Supplemental Data Integrating omics and alternative splicing i reveals insights i into grape response to high temperature Jianfu Jiang 1, Xinna Liu 1, Guotian Liu, Chonghuih Liu*, Shaohuah Li*, and Lijun Wang* Zhengzhou Fruit Research Institute (J.J., C.L.), Chinese Academy of Agricultural Sciences, Zhengzhou 450009, China; and Institute of Botany (X.L., G.L., S.L., L.W.), Chinese Academy of Sciences, Beijing 100093, China 1 These authors contributed equally to the article. *Address correspondence to ljwang@ibcas.ac.cn, shhli@ibcas.ac.cn and liuchonghuai@caas.cn
Supplemental Figure S1. Cluster analysis for all transcripts in grape leaves under four different temperatures (25 o C, 35 o C, 40 o C and 45 o C). T25_1, T25_2 and T25_3 indicate three replicates of 25 o C treatment. Representations of the other treatments were similar to 25 o C treatment.
25 o C 35 o C 40 o C 45 o C 25 o C 35 o C 40 o C 45 o C Supplemental Figure S2. Summary number of differentially expressed transcripts in grape leaves under 35 o C, 40 o C and 45 o C compared to 25 o C at a fold change larger than 2 and false discovery rate (FDR) less than 0.05.
Quantitative Proteome Temperature Repeat1/2/3 25 35 40 45 Proteome extract Trypsin digestion Sample Preparation RNA-Sequencing Gene expression Alternative splicing itraq-labeling 114 115 116 117 Combine Data analysis and integration RAW MGF X! Tandem 2D-HPLC-MS/MS MaxQuant Protein Quantitation Uniprot database + 3-frame RefSeq transcripts Proteotranscriptomics analysis Functionally integrated Omics Supplemental Figure S3. Flow chart of RNA-sequencing and quantitative proteome for grape leaves.
Supplemental Figure S4. Venn diagram of non-redundant Uniprot annotated proteins identified with at least 2 unique peptides (protein and peptide FDR < 0.01) in grape leaves. EXP1, EXP2 and EXP3 indicate three replicates of each treatments of 25 o C, 35 o C, 40 o C and 45 o C. The overlapping regions correspond to the number of proteins present at more than one temperature treatment.
Up-regulation Down-regulation Supplemental Figure S5. Up and down-regulated biological pathways of differentially expressed proteins in grape leaves under 35 o C compared to 25 o C.
Up-regulation Down-regulation Supplemental Figure S6. Up and down regulated biological pathways of differentially expressed proteins in grape leaves under 40 o C compared to 25 o C.
Supplemental Figure S7a. Up-regulated biological pathways of differentially expressed proteins in grape leaves under 45 o C compared to 25 o C.
Supplemental Figure S7b. Down-regulated biological pathways of differentially expressed proteins in grape leaves under 45 o C compared to 25 o C.
160 140 R² = 0.7884 qrt-pcr R 120 100 80 60 40 20 0 0 50 100 150 200 250 RNA-sequencing Supplemental lfigure S8. Linear correlation analysis (r 2 = 0.7884) between qrt-pcr and RNA-sequencing results for 35 genes in grape leaves under four temperatures (25 o C, 35 o C, 40 o C and 45 o C). The X-axis refers to fold change values from RNA-sequencing data; the Y-axis refers to fold change values from qrt-pcr data.
25 o C-1 25 o C-1 25 o C-2 25 o C-2 25 o C-3 25 o C-3 40 o C-1 45 o C-1 40 o C-2 45 o C-2 40 o C-3 45 o C-3 Fully spliced transcript Exon Intron Exon Exon Intron Exon IR transcript Supplemental Figure S9. The sashimi plots of a HSP90.1 transcript with an IR event in grape leaves under 40 o Cand 45 o Ccomparedto25 o C. Each treatment has three replicates. 25 o C-1, 25 o C-2 and 25 o C-3 represent three replicates of the control 25 o Cfor2h.40 o C-1, 40 o C-2 and 40 o C-3 represent three replicates of the 40 o Cfor2h.45 o C-1, 45 o C-2 and 45 o C-3 represent three replicates of the 45 o C for 2 h. Sashimi plots (stand-alone) alone) for alternatively spliced exon and flanking exons in samples. Per-base expression is plotted on y-axis of Sashimi plot, genomic coordinates on x-axis. Arcs represents splice junctions connecting exons and displays the number of reads split across the junction (junction depth). mrna isoforms quantified are shown on bottom (exons in black, introns as lines with arrow heads).
