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1. HIGH-THROUGHPUT PHENOTYPING  FOR NITROGEN USE EFFICIENCY IN CEREALS ICAR - INDIAN AGRICULTURAL RESEARCH INSTITUTE DIVISION OF PLANT PHYSIOLOGY Erect leaf stay-green ? Root system NO 3 - NH 4 + NGUYEN TRUNG DUC DR. SUDHIR KUMAR DR. DHANDAPANI RAJU DIVISION OF PLANT PHYSIOLOGY ICAR - INDIAN AGRICULTURAL RESEARCH INSTITUTE

2. Outline Introduction NUE and Efforts To Improve NUE Why Has No Progress Been Made? Can Modern Phenomics Help? Case Studies Conclusion And Ongoing Investigations

3. 1. Introduction

4. Nitrogen: The most abundant mineral element in a plant The most abundant element in the earth’s atmosphere The 4 th most abundant element in a plant (after C, H and O) Often the limiting nutrient for plant growth Nitrogen is one of the three major macronutrients found in most fertilizers N is in amino acids (proteins), nucleic acids (DNA, RNA), chlorophyll, and countless small molecules Blank, L.M. (2012). The cell and P: From cellular function to biotechnological application. Curr. Opin. Biotech. 23: 846 – 851 . From: Buchanan, B.B., Gruissem, W. and Jones, R.L. (2000) Biochemistry and Molecular Biology of Plants . American Society of Plant Physiologists.

5. Nitrogen metabolism: Uptake, assimilation and remobilization Uptake NO 3 - NH 4 + NH 4 + NO 3 - Nitrate reductase NO 2 - Nitrite reductase Glutamine synthetase (GS) Glutamate Glutamine Incorporation into amino acids and other nitrogen-containing compounds Amino acid recycling, photorespiration Carbon pools TCA cycle 2-oxoglutarate Glutamate Glutamine-2-oxoglutarate aminotransferase (GOGAT) Assimilation Remobilization Assimilation NH 4 + R-NH 2 N 2 Adapted from Xu, G., Fan, X. and Miller, A.J . (2012). Plant nitrogen assimilation and use efficiency. Annu . Rev. Plant Biol. 63: 153-182 .

6. 2. NUE and Efforts To Improve NUE

7. Multiple components of NUE Good AG, Shrawat AK, Muench DG (2004) Can less yield more? Is reducing nutrient input into the environment compatible with maintaining crop production? Trends Plant Sci 9(12):597–605 Eqn Term Formula Definition Comments 1 Nitrogen use e f ficiency NUE= Sw ÷ N Sw , sh o ot weight (DW); N, nitrogen c o ntent of shoots (DW) Does not account for biomass increases 2 Usage index U I = Sw × ( Sw ÷ N) Sw, sh o ot weight; N, n itrogen in shoots Takes into account absolute biomass increase 3 Nitrogen use e f ficiency (grain) NUE= Gw ÷ Ns Gw, g rain weight; Ns, nitrogen supply (g per plant) Reflects increased yield per unit applied nitrogen 4 Uptake e f ficiency UpE = Nt ÷ Ns Nt, total nitrogen in plant; Ns, nitrogen s u pply (g per plant) Measures e f ficiency of uptake of nitrogen into plant 5 Utilization e f ficiency UtE = Gw ÷ Nt Gw , g rain weight; Nt , total nitrogen in plant Fraction of nitrogen convert e d to grain 6 Agronomic e f ficiency AE=( Gw F - Gw C ) ÷ N F N F , nitrogen fertilizer applied; Gw F , grain weight with fertilizer; Gw C , grain weight of unfertilized c o ntrol Measures the e f ficiency of converting applied nitrogen to grain yield 7 Apparent nitrogen recovery AR=(N F uptak e - N C uptake) N F × 100 N F uptake = plant nitrogen (fertilizer); N C u p tak e = plant nitrogen ( n o fertilizer); N F = Nitrogen fertilizer applied Measures the e f ficiency of capture of n itrogen from soil 8 Physiological e f ficiency PE = ( Gw F - Gw C ) ÷ (N F uptak e - N C uptake) Gw F , grain weight (fertilizer); Gw C , grain weight (no fertilizer) Measures the e f ficiency of capture of p lant nitrogen in grain yield

