11th Int. Wheat Gluten Workshop, Beijing August 2012       Monitoring of storage proteins : accumulation and impact of    ...
Content                                                       Wheat proteomicsIntroductionWhat we’ve learned fromwheat pro...
Content                          Some studies of kernel responses to Heat stressIntroduction              Physiology and b...
Content                            Proteomic of endosperm soluble proteins : material Introduction What we’ve learnedfrom ...
Content                         Proteomic of endosperm soluble proteins : some results Introduction What we’ve learnedfrom...
Content                Proteomic of endosperm soluble proteins : protein classification and                               ...
Content            Proteomic of endosperm soluble proteins : proteome map of stress defence Introduction                  ...
Content            Proteomic of endosperm soluble proteins : predicted subcellular localization Introduction              ...
Content            Proteomic of storage proteins : Main protein spots and profiles Introduction What we’ve learnedfrom whe...
Content              Proteomic of storage proteins : accumulation of major protein types Introduction What we’ve learned  ...
Content                          Proteomics of medium thermal stressed kernel: cv Arche 28/15 vs Arche Introduction       ...
Content                 Proteomics of medium thermal stressed kernel:2DE comparison for total Introduction                ...
Content                 Proteomics of medium thermal stressed kernel:2DE comparison for total Introduction                ...
Content                 Proteomics of medium thermal stressed kernel:2DE comparison for total Introduction                ...
Content                      Proteomics of medium thermal stressed kernel : Introduction                 2DE Mapping of th...
Content                 Proteomics of medium thermal stressed kernel :Evolution of the total Introduction                a...
Content                Proteomics of medium thermal stressed kernel : Evolution of the ratio Introduction            gliad...
Content                Proteomics of medium thermal stressed kernel : Evolution of the ratio Introduction            gliad...
Content Introduction What we’ve learnedfrom wheat proteomicsProteomics ofdevelopping kernel-The endosperm     soluble prot...
Content                                 Conclusion 1- on wheat storage proteins stability:                                ...
Content                 Conclusion 2- on wheat storage proteins response to genome variations :                           ...
Content                Conclusion 2- on wheat storage proteins response to genome variations :                            ...
Content                 Conclusion 3: Next step: Deciphering the regulation networkIntroductionWhat we’ve learned fromwhea...
Content             Conclusion : 4- about the genetic and environmental factors associated to                             ...
ContentIntroduction                          The Unfolded Protein Response (UPR mammalian model)What we’ve learned fromwhe...
ACKNOWLEDGEMENTSAyesha TASLEEM-TAHIREmmanuelle BANCELCatherine RAVELPierre MARTRE                                         ...
Thank you
Experiment 3          Quality variations of Arche placed in two conditions during grain formation:                      St...
Experiment 3    Quality variation of Tamaro placed in two conditions during grain formation: Stressed                     ...
Monitoring of storage proteins: accumulation and impact of high temperature in wheat kernel using proteomics
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Monitoring of storage proteins: accumulation and impact of high temperature in wheat kernel using proteomics

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International Gluten Workshop, 11th; Beijing (China); 12-15 Aug 2012

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Monitoring of storage proteins: accumulation and impact of high temperature in wheat kernel using proteomics

