constant temperature and then incubated at 32°C for 24 h, after which the survival rate was calculated. B) Examination of heat temperature as a heat tolerance. Worms cultivated at 15°C, 20°C, and 25°C, respectively, were subjected to either 30°C, 32°C, or 34°C heat stimulation for 24 h. 30°C heat stimulation exerted no effect on survival, whereas 32°C and 34°C increased the survival as the rearing temperature increased. Number of assays >11 (n = 1,198, 814, and 642 worms for 30°C; n = 899, 971, and 1,207 worms for 32°C; n = 930, 918, and 1,160 worms for 34°C). C, D) emb-4 mutant exhibited abnormal heat and cold tolerance. WT and emb-4 as genotypes in the horizontal axis indicate wild-type and emb-4(hc60) mutant, respectively, and cultivation temperatures are indicated at bottom of the charts. When worms were cultivated at 20°C and 25°C, the survival rates of emb-4 mutant in the heat tolerance assay were significantly lower than those of the wild-type strain, but in the cold tolerance assay, its survival rates were significantly higher than those of the wild-type strain. Number of assays ≥9 (n = 382, 511, 662, 479, 918, and 444 worms). E) Schematic illustration of EMB-4, LHT1/MAC7, and AQR proteins. Caenorhabditis elegans EMB-4, plant LHT1/MAC7, and human AQR all have a RNA helicase domain, a DNA helicase domain, and a linker-domain that links them together. These domain structures were predicted by a database, InterPro 99.0 EMBL-EBI (https://www.ebi.ac.UK/interpro/). F, H) Both emb-4(hc60) and emb-4(qm31) mutants exhibited similar abnormalities in the cold and heat tolerance assays. Number of assays ≥ 7 ([F] n = 629, 243, and 486 worms, [H] n = 639, 307, and 703 worms). G, I) RNA interference of emb-4 caused weakly abnormal phenotypes in the cold and heat tolerance assays, which were consistent with the phenotypes of emb-4 null mutants. Number of assays ≥ 11 ([G] n = 680, 760, 723, and 628 worms, [I] n = 708, 704, 623, and 538 worms). J) A heterozygous animal with the maternal effect mutation emb-4(qm31) demonstrated normal heat tolerance. All the animals were grown at 20°C. Number of assays ≥ 4 (n = 660, 246, and 216 worms). K) Cold tolerance in the emb-4(qm31) heterozygote. All the animals were grown at 20°C. Number of assays ≥ 4 (n = 920, 356, and 216 worms). The error bars indicate SEMs. n.s. P ≥ 0.05; *P < 0.05; **P < 0.01. Comparisons were performed using Welch's t tests (C, D) or one-way ANOVA followed by Dunnett's post hoc tests (F–I), or followed by Tukey Kramer's post hoc tests (J, K).