Long distance iron transport and metabolism changes in iron deficient plants

My thesis presentation

Transcript

1. Long distance iron transport and metabolism changes in iron deficient plants Rubén Rellán Álvarez PhD Thesis Javier Abadía and Ana Álvarez PhD Advisors
2. Introduction Outline Conclusions Objective 1: Develop Analytical Methodologies to Study Fe- Long Distance Transport Objective 2: Study the Metabolite Profile of Fe-deficient Plants Summary
3. Plant Fe deficiency Introduction
4. Chlorophyll synthesis Photosynthetic electron transport Respiration DNA and hormone synthesis Fe is essential for plants Plant Fe deficiency Introduction
5. Chlorophyll synthesis Photosynthetic electron transport Respiration DNA and hormone synthesis Fe is essential for plants Fe deficiency Low Fe soil bioavailability Fe chlorosis paradox Agriculture limiting factor 30% of humans are Fe-deficient Plant Fe deficiency Introduction
6. Chlorophyll synthesis Photosynthetic electron transport Respiration DNA and hormone synthesis Fe is essential for plants Fe deficiency Fe can be toxic Low Fe soil bioavailability Fe chlorosis paradox Agriculture limiting factor 30% of humans are Fe-deficient Participates in redox reactions ROS production Is usually complexed Plant Fe deficiency Introduction
7. Chlorophyll synthesis Photosynthetic electron transport Respiration DNA and hormone synthesis Fe is essential for plants Fe deficiency Fe can be toxic Low Fe soil bioavailability Fe chlorosis paradox Agriculture limiting factor 30% of humans are Fe-deficient Participates in redox reactions ROS production Is usually complexed Plant Fe deficiency pH Sensitive Introduction
8. Chlorophyll synthesis Photosynthetic electron transport Respiration DNA and hormone synthesis Fe is essential for plants Fe deficiency Fe can be toxic Low Fe soil bioavailability Fe chlorosis paradox Agriculture limiting factor 30% of humans are Fe-deficient Participates in redox reactions ROS production Is usually complexed Plant Fe deficiency Homeostasis pH Sensitive Introduction
9. xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction
10. xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Strategy I Reduction Strategy II Chelation
11. xylem root Fe(III)-X FRO Fe(II) IRT Fe(II) leaf Phe, OA, PDR H+ AHA Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Flavins
12. xylem root Fe(III)-X FRO Fe(II) IRT Fe(II) Fe(III)-PS YSL Fe(III)-PS leaf Phe, OA, PDR H+ AHA PS TOM Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Flavins
13. xylem root Fe(III)-X FRO Fe(II) IRT Fe(II) Fe(III)-PS YSL Fe(III)-PS leaf Phe, OA, PDR H+ AHA Fe-NA ? PS TOM Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Fe-NA Introduction Flavins
14. xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction
15. xylem root leaf Transpor Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Xylem Unloading Xylem Loading
16. xylem root Cit FRD Cit IREG Fe Fe leaf Fe-NA YSL Transpor Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Xylem Unloading
17. Fe-Cit ? xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Xylem Unloading
18. Fe-Cit ? xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez Introduction
19. Fe-Cit ? xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez Bottleneck Introduction
20. Fe-Cit ? xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root Fe(III)-X FRO YSL ZIP leaf hν Fe(II) Fe(II) IRT Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez Bottleneck Introduction
21. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez Introduction
22. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction
23. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
24. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
25. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
26. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
27. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
28. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
29. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
30. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
31. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
32. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
33. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
34. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
35. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
36. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
37. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
38. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
39. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
40. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
41. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
42. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
43. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
44. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
45. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
46. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
47. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
48. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
49. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
50. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
51. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
52. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
53. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
54. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez 80% of root acquired Fe is in the chloroplasts Introduction What are the Fe forms involved in Fe long distance transport?
55. xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction
56. xylem root Fe(III)-X FRO Fe(II) IRT Fe(II) leaf Phe, OA, PDR H+ AHA Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Flavins
57. xylem root Fe(III)-X FRO Fe(II) IRT Fe(II) Fe(III)-PS YSL Fe(III)-PS leaf Phe, OA, PDR H+ AHA PS TOM Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Flavins
58. xylem root Fe(III)-X FRO Fe(II) IRT Fe(II) Fe(III)-PS YSL Fe(III)-PS leaf Phe, OA, PDR H+ AHA Fe-NA ? PS TOM Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Fe-NA Introduction Flavins
59. Fe-Cit ? xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction
60. Fe-Cit ? xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root Fe(III)-X FRO YSL ZIP leaf hν Fe(II) Fe(II) IRT Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction
61. xylem vessel xylem parenchima cell apoplastic space mesophyll cell xylem root leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction
62. Metabolic Changes induced by Fe deficiency Introduction
63. Metabolic Changes induced by Fe deficiency Introduction
64. Metabolic Changes induced by Fe deficiency Leaves Introduction
65. Photosynthetic Rates C Fixation Chlorophyll Metabolic Changes induced by Fe deficiency Leaves Introduction
66. Photosynthetic Rates C Fixation Chlorophyll Metabolic Changes induced by Fe deficiency Leaves Fe deficient plants are C deficient Introduction
67. Photosynthetic Rates C Fixation Chlorophyll Metabolic Changes induced by Fe deficiency Leaves Fe deficient plants are C deficient Introduction Free AAs
68. Photosynthetic Rates C Fixation Chlorophyll Metabolic Changes induced by Fe deficiency Leaves Fe deficient plants are C deficient Introduction Free AAs
69. Metabolic Changes induced by Fe deficiency Glycolysis PEPC López-Millán et al. 2000 Plant Phys 124: 885 López-Millán et al. 2009 J Plant Phys 166: 375 Roots Introduction TCA cycle
70. Metabolic Changes induced by Fe deficiency Glycolysis PEPC López-Millán et al. 2000 Plant Phys 124: 885 López-Millán et al. 2009 J Plant Phys 166: 375 Roots Anaplerotic C root fixation: Carboxylates Introduction TCA cycle
71. Carboxylates López-Millán et al. 2000 Plant Phys 124: 885 Metabolic Changes induced by Fe deficiency Xylem Introduction
72. Carboxylates López-Millán et al. 2000 Plant Phys 124: 885 Metabolic Changes induced by Fe deficiency Xylem C transport Fe transport Citrate Introduction
73. Metabolic Changes induced by Fe deficiency Introduction
74. Metabolic Changes induced by Fe deficiency Introduction What is the metabolite profile of Fe deficient plants?
75. Analytical Strategies Introduction
76. Analytical Strategies Long Distance Plant Iron Transport Integrated mass spectrometry Introduction
77. Analytical Strategies Long Distance Plant Iron Transport Integrated mass spectrometry Fe Deficiency Plant Metabolite Changes Metabolomics Introduction
78. the majority of species of interest in bioinorganic trace analysis have not yet been isolated in sufficient purity to be used as retention or migration time standards. Therefore, it is becoming of paramount importance to employ in parallel a molecule (or moiety)-specific detector to establish the identity of the eluted smaller species, especially ions with a ratio, secondary adsorption and ion-exch separations. These phenomena, initially sance in SE HPLC, are becoming m employed for the separation of organose noarsenic compounds.100–103 SE HPLC has the advantage over othe the high tolerance to biological matr (0.7–1.0 ml min21) and compositions of t are readily tolerated by flame atomic abso emission and mass spectrometers. Anoth of SE HPLC is the possibility of avoiding mobile phase,98,104 and hence the possib matrix in heartcut and lyophilized fractio multifold dilution of the sample during c is the higher the larger are the column d Packing. Separation by SE HPLC sho the analyte’s charge but, in practice, the s displays charged properties so that a mix observed. This makes the choice of pac major categories of packing used hav organic polymers. The former were repo for metal losses when nanogram amounts Fig. 1 Hyphenated techniques available for species-selective analysis of biological materials. 