25 o C-1 25 o C-2 25 o C-3 40 o C-1 40 o C-2 25 o C-1 25 o C-2 25 o C-3 45 o C-1 45 o C-2 40 o C-3 45 o C-3 Exon Intron Exon Exon Intron Exon Fully spliced transcript IR transcript Supplemental Figure S10. The sashimi plots of a HSP25.3 transcript with an IR event in grape leaves under 40 o C and 45 o Ccomparedto25 o C. Each treatment has three replicates. 25 o C-1, 25 o C-2 and 25 o C-3 represent three replicates of the control 25 o Cfor2h.40 o C-1, 40 o C-2 and 40 o C-3 represent three replicates of the 40 o Cfor2h.45 o C-1, 45 o C-2 and 45 o C-3 represent three replicates of the 45 o C for 2 h. Sashimi plots (stand-alone) for alternatively spliced exon and flanking exons in samples. Per-base expression is plotted on y-axis of Sashimi plot, genomic coordinates on x-axis. Arcs represents splice junctions connecting exons and displays the number of reads split across the junction (junction depth). mrna isoforms quantified are shown on bottom (exons in black, introns as lines with arrow heads).
25 o C-1 25 o C-2 25 o C-3 40 o C-1 40 o C-2 40 o C-3 25 o C-1 25 o C-2 25 o C-3 45 o C-1 45 o C-2 45 o C-3 Exon Intron Exon Exon Intron Exon Fully spliced transcript IR transcript Supplemental Figure S11. The sashimi plots of a HSP101 transcript with an IR event in grape leaves under 40 o C and 45 o Ccomparedto25 o C. Each treatment has three replicates. 25 o C-1, 25 o C-2 and 25 o C-3 represent three replicates of the control 25 o Cfor2h.40 o C-1, 40 o C-2 and 40 o C-3 represent three replicates of the 40 o Cfor2h.45 o C-1, 45 o C-2 and 45 o C-3 represent three replicates of the 45 o C for 2 h. Sashimi plots (stand-alone) alone) for alternatively spliced exon and flanking exons in samples. Per-base expression is plotted on y-axis of Sashimi plot, genomic coordinates on x-axis. Arcs represents splice junctions connecting exons and displays the number of reads split across the junction (junction depth). mrna isoforms quantified are shown on bottom (exons in black, introns as lines with arrow heads).
25 o C-1 25 o C-2 25 o C-3 40 o C-1 40 o C-2 40 o C-3 25 o C-1 25 o C-2 25 o C-3 45 o C-1 45 o C-2 45 o C-3 Exon Intron Exon Exon Intron Exon Fully spliced transcript IR transcript Supplemental Figure S12. The sashimi plots of a HSP70.4 transcript with an IR event in grape leaves under 40 o C and 45 o Ccomparedto25 o C. Each treatment has three replicates. 25 o C-1, 25 o C-2 and 25 o C-3 represent three replicates of the control 25 o Cfor2h.40 o C-1, 40 o C-2 and 40 o C-3 represent three replicates of the 40 o Cfor2h.45 o C-1, 45 o C-2 and 45 o C-3 represent three replicates of the 45 o C for 2 h. Sashimi plots (stand-alone) alone) for alternatively spliced exon and flanking exons in samples. Per-base expression is plotted on y-axis of Sashimi plot, genomic coordinates on x-axis. Arcs represents splice junctions connecting exons and displays the number of reads split across the junction (junction depth). mrna isoforms quantified are shown on bottom (exons in black, introns as lines with arrow heads).
