1. Terry Rose, Southern Cross Plant Science/Southern Cross GeoScience Enhancing internal phosphorus use efficiency in crops: concepts and approaches

2. Internal phosphorus use efficiency Three main ways we examine it: 1.Molecular and physiological responses from P starved model plants (e.g. arabidopsis) 2.Investigate mechanisms in highly P-efficient non-crop species 3.Investigate and attempt to exploit genetic variation within a crop species

3. www.scu.edu.au/scps/ Definitions of Internal Phosphorus Use Efficiency Grain yield per unit of P in above-ground tissue (g mg -1 ) - yield formation may be independent of P use - selects against genotypes with low grain yield potential Biomass yield per unit of P in above-ground tissue (g mg -1 ) - inverse of tissue P concentration Critical shoot P concentration for 90% maximum yield (mg g -1 ) - expensive when screening large numbers Shoot biomass/shoot P concentration (g 2 mg -1 )

4. www.scu.edu.au/scps/ Problem caused by differential P uptake 29 rice genotypes grown in low P soil for 50 d with four replicates

5. www.scu.edu.au/scps/ Can screening at equal P uptake help? low-P soil500 µg P hydroponic+P hydroponic Total PTotal DMTotal PTotal DMTotal PTotal DM ShtPUE-0.81 ** -0.65 ** -0.43 ** 0.51 ** -0.30 ** -0.02 SeedP0.100.070.40 ** 0.59 ** 0.10

6. www.scu.edu.au/scps/ Screening large numbers in hydroponics GWAS study needed +P controls for two reasons: 1.Can remove any genotypes that grew poorly in +P from any analyses – poor growth at low- P not related to low P but other artefacts 2.Can map GWAS peaks under +P conditions to find loci related to ‘general vigour’ and not specifically related to P

7. www.scu.edu.au/scps/ Genome-wide association study (GWAS) for PUE Main QTL on chromosomes 1 (indica) and 11 (aus)

8. www.scu.edu.au/scps/ Does internal PUE ranking change with shoot P content? There is no presumption that high internal PUE lines will grow well in the field because they may lack P uptake genes: The aim is to find loci/gene(s) that can be ‘pyramided’ into elite lines in local breeding programs.

9. www.scu.edu.au/scps/ What are the consequences of higher internal PUE ? These authors apply ‘The law of conservation of matter’ to nutrient use efficiency.

10. www.scu.edu.au/scps/ What are the consequences of higher internal PUE ? Source: Rose et al. 2013 Frontiers in Plant Science

11. www.scu.edu.au/scps/ What are the consequences of higher internal PUE ? Obtained same biomass as the wild-type plants with a quarter of the P content in shoots, while seed yield was not reduced. No seed P concentrations shown

12. www.scu.edu.au/scps/ What are the consequences of lower seed P concentrations? Grain quality – milling traits in rice, dough quality in wheat? Human health 1. P deficiency in humans? Unlikely. 2. phytate - Some reports suggest it may have anti-cancer properties, recent review by Kumar et al. (2010) suggests that there is limited evidence for this - Strong evidence for its role in binding micronutrients, so reduction in phytate may be beneficial 3. phospholipids - play a role in grain quality and human health, but may be quite stable (Tong et al. 2014)

13. www.scu.edu.au/scps/ What are the consequences of lower seed P concentrations? Seedling germination and vigour Two lines of enquiry have led to the conclusion that reducing seed P is detrimental to seed germination and vigour. 1.Studies with low phytic acid (lpa) mutants - Low-phytic-acid mutants often have impaired germination and vigour BUT this is because whole genes are often knocked out - The only LPA mutant used in breeding programs (Barley lpa1-1; Bregitzer et al. 2007 Crop Science) has no impact on seedling vigour BUT this mutant has a 10-14% reduction in seed total P due to mutation of a putative sulfate transporter.

