DOI: 10.5281/zenodo.10952750
title: Supplementary data of article "Domestication has altered gene expression and secondary metabolites in pea seed coat".

description: Table S1. Excel- GO_terms_MF_selected_WGCNA_modules.

Table S2. Excel- GO_terms_MF_DEGs_UP_and_DOWN.

Table S3. Excel- GO_terms_MF_DEGs_summary.

Table S4. Excel- List of DEGs involved in flavonoid pathway found in WILD gene set.

Table S5. Protein recoveries calculated for individual pea protein samples. Numbers 1, 2, 3 denote treatment groups corresponding to seed developmental stages (D1, D2 and mature seeds, respectively). Letters a–d denote biological replicates within the treatment groups.

Table S6. Excel- Annotation of proteins differentially expressed in wild and domesticated pea seed coat samples.

Table S7. Primary metabolites identified by spectral similarity library search and/or co-elution with authentic standards in pea seed coats aqua methanolic extracts. Metabolite analysis relied on GC-EI-Q-MS analysis after derivatization of the lyophilized extracts with methoxamine hydrochloride (MOA) and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA).

Table S8. Primary metabolites detected in the aq. methanolic extracts of mature Cameor seed coats demonstrating statistically significant up- and down-regulation in comparison to those of wild JI261.

Table S9. Primary metabolites of mature JI92 seed coats demonstrating statistically significant up- and down-regulation in comparison with those of wild JI261.

Table S10. Primary metabolites of mature JI1794 seed coats demonstrating statistically significant up- and down-regulation in comparison with those of JI261.

Table S11. Primary metabolites of mature JI64 seed coats demonstrating statistically significant up- and down-regulation in comparison with those of JI261.

Table S12. Mass analyzer settings applied for QqTOF-MS experiments in analysis of seed coat (cell wall) hydrolyzates and reference authentic standards.

Table S13. Cell wall-bound metabolites extracted from the seed coats of wild (JI64, JI1794, JI261) and domesticated (Cameor, JI92) peas upon alkali hydrolysis of corresponding isolated and purified cell wall material.

Table S14. Excel- Coordinates of markers in S-plot obtained from OPLS-DA analysis (FIA-ESI-HRTMS, negative ionization, lock mass uncorrected).

Table S15. List of identified significantly differential metabolites rising during seed coat development.

Table S16. List of identified significantly differential metabolites decreasing during seed coat development (positive ionization mode).

Table S17. List of identified metabolites with significantly higher content in wild compared cultivated genotypes in older developmental stages (D5-6).

Table S18. Excel- Expression of genes encoding enzymes of monolignol pathway in seed coats (SC) and embryos (E) of domesticated (Cameor, JI92 and Pisum abyssinicum PI358617) and wild (JI64, JI1794, JI261) peas over five seed developmental stages (13, 17, 20, 23, 28 DAP, labelled as 1-5). PAL: phenylalanine ammonia-lyase, C4H: cinnamate-4-hydroxylase, 4CL: 4-coumaroyl: CoA ligase, HCT: hydroxycinnamoyl CoA:shikimate hydroxycinnamoyltransferase, COMT: caffeic acid O-methyltransferase, CSE: caffeoyl shikimate esterase, CAD: cinnamyl alcohol dehydrogenase, CCR: cinnamoyl CoA reductase, CCoAMT: caffeoyl CoA-3-methyltransferase, F5H: ferulate-5-hydroxylase

Table S19. Studied metabolites of phenylpropanoid pathway.

Table S20. Instrument settings used in the proteomics LIT-Orbitrap-MS and -MS/MS experiments.

Table S21. Procedures and specific settings for data processing and post-processing of the proteomics data.

Table S22. Gas chromatographic (GC) separation conditions and electron ionization-quadrupole-mass spectrometry (EI-Q-MS) settings for GC-EI-Q-MS analysis of the primary metabolites in pea seed coats.

Table S23. Chromatographic conditions used for UHPLC separation of seed coat (cell wall) hydrolyzates and reference authentic standards.

Table S24. Variable parameters of MS/cIMS/MS measurements.

Figure S1. The dynamics of gene expression between studied developmental stages within all genotypes (a) or among genotypes in particular developmental stages (b).

Figure S2. Twelve representative groups of transcription factors described within 20 gene modules of pea SC. Visualized by Cytoscape 3.9.0.

Figure S3. SDS-PAGE electropherograms of the total protein fractions isolated from the seed coats of JI92 (a, c, e) and JI64 (b, d, f) seeds before and after tryptic hydrolysis. Numbers 1, 2, 3 denote seed developmental stages: DS1, DS2 and mature seeds, respectively. Letters a-d denote biological replicates. The aliquots (10 μg) of samples before hydrolysis (a, b), the incompletely digested aliquots left on filter unit after peptide elution (c, d) and aliquots of tryptic hydrolysates (corresponding to 5 μg of protein), (e, f) were loaded on gels. Inter-gel normalization relied on the total density of the Protein Ladder (PageRuler™ Prestained Protein Ladder #26616, 10–180 kDa) lane (St); the ND (non-digested) sample represents a reference protein not subjected to hydrolysis.