966 Analyst, 2000, 125, 963–988 Meija et al. 2006 TrAC 25: 44 Szpunar 2000 Analyst 125: 963 Szpunar 2005 Analyst 130: 442 HPLC ICP-MS ESI-TOFMS Integrated mass spectrometry Introduction
79. the majority of species of interest in bioinorganic trace analysis have not yet been isolated in sufficient purity to be used as retention or migration time standards. Therefore, it is becoming of paramount importance to employ in parallel a molecule (or moiety)-specific detector to establish the identity of the eluted smaller species, especially ions with a ratio, secondary adsorption and ion-exch separations. These phenomena, initially sance in SE HPLC, are becoming m employed for the separation of organose noarsenic compounds.100–103 SE HPLC has the advantage over othe the high tolerance to biological matr (0.7–1.0 ml min21) and compositions of t are readily tolerated by flame atomic abso emission and mass spectrometers. Anoth of SE HPLC is the possibility of avoiding mobile phase,98,104 and hence the possib matrix in heartcut and lyophilized fractio multifold dilution of the sample during c is the higher the larger are the column d Packing. Separation by SE HPLC sho the analyte’s charge but, in practice, the s displays charged properties so that a mix observed. This makes the choice of pac major categories of packing used hav organic polymers. The former were repo for metal losses when nanogram amounts Fig. 1 Hyphenated techniques available for species-selective analysis of biological materials. 966 Analyst, 2000, 125, 963–988 Separation Detection Identification Meija et al. 2006 TrAC 25: 44 Szpunar 2000 Analyst 125: 963 Szpunar 2005 Analyst 130: 442 HPLC ICP-MS ESI-TOFMS Integrated mass spectrometry Introduction
80. the majority of species of interest in bioinorganic trace analysis have not yet been isolated in sufficient purity to be used as retention or migration time standards. Therefore, it is becoming of paramount importance to employ in parallel a molecule (or moiety)-specific detector to establish the identity of the eluted smaller species, especially ions with a ratio, secondary adsorption and ion-exch separations. These phenomena, initially sance in SE HPLC, are becoming m employed for the separation of organose noarsenic compounds.100–103 SE HPLC has the advantage over othe the high tolerance to biological matr (0.7–1.0 ml min21) and compositions of t are readily tolerated by flame atomic abso emission and mass spectrometers. Anoth of SE HPLC is the possibility of avoiding mobile phase,98,104 and hence the possib matrix in heartcut and lyophilized fractio multifold dilution of the sample during c is the higher the larger are the column d Packing. Separation by SE HPLC sho the analyte’s charge but, in practice, the s displays charged properties so that a mix observed. This makes the choice of pac major categories of packing used hav organic polymers. The former were repo for metal losses when nanogram amounts Fig. 1 Hyphenated techniques available for species-selective analysis of biological materials. 966 Analyst, 2000, 125, 963–988 Separation Detection Identification Meija et al. 2006 TrAC 25: 44 Szpunar 2000 Analyst 125: 963 Szpunar 2005 Analyst 130: 442 HPLC ICP-MS ESI-TOFMS Integrated mass spectrometry Introduction
