Rosa Caiazzo, PhD. The 3 rd Plant Genomics Congress May 2015 London, UK

Transcription

1 A systems biology approach to investigate the mechanisms that promote ripening and regulate post-harvest fruit withering in the cherry-like tomato landrace pomodorino del piennolo del Vesuvio Rosa Caiazzo, PhD The 3 rd Plant Genomics Congress May 2015 London, UK

2 Cultivation area Protected Designation Origin VESUVIO

3 Peculiarities of Piennolo cultivation techniques no irrigation and apical buds constantly removed jointless pedicels long shelf life thick skin which limits dehydration 3

4 MICROSCOPIC ANALYSIS Weight (mg) Cu cle development Thickness (μm ) Ailsa craig Fogliariccia Lucariello 4

5 OBJECTIVES To identify differential expressed genes and determine metabolite content during fruit development and post-harvest storage To generate a model of biochemical and molecular mechanisms involved in the post-harvest withering 5

6 STRATEGIES Next-generation sequencing digital gene expression profiling Metabolite content evaluation (HPLC,GC/MS) Optical microscopy analysis of cuticle 6

7 EXPERIMENTAL DESIGN Genotype: Lucariello Tissues: Exocarp and Mesocarp Biological replicates: 3 MG BR RR mature green breaker red ripe 60DPH 150DPH 60 days post-harvest days post-harvest

8 WORKFLOW RNA extraction Library construction & sequencing Quality control on raw reads & adapter trimming Alignment on reference Summarization Normalization DEG call 8

9 WORKFLOW RNA extraction Library construction & sequencing Quality control on raw reads & adapter trimming Alignment on reference Summarization Normalization DEG call 9

10 WORKFLOW RNA extraction Library construction & sequencing Quality control on raw reads & adapter trimming Alignment on reference Summarization Normalization DEG call Directional singleend sequencing on Illumina HiSeq

11 WORKFLOW RNA extraction Library construction & sequencing Quality control on raw reads & adapter trimming Alignment on reference Summarization Normalization DEG call FastQC, FASTX-Toolkit and Trimmomatic 11

12 WORKFLOW RNA extraction Library construction & sequencing Quality control on raw reads & adapter trimming Alignment on reference Summarization Normalization DEG call Reference: SL2.50 TopHat2 12

13 ALIGNMENT ON REFERENCE Statistics on read mapping 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% # reads with multiple matches # unmapped reads MG1 MG2 MG3 BR1 BR2 BR3 RR1 RR2 RR3 60DPH1 60DPH2 60DPH3 150DPH1 150DPH2 150DPH3 MG1M MG2M MG3M BR1M BR2M BR3M RR1M RR2M RR3M 60DPH1M 60DPH2M 60DPH3M 150DPH1M 150DPH2M 150DPH3M # reads uniquely mapped on exocarp tissue # reads uniquely mapped on mesocarp tissue

14 WORKFLOW RNA extraction Library construction & sequencing Quality control on raw reads & adapter trimming Alignment on reference Summarization Normalization DEG call HTSeq-count. Reads were aggregated over genes as biological units 14

15 ALIGNING READS TO THE TOMATO REFERENCE GENOME Results of the mapping of RNA-Seq reads along the reference genome (SL2.50) VS Genomic coordinates of annotated genes (itag 2.40) characterization files mesocarp exocarp E Read Genes Read Genes Read Genes 15

16 ALIGNING READS TO THE TOMATO REFERENCE GENOME Intron only Neighboring exon Gene desert % % % Exons-only Alternative TSS Alternative PolyA Internal exon extention Multiple annotations REGION 16

17 WORKFLOW RNA extraction Library construction & sequencing Quality control on raw reads & adapter trimming Alignment on reference Summarization Normalization DEG call EdgeR Trimmed mean of M-value 17

18 NORMALIZATION Raw Data Normalized Data Cu Exocarp cle Mesocarp Cu Exocarp cle MG BR RR 60DPH 150DPH Mesocarp 18

19 MG3 MG2 MG1 CORRELATION AMONG BIOLOGICAL REPLICATES MG1 0.8 < r < 1 r=0.87 MG2 r=0.85 r=0.88 MG3 19

20 WORKFLOW RNA extraction Library construction & sequencing Quality control on raw reads & adapter trimming Alignment on reference Summarization Normalization DEG call EdgeR pdj<0.05 & LogFC -2 e 2 20

