docPresentation in PDF
download 
Report Transcription
Presentation in PDF
Genes associated with Embryogenesis and Abiotic Stress Tolerance in Wheat Paramjit Khurana Department of Plant Molecular Biology University of Delhi South Campus New Delhi-110021, India Broad Areas & Objectives Molecular Biology of Embryogenesis Mechanism of Abiotic Stress Tolerance Genetic Transformation for Crop Improvement Comparative/Functional Genomics All grain is seed, and all seed is EMBRYO ! Plant Embryogenesis Zygotic vs. Somatic Embryogenesis • Proves the concept of Totipotency • Defies Cell Commitment & Genetic Programming • Implies Cellular Plasticity • Similarity with Zygotic Embryogenesis • Model system for Embryogenesis • Practical Applications !! Plant Embryogenesis Triticum aestivum …..bread wheat • Short-term auxin treatment • Short-term response • Efficient regeneration • Genetic transformation studies • Developmental Biology of SE ….Plant Biotech. (2003) Molecular Analysis of Somatic Embryogenesis Control MS basal (10 days) 2,4-D (24 h) 2,4-D (1 d) No. No. of analysed of analysed ESTs ESTs 1196 1196 No. No. of assembled of assembled ESTs ESTs 813 813 No. No. of singlets of singlets 383 383 No. No. of contigs of contigs 312 312 Average Average contig contig length length (bp) (bp) 782 782 % of%the of library the library ESTs ESTs 83 83 Maximun Maximun no. no. of ESTs of ESTs perper contig contig 25 25 502 64 Control 201 55 414 67 91 170 57 385 10 days on MS (SE) ….Plant Molecular Biology (2007) Characterization of TaIAA1 in Wheat Hoechst dye GUS buffer • Auxin induced • BR stimulated • Calcium-mediated • Nucleus localized • Light down-regulated both the NLSs + GUS a b SV40 type NLS + GUS c d bipartite NLS + GUS e KR f RSFRR KRLRIMK nls1-2 only GUS nls1-3 nls A-3 GUS nucleus GUS nucleus GUS nucleus/cytoplasm g ….J. Expt. Bot. (2006) Somatic Embryogenesis Related Kinase (SERK) gene family in Wheat & Rice 6 TaSERK1 4 OsSERL1 OsSERL2 OsSERL3 OsSERL4 OsSERL5 6 4 OsSERL6 TaSERK 2 OsSERL7 OsSERL8 OsSERL9 OsSERK1 Cysteine pairs 6 Signal seq. MER BSH MEH MEC LBE LBC ME DS MSS Leucine zipper anther ovary root TaSERK3 leaf 4 2 OsSERK2 SPP motif Protein Kinase LRR Transmemb. region ….Plant Cell Reports (2008) ….Int. J. Plant Genomics (2009) Molecular Network for Somatic Embryogenesis ….the present senario! ….Plant Molecular Biology (2007) Molecular Network for Somatic Embryogenesis GST ….Plant Molecular Biology (2007) Auxin & Induction of Somatic Embryogenesis Somatic Embryogenesis Heat Stress and Wheat Productivity …. Grain starch !! Grain filling enzymes ..soluble starch synthases ..starch branching enzymes ..ADP glucose pyrophosphorylase ..etc. • Every degree increase in temperature (>20oC) corresponds to a 4% drop of grain weight ! ∆ Per day loss = per quintal /hectare drop. Subtractive cDNA library…. Northern analysis of select ESTs from Seedling and Developing Seed Library C C 37o 42oC 37o 42oC Stress induced protein STi Putative Se binding protein Put. transmembrane protein F-box domain Calmodulin binding protein Putative B2 protein Lipid transfer protein Putative placental protein Stress responsive protein Luminal BiP HSP26.6 MIPS MIPS Unknown protein HSP 18 Helicase like protein Calcineurin Stress-induced protein STi Rubisco activase HSF Calmodulin Myb transcription factor Unknown Unknown protein Unknown Unknown rRNA rRNA Compromised Fertility during Heat Stress ! ∴ necessary to target the reproductive organs as well ! Semi-quantitative RT-PCR analysis of some ESTs from Tau (Unfertilized Flower FSH) library Flowe Anthe Carpel r r C T C T C T L-ascorbate peroxidase HSP18 HSP23.5 Unknown protein Chitinase class III Peptydil prolyl isomerase Eukaryotic translation initiation factor 5A3 Putative translation factor Zinc finger C 3 HC 4 type Unknown protein Translation elongation factor 1 alpha Lipid binding protein precursor 2 (LTP2) bZIP transcription factor HSP 70C TaHSP82 TaCAM3-1 Cold acclimation protein 14-3-3 protein Unknown protein Actin Expression analysis of MADS box gene in different wheat tissues by real time PCR . 