  1. 1. 11th Int. Wheat Gluten Workshop, Beijing August 2012 Monitoring of storage proteins : accumulation and impact of high temperature in wheat kernel using proteomics A TASLEEM-TAHIR, E. BANCEL, P. MARTRE and G. BRANLARD INRA 63039 Clermont Ferrand France branlard@clermont.inra.fr
  2. 2. Content Wheat proteomicsIntroductionWhat we’ve learned fromwheat proteomics Proteome : all the proteins expressed in a cell (an organite or a tissue) at aProteomics of given timedevelopping kernel Wheat kernel proteomics on:-The endosperm soluble proteins Storage proteins (since several decades)- The storage proteins Specific proteins Puroindolines, Thioredoxins, GluteninsProteomics of heatstressed kernel Amphiphilic proteins, Albumins and Globulins - Heat effect Proteins associated to starch granules and plastidesConcluding remarks Aleurone layer proteins- Regulations of WSP Aneuploid and isogenic lines 2x, 4x, 6x wheats Kernel development Kernel response to Heat stress
  3. 3. Content Some studies of kernel responses to Heat stressIntroduction Physiology and biochemistryWhat we’ve learned from Bullar and Jenner 1985wheat proteomics Blumenthal et al. 1990 Composition and quality variations High T.: Starch synthesisProteomics of Peterson et al. 1992 Grain Protein contentdevelopping kernel Blumenthal et al. 1993, 1998 Stone et al. 1994 Glutenins, Gliadins-The endosperm soluble Wardlaw and Wrigley 1994 Grain filling time proteins Craft et al. 1996 Gibson et al. 1998 Dough strength- The storage proteins Uhlen et al. 1998 QTLs analysis Jordan et al 2007Proteomics of heat Proteomicsstressed kernel Skylas et al. 2000, 2002 Majoul et al. 2003, 2004 - Heat effect Vensel et al. 2005 Dupont et al. 2006 High T.: Enzymes associated toConcluding remarks Hurkman et al. 2009 Laino et al. 2010 - Energy metabolism- Regulations of WSP Transcriptomics - Starch synthesis Dupont and Altenbach 2003 Altenbach and Kothari 2004 - Stress responses (HSPs) Altenbach et al. 2009 - Protein synthesis Kawaura et al. 2005 Laudencia-Chingcuanco et al. 2007 - Signal transduction McIntosh et al. 2007 Wan et al. 2008
  4. 4. Content Proteomic of endosperm soluble proteins : material Introduction What we’ve learnedfrom wheat proteomics Triticum aestivum L. cv RécitalProteomics ofdevelopping kernel °C days-The endosperm soluble proteins 0 57 93 152 192 247 309 343 401 457 497 547 597 655 706 764 804 857 903 957 1006- The storage proteinsProteomics of heatstressed kernel Binocular Wheat grain Outer Pericarp & Embryo removed PL ESM - Heat effectConcluding remarks - Regulations of WSP 4
  5. 5. Content Proteomic of endosperm soluble proteins : some results Introduction What we’ve learnedfrom wheat proteomics 152°Cd 1006°CdProteomics ofdevelopping kernel-The endosperm soluble proteins- The storage proteinsProteomics of heatstressed kernel - Heat effectConcluding remarks - Regulations of WSP 950 significantly varying spots 487 identified 17 biochemical functions 9 expression profiles Tasleem Tahir A et al., J. Proteome Res. 2012, 11, 2754−2773 5
  6. 6. Content Proteomic of endosperm soluble proteins : protein classification and expression profiles Introduction What we’ve learnedfrom wheat proteomics Carbohydrate M. Stress / defenseProteomics of cytoskeletondevelopping kernel Cell division-The endosperm Protein turn-over soluble proteins Protein synthesis Protein transport- The storage proteins Transcription / translation Amino acid M.Proteomics of heat Lipid M.stressed kernel Protein folding - Heat effect ATP Interconversion Signal transductionConcluding remarks Cell wall - Regulations of WSP Folic acid M. Storage proteins Unknown proteins 6 Tasleem Tahir A et al., J. Proteome Res. 2012, 11, 2754−2773
  7. 7. Content Proteomic of endosperm soluble proteins : proteome map of stress defence Introduction related proteins What we’ve learned kDafrom wheat proteomics 82Proteomics of 63developping kernel-The endosperm P1 soluble proteins P2 P3- The storage proteins 39 P4 P5Proteomics of heat P6stressed kernel 21 P7 - Heat effect P8 P9Concluding remarks 15 - Regulations of WSP In early stages proteins, Catalase, ascorbate reductase, were abundant on acidic side pI 4.8 6.1 7.8 9.5 At later stages proteins (Serpins, alpha amylase and xylanase inhibitor) 7 on basic side