81. the majority of species of interest in bioinorganic trace analysis have not yet been isolated in sufficient purity to be used as retention or migration time standards. Therefore, it is becoming of paramount importance to employ in parallel a molecule (or moiety)-specific detector to establish the identity of the eluted smaller species, especially ions with a ratio, secondary adsorption and ion-exch separations. These phenomena, initially sance in SE HPLC, are becoming m employed for the separation of organose noarsenic compounds.100–103 SE HPLC has the advantage over othe the high tolerance to biological matr (0.7–1.0 ml min21) and compositions of t are readily tolerated by flame atomic abso emission and mass spectrometers. Anoth of SE HPLC is the possibility of avoiding mobile phase,98,104 and hence the possib matrix in heartcut and lyophilized fractio multifold dilution of the sample during c is the higher the larger are the column d Packing. Separation by SE HPLC sho the analyte’s charge but, in practice, the s displays charged properties so that a mix observed. This makes the choice of pac major categories of packing used hav organic polymers. The former were repo for metal losses when nanogram amounts Fig. 1 Hyphenated techniques available for species-selective analysis of biological materials. 966 Analyst, 2000, 125, 963–988 Separation Detection Identification Meija et al. 2006 TrAC 25: 44 Szpunar 2000 Analyst 125: 963 Szpunar 2005 Analyst 130: 442 HPLC ICP-MS ESI-TOFMS Integrated mass spectrometry Introduction
82. the majority of species of interest in bioinorganic trace analysis have not yet been isolated in sufficient purity to be used as retention or migration time standards. Therefore, it is becoming of paramount importance to employ in parallel a molecule (or moiety)-specific detector to establish the identity of the eluted smaller species, especially ions with a ratio, secondary adsorption and ion-exch separations. These phenomena, initially sance in SE HPLC, are becoming m employed for the separation of organose noarsenic compounds.100–103 SE HPLC has the advantage over othe the high tolerance to biological matr (0.7–1.0 ml min21) and compositions of t are readily tolerated by flame atomic abso emission and mass spectrometers. Anoth of SE HPLC is the possibility of avoiding mobile phase,98,104 and hence the possib matrix in heartcut and lyophilized fractio multifold dilution of the sample during c is the higher the larger are the column d Packing. Separation by SE HPLC sho the analyte’s charge but, in practice, the s displays charged properties so that a mix observed. This makes the choice of pac major categories of packing used hav organic polymers. The former were repo for metal losses when nanogram amounts Fig. 1 Hyphenated techniques available for species-selective analysis of biological materials. 966 Analyst, 2000, 125, 963–988 Separation Detection Identification Meija et al. 2006 TrAC 25: 44 Szpunar 2000 Analyst 125: 963 Szpunar 2005 Analyst 130: 442 HPLC ICP-MS ESI-TOFMS Integrated mass spectrometry What information can we get using HPLC-ESI-TOFMS? Introduction
83. 3D Compound Information Introduction
84. 5.9 min 0 5 10 15 20 25 Time [min] 0 50 100 150 200 250 Intens. Intens. Intens. Separation (by HPLC) 5.9 min 3D Compound Information Retention Time Introduction
85. 5.9 min 0 5 10 15 20 25 Time [min] 0 50 100 150 200 250 Intens. Intens. Intens. mass/charge (m/z) Identification (by TOFMS) 330.4 344.4 398.5 412.5 440.5 500.5 572.6 -MS, 3.0-19.1min #(89-573) 0 200 400 600 800 Intens. 300 350 400 450 500 550 m/z Separation (by HPLC) 5.9 min 3D Compound Information Retention Time Introduction
