Journal: Nature Communications

Article Title: A naturally occurring SNP modulates thermotolerance divergence among grapevines

doi: 10.1038/s41467-025-60209-2

Figure Lengend Snippet: a Thermotolerance evaluation of grape germplasms. Materials used include 23 accessions of V. vinifera ( Vv ), 86 accessions of interspecific hybrids between V. vinifera and wild species ( Vv × Ws ), and 12 accessions of wild species ( Ws ). F v / F m values represent the potential maximum quantum yield of primary photochemistry in leaves, n = 23/86/12 plants. b Manhattan plot of GWAS for F v / F m indicating thermotolerance. The red line denotes the threshold (-log 10 [1/328,870]): 5.5, based on the Bonferroni test. c Quantile-quantile plot for the GWAS based on thermotolerance of 121 grapevine accessions under a mixed linear model (MLM). -Log 10 -transformed observed P values are plotted against -Log 10 -transformed expected P values. d Heat map of 16 up-regulated genes from the integrated analysis of GWAS and RNA-seq (fold change ≥1.5) in heat tolerant V. davidii ‘Tangwei’ (TW) and heat sensitive V. vinifera ‘Jingxiu’ (JX) under different high temperatures. e The relative expression levels of the above 16 candidate genes in the leaves of TW and JX grapevines subjected to 42 °C for 0, 1, 2, 4, and 8 h. f Analysis of candidate VIT_204s0023g00470 expression in various organs and tissues of TW and JX. Then we named VIT_204s0023g00470 as TTC4 (thermotolerance on chromosome 4). g Analysis of TTC4 TW and TTC4 JX transcriptional activity in maize protoplasts . The coding sequences of both TTC4 TW and TTC4 JX were fused with the Gal4 binding domain of the Gal4BD vector, respectively. In ( a ), different lowercase letters indicate statistically significant differences at P = 0.05 by one-way ANOVA with Duncan’s multiple-range test, and data represent means ± standard deviation (SD). In ( b , c ), n = 121; In ( f , g ), asterisks indicate statistical significance by two-tailed Student’s t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001), data represent means ± SD ( n = 3 biological replicates). Source data is provided as a Source Data file.

Article Snippet: Fig. 3 TTC4 directly activates the expression of HSP18.1 and APX3. a-c , Genome-wide identification of TTC4 binding sites using DNA affinity purification combined with high-throughput Illumina sequencing (DAP-seq). a Metaplot of TTC4 binding sites.

Techniques: Transformation Assay, RNA Sequencing, Expressing, Activity Assay, Binding Assay, Plasmid Preparation, Standard Deviation, Two Tailed Test

Journal: Nature Communications

Article Title: A naturally occurring SNP modulates thermotolerance divergence among grapevines

doi: 10.1038/s41467-025-60209-2

Figure Lengend Snippet: The phenotype ( a ), the relative electrolyte leakage ( b ) and the F v / F m values ( c ) of leaves from empty vector (EV), overexpressing TTC4 TW (OE- TTC4 TW ) and overexpressing TTC4 JX (OE- TTC4 JX ) V. vinifera ‘Jingxiu’ (JX) plantlets following a 330-min treatment at 25 °C or 42 °C. d-f The phenotype ( d ), the relative electrolyte leakage ( e ) and the F v / F m values ( f ) of leaves from empty vector (EV) and RNAi of TTC4 (Si TTC4 ) V. quinquangularis ‘Yeniang 2’ (Y2) plants following a 180-min treatment at 25 °C or 42 °C. g, h The phenotype ( g ) and the fresh weight ( h ) of ‘41B’ ( V. vinifera ‘Chasselas’ × V. berlandieri ) grape suspension cells of empty vector (EV), overexpressing TTC4 TW (OE- TTC4 TW ) and overexpressing TTC4 JX (OE- TTC4 JX ) after the treatments of 25 °C or 45 °C for 120 min followed by 7 d of recovery. Scale bars in panel ( a , d , g ) represent 2.5 cm. The chlorophyll α fluorescence F v /F m values indicate the potential maximum quantum yield of primary photochemistry) of leaves after high temperature treatments. In ( b , c , e , f , h ), asterisks indicate statistical significance by two-tailed Student’s t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001), ns indicates no significant difference. Data represent means ± SD ( n = 3 biological replicates). Source data is provided as a Source Data file.