8. Increasing Nitrogen use efficiency (NUE) A vast amount (>100 mill T) of Nitrogen (N) is applied to crops annually to maximize yield, of which 60% is apply in cereals. (FAOSAT, 2019) Cereals are relatively poor at capturing N fertilizer Only 40-50% of applied N is taken up by the cereal crops (Peoples et al., 1995; Sylvester-Bradley and Kindred, 2009) Pollution - Greenhouse impact (production and usage). - Water (ground water, rivers, oceans) - How can this uptake efficiency be increased? - What limits uptake of N? - Nitrate is the predominant form of N available to most cereal crops - What is the cereal nitrate uptake system? How does it respond to N availability? How does it change throughout the lifecycle? How can it be modified to improved N uptake?

9. Nitrogen losses and i m pact of low NUE Nolan, B.T. and Hitt, K.J. (2006). Vulnerability of shallow groundwater and drinking-water wells to nitrate in the United States. Environ. Sci. Technol. 40: 7834-7840 . Image source: Lamiot; Alexandra Pugachevsky ; NASA Earth Observatory Nitrogen fixation is energy demanding N O N Nitrous oxide (N 2 O) derived from fertilizer is a major greenhouse gas Unhealthful nitrate from agricultural uses pollutes groundwater Lake Erie Cyanabacterial bloom

10. Breeding strategies for enhanced nitrogen use efficiency Chardon, F., Noël, V. and Masclaux-Daubresse , C. (2012). Exploring NUE in crops and in Arabidopsis ideotypes to improve yield and seed quality. J. Exp. Bot. 63: 3401-3412 by permission of Oxford University Press; Martin, A., et al. and Hirel , B. (2006). Two cytosolic glutamine synthetase isoforms of maize are specifically involved in the control of grain production. Plant Cell. 18: 3252-3274. Reprinted by permission from Macmillan Publishers Ltd: Sun, H., et al. (2014). Heterotrimeric G proteins regulate nitrogen-use efficiency in rice. Nat Genet. 46: 652-656 . Hirel , B., Le Gouis , J., Ney, B. and Gallais , A. (2007). The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. J. Exp. Bot. 58: 2369-2387 Traits of an idealized plant with high NUE Glutamine synthetase activity is an important component of NUE In rice, a subunit of a heterotrimeric G protein contributes to N-sensitive growth and N assimilation

11. 3. Why Has No Progress Been Made?

12. B r eeding NUE c ultivars Ge n etic v ariability f o r N U E Agro n o m ic traits P h en ological traits Ph ysiological traits Modif y ing p h otosyn t he s is d u rat i on and t i m ing f o r seaso n al varia t ions of reso u rce ava i l a bi l i t y to enhance DM and N y i e lds W heat y i e l d i nc r eases in past l i n k ed with sho r tve g e t a t ive de v e l o p m ent p h ases R e d u ced N co n c in st e m ( n o t l e aves) l e ads N U Eg i m p r o v e m ent High bio m ass ac c u m ula t ion d u ring the g r a i n fi l l i ng period is hig h ly i m p o rtant f o r N U Eg

13. Physiol o gi c al tr a its (PT) Si m ple o r C o m plex ??? S o m e t i m e d i f fi c ult to m e a sure Ne e d special eq uip m ent Require subst a nti a l t im e P hotos y nthetic traits - F v /Fm ratio Repeat a bili t y ? Req uired PT are hig h ly i nfluenced by en v ironm e nt Sensit i ve n o t o n ly to abiotic s t re s s e s ( s a l t, h e a t a nd dr o u g ht) b u t to biotic s t re s s e s (Di s e a s e s) (Si m ply co m ple x )