VIT_16s0050g01150.1 (HSP90.1) VIT_16s0098g0060.1 (HSP25.3) 25 o C 35 o C 40 o C 45 o C 一 361 bp IR transcript 一 239 bp Fully spliced transcript 672 bp IR transcript 一 592 bp Fully spliced transcript VIT_17s0000g07190.1(HSP101 ) VIT_08s0007g00130.1(HSP70.4) Actin 一 615 bp IR transcript 3 一 505 bp Fully splicedt ranscript5n/ 一 1705 bp IR transcript 一 917 bp Fully spliced transcript 一 82 bp HSP101 25/35/40/45 / / Supplemental Figure S13. RT-PCR analysis of IR splice variants of HSP90.1, HSP25.3, HSP101 and HSP70.4 in grape leaves under 25 o C, 35 o C, 40 o C and 45 o C for 2 h. The forward and reverse primers were designed from the upstream and downstream exons of the retention intron based on each IR event, respectively.
Sample preparation and RNA -sequencing Sequencing reads Genome Map to genome and annotated genes Compute reads distribution Alternative splicing ES IR A5S events S Intron Exon A3SS MXE MXE Differential splicing events under 35 o C, 40 o C and 45 o C compared to25 o C were selected Occurrence of splicing events was showed with Sashimi plots generated by MISO Supplemental Figure S14. Flow chart of alternative splicing analysis for grape leaves under different temperatures
Supplemental Figure S15. The schematic diagrams illustrating the read counts and effective lengths of different categories of alternative splicing events. The alternative splicing events of skipped exons, alternative 5 splice sites, alternative 3 splice sites, and retained introns have two splice junctions for the inclusion isoform and one splice junction for the skipping isoform. The mutually exclusive exons have two splice junctions for the inclusion isoform of the first exon and two splice junctions for the skipping isoform of the first exon (i.e., the inclusion isoform of the second exon). The exon body reads are RNA-Seq reads mapped to the genomic regions of the target exons. The rmats model allows users to use either the splice junction counts plus the exon body counts or the splice junction counts alone as the input. This figure is from Shen et al. (2014).
Supplemental Table S1. Statistics of all AS events in grape leaves under 35 o C, 40 o C and 45 o C compared to 25 o C. 35 o C 40 o C 45 o C IR 7407 7524 7452 ES 5080 6262 5987 A5SS 1757 1883 1816 A3SS 3163 3253 3016 MXE 249 342 288 Total 17656 19624 18559 AS: alternative splicing; IR: intron retention; ES: exon skipping; MXE: mutually exclusive exons; A5SS: alternative 5 splice sites; A3SS: alternative 3 splice sites
Supplemental Table S2. Statistics of all genes with significantly differential AS events in grape leaves under 35 o C, 40 o C and 45 o C compared to 25 o C. 35 o C 40 o C 45 o C Upregulated Downregulated Upregulated Downregulated Upregulated Downregulated IR 46 35 334 115 413 57 ES 22 51 63 269 7 166 A5SS 8 12 81 49 22 26 A3SS 21 9 93 49 28 18 MXE 1 1 13 9 6 5 Total 98 108 584 491 476 272 206 1075 748 AS: alternative splicing; IR: intron retention; ES: exon skipping; MXE: mutually exclusive exons; A5SS: alternative 5 splice sites; A3SS: alternative 3 splice sites