86. 14.8 min 0 5 10 15 20 0 200 400 410 412 413 414 7. -MS, 13.0-15.0min 0 1000 2000 3000 Intens. 408 409 410 411 412 413 414 415 m/z 410 412 413 414 9. -MS, 15.1-16.9min 0 2000 4000 6000 Intens. 408 409 410 411 412 413 414 415 m/z 0 500 1000 1500 2000 Intens. 408 4 m/z 5.9 min 0 5 10 15 20 25 Time [min] 0 50 100 150 200 250 Intens. Intens. Intens. mass/charge (m/z) Identification (by TOFMS) 330.4 344.4 398.5 412.5 440.5 500.5 572.6 -MS, 3.0-19.1min #(89-573) 0 200 400 600 800 Intens. 300 350 400 450 500 550 m/z Separation (by HPLC) 5.9 min 3D Compound Information Isotopic Pattern Retention Time Introduction
87. 14.8 min 0 5 10 15 20 0 200 400 410 412 413 414 7. -MS, 13.0-15.0min 0 1000 2000 3000 Intens. 408 409 410 411 412 413 414 415 m/z 410 412 413 414 9. -MS, 15.1-16.9min 0 2000 4000 6000 Intens. 408 409 410 411 412 413 414 415 m/z 0 500 1000 1500 2000 Intens. 408 4 m/z 5.9 min 0 5 10 15 20 25 Time [min] 0 50 100 150 200 250 Intens. Intens. Intens. mass/charge (m/z) Identification (by TOFMS) 330.4 344.4 398.5 412.5 440.5 500.5 572.6 -MS, 3.0-19.1min #(89-573) 0 200 400 600 800 Intens. 300 350 400 450 500 550 m/z Separation (by HPLC) 5.9 min 3D Compound Information Isotopic Pattern Retention Time Isotopic Pattern Introduction
88. CHO Isotopic Pattern Fe Isotopic Pattern Isotopic Pattern Introduction
89. CHO Isotopic Pattern Fe Isotopic Pattern Isotopic Pattern Introduction 12C 13C 54Fe 56Fe
90. Isotopic Pattern Introduction
91. Isotopic Pattern Introduction
92. CHO + 1 Fe CHO + 3 Fe Isotopic Pattern Introduction
93. CHO + 1 Fe CHO + 3 Fe Isotopic Pattern Introduction We can determine the molecular formula using the isotopic pattern and the exact m/z
94. CHO + 1 Fe CHO + 3 Fe Isotopic Pattern Introduction We can determine the molecular formula using the isotopic pattern and the exact m/z
95. CHO + 1 Fe CHO + 3 Fe Isotopic Pattern Introduction We can determine the molecular formula using the isotopic pattern and the exact m/z
96. CHO + 1 Fe CHO + 3 Fe Isotopic Pattern Introduction We can determine the molecular formula using the isotopic pattern and the exact m/z
97. CHO + 1 Fe CHO + 3 Fe Isotopic Pattern Introduction We can determine the molecular formula using the isotopic pattern and the exact m/z
98. Lan et al. 2011 Plant Phys In Press Rodríguez-Celma et al 2011 J Protem Res Under Review Li et al 2008 Proteomics 8: 2229 Introduction Fe Deficiency Plant Metabolite Changes Metabolomics
99. DNA Genomics RNA Transcriptomics Proteins Proteomics Metabolites Metabolomics Lan et al. 2011 Plant Phys In Press Rodríguez-Celma et al 2011 J Protem Res Under Review Li et al 2008 Proteomics 8: 2229 Introduction Fe Deficiency Plant Metabolite Changes Metabolomics
100. Fiehn et al. 2008 Plant J 53: 691 Fiehn et al. 2007 Metabolomics 3: 195 Fiehn et al. (2008) Plant J 53: 691 Metabolomics Workflow Introduction
101. Fiehn et al. 2008 Plant J 53: 691 Fiehn et al. 2007 Metabolomics 3: 195 Fiehn et al. (2008) Plant J 53: 691 Metabolomics Workflow Sops Experimental Design (SetupX) Sample Preparation Wet lab work MS Data Acquisition Export, align annotate Data Annotation (BinBase) Transformation Statistics Data Analysis Statistics norm & tansform classes Biosource control mutant organ 1 organ 2 organ 1 organ 2 dose 1 Class Class Class Class dose 2 Class Class Class Class dose 1 Class Class Class Class dose 2 Class Class Class Class Growth history treatm 1 treatm 2 GC Separation Introduction
102. Objectives Study of metal-nicotianamine complexes by ESI-TOFMS Iron-citrate xylem transport HPLC-MS determination of organic acids Objective 1: Analytical Methodologies to Study Fe-Long Distance Transport
