作者： Matthew A. Escobar, Daniela A. Geisler and Allan G. Rasmusson

1. Reorganization of the alternative pathways of the Arabidopsis respiratory chain by nitrogen supply：opposing effects of ammonium and nitrate 作者：Matthew A. Escobar, Daniela A. Geisler and Allan G. Rasmusson 出處：The Plant Journal (2006) 45, 775–788

2. Introduction

3. The nitrogen cycle

4. Introduction • nitrogen：nitrate (NO3- ) or ammonium (NH4+ ) • secondary metabolites (二次代謝物) • nitrate (cytosol using 1 NADH) > ammonium (plastid using 3 NADPH). • glutamine synthetase / glutamine：2-oxoglutarate aminotransferase system (GS/GOGAT) (穀氨酰胺合成酶/穀氨酸合成酶).

5. Glutaminesynthetase

6. GS/GOGAT system GDH需要 高濃度氨 葉/根細胞質 or 葉綠體 葉片葉綠體 or 根質體

7. Glutamate synthase cycle • GS/GOGAT requirement for 2-oxoglutarate (2-OG) as a carbon skeleton ties the process of N assimilation to cellular respiration.

8. Electron transport chain

9. alternative oxidases (AOXs) circumvent type II NAD(P)H dehydrogenases circumvent respiratory uncoupling proteins circumvent ATP synthase Electons from NADH oxygen Electons from succinate • In contrast, type II NAD(P)H dehydrogenases and AOXs do not pump protons, and proton flow through uncoupling proteins is not coupled to ATP synthesis. Rasmusson et al., 2004; Vanlerberghe & McIntosh, 1997; Vercesi, 2001

10. Introduction • Respiratory bypass proteins： 1. thermogenesis(生熱) (Siedow & Day, 2000) 2. preventionof reactive oxygen species formation (Fernie et al., 2004; Møller, 2001) 3. dissipation of excessredox equivalents (Raghavendra& Padmasree, 2003) • Rapid adjustmentsin the respiratory chain： insensitive to external factors (Escobar et al., 2004; Finnegan et al., 1997; Simons et al., 1999; Svensson and Rasmusson, 2001; Yu et al., 2001).

11. Introduction • Genesform Arabidopsis thaliana： type II NAD(P)H dehydrogenase：nda1-2, ndb1-4 & ndc1 AOX：aox1a-d & aox2 uncoupling protein：ucp1-2 and ucp4 (Hanak and Jezek, 2001; Michalecka et al., 2003; Saisho et al., 1997; Thirkettle-Watts et al., 2003; Watanabe et al., 1999) • Steady-state transcript are very low： Northern analyses & microarrays (Escobar et al., 2004) • nitrate & ammonium have strong and opposing effects on several respiratory bypasses at the mRNA, protein, and activity levels.

12. Experimental procedures

13. Plant material and culture conditions • Arabidopsis (ecotype Columbia)： liquid cultures (125 rpm, 22℃, 16-h photoperiod). • nutrient solution (Somerville & Ogren, 1982), but half strength 9-day-old seedlings 8-day-old seedlings Rinsed in sterile water Rinsed in sterile water modified nutrient medium lacking N KNO3 →KCl and Ca(NO3)2 →CaCl2 modified nutrient medium 4.5 mM KNO3 or 2.25 mM (NH4)2SO4 3 days 2 days resupplied with 9 mM KNO3, 4.5 mM (NH4)2SO4,or 4.5 mM K2SO4 rinsed again and transferred to the same medium (controls or switch) harvested after 2, 8, or 24 h harvested 8 h

14. RNA isolation, cDNA synthesis and real-time PCR analysis Rotor Gene 2072 Real-Time Cycler (Corbett Research, Sydney, Australia) RevertAid H Minus First Strand cDNA synthesis Kit (Fermentas, St Leon-Rot, Germany) Escobar et al., 2004 to detect (ndb4 and aox2) Escobar et al. 2004

15. Mitochondrial isolation • mitochondria were isolated using the method of Kruft et al. (2001). • isolating importcompetent Arabidopsis chloroplasts (Schulz et al., 2004). • Potato leaf mitochondria were isolated as previously described (Svensson & Rasmusson, 2001). • Total protein concentration in mitochondrial preparations was determined by the bicinchoninic acid protein assay (Sigma-Aldrich, St. Louis, MO, USA). • Chlorophyll concentration was determined by acetone extraction (Arnon, 1949).

