Engineering c4 rice

Engineering c4 rice
Engineering C4 rice C3 C4 XI Conferencia Internacional de Arroz para America Latina y el Caribe 21-24 Septiembre 2010 Cali, Colombia © WPQ
Green Revolution Slows World Rice Yield (1961-2010) (1961 2010) Data Source: FAO Average yield (t ha-1) Average yearly increase over previous 10 years (kg ha-1) 5.0 5 0 200 4.0 160 3.0 120 2.0 80 1.0 1 0 40 0.0 0 1955 1965 1975 1985 1995 2005 2015 Year © WPQ
The relationship between rice production and population f A i rice consumers (1961 2004) l ti for Asian i (1961-2004) Data Source: UN and FAO Production (Mt) 900 800 4.56 B 700 2050 600 500 400 300 200 100 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Population (Billion) © WPQ
A Second Green Revolution? Final dry weights of crops in C3 and C4 groups correlated with the length of growing season (Monteith, 1978) Standing dry weight at harvest (t/ha) C3 kale Maize C4 Grain Yield = 13.9 t ha-1 44 DAG 1 80 sugar beet potatoes rice cassava 60 C4 oil palm C4 bulrush millet 40 maize sorghum sugarcane C3 napier grass 20 IRRI Expt-rice IRRI Expt-maize 0 0 100 200 300 Length of growing season (days) Rice C3 Grain Yield = 8.3 t ha-1 42 DAT © WPQ
C4 RICE C4 rice could: i ld •increase rice yield by 50% •double water-use efficiency •improve nitrogen use efficiency nitrogen-use C4 photosynthesis is one of the few evolutionary mechanisms that could deliver these superior combination of b th i bi ti f benefits. fit © WPQ
C4 would confer benefits on all of the global rice ecosystems Source: Ric Alm n c (Maclean t l S u c : Rice Almanac (M cl n et al, 2002) IRRIGATED RAINFED Area = 79 M ha Area = 36 M ha Production = 75% Production = 18% UPLAND DEEP WATER Area = 19 M ha Area = 12 M ha Production = 4% Production = 3% © WPQ
C4 Supercharges Photosynthesis Using A Two Compartment CO2 Concentrating Mechanism C3 Photosynthesis C4 Photosynthesis 3 Phosphoglycerate c c c © WPQ
C4 photosynthesis involves alterations to biochemistry, cell biology and leaf anatomy CO2 Mesophyll Cell HCO3- 1 OAA 2 PEP 3 5 Pyruvate Malate = C4 4 CO2 RuBisCO Bundle Sheath Cell Many of the genes that control these processes are unknown © WPQ
Evolutionary Change Genetic alterations C3 + Anatomy Change + Biochem Change + Fine Tuning = C4 © WPQ
Despite its complexity C4 has complexity, evolved independently ~60 times It can’t be that difficult?! © WPQ
© WPQ
The Timeline for C4 Rice It will likely take a minimum of 15 years of coordinated research carried out in the laboratories of the C4 Rice Consortium to deliver C4 rice to plant breeders in the developing world. 3 years 3 years Gene 5 years discovery Transform 4 years and rice to Optimize molecular express Breed C4 C4 function toolbox Kranz transgenic in development anatomy s transgenic and the C4 into local rice Characterize metabolic varieties regulatory enzymes controls © WPQ
To determine the feasibility of replicating the two-cell C4 photosynthetic pathway in rice: p y p y Challenge 1 Leaf Anatomy Challenge 2 Cell Biochemistry © WPQ
Molecular Engineering Team Julian Hibberd Ajay Kohli Jane Langdale Inez Slamet-Loedin Peter Westhoff Transgenics; over expression, RNAi reduction, characterisation of expression reduction transgenics Identification of regulatory switches, micro dissection of leaf BSC, MC primordia BSC MC, primordia, transcriptome and genome sequencing Identification of promoters and their regulation to give accurate cell specific and developmenal expression