  8. 8. Content Proteomic of endosperm soluble proteins : predicted subcellular localization Introduction of stress defence proteins What we’ve learnedfrom wheat proteomics Predicted Sub-cellular Localization of SD ProteinsProteomics of Number of identified proteinsdevelopping kernel 45 Peroxisome 40-The endosperm Plastid 35 soluble proteins 30 Nucleus- The storage proteins 25 Mitochondria 20Proteomics of heat 15 Extracellularstressed kernel 10 Endoplasmic Reticulum - Heat effect 5 0 cytosolConcluding remarks 1 2 3 4 5 6 7 8 9 Expression profiles - Regulations of WSP Presence of SD proteins in these localizations (Extr, P, ER) at grain maturity help for grain protection Tasleem Tahir A et al., J. Proteome Res. 2012, 11, 2754−2773
  9. 9. Content Proteomic of storage proteins : Main protein spots and profiles Introduction What we’ve learnedfrom wheat proteomicsProteomics of 0 57 93 152 192 247 309 343 401 457 497 547 597 655 706 764 804 857 903 957 1006developping kernel 3 IPGE 11 Type Spots Profiles-The endosperm HMW 22 3 soluble proteins LMW 47 2- The storage proteins S a- b-Gli 15 1Proteomics of heat dstressed kernel s g- Gli 11 2 P - Heat effect a w- Gli 9 2Concluding remarks g e - Regulations of WSP
  10. 10. Content Proteomic of storage proteins : accumulation of major protein types Introduction What we’ve learned mg protein/grain 0.7from wheat proteomics 0.6 alpha-betaProteomics of 0.5 gammadevelopping kernel omega 0.4-The endosperm 0.3 soluble proteins 0.2 ratio- The storage proteins 0.1 0.8 0.7 0 192 247 343 457 547 655 764 1006 0.6Proteomics of heat stages of development °Cd 0.5stressed kernel 0.4 mg protein/grain 1.6 0.3 - Heat effect 1.4 0.2 HMW/LMW HMW gli/gluConcluding remarks 1.2 LMW 0.1 1 0 - Regulations of WSP 0.8 192 247 343 457 547 655 764 1006 stages of development °Cd 0.6 0.4 0.2 0 192 247 343 457 547 655 764 1006 stages of development °Cd
  11. 11. Content Proteomics of medium thermal stressed kernel: cv Arche 28/15 vs Arche Introduction control 23/11 (°C Day/ Night) What we’ve learnedfrom wheat proteomicsProteomics ofdevelopping kernel-The endosperm soluble proteins- The storage proteinsProteomics of heat S1 S2 S3 S4 S5 S6 S7 Harvest Day / Nightstressed kernel 28 °C / 15 °C - Heat effect 23 °C / 11°CConcluding remarks Anthesis 10 20 30 40 50 DAP- Regulations of WSP Stressed 218 288 382 476 546 640 781 Cumulative average Control 163 233 376 443 545 646 778 day temperature Total protein extraction from whole developing seed 4-6 2DEs (IPG pH 3-10 x SDS PAGE) for each sampling
  12. 12. Content Proteomics of medium thermal stressed kernel:2DE comparison for total Introduction proteins cv Arche stressed ( 28/15) compared to control (23/11), Stage S7 What we’ve learnedfrom wheat proteomics 3 IPGE NL 11 97Proteomics of 57 up-regulateddevelopping kernel spots-The endosperm soluble 66 proteins- The storage proteins 40Proteomics of heatstressed kernel - Heat effect 26Concluding remarks- Regulations of WSP 22 14
  13. 13. Content Proteomics of medium thermal stressed kernel:2DE comparison for total Introduction proteins cv Arche stressed ( 28/15) compared to control (23/11), Stage S7 What we’ve learned IPGE NLfrom wheat proteomics 3 11 97Proteomics of 25 down-developping kernel regulated spots-The endosperm soluble 66 proteins- The storage proteins 40Proteomics of heatstressed kernel - Heat effect 26Concluding remarks- Regulations of WSP 22 14
  14. 14. Content Proteomics of medium thermal stressed kernel:2DE comparison for total Introduction proteins cv Arche stressed ( 28/15) compared to control (23/11), Stage S7 What we’ve learnedfrom wheat proteomics IPGE NL 3 1Proteomics of 97 5 induced spots 1developping kernel-The endosperm soluble 66 proteins- The storage proteins 40Proteomics of heatstressed kernel - Heat effect 26Concluding remarks- Regulations of WSP 22 14