14. Important P T s C a n o py te m p e rat u re - Infrared thermo m eter St o m a tal c o n d u c tan c e – Por o meter L e a f w a ter potential - S c h o lan d er pre s s u re c h am b er Os m otic a d ju s tment - V a p or pr e ss ure o s mo m eter C h lor o p h yll c o nt e nt - Chlorop h yll meter High Chl in l o w NUE lines under rec om m en d ed do s e Cr o p gro u nd c o v er - Digital c a mera L e af thi c k n e s s - Hig her th i c k ne s s in l o w NUE L i g ht i n t e r c e p t i o n - Cep t om eter L e af a re a i n d e x - L eaf a r ea me t er Gas ex c h a n g e - Inf r are d gas an a lyz er Ch l o r o p h y ll flu o r e s c e n c e - Ch l o r o p h y ll flu o r ome t er I n - se a s on b i om ass (4 X) – No s p e c i a l in stru m ent W a t er s ol u b l e ca r b o h yd r a t es - Sample mill; ne a r i n frare d refle c t a n ce s p e c t ro s c o p e So i l c o r i n g f o r mo i s t u r e - e l e ctr i c pe r cus si o n h a mmer Y i eld - Plot combi n e h a rv e s ter

15. Physi olo g ic al pe r spective to i mp r o v e N UE Dela y ed senescence increase the d u ration o f g rain filling and y ield u n der l i mit i ng N . Mai n taini n g g reen leaf a rea lo ng e r , p articularly after ant h esi s , i s an o ther way to incr e a s e crop yield, a nd p o ss i bly crop N if N u p take is also m a intained . Ge n o ty p es with hi g h stem b iomass fa vou rs N s tora g e and su b seq u ent transl o cation to t h e g rain – I m pr o ve N U pE I ncre a s i ng the f ra c t i on of pre - anthes i s crop DM re m o b ili z ed d uri n g grain fil l ing m ay also b e a way to increase the D M h arvest in d ex and th u s crop N U E .

16. 4. Can Modern Phenomics Help?

17. A taxonomy of phenotypes  Das Choudhury, S., Samal , A., and Awada , T. (2019). Leveraging Image Analysis for High-Throughput Plant Phenotyping. Frontiers in Plant Science 10:508

18. High-throughput plant phenotyping and data accumulation Rahaman , M., Chen, D., Gillani , Z., Klukas , C., & Chen, M. (2015). Advanced phenotyping and phenotype data analysis for the study of plant growth and development.  Frontiers in plant science ,  6 , 619.

19. NANAJI DESHMUKH PLANT PHENOMICS CENTRE, IARI, NEW DELHI Plant cultivation in moving field Weighing and water stations Imaging platforms

20. Case Study: Phenotyping for Nitrogen Use Efficiency : Rice Genotypes Differ in N-Responsive Germination, Oxygen Consumption, Seed Urease Activities, Root Growth, Crop Duration, and Yield at Low N Sharma, N., Sinha, V. B., Gupta, N., Rajpal , S., Kuchi , S., Sitaramam , V., Parsad , R., and Raghuram , N. (2018). Phenotyping for Nitrogen Use Efficiency: Rice Genotypes Differ in N-Responsive Germination, Oxygen Consumption, Seed Urease Activities, Root Growth, Crop Duration, and Yield at Low N. Frontiers in Plant Science 9:1452

21. Role of Root in N uptake Nitrogen uptake efficiency Nitrogen utilization efficiency Root system architecture Root exudates Rhizosphere microbiota Symbioses P P N N NH 3 Transporters and pumps Intercellular transport efficiency X R-X Assimilation and remobilization efficiency Regulatory and control networks GERMINATION OXYGEN CONSUMPTION SEED UREASE ACTIVITIES ROOT GROWTH CROP DURATION YIELD