103. 1. To study the formation of metal complexes with nicotianamine as affected by pH, ligand and metal exchange by means of electrospray time- of-flight mass spectrometry (ESI-TOFMS) Study of metal-nicotianamine complexes by ESI-TOFMS Objective Nicotianamine (NA) Fe-NA
104. xylem root leaf Study of metal-nicotianamine complexes by ESI-TOFMS
105. pH 7-7.5 pH 5-6.5 pH 7-7.5 xylem root leaf pH changes Study of metal-nicotianamine complexes by ESI-TOFMS
106. Mat and Met pH dependance Ligand Exchange (Citrate) Metal Exchange pH 5.5 pH 7.5 DI-ESI-MS In Silico pH 5.5 pH 7.5 Cu Zn Cu Zn Fe(II)-NA X X X X X X X X Fe(III)-NA X X Mn(II)-NA X X Zn(II)-NA X X Ni(II)-NA X X Cu(II)-NA X X Study of metal-nicotianamine complexes by ESI-TOFMS
107. Study of metal-nicotianamine complexes by ESI-TOFMS Materials and Methods Direct Infusion DI Metal-NA
108. Study of metal-nicotianamine complexes by ESI-TOFMS Materials and Methods Direct Infusion DI ESI Identifying System Components Bruker Daltonik GmbH 2.2.2 Syringe pump A small syringe pump (see Figure 2-3) is included with the micrOTOF-Q system to provide for the introduction of samples directly to either the electrospray or APCI ion sources. The syringe pump is supplied with a 250
     l syringe. Smaller and larger syringes can also be used. Figure 2-3 Syringe pump coupled to the Apollo source 2-4 micrOTOF-Q User Manual, Version 1.1 TOFMS Metal-NA
109. Study of metal-nicotianamine complexes by ESI-TOFMS Materials and Methods Direct Infusion DI ESI Identifying System Components Bruker Daltonik GmbH 2.2.2 Syringe pump A small syringe pump (see Figure 2-3) is included with the micrOTOF-Q system to provide for the introduction of samples directly to either the electrospray or APCI ion sources. The syringe pump is supplied with a 250
     l syringe. Smaller and larger syringes can also be used. Figure 2-3 Syringe pump coupled to the Apollo source 2-4 micrOTOF-Q User Manual, Version 1.1 TOFMS Metal-NA Citrate, Cu, Zn Ligand and Metal Exchange
110. Metal-NA complexes can be studied by ESI-MS Results and Discussion Study of metal-nicotianamine complexes by ESI-TOFMS Rellán-Álvarez et al. 2008 Rapid Commun Mass Spectrom 22: 15
111. Metal-NA complexes can be studied by ESI-MS Results and Discussion Study of metal-nicotianamine complexes by ESI-TOFMS Rellán-Álvarez et al. 2008 Rapid Commun Mass Spectrom 22: 15 Experimental Data Experimental Data Theoretical Data Theoretical Data
112. DI-ESI-MS Fe-NA complexes are pH dependent In Silico Intensity pH Concentration Study of metal-nicotianamine complexes by ESI-TOFMS Results and Discussion Rellán-Álvarez et al. 2008 Rapid Commun Mass Spectrom 22: 15
113. DI-ESI-MS Fe-NA complexes are pH dependent In Silico Intensity pH Concentration Study of metal-nicotianamine complexes by ESI-TOFMS Results and Discussion Rellán-Álvarez et al. 2008 Rapid Commun Mass Spectrom 22: 15
114. DI-ESI-MS Fe-NA complexes are pH dependent In Silico Intensity pH Concentration Study of metal-nicotianamine complexes by ESI-TOFMS Results and Discussion Rellán-Álvarez et al. 2008 Rapid Commun Mass Spectrom 22: 15
115. Fe(II)-NA ligand exchange with citrate Study of metal-nicotianamine complexes by ESI-TOFMS Results and Discussion Rellán-Álvarez et al. 2008 Rapid Commun Mass Spectrom 22: 15 +Citrate +Citrate
116. Fe(II)-NA ligand exchange with citrate Study of metal-nicotianamine complexes by ESI-TOFMS Results and Discussion Rellán-Álvarez et al. 2008 Rapid Commun Mass Spectrom 22: 15 Relative abundance m/z pH 7.5 pH 5.5 +Citrate +Citrate
