Natural variations of SLG1 confer high-temperature tolerance in indica rice

Abstract

With global warming and climate change, breeding crop plants tolerant to high-temperature stress is of immense significance. tRNA 2-thiolation is a highly conserved form of tRNA modification among living organisms. Here, we report the identification of SLG1 (Slender Guy 1), which encodes the cytosolic tRNA 2-thiolation protein 2 (RCTU2) in rice. SLG1 plays a key role in the response of rice plants to high-temperature stress at both seedling and reproductive stages. Dysfunction of SLG1 results in plants with thermosensitive phenotype, while overexpression of SLG1 enhances the tolerance of plants to high temperature. SLG1 is differentiated between the two Asian cultivated rice subspecies, indica and japonica, and the variations at both promoter and coding regions lead to an increased level of thiolated tRNA and enhanced thermotolerance of indica rice varieties. Our results demonstrate that the allelic differentiation of SLG1 confers indica rice to high-temperature tolerance, and tRNA thiolation pathway might be a potential target in the next generation rice breeding for the warming globe.

Fig. 1. SLG1 is a positive regulator of thermotolerance.

a, b Phenotypes of whole plant and grain shape of slg1, WT, and the transgenic T₂ lines of slg1-C, SLG1-RNAi, and SLG1-OE. C, complementation; OE, overexpression; RNAi, RNA interference. Bars = 10 cm (a) and 1 cm (b). c Fine mapping of the SLG1 gene. A single-nucleotide substitution at the splicing acceptor site of the fifth intron of LOC_Os12g39840 in slg1 was indicated. d SLG1 and RCTU1 show high-temperature-inducible expression. Total RNA was extracted from the shoots of 2-leaf-stage seedlings at the indicated time-points. The expression levels were determined by qPCR, and the expression level at time 0 was set to 1. Actin was used as the internal control. Data are means ± SD (n = 3 biological replicates). e–g SLG1 is a positive regulator of thermotolerance. Phenotypes of WT, slg1, SLG1-RNAi, and SLG1-OE plants before and after treatment are shown. Bars = 5 cm. h Dysfunction of RCTU1 shows thermosensitive phenotype similar to the slg1 mutant. Phenotypes of RCTU1-RNAi plants on the background of slg1 and WT are shown before and after treatment. Bar = 5 cm. The source data underlying (d) are provided as a Source data file.

Fig. 2. SLG1-mediated thermotolerance is positively correlated with thiolated tRNA level.

a Comparison of thiolated tRNA level between slg1 and WT. Shoots of 2-leaf-stage seedlings under normal conditions were sampled. The slow-migration band indicates thiolated tRNAs in the presence of APM. The loaded amount of tRNA was 1, 2, and 5 µg for each lane, respectively. b Northern blot analysis. The loaded amount of tRNA was 1 µg, which was extracted from 2-leaf-stage normal-grown seedlings. Three tRNA isoacceptors of tK^UUU, tE^UUC, and tQ^UUG were used to detect the specifically thiolated tRNAs. The bands observed in tK (arrowhead) might be indicative of tRNA halves or tRNA degradation. c tRNA thiolation process was adversely affected by high-temperature stress. Shoots of 2-leaf-stage seedlings grown under high-temperature (45 °C, 44 h) and normal conditions were sampled, respectively. The loaded amount of tRNA was 1, 2, and 5 µg for each lane, respectively. NC, normal conditions; HC, high-temperature conditions. d–f Comparison of thiolated tRNA level. Shoots of 2-leaf-stage seedlings under high-temperature (45 °C, 44 h; g) or normal conditions (h, i) were sampled, respectively. The loaded amount of tRNA was 1, 2, and 5 µg (d), 3 µg (e), 1 and 3 µg (f) for each lane, respectively. The experiments in (a–f) were repeated three times with similar results. Source data are provided as a Source data file.

