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 63100 Clermont-Ferrand France
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
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 2007 60 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
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
Introduction Composition of Wheat grain 63-74.5% 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 97 338 10 Manganese 56 78 5.5 Total Phosphate (%) 0.056 3.17 0.08 Thiamine 0.6 16.5 0.42 Niacine 20-25.7 613-741 8.79 Pyridoxine 6 36 - Riboflavine traces 10 0.28 (Pomeranz, 1988) PL having AL are the most nutrious tissue of wheat grain
Principal phases of Wheat grain development Introduction 24-48 hr 4 DAP 6-8-10 DAP DAP: Days after pollonation 21-30 DAP 11-16 DAP 17-21 DAP 30-40 DAP http://www.wheatbp.net Transcriptomic and cytological studies Wan et al; 2008 Beaugrand et al; 2004
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
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
Material and methods MATERIAL SELECTION & PREPARATION Triticum aestivum cv Récital Standard growth conditions: 15 stages 0 59 109 153 204 247 295 350 397 455 501 546 594 658 700 ( Cd) 30-50 grains dissected/stage Wheat grain Embryo and brush removed Outer Pericarp Removal Periphral layers
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
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
Results Protein expression changes by 2DE of PLs MW kda 80 pi 4.5 5.5 6.5 7.5 8.5 MW kda 80 pi 4.5 5.5 6.5 7.5 8.5 60 60 40 40 20 20 15 15 153 Cd 700 Cd Reference gel for image analysis (mixture of 3 different stages)
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
Results Expression Profiles ; Cluster analysis Dendrogram of columns 1 1 2 a b a b X 1 2 0 59 109 153 204 247 295 350 397 455 501 546 594 658 700 5 main profiles with 4 phases of development X X 3 1: a : 0-59 Cd b: 109-247 Cd 2 a: 295-455 Cd b: 501-700 Cd 4 5 X X X
Results Protein Classification Profile 1 (46 spots) MW kda 80 pi 4.5 5.5 6.5 7.5 8.5 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 20 15 Methionine Glutathion 1400 1200 1000 800 600 400 200 0 109 153 204 247 295 350 397 455 501 546 594 658 700
Results Expression Profiles ; Cluster analysis MW kda pi 4.5 5.5 6.5 7.5 8.5 80 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%) 20 15 5000 4000 3000 2000 1000 0 109 153 204 247 295 350 397 455 501 546 594 658 700 Globulin and globulin like proteins Xylanase inhibitors chitinases Vensel 2005 McIntosh 2007
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
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 P0 0-59 Cd P1 109-350 Cd P2 109-455 Cd P3 153-594 Cd 0 59 109 153 204 247 295 350 397 455 501 546 594 658 700 P4 350-700 Cd
SUMMARY Metabolism PS Storage proteins Genetic Information and processing BSM PT MT ST Defence related proteins Defence structuration by aleurone for embryo and endosperm
PERSPECTIVES Analysis of individual peripheral layers (particularly aleurone layer) For better quality of wheat grain (nutrious and health friendly )
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
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
ACKNOWLEDGEMENT Gerard Branlard Isabelle Nadaud Emmanual Bancel Marielle Merlino Annie Faye Clement Debiton Christoph Chambon Didier Marrion Christine Girrous