Page S-1 Supporting Information (SI) itraq and RNA-Seq analyses provide new insights into regulation mechanism of symbiotic germination of Dendrobium officinale seeds (Orchidaceae) Juan Chen 1, Si Si Liu 1, Annegret Kohler 2, Bo Yan 1, Hong Mei Luo 1, Xiao Mei Chen 1, Shun Xing Guo 1 * 1 Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100193, P. R. China 2 UMR 1136 INRA/Université de Lorraine, Interactions Arbres/Micro-organismes, INRA, Institut National de la Recherche Agronomique, Centre INRA de Nancy, 54280 Champenoux, France. Figure S-1. Examples of tandem mass spectra of itraq for the differentially expressed proteins (DEPs) with low number of peptide. Five DEPs during seed germination of D. officinale were selected from Table S9 and Table S10 in Supporting Information (marked **), including Dendrobium_GLEAN_10057503, Dendrobium_GLEAN_10089327, Dendrobium_GLEAN_10117734, Dendrobium_GLEAN_10122630 and Dendrobium_GLEAN_10052315. Left column is the tandem mass spectra of the proteins and S1
right column is the region containing the reporter ions of spectra (m/z:113-121) associated with the spectrum of proteins. Figure S-2. RNA-Seq and itraq 8-plex workflow of asymbiotic and symbiotic germination of D. officinale seeds. Stage2: early germination; Stage3: protocorm stage; Stage4: seedling stage; A1, A2, A3 represent developmental stage 2, stage3, stage4 in asymbiotic germination and S1, S2, S3 represent stage2, stage 3, and stage 4 in symbiotic germination, C0 represent ungermination seed. Figure S-3. Comparative analysis of seed germination at all stages over 12 weeks time of D. officinale. A. Seed development in symbiotic condition. B. Seed development in asymbiotic conditions. C. A quantitive proportion of seeds at all stage compared total germination seed in 12 th week. AG: asymbiotic germination; SG: symbiotic germination. Figure S-4. Comparison of expression ratios from transcriptomic (y-axis) and protomic (x-axis) profiling based on differentially expressed proteins and correlated genes at each compared group. Log2 expression ratios were calculated in adjacent development stage during asymbiotic (left columns) and symbiotic germination (middle columns) and at the same stage between symbiotic and asymbiotic germination (right columns). Red point represents both proteins and genes are differentially expressed. AG: asymbiotic germination; SG: symbiotic germination. Figure S-5. Summary of correlation coefficient of quantitative correlation results (6 types) in each compared group. 6 types: correlation result of all quantitative protein and the mrna (blue); the differentially expressed proteins and genes with the same trend (green); the differentially expressed proteins and genes with the opposite trend (yellow); both proteins and S2
RNA no change (orange); proteins no change but genes differentially expressed (red); proteins differentially expressed but genes no change (pale red). A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic germination and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. Table S-1. Labeling samples information of Dendrobium officinale seeds in our itraq experiments. Table S-2. Mascot search parameters used in protein identification by itraq analysis of D. officinale seeds. Table S-3. Quantification parameters used in our relative quantification of protein by itraq analysis of D. officinale seeds. Table S-4. Detailed information of 2256 plant proteins identified by itraq during seed germination of D. officinale. Table S-5. 308 differentially expressed proteins (DEPs) in at least one more germination stage during asymbiotic and symbiotic germination of D. officinale. A protein was considered to be DEP if it met fold change 1.2 or 0.83 and Q value < 0.05. A1, A2, A3 represent developmental stage 2, stage3, stage4 in asymbiotic germination and S1, S2, S3 represent stage2, stage 3, and stage 4 in symbiotic germination, C0 represents ungerminated seed. AG: asymbiotic germination; SG: symbiotic germination. Red color: A protein was up-regulated expression in the given compared group; green color, a protein was down-regulated and gray color: protein no significant expression change at the given compared group. Table S-6. 229 differentially expressed proteins (DEPs) in at one more development stage during symbiotic germination compared to asymbiotic germination of D. officinale. A protein was considered to be DEP if it met fold change 1.2 or 0.83 and Q value < 0.05. Red S3
