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Integrating diverse datasets to understand photosynthetic development in C3 and C4 plants Tom Brutnell Boyce Thompson Institute Why iPlant and not iBrain? • World population is expected to reach 9 billion by 2050 (currently approx. 7 billion) = more mouths to feed, cars to fuel and bodies to clothe • We are near or past peak oil production = food production is more expensive (fertilizer and tractors) • Global warming is reshaping land use (bioenergy) and contributing to a more unpredictable environment Urgent need to develop the NextGen of food, feed and bioenergy crops Global food security • In 46 developing countries food prices are higher than 12 months ago, despite global recession • In sub-Saharian Africa price of maize, millet and sorghum are at alltime highs • Major agronomic disaster (e.g. drought in China) could have serious consequences on food supply Photosynthesis: C3 to C4 C5 BS 2xC3 M From website of Donald Ort http://www.life.uiuc.edu/pru/labs/ort.html Models for C4 photosynthetic development C3 state C4 state A) repression of gene expression through novel trans-factor B) Enhanced expression in BS or M through novel cis-element Models for C4 photosynthetic development C3 state C4 state C) Destabilization of protein due to novel cellular environment (e.g. redox, missing complex) D) Expression directly or indirectly linked to metabolite Y Using monocot leaf gradient to find networks and regulatory points for C4 Tests Modify regulatory points Perturb environment Modeling Expression/regulation Metabolism Comparisons developmental stages BS vs. M cells (LCM) C3 and C4 Developmental inventories transcripts proteins metabolites physiology anatomy Physiology & cell biology—Bob Turgeon (Cornell) Photosynthesis—Richard Peterson (CAES) Transcriptomics—Tom Brutnell (Boyce Thompson Inst) Proteomics—Klaas van Wijk (Cornell) Laser microdissection—Tim Nelson (Yale) Metabolites—Alisdair Fernie (MPI-Potsdam Golm) Statistics—Peng Liu (Iowa St U) Informatics—Qi Sun (Cornell), Pankaj Jaiswal (Oregon St ) Modeling, systems—Chris Meyers (Cornell) Where should we sample? Maize inbred B73, 9 dap L3 L2 Ligule of L2 L3 L1 Standardization of the base-to-tip leaf gradients • extent of growth (time) • anatomical features • gene markers • sink-source transition Anatomical gradient: Maize L3 Base Maize L3: -1 to 0 cm past L2 ligule Maize L3: 4 to 5 cm past L2 ligule M BS Maize L3: 1 cm from tip BS M Locating sink-source transition Sink-source transition is site of global changes in physiology, expression, anatomy Label from L3 tip Label 5 min Chase 1 hour from L1 Max chl accumulation Plastid diffn complete Sink-Source transition complete L2 ligule Zone of import limited to below L2 ligule • L2 ligule is an accurate physical marker for sink-source transition in L3 • Ligule site confirmed accurate for both maize and rice L3 Gene expression markers along gradient 3 2 Calibrated gradient with 20 expression markers (RT-PCR) PPdK • PS genes increase from S-S transition 1 0 20 16 12 8 4 0 Sheath CycD2 • Cell cycle genes only near base Developmental Inventories—Common samples for all inventories Leaf Section 1 Section 4 Section 9 Section 14 Source-sink Histology LCM RNA isolated RNA isolation and amplification Metabolite extraction Total RNA Primary metabolites Bundle sheath cell Mesophyll cell mRNA aRNA Secondary metabolites Small RNA