Author : Surya Kant, Yong-Mei Bi and Steven J. Rothstein

1. Understanding plant response to nitrogen limitation for the improvement of crop nitrogen use efficiency 組員: 彭元慶 林柏齡 郭宇翔 陳傑君 Author : Surya Kant, Yong-Mei Bi and Steven J. Rothstein

2. Content • Introduction • Physiological and molecular componentsgoverning NUE • Understanding plant response to N limitation with physiological and molecular approach • Future prospect

3. Need for genetic improvement of nitrogen use efficiency (NUE) • Population growth • Cost of N input Energy-intensive Marginal effect of N use N waste • Genetic affect http://www.camelclimatechange.org/topics/view/66030/ http://www.investmentu.com/2012/February/investing-in-agriculture.html

4. definition ofnitrogen use efficiency (NUE) Yield per unit of N available in the soil Allen et al., 2004

5. Physiological and molecular componentsgoverning NUE • Two general stages for N use in the plant life cycle • Vegetative stage N uptake and assimilation • Reproductive stage N remobilization Chardon et al., 2012

6. uptake and assimilation • Nitrate transport (NRT):In Arabidopsis, three families of nitrate transporters NRT1, NRT2, and chloride channel (CLC )with 53 NRT1, 7 NRT2, and7 CLC genes are identified. • The ammonium derived from nitrate or from direct ammonium uptake by Ammonium transporters(AMT) NRT1.1, NRT1.2, NRT2.1, andNRT2.2

7. Markus et al..2009 Xuet al., 2011

8. uptake • NRT1.1 is adualaffinitynitrate transporter NRT1.1 is a dualaffinitynitrate transporter Markus et al..2009

9. CHL1 Functions as aNitrate Sensor in Plants Cheng-Hsun Ho, Shan-Hua Lin, Heng-Cheng Hu, and Yi-Fang Tsay cell.2009.07.004

12. N uptake(supplement) • Nitrate&ammonium之間的調控與平衡 Anthony D.M. et al.,2002

13. signaling Regulation of gene (Black) Extemal input (Grey ) Response (Blue) Markus et al..2009

14. N assimilation • Glutamine synthetase cytosol (GS1) plastids (GS2) • GOGAT Fdx-GOGAT NAD(P)H-GOGAT GS1.3 In maize led to an increase of 30% in kernel number (Martinet al., 2006) Cytosolic GS1 Enhanced N accumulation capacity in shoots and grainsinwheat Jed P. Sparks 2009

15. N uptake and assimilation Xuet al., 2011

16. N remobilization • Step1: The protein degradation 1.Chloroplast degradation pathway 2.Vacuolar and autophagic pathway 3.Ubiquitin-26S proteasome pathway • Step2: Amino acid remobilizing

17. Chloroplast degradation pathway Lui et al., 2008

18. Vacuolar and autophagic pathway D. E. Martı´nez,2008 Sigrun et al., 2010

19. Ubiquitin-26S proteasome pathway Lui et al., 2008 http://www.angenetik.fu-berlin.de/hellmann_eng.html

20. N remobilization • After proteindegradation all the amino acids are remobilized, glutamine, asparagine followed by glutamate, aspartate, serine, and alanine are predominant in phloem sieve tube

21. Amino acid permeases (AAP) • lysine/histidine transporter (LHT) • cationic amino acid transporters (CAT) • proline transporters (ProT) • aromatic and neutral amino acid (ANT) • transporters (ANT) • oligopeptide transporters (OPT)

22. N remobilization • Much evidence supports the role of cytosolic GS1 in the efficient remobilization of amino acids forsenescing leaves towards grain-filling

23. Morphological response of root system to N supply • Signal for development processes • genetic and environmental factors

24. Signal for development processes • N concentration affects root development, root architecture, and the root-to-shoot ratio • low N supply generally leads to decreased root growth, suppression of lateral root initiation, increase in the C/N ratio within the plant, reduction in photosynthesis, and early leaf senescence

25. Signal for development processes • TheANR1 dual regulation of lateral roots by nitrate is developmental stage dependent (Zhang and Forde, 1998, 2000; Zhang et al., 1999). • NRT2.1acts as a positive regulator for lateral root initiation under N limitation conditions (Remans et al. 2006b) • By contrast, reported a repressive role in lateral root initiation (Little et al. 2005)

26. Plant’s adaptive response to N limition • Increase in N uptake by high affinity transporters • Remobilization of N from older to younger leaves and reproductive part • Retardation of growth and photosynthesis. • Increased anthocyanin accumulation.