22. Rate of Germination Varies With Genotypes

23. Rate of Germination Varies With Genotypes

24. Analysis of variance of germination rates of 21 rice genotypes under different N regimes

25. N Delays Germination in a Genotype-Dependent and N-Source-Dependent Manner

26. Ranking of 21 Indica rice genotypes based on the extent of N-induced delay in germination

27. Slow Germinating Genotypes Are Most N-Responsive vis-à-vis Fast Germinating Genotypes

28. Biplot Analysis Validates Contrasting Genotypes for N-Response

29. Field Data Reveal Significant Relationship Between Crop Duration and Yield Grain yield (t/ha) Crop duration (Days) Genotypes N-0 (no nitrogen) N100 (N-100 kg/ha) Aditya 2.93 4.44 120 Swarnadhan 3.63 5.55 120 Rasi 3.13 4.85 120 Jaya 3.86 6.03 130 Varadhan 3.80 6.13 130 Ravi 3.08 4.64 120 Swarna 4.07 5.06 150 Suraksha 3.08 4.94 135 Vibhava 3.35 5.40 125 Vikas 3.45 5.48 115 Krishna Hamsa 3.52 4.81 125 Sampada 4.02 5.50 140 Prasanna 2.91 3.73 105 Pusa Basmati 3.65 4.86 130 Triguna 3.56 4.53 125

30. Germination Rates Are a Function of Oxygen Consumption Rates

31. Germinating Seeds Have Endogenous Urease Activity

32. Urea Inhibits Root Growth More Significantly Than Shoot Growth

33. Little progress has been made in improving NUE of cereals (Garnett et al. 2015). This is despite the fact that the genomes of important cereal crops have been sequenced. Furthermore, genes, loci of interest, and regulatory networks influencing NUE have been identified but, as yet, no improved NUE cereals have been released commercially. Deepening genetic understanding may have provided false hope that improving cereal NUE could be easily achieved. The NUE research carried out in the field and in controlled environments, although not yet leading to germplasm with improved NUE, has helped us better understand the complexity of the trait and, in particular, the major GxE interaction. Modern phenomics as detailed here gives us the opportunity to better characterize the environment, plant responses to the environment and, combined with continually increasing genetic information, offers the opportunity to make real progress in improving NUE. Conclusion And Ongoing Investigations

34. REFERENCE Evenson RE, Gollin D (2003) Assessing the impact of the green revolution, 1960 to 2000. Science 300(5620):758–762. Furbank RT, Tester M (2011) Phenomics–technologies to relieve the phenotyping bottleneck. Trends Plant Sci 16(12):635–644. Garnett T, Plett D, Heuer S, Okamoto M (2015). Genetic approaches to enhancing nitrogen-use efficiency (NUE) in cereals: challenges and future directions. Funct Plant Biol 42(10):921–941 Golzarian M, Frick R, Rajendran K, Berger B, Roy S, Tester M, Lun D (2011) Accurate inference of shoot biomass from high-throughput images of cereal plants. Plant Methods 7 Honsdorf N, March TJ, Berger B, Tester M, Pillen K (2014) High-throughput phenotyping to detect drought tolerance QTL in wild barley introgression lines. PLoS ONE 9(5):e97047 Neilson EH, Edwards A, Blomstedt C, Berger B, Møller BL, Gleadow R (2015) Utilization of a high-throughput shoot imaging system to examine the dynamic phenotypic responses of a C4 cereal crop plant to nitrogen and water deficiency over time. J Exp Bot eru526 Sharma, N., Sinha, V. B., Gupta, N., Rajpal , S., Kuchi , S., Sitaramam , V., Parsad , R, and Raghuram , N. (2018). Phenotyping for nitrogen use efficiency (NUE) I: Rice genotypes differ in N-responsive germination, oxygen consumption, seed urease activities, root growth, crop duration and yield at low N. Frontiers in plant science, 9, 1452. Steffens, B., and Rasmussen, A. (2016). The physiology of adventitious roots. Plant Physiol. 170, 603–617. doi : 10.1104/pp.15.01360. Rajjou , L., Duval, M., Gallardo, K., Catusse , J., Bally, J., Job, C., et al. (2012). Seed germination and vigor . Annu . Rev. Plant Biol. 63, 507–533. doi : 10.1146/ annurev-arplant-042811-105550. Yang W, Guo Z, Huang C, Duan L, Chen G, Jiang N, Fang W, Feng H, Xie W, Lian X (2014). Combining high-throughput phenotyping and genome-wide association studies to reveal natural genetic variation in rice. Nat Commun 5.