16. Western analysis • Mitochondrial protein (20 lg/lane) • Molecular masses were determined with pre-stained markers (Bio-Rad, Hercules, CA, USA) (Michalecka et al 2004).

17. Activity measurements • Malate dehydrogenase activity and latency was determined spectrophotometrically (Møller et al., 1987) on a Shimadzu UV-160A spectrophotometer (Kyoto, Japan). • Oxidation of NAD(P)H in intact mitochondria was measured with an Aminco DW2a dual wavelength spectrophotometer at 340–400 nm. 蘋果酸

18. A screen to identify nitrogen-regulated respiratory bypass genes: 9-day-old seedling (in complete nutrient medium) N-free medium for 3 days KNO3 (NH4)2SO4 K2SO4 analysed by real-time RT-PCR

19. real-time RT-PCR screen for nitrogen (N)–regulated respiratory genes Negative control Positive control The aox1a, aox1d, aox2, nda2, ndb2 and ndb4 genes, whose transcript abundance varied >2.5-fold by ammonium.

20. The kinetics of nitrogen effects on respiratory bypass genes: 9-day-old seedling (in complete nutrient medium) N-free medium for 3 days KNO3 (NH4)2SO4 K2SO4 Transcript levels analysis after 2, 8, and 24 h

21. Rapid and sustained effects of nitrate and ammonium on expression of genes encoding respiratory bypass proteins ammonium ＞ nitrate: AOX1a, AOX1d, AOX2, NDB2, and NDB4

22. The organ specificity of nitrogen effects on respiratory bypass genes: 9-day-old seedling (in complete nutrient medium) N-free medium for 3 days KNO3 (NH4)2SO4 K2SO4 After 8 h Transcript levels analysis from leaves and roots

23. Organ specificity of N-mediated changes in expression of respiratory bypass genes. Negative control (K2SO4)

24. Expression of respiratory bypass genes under non-nitrogenlimited conditions: 8-day-old seedling (in complete nutrient medium) (NH4)2SO4 KNO3 after 2 days after 2 days KNO3 (NH4)2SO4 KNO3 (NH4)2SO4 after 8 h Transcript levels analysis

25. Nitrogen source effects on steady-state transcript levels of alternative oxidase (AOX) and type II NAD(P)H dehydrogenase genes. Nitrate to ammonium↑:AOX1a, AOX1d, and NDB4 Ammonium to nitrate↓: AOX1a, AOX1d, AOX2, NDA2, NDB2, and NDB4

26. Medium PH changes caused by nitrate and ammonium in Arabidopsis liquid culture

27. Transcript level in the presence of 5 mM MES buffer

28. PH maintained at several variation

29. Transcript level in the presence of Na2WO4

30. Transcript level in the presence of GS inhibitor

31. Induction of AOX in seedlings after a switch from nitrate to ammonuim

32. Western analysis of AOX protein in isolated mitochondria

33. Discussion

34. External type II NAD(P)H dehydrogenases have a high Km compared with nitrate reductase (approx. 30 μm:1.4 μm) • allowing reductant exported from the mitochondria and plastids to be utilized for nitrate reduction

35. 2. All three of the respiratory bypass genes downregulated by nitrate (nda2, aox1a and aox1d) are relatively abundant in leaves which correlates with the leaf being the primary site of nitrate assimilation

36. More respiratory oxidation of matrix NADH and less export of redox equivalents to the cytosol • Type II NAD(P)H dehydrogenases NDB2 and NDB4, and the root-specific AOX2 are probably most physiologically relevant in ammonium metabolism under natural conditions

38. The patterns of ammonium- and nitrate-mediated changes in the alternative respiratory pathways are entirely consistent with a role in the maintenance of whole cell redox homeostasis during N assimilation (Barneix et al., 1984; Lambers, 1980)

39. Hints! • transcript abundance was monitored at 2, 8 and 24 h after addition of nitrate, ammonium, or potassium sulphate (Fig1處理方式) • aox1d, which had a delayed response to ammonium. • nda2 expression was suppressed by nitrate • aox2 and ndb2 expression was induced by ammonium, • aox1a and aox1d was both induced by ammonium and suppressed by nitrate

40. nda2, aox1a and aox1d were higher in leaves, while the ndb2, ndb4 and aox2 transcripts were more abundant in roots