21 WORKFLOW RNA extraction Library construction & sequencing Quality control on raw reads & adapter trimming Alignment on reference Summarization Normalization DEG call DESeq pdj <0.05 & LogFC -2 e 2 21

22 DEG called with EdgeR & DESeq EXOCARP E MG BR 464 MG RR D 1087 MG 60DPH MG 150DPH BR RR BR 60DPH 838 BR 150DPH RR 60DPH RR 150DPH E E E E E E E E E 60DPH 150DPH D D D D D D D D D MESOCARP E MG BR 1178 MG RR 1094 MG 60DPH 1104 MG 150DPH 1166 BR RR 823 BR 60DPH BR 150DPH RR 60DPH RR 150DPH E E E E E E E E E DPH 150DPH D D D D D D D D D D E =EdgeR D =DESeq

23 DEG EXOCARP

24 DEG MESOCARP involved in the withering?

25 GO enrichment analysis (mesocarp): RR vs 60DPH Response to desiccation Response to water deprivation Hexose metabolic process Monosaccharide metabolic process Response to acid chemical Response to water Response to inorganic substance Response to oxygen-containing compound Response to chemical Response to abiotic stimulus Response to stress Response to stimulus Terpenoid metabolic process Terpenoid biosynthetic process Coenzyme biosynthetic process Isoprenoid biosynthetic process Isoprenoid metabolic process Cofactor biosynthetic process Cell redox homeostasis Cellular homeostasis Homeostatic process Regulation of biological quality Organic acid metabolic process Carboxylic acid metabolic process Oxoacid metabolic process Single-organism process

26 GO enrichment analysis (exocarp): RR vs 60DPH Glutamine family amino acid metabolic pr... Response to desiccation Response to water deprivation Lipid catabolic process Response to abiotic stimulus Response to acid chemical Response to chemical Response to inorganic substance Response to oxygen-containing compound Response to water Single-organism catabolic process Cation transport Ion transport Metal ion transport Carbohydrate metabolic process Coenzyme biosynthetic process Cofactor biosynthetic process Single-organism organelle organization Organelle organization Organic acid metabolic process Carboxylic acid metabolic process Oxoacid metabolic process 22 Single-organism cellular process Single-organism metabolic process 26 Single-organism process

27 Metabolism overview (exocarp): RR vs 60DPH

28 Functional classification of DEG (exocarp):rr vs 60DPH Biodegradation of xenobiotics S-assimilation C1-metabolism Micro RNA, natural antisense Co-factor and vitamine Fermentation Polyamine metabolism TCA / organic transformation Glycolysis N-metabolism OPP Mitochondrial electron Metal handling Tetrapyrrole synthesis Nucleotide metabolism Redox Minor CHO metabolism Major CHO metabolism Amino acid metabolism Photosinthesis Lipid metabolism DNA Cell Secondary metabolism Development Cell wall Hormone metabolism Transport Stress Other MISC Signalling RNA Protein -Lipid Metabolism -Secondary metabolism -Development -Cell wall -Hormone metabolism -Transport -Stress Down UP Number of genes

29 Functional classification of DEG(mesocarp):RR vs 60DPH Polyamine metabolism Gluconeogenesis / glyoxylate cycle Co-factor and vitamine metabolism C1-metabolism TCA / organic transformation Glycolysis OPP N-metabolism Fermentation Mitochondrial electron transport Nucleotide metabolism Metal handling Tetrapyrrole synthesis Minor CHO metabolism Redox Major CHO metabolism Photosinthesis Amino acid metabolism DNA Lipid metabolism Cell Secondary metabolism Cell wall Development Hormone metabolism Transport Stress Other MISC Signalling RNA Protein -Lipid metabolism -Secondary metabolism -Development -Cell wall -Hormone metabolism -Transport -Stress down Down Up UPregulated Number of genes