  15. 15. Content Proteomics of medium thermal stressed kernel : Introduction 2DE Mapping of the gliadins , HMW-GS and LMW-GS spots What we’ve learned IPGE 3 11from wheat proteomics 97Proteomics ofdevelopping kernel-The endosperm soluble 66 proteins- The storage proteins 40Proteomics of heatstressed kernel - Heat effectConcluding remarks 26- Regulations of WSP 22 14
  16. 16. Content Proteomics of medium thermal stressed kernel :Evolution of the total Introduction amount (sum of spots) of gliadins and glutenins in the kernel of cv Arche What we’ve learned placed in two conditions during grain formation: Stressed 28°C/15°C;from wheat proteomics Control: 23°C/11°C (day/night)Proteomics of proteins µg/graindevelopping kernel 9000-The endosperm soluble Total proteins : Control 8000 proteins 7000- The storage proteins Total proteins : Stressed 6000 Total proteins C 5000 Total proteins SProteomics of heat Gliadins Cstressed kernel 4000 Gliadins S Gliadins : Control Glutenins C Glutenins S 3000 - Heat effect Gliadins : S 2000Concluding remarks Glutenins : C 1000 Glutenins : S- Regulations of WSP 0 0 1 2 3 4 5 6 7 8 Stressed 218 288 382 stages 476 546 640 781 Cumulative average Control 163 233 376 443 545 646 778 day temperature
  17. 17. Content Proteomics of medium thermal stressed kernel : Evolution of the ratio Introduction gliadins/glutenins in kernel for two thermal conditions Stressed: 28°C/15°C; What we’ve learned Control: 23°C/11°Cfrom wheat proteomicsProteomics ofdevelopping kernel Gliadins/Glutenins-The endosperm soluble 2.500 proteins 2.000- The storage proteins 1.500 gliadins/glutenins CProteomics of heat gliadins/glutenins S 1.000stressed kernel 0.500 - Heat effect 0.000Concluding remarks 0 100 200 300 400 500 600 700 800 900 thermal time °C/j- Regulations of WSP
  18. 18. Content Proteomics of medium thermal stressed kernel : Evolution of the ratio Introduction gliadins/glutenins in kernel for two thermal conditions Stressed: 28°C/15°C; What we’ve learned Control: 23°C/11°Cfrom wheat proteomicsProteomics ofdevelopping kernel Ratio gliadins/glutenins TAMARO Gliadins/Glutenins 2.5-The endosperm soluble proteins 2.0- The storage proteins 1.5 gliadins/glutenins CProteomics of heat gliadins/glutenins S 1.0stressed kernel 0.5 - Heat effectConcluding remarks 0.0 150 250 350 450 550 650 750 850 thermal time °C/j- Regulations of WSP
  19. 19. Content Introduction What we’ve learnedfrom wheat proteomicsProteomics ofdevelopping kernel-The endosperm soluble proteins- The storage proteins Some concluding remarks on Wheat storage protein regulationProteomics of heat from different proteomics datastressed kernel - Heat effectConcluding remarks - Regulations of WSP
  20. 20. Content Conclusion 1- on wheat storage proteins stability: Evolution of the ratio gliadins/glutenins in kernel for two thermal conditions Introduction What we’ve learned Stressed: 28°C/15°C; Control: 23°C/11°Cfrom wheat proteomicsProteomics ofdevelopping kernel Arche : medium Quality Tamaro : Very High Quality-The endosperm soluble proteins Gliadins/Glutenins cv Arche Gliadins/Glutenins cv Tamaro Ratio gliadins/glutenins TAMARO- The storage proteins2.500 2.52.000 2.0Proteomics of heat1.500 1.5 gliadins/glutenins C gliadins /glutenins Cstressed kernel gliadins/glutenins S gliadins /glutenins S1.000 1.0 - Heat effect0.500 0.5Concluding remarks0.000 0 100 200 300 400 500 600 700 800 900 0.0 150 250 350 450 550 650 750 850 thermal time °C/j thermal time °C/j - Regulations of WSP Differences between cultivars associated to gene regulation
  21. 21. Content Conclusion 2- on wheat storage proteins response to genome variations : ex 1: 1BL/1RS translocation vs normal 1BLIntroductionWhat we’ve learned fromwheat proteomics Line 1B Line 1BL/1RSProteomics ofdevelopping kernel-The endosperm soluble proteinsGli-B1 Glu-B3- The storage proteinsProteomics of heatstressed kernel ? LEA Gli-D1 g-gliadin Gli-R1? a-Amyl - Heat effect Inhibitor with 9 cysteinsConcluding remarks - Regulations of WSP « Silenced gene », strongly up regulated for SP accumulation, and revealed through selection for high quality Gobaa S., et al. Proteomics 2007, 7 : 4349-4357.