Fig. 3. SLG1 is differentiated in Asian cultivated rice.

a Haplotype analysis of SLG1 promoter and coding regions from 4219 Asian cultivated rice accessions. Red letters indicate different nucleotides, and gray-marked letters indicate non-synonymous substitutions. b Distribution frequency of the five SLG1 haplotypes in diverse Asian cultivated rice collection. The number of cultivars for each haplotype was given from left to right below each subpopulation. The haplotype with the largest number was highlighted in red. The source data underlying (b) are provided as a Source data file.

Fig. 4. SLG1^Ind confers higher thermotolerance than SLG1^Tej.

a Chromosome maps of NIL-SLG1^Ind and NIL-SLG1^Tej. Red rectangles indicate the donor segment containing the SLG1 locus. NIL-SLG1^Ind indicates KY131 (temperate japonica) with SLG1^Ind allele from ZF802 (indica), and NIL-SLG1^Tej indicates ZF802 with SLG1^Tej allele from KY131. b Comparison of thermotolerance between SLG1^Ind and SLG1^Tej alleles. Two-leaf-stage seedlings were treated with 45 °C for 56 h for KY131 and NIL-SLG1^Ind, and 64 h for ZF802 and NIL-SLG1^Tej, respectively. The seedlings were then recovered under normal conditions for 10 days. Data are means ± SD (n = 3 biological replicates, with 80–96 seedlings per biological replicate). **P < 0.01, two-tailed Student’s t-test. Bar = 5 cm. The source data underlying (b) are provided as a Source data file.

Fig. 5. SLG1^Ind-complemented plants show strong thermotolerance and high thiolated tRNA level.

a, b Comparison of thermotolerance among IPIC, IPJC, JPIC, and JPJC plants. Three independent homozygous IPIC, IPJC, JPIC, and JPJC lines were selected and treated with 45 °C for 56 h and then recovered under normal condition for 10 days. Data are means ± SD (n = 3 independent transgenic lines. Each transgenic line was analyzed with three biological replicates, with 40–56 seedlings per biological replicate). *P < 0.05, **P < 0.01, ***P < 0.001, two-tailed Student’s t-test. Bar = 5 cm. c, d Detection of thiolated tRNAs in slg1, WT, IPIC, and JPJC transgenic lines based on LC/MS-QQQ- (c) and APM- (d) methods. Shoots of 2-leaf-stage seedlings under normal growth conditions were sampled. The loaded amount of tRNA was 3 µg for each lane (d). Data are means ± SD (n = 3 biological replicates). ****P < 0.0001, two-tailed Student’s t-test. The experiment in (d) was repeated three times with similar results. The source data underlying (b–d) are provided as a Source data file.

Fig. 6. SLG1^Ind enhances thermotolerance at the reproductive stage.

a, b The slg1 mutant is highly sensitive to high temperature at the reproductive stage. Seed-setting rate was determined after treatment under 14 h light (40 °C)/10 h dark (31 °C) for 5 days, and recovered under normal conditions until seed maturation. Data are means ± SD (n = 10 panicles with three biological replicates). ****P < 0.0001, two-tailed Student’s t-test. Bar = 5 cm. c, d Comparison of thermotolerance among IPIC, IPJC, JPIC, and JPJC plants. Eight independent homozygous IPIC, IPJC, JPIC, and JPJC lines were treated with 14 h light (40 °C)/10 h dark (31 °C) for 5 days and 7 days, respectively, and then recovered under normal conditions until seed maturation. The x-axis indicates different transgenic lines. Data are means ± SD (n = 5 panicles with three biological replicates). Bar = 5 cm. The source data underlying (b), (d) are provided as a Source data file.

Fig. 7. Selection model of the SLG1 alleles during domestication.

SLG1 is diversified among wild rice accessions. The intimate association of tRNA thiolation and thermotolerance led to the artificial selection of the SLG1^Ind allele for the adaptation of indica rice to geographical regions with high environmental temperature. Interestingly, the SLG1^Tej allele was also subjected to selection during japonica domestication, which might be due to the dynamic response of tRNA thiolation to various environmental stresses.