Mod odify fyin ing rib ribulose-1,5-bis isphosphate car arboxylase/oxygenase (Rubis isco) protein in tu turnover as as a a novel l mechanis ism to enhance wheat yie yields an and enhance clim climate resi silie lience. Rese search Teams Leeds: Sarah Alomrani, Mary McKay and Christine Foyer Lancaster: Ana Karla Lobo, Doug Orr, Marta Oñate , Elizabete Carmo-Silva Investigating the role of proteases and their naturally-occurring inhibitors in plants (T (Tobacco, So Soybean, , Ar Arabidopsis s and and whe heat)
Improving photosynthesis to increase crop yields Target for improvement Predicted Reference increase in photosynthesis Light capture and recovery from photoprotection 40% Long et al. 2006 Increased stomatal and mesophyll conductance 5% Reynolds et al. 2009 Increased RuBP regeneration 10% Reynolds et al. 2009 Rubisco with increased specificity for CO 2 and 60% Reynolds et al. 2009 21% Taylor & Long, 2017 increased carboxylation rate ( k cat ) Optimised Rubisco regulation 10% Reynolds et al. 2009 CO 2 concentrating mechanism (C 4 -plant) 50% Reynolds et al. 2009 CO 2 concentrating mechanism (cyanobacteria) 60% McGrath & Long 2014 Long et al. (2006) Plant Cell Environ 29:315; Reynolds et al. (2009) J Exp Bot 60:1899; McGrath & Long (2014) Plant Physiol 164:2247; Taylor & Long (2017) 372:20160543
Plant Cysteine Proteinase Inhibitors: Cystatins • Inhibit cysteine proteinases (bind tightly and reversibly to papain-like cysteine proteinases) • Oryzacystatin I (OC-I) is a rice cystatin that was originally used in potato to increase resistance to Colorado potato beetle Cystatins have many functions in plants: Seed germination Defenses against insects , nematodes and viruses Abiotic stress tolerance Regulation of plant development Leaf senescence R. Bessin, Univ. of Kentucky
Oryzacystatin I (OC-I) expression in tobacco delays senescence and enhances biomass 7 weeks 15 weeks T4/5 T4/3-2 T4/3-1 GUS WT 10 weeks WT T4/5 T4/5 WT T4/5 T4/3-1 T4/3-2
Protein Accumulates in OCI-expressing tobacco leaves at 14 weeks. Proteomics analysis revealed accumulation of chloroplast proteins particularly Rubisco and Rubisco activase Protein Chlorophyll 70 5 4 60 3 2 50 1 0 40 g mg-1 DW -1 5 30 4 3 20 2 1 10 0 Bottom leaf Top leaf Bottom leaf Top leaf 0 -1 (senescent) (senescent) Bottom of plant Top of plant WT T4/5
Rubisco activities are higher in OCI expressing plants. Rubisco is protected from stress-induced degradation. WT T4/5 21 21 18 18 164 kDa 15 15 CO 2 assimilation rate CO 2 assimilation rate 12 12 -2 s -1 ) -2 s -1 ) (µmol m 9 (µmol m 9 55 kDa 6 6 3 3 0 0 -3 -3 0 200 400 600 800 1000 1200 1400 0 200 400 600 800 1000 1200 1400 Intercellular CO 2 concentration -1 ) (µmol mol
OCI expression in soybean M 55-4, 55-8 55-27 56-2 56-7 56-12 57-3 57-4 57-7 +Ctrl +Ctrl WT OC-I Plasmid Gen Promoter Target e 9 8 pTF101.1-OC- OC-I 35S promoter Cytosol 7 I Relative expression 6 pTF101.1- OC-I Leg promoter Nodules 5 LegOC-I 4 3 pTF101.1- OC-I 35S with PRK Chloroplast PRKOC-I signal 2 1 pTF101.1-BBI BBI 35S promoter Cytosol 0 Control 55 56 57 pTF101.1-TI Serp 35S promoter Cytosol in
Tobacco OCI expression a b influences seed production Soybean c d
Wheat Transformation Embryo OCI expression in wheat preparation and DNA delivery Callus Production Plant regeneration Seed production
OCI-expressing wheat lines OCI overexpression wheat lines have been produced with construct pRMH052. 37 transformed plants and 4 control lines were selected for further production based on copy number using the Taqman assay PCRs were carried out to confirm the presence of the gene of interest Tissue fromT0 plants was collected for analysis by Southern blots, expression analysis etc
T2 transgenic lines (CW47: A and CW48: B) and wild type plants (C) at 6 weeks. Plants were grown on soil in long day photoperiod (LD- with 16 h day/8 h night) at 20°C, 60% of humidity at 350 uEm-2 s-light intensity. Scale bar 3 cm.
Can we manipulate CA1Pase levels to improve wheat yields & climate resilience? 1. CA1Pase Rubisco CA1P phosphatase CA1P phosphatase activase Rubisco mainly in Inhibitor the active form; Increased crop Rubisco-Inhibitor Rubisco Sugar + Pi yields under optimal Inactive enzyme Active enzyme conditions Inhibitor Phosphorylation 2. CA1Pase X Rubisco Rubisco mainly in CA1P phosphatase activase Inhibitor the inactive form; Increased protection Rubisco-Inhibitor Rubisco from proteolysis Sugar + Pi Inactive enzyme Active enzyme under stress Inhibitor Phosphorylation Adapted from Parry et al. (2008) J Exp Bot 59:1569
Well-watered plants 8 days of water withdrawal Wt CA1Pase CA1Pase Wt CA1Pase CA1Pase Well-watered plants 16h after re-watering (recovery) Wt CA1Pase CA1Pase Wt CA1Pase CA1Pase
Plant growth Drought treatment & sampling Protein extraction & assays Final harvest and yield determination
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