library Metabolites profiling RNA-seq library Solexa libraries sequencing qPCR validation Solexa expression analysis Proteomics Systems biology NextGen Sequencing 16 NextGen Sequencing 17 Developmental Inventories—dynamics of individual RNA-seq profiles • Resolves closely related members of a gene family • Identifies alternative splicing patterns • Allele-specific transcription • Less dependent on gene annotation • More dynamic fold change Splice variants by stage novel transcript MapMan Pathway Viewer- see Bjoern Base Tip Adapted MapMan view: does not show multiple stages, cell types, C4 Mesophyll Cell Bundle Sheath Cell PEP PEP MEP3 RPE Xyl-5P RPI TKL OAA OAA MEP1? TPI TPI NADP+ NADP-MDH Mal DAP Maltose DIT1 ASP PPDK: Pyruvate Orthophospho-Dikinase PRP: PPDK – regulatory protein PEPCase: PhosphoenolPyruvate Carboxylase CA: Carbonic Anhydrase NADP-MDH: NADP – malate dehydrogenase TPT: Phosphate/triose-phosphate translocator SBP NADPH Sucrose Starch Synthesis OAA Glu NADP+ AKG Mal FBA MEX1 AKG ASP-TS Glu TKL FBP DAP NADPH OAA ASP-TS GAPB Pyr ATP PRK PGK GAPA AMP Mitochondrion HCO3- PGK CO2 NADP-ME CA RCA Rubisco PGA PGA PEP PEPCase CO2 PPT ? PPDK PRP Chloroplast AMP Chloroplast ATP ASP DIT2 ATP ADP AATP1 Pyr MEP4 PEPCK ATP ADP Pyr Mal ASP NADP-ME: NADP – Malic enzyme PPT: Phosphate/PhosphoenolPyruvate translocator DIT: 2-oxoglutarate/malate translocator MEP: Envelope protein PEPCK: PhosphoenolPyruvate carboxykinase Rubisco: Ribulose-1,5-bisphosphate carboxylase/oxygenase RCA: Rubisco activase PGK: Phosphoglycerate kinase PRK: Phosphoribulokinase TPI: Triosephosphate isomerase RPI: ribose-5-phosphate isomerase GAPA: Glyceraldehyde-3-phosphate dehydrogenas TKL: Transketolase GAPB: Glyceraldehyde-3-phosphate dehydrogena SBP: Sedoheptulose-1,7-bisphosphatase ASP-TS: Aspartate Aminotransferase FBA: Fructose-bisphosphate aldolase DAP: dihydroxyacetone phosphate Lin 02/2009 δ α β AT P α PsbC δ α β AT P PSII H2 +O2 α PsaF PC PSI Lumen stroma cytosol H+ NDH complex H+ Cyt b6f complex PC PsaA PsaB PsaN PsaF PSI Lhca 1-4 ISPa 3H+ C, E, H, J, K IV M, I, L, O, D, G PQH2 Cyt f NdhA NdhB ISPb ATP synthase PsaN FD Cyt b6 a PsaB N M H NdhL NdhJ NdhD NdhF C12 H+ Cyt b6f complex O γ thylakoid membrane ISPa PsaA FNR2 bb ε PsbR ISPb Lhca 1-4 ADP + Pi PsbQ IV C, E, H, J, K H2O PsbO M, I, L, O, D, G 3H+ PsbP Cyt f PsbA PsbD Cyt a PsbB b6 C12 FD L, S, N, H, M, X γ ATP synthase Bundle Sheath PQH2 bb ε thylakoid membrane FNR1 hv J, K, W, I, E, F Lhcb 1-6 Mesophyll ADP + Pi NADP+ NADPH Lumen stroma cytosol Lin 02/2009 PageMan view of developmental gradient, BS/M specificity gradient BS/M Down in M Up in BS C4 pathway viewer with plasmodesmata, transporters, etc TFs clustered by developmental dynamics base -1 4 tip A B C D E F G H I TF types in dynamic classes A B C D E F G H I XXXX Input gene ID 0h 6h 12h 18h 24h Gene expression base -1 +4 tip “a slider view” 0h 6h 12h 18h 24h Gene expression base -1 +4 tip “a slider view” 0h 6h 12h 18h 24h Gene expression base -1 +4 tip “a slider view” 0h 6h 12h 18h 24h Gene expression base -1 +4 tip “a slider view” 0h 6h 12h 18h 24h Molecular inventories correspond well to leaf gradient features Plastid differentiation Cell division Cell wall Polymers Plasmodesmata Cell patterning Respiration Transcr. factors PS light & dark reactions (C4) PS Pigments PS Activities PS metabolites Plastid number Transcription factors L3 base tip Sink-Source transition L2 ligule