200 180 160 140 ▼ 120 ▼ 100 80 60 ▲ 40 20 C HS BR SA PEG ABA NaCl cold C HS 4D Rec BR SA PEG ABA NaCl cold CaCl2 C HS C HS C HS C HS C HS C HS C HS C HS Mature 0 7DAA 10DAA 15DAA Root Shoot MADS-BOX : pB4Nu Spike Flower Anther Carpel Wheat transformation 20DAA Developing Seed Ta HSF, a developing seed specific protein Shoot C 37 Developing Seed 42 C 37 Root 42 C 37 42 90 80 70 60 50 40 30 20 10 C HS BR SA PEG ABA NaCl cold C HS 4D Rec BR SA PEG ABA NaCl cold CaCl2 C HS C HS C HS C HS C HS C HS C HS C HS Mature 0 7DAA 10DAA 15DAA Root Shoot Agl : pMDC32 : HSF Spike Flower Anther Carpel 20DAA Developing Seed Arabidopsis transformation (T3) Salt Cold Drought 11-20 DAP 21-30 DAP 5-10 DAP 0- 2 DAP 2- 4 DAP 30 cm 20 cm Panicle Developing Seed 10 cm 7D Seedling Genome-wide Expression Analysis of Rice HSF‘s C2b B1 A9 A2e C1b A4b A5 A4d A3 C2a A1a A2c B2a A7 B2c A2d A2b A2a B2b B4b A2f C1a B4c B4d B4a Model of Environmental Stressors & Differential HSF functions Ta_HSP 26, a temperature and developing seed specific protein 600 500 400 300 200 100 C HS BR SA PEG ABA NaCl cold C HS 4D Rec BR SA PEG ABA NaCl cold CaCl2 C HS C HS C HS C HS C HS C HS C HS C HS Mature 0 7DAA 10DAA 15DAA 20DAA Root Shoot Spike Flower Anther Carpel Developing Seed Growth of WT and TaHSP26 over-expressing Arabidopsis under constant high temperatures of 35oC WT 6.3 D WT 6.3 WT 6.3 6.3 WT 6.3 AS-HSP26 Arabidopsis after 2 hr of non-lethal 39oC HS ..... at control conditions A WT B A WT AS6 AS6 Fv/Fm C Heat Shock (39oC) Recovery (22oC) B Microarray analysis of TaHSP26 Arabidopsis lines Transcription Factors WT OE Signal Transduction Hormone Signalling Special Interest Cell wall WT OE WT OE WT OE WT OE Transcription factors 50 30 183 Others Cell wall components 27 16 16 Signal transduction comps. Hormone signaling Genes of importance/special interest TaHSP26, P26 promoter activity in under Heat Stress C A B C 24h 10’ 20’ 30’ 40’ 50’ 60’ C 10’ 20’ 30’ 40’ 50’ 60’ 2h 24 h 2h Quantitative (A) and semi-quantitative (B) RT-PCR for GUS transcript, respectively, and Gus assay showing induction of governed by P26 promoter in transgenic Arabidopsis plants at different time points (C). Promoter motif analysis through PLACE tool Early responsive to dehydration (3) TATA Abiotic and biotic stresses (4) Heat shock protein (3) Responsive to water stress (8) Abiotic stresses (4) rd22 (13) HSP70A (3) TaHSP26, P26 promoter activity during Germination and Abiotic Stress Control 35oC 1day Cold (4oC) 2day 3day NaCl (150 mM) 4day Mannitol (2%) Under various stresses for 24 hr. Under heat stress during germination Ta MIPS2, expression in vegetative and generative tissues 2504000 3500 200 3000 ▲ 2500 150 2000 1001500 1000 50 ▼ 500 Root Root Shoot Shoot 7DAA 10DAA 15DAA 20DAA Spike Flower Anther Carpel Developing Seed ….cloned full length and used for Arabidopsis transformation ! Mature C HS C HS HS C HS C C HS C HS HS C HS C CaCl2 Cont HS C BR HS SA BR PEGSA ABA PEG NaCl ABA cold NaCl Contcold HS C RecHS BRRec 4D SABR PEGSA PEG ABA ABA NaCl NaCl cold CaCl2cold 0 0 TaMIPS1 C HS BR SA PEG ABA NaCl cold C HS 4D Rec BR SA PEG ABA NaCl cold CaCl2 C HS C HS C HS C HS C HS C HS C HS C HS Mature 500 450 400 350 300 250 200 150 100 50 0 Real time-PCR analysis of TaMIPS in different wheat tissues representing major growth stages and abiotic stresses Root 7DAA 10DAA 15DAA 20DAA Developing Seed Shoot 4000 3500 TaMIPS2 3000 2500 2000 1500 1000 500 C HS BR SA PEG ABA NaCl cold C HS 4D Rec BR SA PEG ABA NaCl cold CaCl2 C HS C HS C HS C HS C HS C HS C HS C HS Mature 0 7DAA 10DAA 15DAA 20DAA Root Shoot Developing Seed Ta STi expression in seedling and developing seeds 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 Shoot Developing Seed Domain organization Mature HS C 20DAA HS C 15DAA HS C 10DAA HS HS C HS C HS C HS C CaCl2 cold NaCl ABA PEG SA BR 4D Rec HS C cold NaCl ABA PEG SA HS C BR Root C 7DAA 0 Origin of Bread Wheat and importance of Wild Relatives to conclude…. Somatic embryogenesis can be used to study plant embryogenesis. Heat stress is a complex, regulated, network of genes participating in multiple pathways; sensitivity to heat stress varies at different growth stages, early embryogenesis being critical. Chloroplastic small HSP26 is highly inducible at various developmental stages, protecting PSII from heat induced damage, and has a crucial role in seed development. Apart from high temperature, wheat HSP26 promoter is also induced by other abiotic stresses and regulates expression in a time and tissue dependent manner (patented). Wheat HSF2a, MIPS, STi and other genes are being functionally characterized. Functional and Comparative genomics of genes of interest! “Whether the weather be fine, Whether the weather be not, Whether the weather be cold, Whether the weather be hot, We'll weather the weather, Whatever the weather, Whether we like it or not” Mahalakshmi, Bhumica Singla, Harsh Chauhan, Neetika Khurana….