Fruit-omics: from the 2DE era, over shotgun MS, towards single cell omics. Sebastien Carpentier, PhD Principal scientist Program leader banana research International Musa Germplasm Transit Center Make use of available biodiversity! Experimental set up: evaluation and characterisation Adapted from Keiichi Mochida and Kazuo Shinozaki 2010 Plant ➔ protein extraction (Carpentier et al 2005) Non sequenced varieties ➔protein identification (Buts et al. 2014 (spectral library searches); variety specific mRNAseq databases Polyploid species ➔multiple alleles and isoforms ➔protein reconstruction (Vertommen et al. 2011; Carpentier et al 2020) Overview of challenges for non-model organisms Optimization of protein extraction protocol • Preparation of protein extracts from recalcitrant plant tissues: an evaluation of different methods for two-dimensional gel electrophoresis analysis. • Carpentier et al. Proteomics 2005, 5, 2497-2507. • Phenol extraction: inactivation and removal of interfering compounds • Precipitation: inactivation and removal of interfering compounds and concentration of the proteins Banana Biodiversity AA x AB x BB AAA AAB ABB Illustration of specific proteins in AAA and ABB and imbalance A specific proteins B specific proteinsPGK 1165 Chit 1648 OSR 1339 ASR 1911 PGK 1162 PGK 1179 OSR 1366 ASR 1907 PCA analysis -20 -15 -10 -5 0 5 10 15 20 -30 -20 -10 0 10 20 30 P C 2 PC1 BBB ABB AAB AAB AAA AAA ASR: Abscisic stress ripening protein ABB AAA BBAA AAAh ASR upregulated in stress (Carpentier et al 2010 JPR) 4 da ys 14 d ay s 8 da ys 2 da ys St an da rd iz ed L og A bu nd an ce 0.11 0.1 0.09 0.08 0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 -0.01 -0.02 -0.03 -0.04 -0.05 2D optimization Buts et al. 2014 Phytochemistry 12 3 4 Quadrants Kissel et al 2015 Results PLS transcriptome Zorrilla et al. 2016 Sci Rep Genes of interest Stress sensitive markers AAA AAA ABB Genes of interest Tolerance specific markers Appearance AAA AAA ABB Proteomics in complex eukaryotic sample 234 10E3 peptides 30% of the peptides was selected for MSMS with an identification rate of 16% (2749 unique proteins) mRNA seq 0 2000 4000 6000 8000 10000 12000 14000 16000 Fr eq ue nc y Number of reads Histogram Frequency 18*10E6 reads belonging to 37577 different genes Complementarity mRNA seq proteomics 3627 11 331 Quantitatively, 342 proteins (12%) and 3638 mRNAs (10%) were considered as differential. Eleven were in common. Allele specific peptides PC1 PC2 AAA, AAAh ABB Score plot (sPLS) Allele specific peptides - verification m/z ret. time int. m/z ret. time int. AAA AAA ABB int. ABB AAA Specific abundance -log(p) PC1 p=0.05 AAA, AAAh ABB 978 specific peptides 705 ABB253 AAA Proteogenomics Ma02_t23730 y10AAA = y10ABB 1145,63 m/z y11AAA ≠ y11ABB Δ = 9 Da Δ = 9 Da ❖ Causative SNP? LC-MSMS 100% read alignment SNP Ma02_p15060 Ma11_p17540 Proteogenomics RNA-seq LC-MSMS • SNP promotor • epigenetics transcript~proteome J van Wesemael et al 2018 van Wesemael et al 2018 Cenci et al. 2019 GO-term Description P (Fisher) GO:0006096 Glycolytic process 0.003 GO:0006457 Protein folding 0.015 GO:0006950 Response to stress 0.018 COST vision paper Phenotyping and timing are crucial Geilfus et al 2017 50 mM NaCl 75 mM NaCl Geilfus et al 2017 Fruit proteomics and metabolomics Source: LIPI 2 different plantain varieties (AAB) Campos et al.2022 J Exp Bot. Highest activities were observed for fluxes involved in respiration, glycolysis, and the TCA cycle Metabolic flux modelling Campos et al.2022 Campos et al.2022 Campos et al.2022 Campos et al.2022 Campos et al.2022 high respiration ➔ cell division Campos et al.2022 J Exp Bot. Campos et al.2022 Slide courtesy of Kim-Ahn Le Cao Guard cell proteomics -15,0000 -10,0000 -5,0000 0,0000 5,0000 10,0000 15,0000 20,0000 -25,0000 -20,0000 -15,0000 -10,0000 -5,0000 0,0000 5,0000 10,0000 15,0000 20,0000 min 1h plus 15 min plus 1h Geilfus et al 2018 Dittriech et al 2019 Eyland et al 2023 Guard cell transcriptomics Gambart et al submitted Guard cell proteomics Gambart et al summitted Guard cell proteomics Gambart et al submitted 0,0000 0,0001 0,0002 0,0003 0,0004 0,0005 0,0006 0,0007 0,0008 0,0009 -30 -20 -10 0 10 20 30 40 Tr an sp ira tio n PC2 A Sample Information Acetyl ation B C E D F Sijun Zheng et al in preparation Acknowledgments: Ives Gibon Christoph-Martin Geilfus Sijun Zheng Romina Pereschi Thanks! The banana Team alliancebioversityciat.org https://alliancebioversityciat.org/ Slide 1: Fruit-omics: from the 2DE era, over shotgun MS, towards single cell omics. Slide 2: International Musa Germplasm Transit Center Slide 3: Experimental set up: evaluation and characterisation Slide 4: Overview of challenges for non-model organisms Slide 5: Optimization of protein extraction protocol Slide 6 Slide 7: Banana Biodiversity Slide 8: Illustration of specific proteins in AAA and ABB and imbalance Slide 9: PCA analysis Slide 10 Slide 11: ASR upregulated in stress (Carpentier et al 2010 JPR) Slide 12: 2D optimization Slide 13 Slide 14 Slide 15: PLS transcriptome Slide 16: Genes of interest Slide 17: Genes of interest Slide 18: Proteomics in complex eukaryotic sample Slide 19: 234 10E3 peptides Slide 20: mRNA seq Slide 21: Complementarity Slide 22 Slide 23: Allele specific peptides Slide 24: Allele specific peptides - verification Slide 25: Specific abundance Slide 26: Proteogenomics Slide 27: Proteogenomics Slide 28 Slide 29 Slide 30 Slide 31 Slide 32 Slide 33: COST vision paper Slide 34 Slide 35: Phenotyping and timing are crucial Slide 36 Slide 37 Slide 38: Fruit proteomics and metabolomics Slide 39: 2 different plantain varieties (AAB) Slide 40: Metabolic flux modelling Slide 41 Slide 42 Slide 43 Slide 44 Slide 45 Slide 46 Slide 47 Slide 48 Slide 49 Slide 50 Slide 51 Slide 52 Slide 53 Slide 54: Guard cell proteomics Slide 55 Slide 56 Slide 57 Slide 58: Guard cell transcriptomics Slide 59: Guard cell proteomics Slide 60: Guard cell proteomics Slide 61 Slide 62 Slide 63 Slide 64