  22. 22. Content Conclusion 2- on wheat storage proteins response to genome variations : ex 2 : nullisomic lines vs normal linesIntroductionWhat1we’ve 3 4 5 from 7 2 learned 6 IPGEwheat proteomics 3 10 Mr Nulli1A 1 (kDa) A * 3 2 7 5 6 100Proteomics of 9 11 13developping kernel 12 14 184 w1B B 18 17 15 22 321 w1B-The endosperm 25 24 26 soluble proteins D * * 28 32 30 29 64 35- The storage proteins 47 54 S 81 37 * * 69 56 89 73 52 582 g1B D S 87 105 648 b6BProteomics of heat 121 120 125 -stressed kernel P * 126 128 127 A 39 129 131 228 w1B - Heat effect 41 49 40 32 G 62 20 LMW 1DConcluding remarks E * 71 65 72 75 79 77 85 - Regulations of WSP Compensation revealing that regulation of SP accumulation 90 97 91 92 94 141 256 a6A 166 a6A 100 104 107 involves complex interactions between homoeologous loci. 108 111 110 142 a6A 115 114 118 112 What are the cis-acting transcription factors? 113 116 20 146 62 g1B Dumur J. et al. Proteomics, 2004; 4: 2685-2695.
  23. 23. Content Conclusion 3: Next step: Deciphering the regulation networkIntroductionWhat we’ve learned fromwheat proteomics Model* for Regulation of Barley Storage ProteinProteomics ofdevelopping kernel-The endosperm Proximal promoters Distal promoter sequence soluble proteins- The storage proteinsProteomics of heatstressed kernel - Heat effectConcluding remarks - Regulations of WSP 8 TFs capable to be linked to SP promoters 3 orthologs each 24 candidate genes * From : Diaz et al. 2002; Rubio-Samosa et al.2006; Moreno-Risueno et al., 2008
  24. 24. Content Conclusion : 4- about the genetic and environmental factors associated to gluten polymer sizeIntroductionWhat we’ve learned from For developping wheat tolerant to heat stress, the expressionwheat proteomics and regulation of some major enzymes need to be better knownProteomics ofdevelopping kernel - For energy and starch metabolisms ATP synthase ; RuBisCo-The endosperm PPase, SSS, Beta-amylase soluble proteins - For protein accumulation- The storage proteins eIF4E ; PDIs ; HSPs 90 ; HSPs 70 etc..Proteomics of heat - For response to oxydative stressstressed kernel O2-, superoxide anion - Heat effect H2O2, hydrogen peroxide Endosperm cell apoptosis •OH, hydroxyl radicalConcluding remarks ROO•, peroxy radicals - Regulations of WSP GSS, CAT, GPX, etc.. Grain Hardness impacts the gluten polymer size Lesage, V et al., J Cereal Sci 2011, 53, 231-238. Lesage, V. et al., J Exp Botany, 2012, 63,1001-1011.
  25. 25. ContentIntroduction The Unfolded Protein Response (UPR mammalian model)What we’ve learned fromwheat proteomicsProteomics ofdevelopping kernel ROS • Increase of: • Chaperone synthesis-The endosperm soluble proteins • Anti-oxidant response ER • ER Associated degradation- The storage proteins Decrease of translationProteomics of heatstressed kernel - Heat effect NucleusConcluding remarks - Regulations of WSP Protein transport via ER Schröder and Kaufman, 2005. Annu. Rev. Biochem.
  26. 26. ACKNOWLEDGEMENTSAyesha TASLEEM-TAHIREmmanuelle BANCELCatherine RAVELPierre MARTRE 26
  27. 27. Thank you
  28. 28. Experiment 3 Quality variations of Arche placed in two conditions during grain formation: Stressed 28°C/15°C; Control: 23°C/11°C (day/night) Arche 100 Stressed Control P(mm) 50 0 0 50 99 149 L(mm)
  29. 29. Experiment 3 Quality variation of Tamaro placed in two conditions during grain formation: Stressed 28°C/15°C; Control: 23°C/11°C (day/night) Tamaro 150 Stressed Control 100 P(mm) 50 0 0 50 99 149 L(mm)

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