color means up-regulated proteins, green color means down-regulated protein and gray color means no significant change in the given compared group, respectively. Table S-7. Key parameters used for correlation analysis between transcriptome and proteome of seed germination of D. officinale. Table S-8. Correlation analysis of the transcriptome and proteome reveals the number relationship of proteins and genes on identification, quantitative and differentially expressed aspects. Table S-9. 66 co-up-regulated proteins at transcriptomic and proteomic level across at least one more adjunct developmental stage during asymbiotic or symbiotic germination of D. officinale. A protein was identified as co-up-regulated protein if it met: i) fold change 1.2 (itraq) and 2.0 (RNA-Seq); ii) Q value < 0.05(iTRAQ) and FDR < 0.001(RNA-Seq). Yellow color: co-up-regulated proteins; red color: up-regulated proteins only in itraq; orange color: up-regulated genes only in RNA-Seq; gray color: proteins and genes without significant expression change. ** The tandem mass spectra were showed in Figure S1 in Supporting Information. Table S-10. 32 Co-up-regulated proteins at transcriptomic and proteomic level during symbiotic germination compared to asymbiotic germination of D. officinale. A protein was identified as co-up-regulated protein if it met: i) fold change 1.2 (itraq) and 2.0 (RNA-Seq); ii) Q value < 0.05(iTRAQ) and FDR < 0.001(RNA-Seq). Yellow color: co-up-regulated proteins; red color: up-regulated proteins only in itraq; orange color: up-regulated genes only in RNA-Seq; gray color: proteins and genes without significant expression change. * gene expression was validated by qpcrgenes for RT-PCR. **The tandem mass spectra were showed in Figure S1 in Supporting Information. S4
S5
Figure S-1. Examples of tandem mass spectra of itraq for the differentially expressed proteins (DEPs) with low number of peptide. Five DEPs during seed germination of D. officinale were selected from Table S9 and Table S10 in Supporting Information (marked **), including Dendrobium_GLEAN_10057503, Dendrobium_GLEAN_10089327, Dendrobium_GLEAN_10117734, Dendrobium_GLEAN_10122630 and Dendrobium_GLEAN_10052315. Left column is the tandem mass spectra of the proteins and right column is the region containing the reporter ions of spectra (m/z:113-121) associated with the spectrum of proteins. Figure S-2. RNA-Seq and itraq 8-plex workflow of asymbiotic and symbiotic germination of D. officinale seeds. Stage2: early germination; Stage3: protocorm stage; Stage4: seedling stage; A1, A2, A3 represent developmental stage 2, stage3, stage4 in asymbiotic S6
germination and S1, S2, S3 represent stage2, stage 3, and stage 4 in symbiotic germination, C0 represent ungermination seed. Figure S-3. Comparative analysis of seed germination at all stages over 12 weeks time of D. officinale. A. Seed development in symbiotic condition. B. Seed development in asymbiotic conditions. C. A quantitive proportion of seeds at all stage compared total germination seed in 12 th week. AG: asymbiotic germination; SG: symbiotic germination. S7
Figure S-4. Comparison of expression ratios from transcriptomic (y-axis) and protomic (x-axis) profiling based on differentially expressed proteins and correlated genes at each compared group. Log2 expression ratios were calculated in adjacent development stage during asymbiotic (left columns) and symbiotic germination (middle columns) and at the same stage between symbiotic and asymbiotic germination (right columns). Red point represents both proteins and genes are differentially expressed. AG: asymbiotic germination; SG: symbiotic germination. S8
Figure S-5. Summary of correlation coefficient of quantitative correlation results (6 types) in each compared group. 6 types: correlation result of all quantitative protein and the mrna (blue); the differentially expressed proteins and genes with the same trend (green); the differentially expressed proteins and genes with the opposite trend (yellow); both proteins and RNA no change (orange); proteins no change but genes differentially expressed (red); proteins differentially expressed but genes no change (pale red). A1, A2, A3 represent developmental stage 2, stage 3, stage 4 in asymbiotic germination and S1, S2, S3 represent stage 2, stage 3, and stage 4 in symbiotic germination, C0 represent ungerminated seed. S9