METABOLOMICS AND PROTEOMICS OF SUCROSE ACCUMULATION IN SUGARCANE

Transcrição

1 METABOLOMICS AND PROTEOMICS OF SUCROSE ACCUMULATION IN SUGARCANE Proc. 2008/ Coordinator: Carlos A. Labate

2 What are the regulatory factors controling the sucrose accumulation during sugarcane maturation? What is the best approach to identify the main regulatory components?

3 SYSTEMS BIOLOGY APPROACH PHYNOTYPE Roberts (2002) PMB, 143:

4 Metabolite and Protein Extraction Rational Leaves Immature Internodes (1 and 2) and mature Internodes (5 and 9) TRANSCRIPTOMICS METABOLOMICS PROTEOMICS GC-TOF-MS LC/MS-MS 2-DE gels MudPit Metabolite Data Bank Bioinformatics Protein Data Bank Bioinformatics SUCEST-FUN Data Bank NETWORK ANALYSES AND SYSTEMS BIOLOGY GENE DISCOVERY HIGH BRIX SUGARCANE

5 PROTEOMICS APPROACH 1. Protein identification in young and mature stems at different intervals during maturation 2. Changes in leaf nuclear proteome during maturation 3. Identification of changes in phosphoproteome of stems and leaves during maturation 4. Cell wall proteomics of leaf and stems

6 Sucrose accumulation during sugarcane maturation

7 Proteomics 2DE Gels from stems 7 and 9

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10 Nuclear Proteome Plantas com 1 e 4 meses de idade O tecido é picotado em éter etílico O éter é retirado O tecido é lavado em tampão de extração O tampão é retirado O núcleo é representado por uma camada escura, localizada entre o Percoll 80% e o 35% O gradiente de Percoll é centrifugado no Swing Bucket, utilizando o break off A solução obtida é submetido à extração com Percoll O tecido homogeneizado é filtrado em Myracloth O tecido é homogeneizado em tampão de extração durante 3 ciclos de 30 segundos Proteínas Nucleares Sucest Separação em Gel 1D A fração obtida é submetida novamente à centrifugação junto com uma camada de 6 ml de Percoll 35% A fração de núcleos obtida é submetida à extração de proteínas utilizando o reagente TRI Seqüenciamento por espectrometria de massas Validação por sites como: BaCCello, WolfPsort e SherLoc

11 Nucleus isolation Microscopia DAPI Orceína Acética Western-blot Histona H1.4 PCNA (proliferating cell nuclear antigen) PEPCase (fosfoenolpiruvato carboxilase) OEE1 (oxygen evolving enhancer 1) Rubisco Núcleo PEPCase Rubisco Total Nuclear OEE1 Núcleo Histona H1.4 PCNA 50 µm

12 Results 20 bandas/gel 3085 clusters identificados 41,62% considerados nucleares por 3 ou mais programas de predição Categorias importantes: Montagem de nucleossomo (histonas) 26,13% (categoria mais representativa) Regulação da transcrição 0,46% (fatores de transcrição bzip, bhlh) Metabolismo do DNA 0,29% (proteína de reparo/ tolerância de dano no DNA, proteína de recombinação meiótica) Metabolismo de RNA 0,29% (proteína argonauta, ribonucleoproteínas, RNA helicases, fator de poliadenização) 2% 11% 3% 1% 1% 11% 0,06% 2% 4% 2% 2% 2% 0,23% 0,13% 3% 15% 0,29% 0,29% 26,13% 1% 9% 3% 0,13% 2% 0,46% 0,2% 0,1% Metabolismo de nucleotídeos 0,23% (ribonuclease)

13 PHOSPHOPROTEOMICS OF SUGARCANE Titansphere Phos-TiO Kit (GL Sciences) Peptides complex Peptides Purification Digestion Phosphoprotein Enrichment Protein complex Phosphopeptides were separated by MudPit (Multidimensional Protein Identification Technology) and identified by mass spectrometry (LC-MS/MS) Protein Extraction Tissue maceration Tissue

14 RESULTS Fosfosproteômica: 43 proteínas: bioenergetics (C-compound and carbohydrate metabolism; photosynthesis); transport; protein metabolism (proteolysis; protein synthesis; protein targeting, sorting and translocation); nucleotide metabolism; amino-acid metabolism; lipid, fatty-acid and isoprenoid metabolism; secondary metabolism; cellular communication / signal transduction; stress response (cell rescue activities); cellular dynamics (biogenesis, organisation and structure of the cell; cell growth).

15 Feiz et al., Cell Wall Proteomics of leaves and stems Isolation of proteins using sucrose gradient centrifugation and extração with 0,2 M CaCl 2 2M LiCl

16 Preliminary data Colmos de cana com 2 meses. Método: INFILTRAÇÃO cell wall proteins 64 (48,48%) 68 (51,52% ) contaminants Proteins related to lipid metabolism Colmos de cana com 2 meses. Método: EXTRAÇÃO 83 (64,84%) 45 cell wall (35,16%) contaminants Oxido-reductases (7,81%) (6,25%) 4 2 (3,13%) 5 (9,37% ) 6 14 (21,88% ) Proteins acting on cell wall polysaccharides Oxido-reductases Miscellaneous Proteases (6,67%) (8,89%) (11,11%) (2,22%) (33,33%) 15 Proteins related to lipid metabolism Proteins with interacting domains (with proteins or polysaccharides) Unknown 7 (10,94%) 9 (14,06%) 17 (26,56%) Proteins with interacting domains (with proteins or polysaccharides) Unknown 7 (15,56%) 10 (22,22%) Miscellaneous Proteases Signaling Artigo em preparação Proteins acting on cell wall polysaccharides

17 WHAT WE HAVE IN TERMS OF INSTRUMENTS FOR PROTEOMICS AND METABOLOMICS? WHAT WE ARE LOOKING FOR COLLABORATION? BIOINFORMATICS FOR INTEGRATION OF THE DATA

18 Synapt G2 HDMS Label free quantitative proteomics - Phosphoproteomics, Glycoproteomics

19 Label free Proteomics

20 MALDI-TOF/TOF e nano LC 2D for complex samples Proteomics, Lipidomics, Fosfoproteomics, Glicoproteomics, Carbohydrates, Metabolomics

21 METABOLOMICs Secondary metabolites; Lipidomics; Glicoproteomics, etc

22 Pegasus 4D GCxGC-TOFMS Comprehensive Two-Dimensional Gas Chromatography with Time-of-Flight Mass Spectrometer Primary metabolites

23 Thank You