Supplemental Table S3. qrt-pcr primers used in this study. Gene ID Forward primer Reverse primer VIT_10s0003g00260.1 TCACACAACCACAGCCTAC CACCCATCTTCTGCTTTGCT VIT_12s0035g01910.1 AGCCAAGCATCATCCCAATC GGTTTCTGGTGTAGGGCAT VIT_13s0019g02840.1 CGCATTGATTGGAAGGAGAC TCGGTGCCACTTGTCATTCT VIT_16s0050g01150.1 GAAGAAAGGCGAATCAAAGACC GCAAGATAGTCCTCCCAGTCAT VIT_13s0019g03000.1 CCAAACTTCTTAGGCGGTC TCCTCCCTTCCTCAACCTC VIT_13s0019g02780.1 CCAAACTTCTTAGGCGGTC TCCTCCCTTCCTCAACCTC VIT_13s0019g02740.1 CCAAACTTCTTAGGCGGTC TCCTCCCTTCCTCAACCTC VIT_13s0019g03170.1 CCAAACTTCTTAGGCGGTC TCCTCCCTTCCTCAACCTC VIT_18s0041g00280.1 ACAAGATGAAGGCTGCTCACT AGAACAAACCGCAAACCCTC VIT_19s0090g00420.1 GCTTTCATTGCCCGTATTTCT GGCTCTTCTTTCCATTCCTCTG VIT_19s0015g00130.1 00130 1 TAACCCACCTCCCTTCTCAC GGCACGCAGGAAACAAGAT VIT_02s0154g00480.1 TGCTTCTCGCTCCTTCAATACC CTGGCTCAAACTTCTCGTCCG VIT_06s0004g04470.1 TCAAGGCAACAGGACTACGC CCTTCCAATCAAACGCTTCGC VIT_18s0089g01270.1 CTCTCTTGTTTCTTCTTTGCTGC ACCTCCTCCTTCCTCAGACC VIT_09s0002g04490.1 GCGTTTCTCATTTCTTCGTTGTC TCTTGGCAGGGTCGGTGAT VIT_00s1490g00010.1 1 TTGGCTGTGACCTCCTCTTC TTGCTCATAGTCCTCCACCTC VIT_07s0005g01080.1 ATCGGGTCTTCTCCTTGGC GGGTTTCACATCCTCTGTTCGT VIT_06s0004g06010.1 GGTATTCTCGCTCACCTTCCc GATTCACGACTCCTTGACCTGC VIT_08s0007g00130.1 CGCCGTCTTATGTTGCGTT GCTTCTCCTCGCCCTTGTAG VIT_08s0007g04000.1 ATCCTGCTGACGGGCTTAG CGTTGTGAACCGAGTCTTTAGG VIT_01s0011g04820.1 GCCTATTCAGTACTATCAGACCAG TCCTCCCTAGTGTAGATCTTACC VIT_04s0008g01490.1 CTTCATCATCGACATGCCGG TTCCTCATAAACTTCCCCACC VIT_04s0008g01500.1 CTTCATCATCGACATGCCGG TTCCTCATAAACTTCCCCACC VIT_04s0008g01520.1 CTTCATCATCGACATGCCGG TTCCTCATAAACTTCCCCACC VIT_04s0008g01530.1 CTTCATCATCGACATGCCGG TTCCTCATAAACTTCCCCACC VIT_04s0008g01590.1 CTTCATCATCGACATGCCGG TTCCTCATAAACTTCCCCACC VIT_04s0008g01510.1 CTTCATCATCGACATGCCGG TTCCTCATAAACTTCCCCACC VIT_16s0022g00510.1 TTCAACACCAACGCCATCC GATCGAACACATCTGAGAAGGAG VIT_17s0000g07190.1 CATTGAAGACTTATGGGCGTG TGATGACCCTCCGAATCTC VIT_11s0037g00510.1 GATGTCACGCCTCTCAGTCTC AGTCTTCCCTTGTCGTTGGTG VIT_11s0016g04080.1 GTTGATACAGAAAGCGAGGTTGG GAGCGACGGACAAGACACT VIT_00s0181g00080.1 AGGGTTTGTTCCAGTCACGC CTGCTTTCTCCTCCACCATTTC VIT_08s0058g00210.1 GTGACGGTTGAGGTTGAAGAG CCATCTTTGCATTTTCAGGAAGTC VIT_13s0019g03090.1 GACCTCTGCCTTCACCAAC CAACCTCAACCTTCACCTCC VIT_19s0085g01050.1 AGAATGAGGTGAAACTGGAGGT CTGAACTGCCTGGAGAAACTG
Supplemental Table S4. The primers of RT-PCR splicing analysis used in this study. Gene ID discription Forward primer Reverse primer VIT_16s0050g01150.1 HSP90.1 GCCTTCCAGGCTGAGATCAATC TGGTCATTCCGATGCCACTG VIT_16s0098g01060.1 Hsp25.3 CAGTTATCAGGTAGCTGCAG ACCAGTGTCTATGGCTTCTC VIT_17s0000g07190.1 HSP101 CATGATGTTAACGGAGACTG TGGAGGAGGGTATTGAATAC VIT_08s0007g00130.1 HSP70.4 TTGGCGTTTATTCTGACACC CAATTTCAATGGTGGTCTGG VIT_04s0008g01110.1 1 HsfA2-I, III TCTCTGATTCAATACCCGAC TTCTTCCCAGGTACTTCAAG VIT_04s0008g01110.1 HsfA2-I, III AGAACCCTTCTTTCGAGTCTGG GACTGGGTGATCATCATGAACC