30 Genes involved in electron transport chain Mesocarp MG/BR BR/RR RR/60 60/150 MG/BR BR/RR RR/60 60/150

31 Genes involved in electron transport chain Metal ion transporter are associated with the cold storage of different fruit (Mellidou et al., 2014) o during post-harvest, cells need to synthesize protective compounds to maintain their cellular compartmentalization and to detoxify accumulated metabolic intermediates

32 Genes involved in the cuticle biosynthesis Exocarp MG/BR BR/RR RR/60 60/150 Mesocarp MG/BR BR/RR RR/60 60/150

33 Wax metabolites 143 metabolites identified Separation between early and late stages Wax metabolites cluster in 3 groups :common pathways 33 33

34 N of wax metabolites WAXES SPECIFIC COMMON NON SPECIFIC 10 0 MG BR RR 60 DPH 150 DPH 34

35 WAXES N wax metabolites MG BR RR 60DPH 150DPH 2 0 alkanes halocarbons esters nitriles ethers heterocycles heterociclics alkenes peroxides amines lipids Chemical class Chemical classes 35

36 WAX SYNTHESIS

37 The β-ketoacyl-coa synthase Involved in the fatty acid elongation process for the production of aliphatic wax components and, consequently, confers resistance to cuticular water loss (Hooker et al., 2002). 37

38 The β-ketoacyl-coa synthase (KCS family) KCS_exocarp MG/BR BR/RR RR/60 60/150 MG/BR BR/RR RR/60 60/150 Gene ID: Solyc05g

39 GDSL esterase/acylhydrolase family Significant reduction in fruit cuticle thickness proportional to the level of GDSL1 silencing (Girard et al., 2012). 39

40 GDSL esterase/acylhydrolase family _exocarp MG/BR BR/RR RR/60 60/150 MG/BR BR/RR RR/60 60/150 Gene ID: Solyc03g Gene ID:Solyc04g

41 PRELIMINARY CONCLUSION The analysis of the cuticle confirmed our hypothesis, that cuticle plays a key role in the post-harvest withering of "piennolo" tomato fruits. Up regulation in post-harvest of genes involved in : o resistance to cuticular water loss o increase of cuticle thickness 41

42 PRELIMINARY CONCLUSION Mobilization of metal ions - Metal ion homeostasis is up to regulate post-harvest disorders (such as internal browning, linked to cell wall modifications, similar to those observed during fruit softening, or the response to biotic stress) 42

43 PRELIMINARY CONCLUSION The alignment along the reference genome proved that tomato genome annotation needs further revisions and RNA-Seq expression profile studies can be influenced by these inaccuracies. 43

44 WORK IN PROGRESS Confirm RNA-Seq data Development of transcript/metabolite correlation matrices 44

45 The working team CONCITA CANTARELLA POST-DOC SARA RICCI RESEARCHER FELLOW SILVANA CANGEMI POST-GRADUATE STUDENT CLAUDIA CIMMINO UNDERGRADUATE STUDENT CRA-ORT Nunzio D Agostino, PI Giuseppe Mennella Mario Parisi Mario Salzano We acknowledge for plant cultivation This work was carried out in the frame of the "GenoPom-pro - Integrating post-genomic platforms to enhance the tomato production chain" project (PON02_00395_ ) and is supported by the PON R&C grant funded by the Italian Ministry of Education, University and Research in cooperation with the European Funds for the Regional Development (FESR). for sequencing service 45

46 THANK YOU! 46

47 47

48

49 CAROTENOIDS Lycopene β-carotene Exocarp Mesocarp MG BR RR 60 DPH 150 DPH MG BR RR 60 DPH 150 DPH Bars with the same letter are not significantly different according to the Tukey HSD test (p 0,05). Lycopene and β-carotene are higher in the cuticle and reach the maximum at the RR stage Lycopene increases in the mesocarp at 150 days post harvest 49

50 ASCORBIC ACID MG BR RR 60 DPH 150 DPH Bars with the same letter are not significantly different according to the Tukey HSD test (p 0,05). Ascorbic acid increases during the berry development and decreases in post-harvest 50