Article Snippet: Fig. 3 TTC4 directly activates the expression of HSP18.1 and APX3. a-c , Genome-wide identification of TTC4 binding sites using DNA affinity purification combined with high-throughput Illumina sequencing (DAP-seq). a Metaplot of TTC4 binding sites.

Techniques: Plasmid Preparation, Suspension, Fluorescence, Two Tailed Test

Journal: Nature Communications

Article Title: A naturally occurring SNP modulates thermotolerance divergence among grapevines

doi: 10.1038/s41467-025-60209-2

Figure Lengend Snippet: a-c , Genome-wide identification of TTC4 binding sites using DNA affinity purification combined with high-throughput Illumina sequencing (DAP-seq). a Metaplot of TTC4 binding sites. The TTC4 binding sites are centered on the promoter. TSS, transcription start site. TES transcription end site. b Distribution of TTC4 binding peaks across genomic features. c DNA logos of enriched DNA binding sites for TTC4 as determined by Homer (version 3). The P -values are shown. d , e Identification of TTC4 binding to the promoter of HSP18.1 and APX3 by yeast one-hybrid assays. The orange triangle represents the core component (TGAC) of W-box located on the promoter of HSP18.1 or APX3 . Based on the location of TGAC, the promoters of HSP18.1 and APX3 were divided into three fragments (P1, P2 and P3), respectively, which were then fused with the placZi2μ vector. The coding sequences of TTC4 TW and TTC4 JX were then fused with the activation domain of pB42AD vector, resulting in TTC4 TW -pB42AD and TTC4 JX -pB42AD. f Electrophoretic mobility shift assays (EMSAs) show TTC4 TW and TTC4 JX could bind to the W-box of P2-2 and P2-4 in the promoter of HSP18.1 , and P2-5 in the promoter of APX3 . Both TTC4 TW and TTC4 JX were fused with HIS and SUMO tags. Cold probe, unlabeled probe. mProbe, mutated probe. g – j Identification of TTC4 activating the expression of HSP18.1 and APX3 by dual luciferase gene reporter assays. The coding sequence of TTC4 TW or TTC4 JX was fused with CaMV35S promoter of pCAMBIA-2300 as effector, respectively. The promoter of HSP18.1 or APX3 was inserted into the pGreenII-0800-LUC vector as reporter. Effectors and reporters were co-transformed into tobacco leaves. The ratio of LUC/REN indicates the relative luciferase activity. In ( g – j ), asterisks indicate statistical significance by two-tailed Student’s t -tests (* P < 0.05, *** P < 0.001). Data represent means ± SD ( n = 3 biological replicates). Source data are provided as a Source Data file.

Article Snippet: Fig. 3 TTC4 directly activates the expression of HSP18.1 and APX3. a-c , Genome-wide identification of TTC4 binding sites using DNA affinity purification combined with high-throughput Illumina sequencing (DAP-seq). a Metaplot of TTC4 binding sites.

Techniques: Genome Wide, Binding Assay, Affinity Purification, High Throughput Screening Assay, Illumina Sequencing, Plasmid Preparation, Activation Assay, Electrophoretic Mobility Shift Assay, Expressing, Luciferase, Sequencing, Transformation Assay, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: A naturally occurring SNP modulates thermotolerance divergence among grapevines