27. Understanding plant responseto N limitation • 1.Transcriptional profiling • 2.Reverse genetics • 3.Forward genetics • 4. Another model plant: Thellungiellahalophila(鹽芥)

28. Transcriptional profiling: • Microarray • Sequence-based transcription profiling technology • next-generation sequencing approaches

29. Reverse genetics • 利用T-DNA 插入正常Arabidopsis某gene片段 • 形成失去某功能的nla mutant • 比較nlatype 和 wild type 在低N環境(3 mM)和正常N環境(10 mM)的生理型態、轉錄程度(whole genome transcript profiling,及生化機制(ex:phenylpropanoid pathway )的不同 • 正常N環境下兩者無明顯差異；低氮環境下有1,272個基因調節方式不同,其中807個 up-regulated,465個down-regulated (Penget al.,2007a). T-DNA

30. Reverse genetics: nla mutant (Peng et al.,2007a)

31. Reverse genetics • In nla mutant plants, the phenylpropanoid pathway wasdisrupted, with substrates from this pathway channelledtowards lignin production and thereby anthocyanin synthesiswas suppressed. (Penget al.,2008)

32. Reverse genetics (Penget al.,2008)

33. Reverse genetics (Penget al.,2008)

34. Reverse genetics Pi limitation induced anthocyanin accumulation and prevented the N limitation-induced early senescence phenotype in the nla mutant (Penget al.,2008)

35. Reverse genetics • NLAmight be controlled bya micro-RNA (miR827) and is an important component forthe integration of phosphorus- and N-limitation responses. (Pant et al.,2009) • NLA gene also has a role in immune responses but as a negative regulator for salicylic acid production (Yaeno and Iba, 2008)

36. Forward genetics • 利用reporter gene 和 activation tagged lines 判斷係由何種組成影響N-limitationresponse. Inducible promoter Reporter gene T-DNA T-DNA

37. Forward genetics • Ex:Girin et al. (2010) used transgenic Arabidopsis plants harbouring a NRT2.1 promoter::LUC reporter gene to screen EMS mutagenized plants and have identified three mutants that appear to be altered in their regulation of nitrate uptake.

38. Forward genetics 阿拉伯芥 (pNRT2.1::LUC) EMS點突變 ( Cto T ) 僅低N大量 表現LUC 高N低N皆大量 表現LUC 低Ninduce LUC gene 轉譯Luciferase 非欲選拔的mutant 三個hni mutant: hni9-1 hni48-1 hni140-1 Luciferase(LUC) 與luciferin反應呈螢光 (Girin et al. 2010) NRT2.1 promoter LUC gene

39. Forward genetics Figure 1. Characterization of NL plants (pNRT2.1::LUC). Plants were grown on vertical agarose plates for 7 d on HN or LN medium supplemented with 1% Suc. A, Bioluminescence imaging and quantification of the LUC activity (values are means of six replicates ± SD). B, Relative accumulation of the LUC transcript (values are means of three replicates ± SD). C, Relative accumulation of the NRT2.1 transcript(values are means of three replicates ± SD). D, High-affinity influx measured in 0.2 mM 15NO3 - (values are means of 10 replicates ± SD). DW, Dry weight.

40. Forward genetics Figure 2. Characterization of NRT2.1 expression in hni mutants. Plants were grown on vertical agarose plates supplemented with 1% Suc for 7 d. A, LUC activity in hni mutants and NL in plants cultivated on HN or LN medium. Values are means of six replicates 6 SD. B, Relative accumulation of the NRT2.1 transcript on HN or LN medium. Values are means of three replicates 6 SD. (Girinet al. 2010)

41. Another model plant:Thellungiellahalophila(鹽芥) (Inanet al. 2004) • Comparison with 阿拉伯芥 • 92%序列相似 • small genomesize: 2倍 • short life cycle :6至8星期 • copious seed number:6,000–8,000 • Ease of transformation

42. Another model plant: 低N環境下,鹽芥 could maintainhigher N content, total amino acids total solubleprotein 低C/N ratio by efficiently acquiring and utilizing nitrate . (Kant et al., 2008).

43. Another model plant: white bars, 4 mM nitrate; light gray bars, 1 mM nitrate; dark gray bars, 0.4 mMnitrate (Kant et al., 2008).

44. Another model plant:Thellungiellahalophila(鹽芥) • The production of BAC and cDNA libraries, and the generation of EST and T-DNA insertion collections will further enhance the power of the Thellungiella system for identifying key components governing N utilization under low N conditions.

45. Candidate gene for improving NUE • Function in the transport of ammonium or nitrate • Regulatory function (ex: some transcription factor genes) • Early nodulin gene( completely unknown function)

46. Candidate gene : GATA transcription factor genes • GATA transcription factor genes regulate N assimilation in plants. (Jarai et al., 1992; Rastogi et al., 1997; Oliveira and Coruzzi, 1999). • GATA factors play a role in NiR gene regulation (Rastogi et al., 1997).

47. Candidate gene : GATA transcription factor genes • GNC(GATA, nitrate-inducible, carbon metabolism-involved).gene is one member of the GATA transcriptional factor gene family. • STP13(Sugar transport protein)is one of the C metabolic genes whose expression levels were tightly influenced by GNC (Bi et al., 2005).

48. Candidate gene : GATA transcription factor genes 55 mM glucose and varying nitrate concentrations Ox:STPoverexpressor line (Bi et al., 2005). GNC STP13 NRT2.2 N

49. Candidate gene : Early nodulin gene( unknown function) • The transgenic plants over-express early nodulin gene (OsENOD93-1) had a significant 10–20%mincrease in the number of spikes and spikelets, and seed yield under both limiting-N and optimum-N conditions, and a significantly higher shoot dry biomass than wild-type plants under limiting-N conditions (Bi et al., 2009) sequence homology of 58.2% to GmENOD93 ( Reddy et al.,1998)

50. Candidate gene : Early nodulin gene( unknown function)