Figure S4. The numbers of tryptic peptides (a), possible proteins (b), and non-redundant proteins (protein groups) (c) identified in domesticated JI92 seed coats at developmental stages D1, D2 and D6. The tryptic digests (n =&thinsp;3) obtained from seed coats were analyzed by nano-high performance liquid chromatography-electrospray ionization linear ion trap-orbital trap mass spectrometry (nanoHPLC-ESI-LIT-Orbitrap-MS) operated in positive DDA mode.

Figure S5. The numbers of tryptic peptides (a), possible proteins (b), and non-redundant proteins (protein groups, c) identified in wild pea JI64 seed coats at D1, D2 and D6 stages. The tryptic digests (n =&thinsp;3), obtained from pea seedlings, were analyzed by nano-high performance liquid chromatography-electrospray ionization linear ion trap-orbital trap mass spectrometry (nanoHPLC-ESI-LIT-Orbitrap-MS) operated in positive DDA mode.

Figure S6. Principal component analysis (PCA) with score plot representation (a) accomplished for seed coat proteins differentially expressed at developmental stages D1 and D2 and in the mature state (D6) and hierarchical clustering with a heatmap representation (b).

Figure S7. Functional annotation (accomplished with the Mercator MapMan v3.6 tool) of the pea seed coat proteins isolated in stage D1. White and black columns denote the functional groups of the proteins, which were more expressed in the developing seeds of domesticated JI92 and wild JI64, respectively.

Figure S8. Functional annotation (accomplished with the Mercator MapMan v3.6 tool) of the pea seed coat proteins isolated in stage D2. White and black columns denote the functional groups of the proteins, which were more expressed in the developing seeds of the domesticated JI92 and wild JI64, respectively.

Figure S9. Prediction of sub-cellular localization of the proteins more expressed in the developing seeds of JI92 and JI64 with the BUSCA prediction tool.

Figure S10. Evaluation of the differences in the metabolic profiles of the mature seeds obtained from the wild JI261 and domesticated Cameor by principal component analysis (PCA).

Figure S11. Representation of the differences in the metabolic profiles of the mature seed coats obtained from the wild JI261 and domesticated Cameor by the t-test with Volcano plot representation (a) and the top 30 differentially abundant metabolites demonstrating the most pronounced differences of corresponding GC-MS signals associated with seed dormancy (b).

Figure S12. Evaluation of the differences in the metabolic profiles of the mature seeds obtained from the wild JI261 and domesticated JI92 by principal component analysis (PCA) with score plot representation (a) and hierarchical clustering with heatmap representation (b).

Figure S13. Principal component analysis (PCA) illustrating distribution of metabolic profiles of mature seed coats of two wild pea genotypes, JI1794 and JI261.

Figure S14. Principal component analysis (PCA) demonstrates the distribution of mature seed coat metabolic profiles of two wild pea genotypes, JI64 and JI261(control).

Figure S15. Evaluation of the differences in the patterns of the cell wall-bound metabolites obtained from mature seed coats of wild JI261 and domesticated Cameor: principal component analysis (PCA) with score plot representation (a), hierarchical clustering with heatmap representation (b) and t-test analysis with the Volcano-plot representation (c).

Figure S16. Statistical analysis (t-test with Volcano plot representation) characterizing the differences between the levels of mature seed coat cell wall-bound metabolites of Cameor compared with those of wild JI261.

Figure S17. Principal component analysis (PCA) illustrates the distribution of mature seed coat metabolic profiles of domesticated JI92 and wild JI261.

Figure S18. Principal component analysis (PCA) shows the distribution of metabolic profiles of mature seed coats of two wild pea genotypes, JI1794 and JI261, control.

Figure S19. Principal component analysis (PCA) demonstrates the distribution of mature seed coat metabolic profiles of two wild genotypes, JI64 and control JI261.

Figure S20. Annotated cell wall-bound metabolites extracted from the seed coats of the dormant wild pea genotype JI261 and the seed coats from several pea genotypes varying in their dormancy (Cameor, JI92, JI64, and JI1794) upon alkali hydrolysis of corresponding isolated and purified cell wall material. 

Figure S21. Ion mobility separation of m/z 299.0841.

Figure S22. Ion mobility separation of m/z 701.1907. 

Figure S23. Ion mobility separation of m/z 619.1041.

Figure S24. Ion mobility separation of m/z 631.1017.

Figure S25. Ion mobility separation of m/z 641.1139.

Figure S26. Ion mobility separation of m/z 771.1346. 

Figure S27. Reconstructed chromatograms of p-hydroxybenzoic and salicylic acids in DS5 of dormant JI64 and domesticated landraces JI92 (LC/HRTMS, negative ionization mode).

Figure S28. Module-trait relationship depiction showing the correlation between expression of the gene modules and the abundance of identified metabolites of the monolignol pathway.