Molecular Engineering - building up C4 biochemistry abundance anscripts Relative Reduce Glycine of decarboxylase in BSC R tra nce pts Increase PEPC c ease C ve transcrip abundan Relativ of in MC abundance transcripts Relative Increase PPDK of in MC BSC =Bundle Sheath Cell MS = Mesophyll Cell
Identify promoter elements to allow cell specific expression in rice Gene Promoter BSC Specific regions of the non-coding DNA sequence MS of C4 genes direct Cell-Specific Cell Specific expression BSC © WPQ
Molecular Physiology Team Bob Furbank Jim Burnell Gerry Edwards Richard Leegood Rowan Sage Tammy Sage Susanne von Caemmerer high throughput screen development, detailed mechanistic physiology of C3 and C4 gene specific antibody production and biochemical characterization of enzymology of transgenics detailed microscopy of C3 and C4 anatomy and characterisation i th mutant and t h t i ti in the t t d transgenic li i lines
Bioinformatics and Systems Biology Team Xinguang Zhu Richard Bruskiewich Chris Myers Tom Brutnell Tim Nelson data d t analysis of sequencing projects t l i f i j t transcriptome/genome i t / modelling the C3 and C4 pathways analysis of maize t l i f i transciptome along l f d i t l leaf developmental l t l gradients in BSC and MC bioinformatics of vascular development in model systems and search f rice analogues d h for l develop the C4 web platform for the consortium
Transcriptome and genome of closely related C3 and C4 species - increasing phylogenetic coverage The 1000 plant transcriptomes project - Gane Wong and Beijing Genome Institute The 100 plant genome project Molluginaceae, Amaranthaceae, Aizoaceae, Chenopodiaceae, Nyctaginaceae, Portulacaceae 26 transcriptomes 20 genomes Euphorbiaceae, Chamocyceae Cleomaceae Boraginaceae, Zygophyllaceae Scrophulariaceae Asteraceae © WPQ
Standardized Maize leaf developmental gradient for 9 day old Leaf 3 Leaf 1 Sink Transition Source Base ‐1 cm +4 cm Tip Nelson and Brutnell N l dB ll Cornell USA © WPQ
Identify development related Adaxial/abaxial polarity HB-PHB, ZmRLD1, REV ARF-ARF3, ARF4 transcription factors YABBY-ZmYAB2, ZmYAB14, ZmYAB15 GARP G2- KAN1, ZmMWP1 Myb- ZmRS2 G1 G2 G3 Stomatal development/movement bHLH-FAMA, MUTE, ICE1 Myb-MYB60, MYB61 GeBP 10 Leaf morphogenesis/development GRF 11 Cell fate: GeBP YABBY-DL1, DL2 MADS-AGL Cell expansion/growth: GRF family Trihelix-GTL Alfin-like 13 Cell differentiation: TCP Myb-LOF1 SBP-ZmLG1, SPL Early = anatomy Trihelix 22 G1 Vascular development: ARF-MP HB-HB15, ZmRS1, KANT7 MADS 11 1 (base) Metabolic process C2C2-YABBY 7 1 Wax/lignin/carbon: AP2/EREBP-SHN1, WRI1NAC NST1 Wa /lignin/carbon AP2/EREBP SHN1 WRI1NAC-NST1 TCP 19 1 2 Signaling Hormone: GRAS-SLR1,GAI1, SCL3 ARF ARR Aux/IAA 17 3 Sugar: bZip-ABF2 zf-HD 11 1 1 Chromatin regulation Alfin-like Middle = cell function SBP 10 2 Secondary cell wall C2C2-GATA 22 2 4 NAC-SND1, SND2 GRAS 23 3 5 HB-KNAT MYB-MYB52, MYB54, MYB63, MYB85 ARF 21 3 5 G2 Lipid (VLCFA): MYB-MYB30 bZIP 42 3 13 ( (transition) ) Light signaling: bHLH-PIL6, PIF3 GRAS-PAT1 bHLH 61 10 18 Leaf morphogenesis/development ARF-ARF19 AS2-ZmRA2 Late = photosynthesis HB 41 9 12 Photosynthetic HSF 11 2 4 Apparatus Circadian GARP G2-ZmG2, ZmGLK C2H2 -zinc 42 23 photoperiod Light s g a g g t signaling DOF CDF3 MYB LHY DOF-CDF3 MYB-LHY AP2/EREBP 25 5 9 C2C2 CO-STO, COL3 DOF-OBP3 WRKY 28 7 9 G3 bHLH-PIL5, PIL6 bZIP-HY5, CPRF2 C2C2-DOF 13 2 6 (tip) Development Photoprotection: C2H2 Zinc-ZAT10 NAC-NAC1, VND7 HB-BEL1 MYB-ZmMybst1 GARP G2-APL MYB 56 15 28 NAC 18 8 17 Photosynthetic Apparatus Photosynthetic Apparatus GARP G2-ZmG2 TCP-PTF1 GARP G2-GLK ARR 4 1 5 Light signaling Light signaling GARP-G2 10 2 13 DOF- OBP3, DAG1 Development BS M AUX/IAA-PAP2 bZIP-HY5 bHLH-PIL5 C2C2 CO-STO 4 5 17 C2C2-CO TCP –TCP5 NAC family GRAS-PAT1 0% 50% 100% © WPQ