117. Fe(II)-NA ligand exchange with citrate Study of metal-nicotianamine complexes by ESI-TOFMS Results and Discussion Rellán-Álvarez et al. 2008 Rapid Commun Mass Spectrom 22: 15 Relative abundance m/z pH 7.5 pH 5.5 +Citrate +Citrate
118. Fe(II)-NA metal exchange with Zn Study of metal-nicotianamine complexes by ESI-TOFMS Results and Discussion Rellán-Álvarez et al. 2008 Rapid Commun Mass Spectrom 22: 15 pH 7.5 pH 5.5 m/z Relative abundance
119. Study of metal-nicotianamine complexes by ESI-TOFMS
120. Concluding Remarks Study of metal-nicotianamine complexes by ESI-TOFMS
121. Concluding Remarks -Metal-NA complexes can be studied using ESI-TOFMS Study of metal-nicotianamine complexes by ESI-TOFMS
122. Concluding Remarks -Metal-NA complexes can be studied using ESI-TOFMS -Small changes in pH can affect metal-NA speciation Study of metal-nicotianamine complexes by ESI-TOFMS
123. Concluding Remarks -Metal-NA complexes can be studied using ESI-TOFMS -Small changes in pH can affect metal-NA speciation -Citrate and metal exchange reactions can easily occur at pH 5.5 but not at 7.5 Study of metal-nicotianamine complexes by ESI-TOFMS
124. Concluding Remarks -Metal-NA complexes can be studied using ESI-TOFMS -Small changes in pH can affect metal-NA speciation -Citrate and metal exchange reactions can easily occur at pH 5.5 but not at 7.5 -NA as a possible Fe chelator in phloem but not in xylem Study of metal-nicotianamine complexes by ESI-TOFMS
125. Concluding Remarks -Metal-NA complexes can be studied using ESI-TOFMS -Small changes in pH can affect metal-NA speciation -Citrate and metal exchange reactions can easily occur at pH 5.5 but not at 7.5 -NA as a possible Fe chelator in phloem but not in xylem -Keypoints when doing metal speciation in plant fluids: Study of metal-nicotianamine complexes by ESI-TOFMS
126. Concluding Remarks -Metal-NA complexes can be studied using ESI-TOFMS -Small changes in pH can affect metal-NA speciation -Citrate and metal exchange reactions can easily occur at pH 5.5 but not at 7.5 -NA as a possible Fe chelator in phloem but not in xylem -Keypoints when doing metal speciation in plant fluids: -Keep the original plant fluid pH Study of metal-nicotianamine complexes by ESI-TOFMS
127. 2. To develop a method for the determination of naturally occurring Fe complexes in xylem sap, using high performance liquid chromatography (HPLC) coupled to ESI-TOFMS and inductively coupled plasma mass spectrometry (ICP-MS) Objective Iron-citrate xylem transport
128. Iron-citrate xylem transport Materials and Methods
129. Iron-citrate xylem transport Materials and Methods Separation Detection and quantification Identification A PRACTICAL GUIDE TO HILIC A tutorial and application book Introduction This guide aims at introducing hydrophilic interaction liquid chromatography (HILIC), which is a technique suitable for separation of very polar and hydrophilic compounds. It deals with the basic theory of HILIC and the practical aspects of this separation mode. This booklet will also introduce the reader to the SeQuant zwitterionic ZIC®-HILIC and ZIC®-pHILIC stationary phases, see Figure 1, and contains a range of application examples for different types of hydrophilic compounds. You can read more about this and lots of other things in this guide. Figure 1: The functional group of the ZIC®-HILIC and ZIC®- pHILIC stationary phases. If your HILIC questions cannot be solved with this compilation SeQuant is at your service. We first recommend you to visit the SeQuant homepage (www.sequant.com), where you always find material update, applications and technical data on our products. If you need additional information the SeQuant staff will be happy to further assist you. Why