PROTEOMIC ANALYSIS OF PERIPHERAL LAYERS DURING WHEAT GRAIN DEVELOPMENT

Transcription

1 BSPR EBI Cambridge, UK PROTEOMIC ANALYSIS OF PERIPHERAL LAYERS DURING WHEAT GRAIN DEVELOPMENT Ayesha TAHIR UMR INRA/UBP 1095 Génétique, diversité et Ecophysiologie des Céréales 234 avenue du Brézet Clermont-Ferrand France

2 CONTENTS Introduction Wheat and Peripheral layers (PLs) Research Targets Sample selection and preparation Results 2-DE PCA analysis Cluster analysis Protein Identification & classification SUMMARY/ Perspectives

3 The bread wheat: a challenging crop species Wheat is the staple food for 35% of the world population 2nd widely grown cereal crop Production: 607 million tons in 2007 Consumption: 620 million tons in days of world stocks in 2007 Annual increase of 2% needed (on constant land area) Significant advances in the understanding of the wheat grain composition must be achieved

4 Introduction Why to study peripheral layers of Wheat Grain? Part of grain weight (% total grain) Proteins Embryo Aleurone layer Pericarp PLs Popineau, 1988 Maternal Origin Several cellular layers 7-9 Aleurone layer, 4 Inner pericarp, Testa, Hyaline Starchy endosperm 3 Albumin et globulin 80 Albumin et globulin Albumin et globulin Gliadines, gluténines Albumin et globulin X Gateway for pathogen attack A. Surget, 2005

5 Introduction Composition of Wheat grain % carbohydrates 12-18% water 8-12% proteins 2.5-3% cellulose 1.5-2% lipids 1.5-2% minerals Constituents (µg/g) Pericarp Aleurone Layer Endosperm Iron Manganese Total Phosphate (%) Thiamine Niacine Pyridoxine Riboflavine traces (Pomeranz, 1988) PL having AL are the most nutrious tissue of wheat grain

6 Principal phases of Wheat grain development Introduction hr 4 DAP DAP DAP: Days after pollonation DAP DAP DAP DAP Transcriptomic and cytological studies Wan et al; 2008 Beaugrand et al; 2004

7 RESEARCH TARGETS Proteomics of peripheral layers (PLs) during wheat grain development To unravel: the major phases of PLs development kinetics of protein expression time course / tissues Identification of: proteins functions metabolic pathways accumulation of constituents (nutritional quality) Research for genes related to nutritional quality of grain

8 CONTENTS Introduction Wheat and Peripheral layers (PLs) Research Targets Sample selection and preparation Results 2-DE PCA analysis Cluster analysis Protein Identification & classification SUMMARY/ Perspectives

9 Material and methods MATERIAL SELECTION & PREPARATION Triticum aestivum cv Récital Standard growth conditions: 15 stages ( Cd) grains dissected/stage Wheat grain Embryo and brush removed Outer Pericarp Removal Periphral layers

10 Material and methods PROTEOMICS: Steps employed for analysis Extraction Reswelling 1 st dimension IEF 3-10 NL (300µg/strip) 2 nd dimension Coloring 3 10 Scanner Image analysis Nonlinear Samespots Cluster anaylsis Permutmatrix 15 stages X 4 rep= 60 gels

11 CONTENTS Introduction Wheat and Peripheral layers (PLs) Research Targets Sample selection and preparation Results 2-DE PCA analysis Cluster analysis Protein Identification & classification SUMMARY / Perspectives

12 Results Protein expression changes by 2DE of PLs MW kda 80 pi MW kda 80 pi Cd 700 Cd Reference gel for image analysis (mixture of 3 different stages)

13 Results Analysis Quality Evaluation Principal Component Analysis (15 stages x 4reps) Statistical Analyse : Normalised Volume 806 significant spots (ANOVA and q-value <0.05) Principal Component Analysis (PCA) PC1 & PC2 = 66% of variance Power analysis (99%) well aligned gels 4 replicates were sufficient

14 Results Expression Profiles ; Cluster analysis Dendrogram of columns a b a b X main profiles with 4 phases of development X X 3 1: a : 0-59 Cd b: Cd 2 a: Cd b: Cd 4 5 X X X

15 Results Protein Classification Profile 1 (46 spots) MW kda 80 pi EM (48%) MOA (7%) TL (2%) AI ( 11%) ST (2%) 60 PF (2%) MDP ( 11%) 40 CM (16%) Chlorophyll a-b binding protein RubisCo enzyme Methionine Glutathion

16 Results Expression Profiles ; Cluster analysis MW kda pi Profile 4 (114 spots) 60 BSM (3%) UP (6%) CSP (14%) MDP (12%) CM (8%) 40 MOA (2%) SP (45%) MT (3%) PS (4%) PF (2%) LM (1%) Globulin and globulin like proteins Xylanase inhibitors chitinases Vensel 2005 McIntosh 2007

17 CONTENTS Introduction Wheat and Peripheral layers (PLs) Research Targets Sample selection and preparation Results 2-DE PCA analysis Cluster analysis Protein Identification & classification Conclusion/ Perspectives SUMMARY / Perspectives

18 SUMMARY 209 proteins identified Classified in 16 different functions 4 phases of grain development Cell division Cell differentiation + start of grain filling Grain filling Grain dehydration 5 main expression profiles of proteins P Cd P Cd P Cd P Cd P Cd

19 SUMMARY Metabolism PS Storage proteins Genetic Information and processing BSM PT MT ST Defence related proteins Defence structuration by aleurone for embryo and endosperm

20 PERSPECTIVES Analysis of individual peripheral layers (particularly aleurone layer) For better quality of wheat grain (nutrious and health friendly )

21 Proteomincs: For Aleurone layer of developing grain 90 3 pi 10 K Da SEM, X 372, Scale 100 microns 10 14% 11% c a T Carbohydrate metabolism AA metabolism, Prot synth. Lipid metabolism Transport 49% 2% S Protease Inhibitor 7% S Oxidative stress 11% 1% 2% 3% Defence Unknown Protein Storage proteins

22 PERSPECTIVES Analysis of individual peripheral layers (particularly aleurone) For better quality of wheat grain (nutrious and health friendly ) Enrichement of knowledge related to cell wall lignification (inner pericarp) Can help for grain size determination Understanding of disease spread and defence mechanisms

23 ACKNOWLEDGEMENT Gerard Branlard Isabelle Nadaud Emmanual Bancel Marielle Merlino Annie Faye Clement Debiton Christoph Chambon Didier Marrion Christine Girrous

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