doi: 10.1038/s41467-025-60209-2

Figure Lengend Snippet: a – d The expression levels of HSP18.1 and APX3 in transient overexpression (OE- TTC4 TW and OE- TTC4 JX , a , b ) and RNA interference (Si TTC4 , c , d ) of TTC4 . e – g The phenotype ( e ), the relative electrolyte leakage ( f ) and the F v / F m values ( g ) of leaves from empty vector (EV), overexpressing HSP18.1 (OE- HSP18.1 ) and overexpressing APX3 (OE- APX3 ) grapevine plantlets following a 360-min treatment at 25 °C or 42 °C. h-j The phenotype ( h ), the relative electrolyte leakage ( i ) and the F v / F m values ( j ) of leaves from empty vector (EV), RNA interference of HSP18.1 (Si HSP18.1 ) and RNA interference of APX3 (Si APX3 ) grapevine plants following a 210-min treatment at 25 °C or 42 °C. Scale bars represent 2.5 cm. The F v / F m values represent the potential maximum quantum yield of primary photochemistry of the leaves. In ( a – d , f , g , i , j ), asterisks indicate statistical significance by two-tailed Student’s t -tests (* P < 0.05, ** P < 0.01, *** P < 0.001), ns indicates no significant difference. Data represent means ± SD ( n = 3 biological replicates). Source data are provided as a Source Data file.

Article Snippet: Fig. 3 TTC4 directly activates the expression of HSP18.1 and APX3. a-c , Genome-wide identification of TTC4 binding sites using DNA affinity purification combined with high-throughput Illumina sequencing (DAP-seq). a Metaplot of TTC4 binding sites.

Techniques: Expressing, Over Expression, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: A naturally occurring SNP modulates thermotolerance divergence among grapevines

doi: 10.1038/s41467-025-60209-2

Figure Lengend Snippet: a An association analysis between genetic variations in TTC4 and F v / F m values using 121 grapevine accessions, n = 121. Significance was determined based on Bonferroni test with a threshold of P ≤ 1.0 × 10 −4.4 (0.1/2255). b Analysis of TTC4 structure. Top, the schematic of the gene structure and two significant allelic variation in TTC4 between TW and JX. The number represents the position of the base. Bottom, the schematic of the luciferase (LUC) reporter gene driven by TTC4 promoter ( pro ), different TTC4 introns ( int1-1 , int1-2 , int2-1 , int2-2 , int3 and int4 ), and TTC4 promoter fusion with different TTC4 introns ( proint1-1 , proint1-2 , proint2-1 , proint2-2 , proint3 and proint4 ). c Analysis of the relative luciferase activity driven by TTC4 TW promoter ( pro TW ), different TTC4 TW introns ( int1-1 TW , int1-2 TW , int2-1 TW , int2-2 TW , int3 TW and int4 TW ), and TTC4 TW promoter fused with different TTC4 TW introns ( pro TW int1-1 TW , pro TW int1-2 TW , pro TW int2-1 TW , pro TW int2-2 TW , pro TW int3 TW and pro TW int4 TW ) in tobacco leaves. d Analysis of the relative luciferase activity driven by TTC4 JX promoter ( pro JX ), different TTC4 JX introns ( int1-1 JX , int1-2 JX , int2-1 JX , int2-2 JX , int3 JX and int4 JX ), and TTC4 JX promoter fused with different TTC4 JX introns ( pro JX int1-1 JX , pro JX int1-2 JX , pro JX int2-1 JX , pro JX int2-2 JX , pro JX int3 JX and pro JX int4 JX ) in tobacco leaves. e Analysis of the relative luciferase activity driven by TTC4 promoter ( pro TW , pro JX ), intron2-2 ( int2-2 TW , int2-2 JX ), and promoter fused with intron2-2 ( pro TW int2-2 TW , pro JX int2-2 JX ) in the same tobacco leaves. f Mutation and complementary analysis of the SNP-C/T (7631) locus of TTC4 intron2-2 in JX plantlets. The introns of TTC4 JX(7631) , TTC4 JX(7631) complementary, TTC4 TW(7631) mutation, and TTC4 TW(7631) were fused with pGreenII-0800-LUC vector, respectively. The constructed vectors were then transformed into JX plantlets. After a 180-min treatment at 25 °C and 37 °C, the relative luciferase activity of transformed JX plantlets was determined. The ratio of LUC/REN represents the relative luciferase activity in panel ( c – f ). In ( c – f ), different lowercase letters indicate statistically significant differences at P = 0.05 by one-way ANOVA with Duncan’s multiple-range test. Data represent means ± SD ( n = 3 biological replicates). Source data is provided as a Source Data file.