Genetic Screening Team Paul Quick Hei Leung Gynheung An Caroline Hsing Erik Murchie Su-May Yu John Sheehy EMS mutagenised rice generation and screening of mutagenised sorghum production of transgenic activation tagged rice populations for non-targeted screening and provision of targeted activation tagged lines gg screening activation tagged rice lines
Phase 1: Gene Discovery y Screen Screen transcriptomes genomes Screen mutagenized g and transgenic lines C3 to C4 lineages Rice activation- activation- Sorghum and maize S h d i C3 and C4 related species tagged lines mesophyll, bundle Model species Arabidopsis, sheath, leaf development Setaria, Brachypodium, Sorghum, Rice Sorghum mutant lines Establish a pool of genetic diversity that confers C4 traits Gene candidates tested transgenically in rice © WPQ
Rice activation tagged lines Ri ti ti t d li ACTIVATION C3 + Anatomy Change + Biochem Change + Fine Tuning = C4 REVERSION Sorghum mutant lines © WPQ
Simple High Throughput Screens © WPQ
Compensation point CO2 response curve Microscopy images Leaf Gross Photosynthesis (mmol CO2 m-2 s-1) 15 Rice Maize 10 Sorghum 5 0 -10 0 10 20 30 40 50 60 70 80 90 100 110 CO2 (ppm) -5 -10 Rice Maize Sorghum © WPQ
Low CO2 Screening Chamber © WPQ
Vein spacing? Identify C4 genes that regulate vein spacing C4 plants -have narrower vein spacing with 7 or more veins per mm C3 plants - have wider vein spacing, there are about 5 veins per mm Currently, these genes are largely unknown Mutate C4 genes – Sorghum or Activate C3 genes - Rice Vein BS M M BS Vein Strategy Vein BS M M M M M M M M BS Vein Mutate C3 plants - Rice © WPQ
Simple and Detailed anatomical characterization of (A) rice and (B) sorghum sorghum. A B © WPQ
Activation tagged lines of rice Leaf Sampling in Taiwan – flag leaf samples collected from each of the 12 replicates of the 5,050 mutant lines Su-May Yu (Academia Sinica, Taiwan) © WPQ
Interesting Rice mutants TRIM # Vein Spacing Tainung67 (WT) 104656 7 + 0.0 vein spacing = 5.5/mm 1 2 3 4 5 6 7 8 9 10 11 108615 7+01 0.1 110321 7 + 0.1 105588 8 + 0.4 106332 6.5 + 0.2 106602 6.5 + 0.4 TRIM Mutant 108615 vein spacing = 7/ i i 7/mm 1 2 3 4 5 6 7 8 91011 12 1314 110124 6.0 0.3 60+03 Frequency of mutation: about 1 in 1000 © WPQ
Some interesting mutants are starting to emerge! Tainung67 7 mesophyll cells p y between veins b i vein vein TRIM Mutant 108615 5 mesophyll cells between veins vein vein © WPQ
Secondary screen - Morphological characterization of high vein density mutants at 7th leaf stage y g 30 cm WT-T67 M104656 M110321 M110124 M105588 1 2 3 4 5 1 2 3 4 5 6 78 1 2 3 4 5 6 78 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 Vein density mutants exhibit shorter plant h i ht V i d it t t hibit h t l t height compared with the wildtype
C4 - Sorghum: Generation of Mutant Resources EMS mutagenised M2 population created 2009 Gamma Irradiated M2 population created 2010
Screening of M2 EMS and Gamma mutant populations of sorghum (2010)
