Rese search Teams Leeds: Sarah Alomrani, Mary McKay and Christine - - PowerPoint PPT Presentation

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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)

Target for improvement Predicted increase in photosynthesis Reference 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 CO2 and increased carboxylation rate (kcat) 60% Reynolds et al. 2009 Optimised Rubisco regulation 10% Reynolds et al. 2009 CO2 concentrating mechanism (C4-plant) 50% Reynolds et al. 2009 CO2 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

Improving photosynthesis to increase crop yields

21% Taylor & Long, 2017

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

• R. Bessin, Univ. of Kentucky

Cystatins have many functions in plants: Seed germination Defenses against insects , nematodes and viruses Abiotic stress tolerance Regulation of plant development Leaf senescence

Oryzacystatin I (OC-I) expression in tobacco delays senescence and enhances biomass

7 weeks

T4/5 T4/3-2 T4/3-1 WT GUS WT T4/5 T4/3-1 T4/3-2

10 weeks

WT T4/5 T4/5

15 weeks

Protein Accumulates in OCI-expressing tobacco leaves at 14 weeks. Proteomics analysis revealed accumulation of chloroplast proteins particularly Rubisco and Rubisco activase

10 20 30 40 50 60 70 g mg-1 DW

• 1

1 2 3 4 5

• 1

1 2 3 4 5  Bottom of plant Top of plant

Bottom leaf (senescent) Top leaf Bottom leaf (senescent) Top leaf

Protein Chlorophyll

T4/5 WT

Rubisco activities are higher in OCI expressing plants. Rubisco is protected from stress-induced degradation.

WT T4/5

55 kDa 164 kDa

Intercellular CO2 concentration (µmol mol

• 1)

200 400 600 800 1000 1200 1400

CO2 assimilation rate (µmol m

• 2 s-1)
• 3

3 6 9 12 15 18 21 200 400 600 800 1000 1200 1400

CO2 assimilation rate (µmol m

• 2 s-1)
• 3

3 6 9 12 15 18 21

OCI expression in soybean

1 2 3 4 5 6 7 8 9 Control 55 56 57 Relative expression

OC-I

M 55-4, 55-8 55-27 56-2 56-7 56-12 57-3 57-4 57-7 +Ctrl +Ctrl WT

Plasmid Gen e Promoter Target pTF101.1-OC- I OC-I 35S promoter Cytosol pTF101.1- LegOC-I OC-I Leg promoter Nodules pTF101.1- PRKOC-I OC-I 35S with PRK signal Chloroplast pTF101.1-BBI BBI 35S promoter Cytosol pTF101.1-TI Serp in 35S promoter Cytosol

a b c d

Tobacco Soybean

OCI expression influences seed production

Wheat Transformation

Embryo preparation and DNA delivery Callus Production Plant regeneration Seed production

OCI expression in wheat

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?

Adapted from Parry et al. (2008) J Exp Bot 59:1569

CA1P phosphatase Inhibitor Inhibitor Sugar + Pi Phosphorylation Rubisco activase Rubisco Active enzyme Rubisco-Inhibitor Inactive enzyme

CA1P phosphatase

Rubisco mainly in the active form; Increased crop yields under optimal conditions

 1. CA1Pase

Rubisco mainly in the inactive form; Increased protection from proteolysis under stress

 2. CA1Pase

CA1P phosphatase Inhibitor Inhibitor Sugar + Pi Phosphorylation Rubisco activase Rubisco Active enzyme Rubisco-Inhibitor Inactive enzyme

X

8 days of water withdrawal 16h after re-watering (recovery) Well-watered plants Well-watered plants

Wt CA1Pase

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CA1Pase

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Wt CA1Pase

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CA1Pase

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Wt CA1Pase

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CA1Pase

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Wt CA1Pase

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CA1Pase

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Plant growth Drought treatment & sampling Protein extraction & assays Final harvest and yield determination