Article Snippet: Fig. 3 TTC4 directly activates the expression of HSP18.1 and APX3. a-c , Genome-wide identification of TTC4 binding sites using DNA affinity purification combined with high-throughput Illumina sequencing (DAP-seq). a Metaplot of TTC4 binding sites.

Techniques: Luciferase, Activity Assay, Mutagenesis, Plasmid Preparation, Construct, Transformation Assay

Journal: Nature Communications

Article Title: A naturally occurring SNP modulates thermotolerance divergence among grapevines

doi: 10.1038/s41467-025-60209-2

Figure Lengend Snippet: a Identification of the direct-regulation factors of TTC4 by yeast one-hybrid assays. The coding sequence of SPL1, SPL3, SPL9, SPL13, SPL14, or GATA25 was fused with the activation domain of pB42AD vector respectively, resulting in SPL1-pB42AD, SPL3-pB42AD, SPL9-pB42AD, SPL13-pB42AD, SPL14-pB42AD, and GATA25-pB42AD. The second intron of TTC4 T(7631) ( int2-2 - TTC4 T(7631) ) or TTC4 C(7631) ( int2-2 - TTC4 C(7631) ) was fused with LacZ reporter gene of placZi2μ vector respectively, resulting in int2-2 - TTC4 T(7631) -placZi2μ and int2-2 - TTC4 C(7631) -placZi2μ. b EMSAs show SPL13 binds to int2-2 - TTC4 C(7631) . SPL13 was fused with HIS and SUMO tags, respectively. Cold Probe, unlabeled probe. mProbe, mutated probe. c, d Identification of SPL13 regulating the expression of TTC4 T(7631) or TTC4 C(7631) by dual luciferase gene reporter assays. The coding sequence of SPL13 was fused with CaMV35S promoter of pCAMBIA-2300 as effector. The second intron of TTC4 T(7631) or TTC4 C(7631) was inserted into the pGreenII-0800-LUC vector as reporter. Effectors and reporters were co-transformed into tobacco leaves. The ratio of LUC/REN means the relative luciferase activity. e-g The phenotype ( e ), the relative electrolyte leakage ( f ) and the F v / F m values ( g ) of leaves from empty vector (EV) and overexpressing SP13 (OE- SPL13 ) of grapevine plantlets following a 300-min treatment at 25 °C or 42 °C. Scale bars represent 2.5 cm. The F v / F m values represent the potential maximum quantum yield of primary photochemistry of the leaves. In ( c, d, f, g ), asterisks indicate statistical significance by two-tailed Student’s t -tests (** P < 0.01, *** P < 0.001), ns indicates no significant difference. Data represent means ± SD ( n = 3 biological replicates). Source data are provided as a Source Data file.

Article Snippet: Fig. 3 TTC4 directly activates the expression of HSP18.1 and APX3. a-c , Genome-wide identification of TTC4 binding sites using DNA affinity purification combined with high-throughput Illumina sequencing (DAP-seq). a Metaplot of TTC4 binding sites.

Techniques: Sequencing, Activation Assay, Plasmid Preparation, Expressing, Luciferase, Transformation Assay, Activity Assay, Two Tailed Test

Journal: Nature Communications

Article Title: A naturally occurring SNP modulates thermotolerance divergence among grapevines