end MACRO SCREEN Discard No Vein density of start 1,000 M2 seed lines Score for pale and albino 5th leaf Vein density/mm ≤7 Yes Possible candidates Phase 1 one leaf Phase 2 all leaves Vein density candidates Detailed leaf anatomy
Leaf vein density of wild type and interesting y yp g EMS mutants of sorghum (BTx623) 1 3 5 7 9 11 13 15 17 2 4 6 8 10 12 14 16 18 1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 a. Wild type, VD = 18 b. Mutant ID no. 161, VD = 14 c. Mutant ID No. 279, VD = 13 © WPQ
Sorghum mutant with increased Sorghum mutant mesophyll cells between veins 5 1 4 2 3 vein vein vein vein 1 2 Sorghum wildtype g m yp
T4R2-028 plant no. 4 T3R1-028 plant no. 18 4-5 mesophyll cells p y 4 mesophyll cells p y 1 2 1 2 3 4 3 4 1 2 3 4 5 4 1 2 3 © WPQ
Crinkly Some vein density changes are associated with phenotypes h t Asymmetric Dwarf Grass-like Pale © WPQ
Conclusions We have assembled a global network of scientists of diverse disciplines to tackle a complex interdisciplinary problem with significant implications for agriculture Transcriptome and genome screens are progressing and T d d have already revealed many candidates for us to test. Much more sequencing is in progress. Some hi h th S high throughput screens are in place for leaf anatomy h t i l f l f t and photosynthetic compensation point. We have already identified in rice and sorghum putative mutant candidates with phenotypic variants i l f t t did t ith h t i i t in leaf anatomy. © WPQ
The IRRI C4 Team Acknowledgements k l d John Sheehy The C4 Rice Consortium BMGF for funding We welcome collaborations and our plan is to expand the consortium as far as possible: • to enhance our current efforts • to bring in new ideas • to introduce additional sources of funding © WPQ
Thank you for listening http://www.amazon.com/books-used-books-textbooks/ C4 Rice web site: http://beta.irri.org/projects15/c4rice
Phenotype 1 45 CO2 response curve 40 35 30 nthesis 25 Rooney 20 ml14-40 Photosyn 15 IR72 10 Ml 14-40 r2 5 0 0 50 100 150 200 250 300 350 -5 -10 Ci (ppm) CO2 Compensation point: 11.36 Vein Density No. of hits with vein density ≤14 = 4 Status: Healthy
Phenotype 2 45 CO2 response curve 40 35 30 nthesis 25 Rooney 20 Ml 26-10 Photosyn 15 ml26-10 IR72 10 5 0 0 50 100 150 200 250 300 350 -5 -10 Ci (ppm) CO2 Compensation point: 11.62 Vein Density No. of hits with vein density ≤14 = 4 Status: Healthy
Radiation Use Efficiency is improved by 50% Source: Sheehy et al 2007 al, Above-ground dry weight (g m-2) 3500 3000 MAIZE y = 4.4x 2500 r2 = 0.98 2000 1500 RICE 1000 y = 2.9x r2 = 0.98 500 0 0 200 400 600 800 Accumulated intercepted PAR (MJ m-2) 2 © WPQ
C (mmol CO2 mol-1 H2O) 0.0 1.5 3.0 4.5 6.0 7.5 F. cro onquisti C3 F. pringlei p C3 Water use efficiency F. robusta r C4-like F. angu ustifolia Annual C4 F. chlor raefolia Type I C3-C4 Perennial C4 Type II C3-C4 F. sono orensis F. an nomala C3-like F. flo oridana F. ramos sissima F. brownii F. palmeri p C4-like F. va aginata greater than C3 plants F. ko ochiana F. bidentis b F. tr rinervia C4 F. austr ralasica Water Use Efficiency is 1.5 to 3 times © WPQ
Nitrogen Use Efficiency is enhanced by 260% Source: Evans and von Caemmerer 2000 Caemmerer, Rate of CO2 assimilation (µmol m-2 s-1) 60 C4 50 Maize Sorghum 40 30 20 C3 Wheat 10 Rice 0 0 20 40 60 80 100 120 140 160 180 200 (mmol m-2) Leaf nitrogen content ( g © WPQ

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