14. www.scu.edu.au/scps/ Seed germination and seedling vigour 2. Studies with low-P seed from P-starved plants -Half a dozen studies with cereal crops where low-P seed have shown reduced germination and seedling vigour compared to high-P seed -These studies do NOT make fair comparisons because the low-P seed came from P-stressed parent plants -Most studies were not conducted with agricultural soils with a history of P fertilisation, so soils were typically highly P-deficient.

15. www.scu.edu.au/scps/ Seed germination and seedling vigour 2. Studies with low-P seed from P-starved plants -Seeds from P-starved plants performed poorly in P-deficient soil but no yield difference in agricultural soil supplied with P fertiliser -Subsequent studies with seed lower in P from environmental effects found no difference in seedling vigour between high- and low-P seed on any soil -Further studies have been conducted and will be the focus of the presentation by Elke Vandamme

16. www.scu.edu.au/scps/ High P input farming systems If seedling vigour can be maintained with lower seed P, then perhaps we could breed for lower seed P concentrations regardless of internal PUE at the vegetative stage. Go for high P uptake and low translocation to grains. May be a useful trait in high-input farming systems where the removal of P in grains is significant and drives the need for continual P fertiliser input World phosphate deposits: FAO data Australian deposits are < 1 % of world P resources. Over 70 % of resources are held by China and Morocco.

17. www.scu.edu.au/scps/ High P input farming systems

18. www.scu.edu.au/scps/ High P input farming systems

19. www.scu.edu.au/scps/ High P input farming systems

20. www.scu.edu.au/scps/ High P input farming systems

21. www.scu.edu.au/scps/ High P input farming systems In an average season in Australia, approximately 60, 000 tonnes of P (the equivalent of over half a million tonnes of super phosphate) is removed off-farm in wheat grain at harvest assuming grain contains 3 mg P/g. Most is exported overseas while some is consumed domestically and contributes to high-P landfill and the pollution of water bodies in Australia. A reduction in grain P concentrations to 2 mg P/g would save about $100 million being removed off farm each year at $5 per kg P.

22. www.scu.edu.au/scps/ Current research project: Global Rice Science Partnership (SCU, JIRCAS, IRRI, AfricaRice) project aims to reduce rice grain P by minimum 20% Approaches for breeding crops with low grain phosphorus Two approaches investigated in the project: 1.Exploiting genotypic variation 2.Molecular approach 3.Mutant approach – not investigated but may be an option later

23. www.scu.edu.au/scps/ GRiSP project: Exploiting genotypic variation Multi-location trials with 20+ rice genotypes over a number of years to look at G x E interactions for grain P concentration 1. Need to identify a genetic component that is independent of grain yield (yield-dilution effect) 2. Need to make sure low grain P isn’t associated with low plant P uptake! Elke Vandamme will be presenting data on this

24. www.scu.edu.au/scps/ GRiSP project: Molecular approach Identify P transporter(s) and use RNAi silencing to reduce gene expression in specific tissue at a specific time Understand regulatory pathway of genes involved in grain P loading and find targets for genetic manipulation

25. www.scu.edu.au/scps/ Summary 1.We have investigated internal PUE at the vegetative stage using a method which screens at equal P uptake and have mapped loci for high internal PUE 2.The consequences of high internal PUE will likely be a reduction in grain P concentration 3.Reducing grain P concentration may be a good option by itself, particularly in high-input systems 4.Further work is needed to ensure lower grain P concentration does not adversely affect grain quality or seedling vigour

26. www.scu.edu.au/scps/ Acknowledgements Southern Cross University Cecile Julia Kwanho Jeong Alicia Hidden Rachel Wood Japan International Research Centre for Agricultural Science Matthias Wissuwa Asako Mori Juan Pariasca-Tanaka Katsuhiko Kondo Africa Rice Elke Vandamme Kazuki Saito IRRI Tobias Kretschmar Funding Agencies Global Rice Science Partnership Japan Society for the Promotion of Science