doi: 10.1038/s41467-025-60209-2

Figure Lengend Snippet: a PCA plots of TTC4 sequences from 121 diverse grapevine accessions including 12 wild species ( Ws ), 86 hybrids between V. vinifera and wild species ( Vv × Ws ) and 23 cultivars of V. vinifera ( Vv ) (Supplementary Data ), n = 12/86/23 plants. b Phylogram of TTC4 generated from the above 121 grapevine accessions. c Nucleotide diversity of TTC4 in the above 121 grapevine accessions. d Neutrality tests of TTC4 in the above 121 grapevine accessions. e Haplotype network of TTC4 illustrated through the minimum-spanning tree in the above 121 grapevine accessions. Circle size is proportional to the number of grapevine accessions with a specific haplotype. Circle colors depict different grapevine species. f The distribution of TTC4 haplotypes (T(7631) and C(7631)) in the above 121 diverse grapevine accessions. g Genotype analysis based on the thermotolerance ( F v / F m values) and the SNP-C/T (7631) in the above 121 accessions. C/C ( n = 58 plants), C/T ( n = 40 plants), and T/T ( n = 23 plants) h Genotype analysis based on the thermotolerance ( F v / F m values) and the SNP-C/T (7631) in 84 accessions (Supplementary Data ). C/C ( n = 62 plants), C/T ( n = 5 plants), and T/T ( n = 17 plants). i Genotype analysis based on the thermotolerance ( F v / F m values) and the SNP-C/T (7631) in the hybrid population of ‘Gupu 1’ ( V. quinquangularis × V. vinifera ) carrying C/T and ‘Cabernet Sauvignon’ ( V. vinifera ) carrying C/C (Supplementary Data ). The red solid circles in C/C and C/T represent the thermotolerance of ‘Cabernet Sauvignon’ and ‘Gupu 1’, respectively. C/C ( n = 52 plants) and C/T ( n = 68 plants). j Genotype analysis based on the thermotolerance ( F v / F m values) and the SNP-C/T (7631) in the hybrid population of ‘Manicure Finger’ ( V. vinifera ) carrying C/C and ‘0940’ ( V. davidii ) carrying T/T (Supplementary Data ). C/C and T/T represent ‘Manicure Finger’ and ‘0940’, respectively. C/C ( n = 6 plants), C/T ( n = 92 plants), and T/T ( n = 6 plants). The F v / F m values represent the potential maximum quantum yield of primary photochemistry of the leaves. In ( b – f ), n = 12/86/23 plants. In ( c , d , g , h , j ), different lowercase letters indicate statistically significant differences at P = 0.05 by one-way ANOVA with Duncan’s multiple-range test, and data represent means ± SD. In ( i ), asterisks indicate statistical significance by two-tailed Student’s t -tests (*** P < 0.001). Data represent means ± SD. Source data are provided as a Source Data file.

Article Snippet: Fig. 3 TTC4 directly activates the expression of HSP18.1 and APX3. a-c , Genome-wide identification of TTC4 binding sites using DNA affinity purification combined with high-throughput Illumina sequencing (DAP-seq). a Metaplot of TTC4 binding sites.

Techniques: Generated, Two Tailed Test

Journal: Nature Communications

Article Title: A naturally occurring SNP modulates thermotolerance divergence among grapevines

doi: 10.1038/s41467-025-60209-2

Figure Lengend Snippet: Under heat stress, the expression level of SPL13 decreases; SPL13 can bind to the TTC4 intron 2 containing the SNP-C(7631) in heat sensitive grapevines, leading to the inhibition of TTC4 C(7631) expression. However, SPL13 is unable to bind to the TTC4 intron 2 containing the SNP-T(7631) in heat tolerant grapevines, thereby not influencing TTC4 T(7631) expression. In addition, the ability of TTC4 T(7631) intron 2 in regulating gene expression is significantly higher than that of the TTC4 C(7631) intron 2. Ultimately, although high temperature induces the expression of TTC4 , TTC4 C(7631) shows a weaker expression level, and has a weak activation effect on heat stress-responsive genes ( HSP18.1 and APX3 ), resulting in corresponding grapevine germplasms exhibiting heat sensitivity. However, TTC4 T(7631) exhibits a higher expression level, and has a strong activation of heat stress-responsive genes. The corresponding grapevine germplasms are heat tolerant. This fine-tuned regulation allows grapevines to respond differentially to heat stress.

Article Snippet: Fig. 3 TTC4 directly activates the expression of HSP18.1 and APX3. a-c , Genome-wide identification of TTC4 binding sites using DNA affinity purification combined with high-throughput Illumina sequencing (DAP-seq). a Metaplot of TTC4 binding sites.

Techniques: Expressing, Inhibition, Gene Expression, Activation Assay