HILIC? Despite the fact that reversed phase liquid chromatography (RPLC) is the overall most applied separation technique, and that it can be used for a variety of applications in junction with the most common detection principles, certain of choice for this purpose, using non-aqueous mobile phases not very friendly to the environment. Yet, under such experimental conditions it is difficult to dissolve polar and hydrophilic compounds. In its place, HILIC is the alternative as the elution order is likewise inverted to RPLC, as illustrated in Figure 2. In other words, solutes that have little or no retention on RPLC columns generally experience strong retention on HILIC columns. Figure 2: Separation of peptides under HILIC and RPLC conditions. Eluents; (HILIC) 60:40 acetonitrile / 10 mM ammonium acetate, pH 7, (RPLC) 5:95 acetonitrile / 10 mM ammonium acetate, pH 7. Legend; (1) Phe-Gly-Gly-Phe, (2) Leu-Gly-Gly, (3) Gly-Gly-Gly. The HILIC technique thus bears similarities with traditional NPLC, but with the important difference that HILIC employs semi-aqueous mobile phases. Consequently, with respect to analyte solubility in the eluent and matrix compatibility, HILIC is superior, as the mobile phase compositions used are comparable to RPLC separations. Typical eluents for HILIC consist of 40-97% acetonitrile in water or a volatile buffer. HILIC is thus a very mass spectrometry (MS) friendly technique, and by changing from RPLC to HILIC a 10-1000 fold increase in sensitivity is HILIC ICP-MS ESI-TOFMS
130. Iron-citrate xylem transport Materials and Methods Justo Giner J. I.García J. A. Rodríguez University of Oviedo Separation Detection and quantification Identification A PRACTICAL GUIDE TO HILIC A tutorial and application book Introduction This guide aims at introducing hydrophilic interaction liquid chromatography (HILIC), which is a technique suitable for separation of very polar and hydrophilic compounds. It deals with the basic theory of HILIC and the practical aspects of this separation mode. This booklet will also introduce the reader to the SeQuant zwitterionic ZIC®-HILIC and ZIC®-pHILIC stationary phases, see Figure 1, and contains a range of application examples for different types of hydrophilic compounds. You can read more about this and lots of other things in this guide. Figure 1: The functional group of the ZIC®-HILIC and ZIC®- pHILIC stationary phases. If your HILIC questions cannot be solved with this compilation SeQuant is at your service. We first recommend you to visit the SeQuant homepage (www.sequant.com), where you always find material update, applications and technical data on our products. If you need additional information the SeQuant staff will be happy to further assist you. Why HILIC? Despite the fact that reversed phase liquid chromatography (RPLC) is the overall most applied separation technique, and that it can be used for a variety of applications in junction with the most common detection principles, certain of choice for this purpose, using non-aqueous mobile phases not very friendly to the environment. Yet, under such experimental conditions it is difficult to dissolve polar and hydrophilic compounds. In its place, HILIC is the alternative as the elution order is likewise inverted to RPLC, as illustrated in Figure 2. In other words, solutes that have little or no retention on RPLC columns generally experience strong retention on HILIC columns. Figure 2: Separation of peptides under HILIC and RPLC conditions. Eluents; (HILIC) 60:40 acetonitrile / 10 mM ammonium acetate, pH 7, (RPLC) 5:95 acetonitrile / 10 mM ammonium acetate, pH 7. Legend; (1) Phe-Gly-Gly-Phe, (2) Leu-Gly-Gly, (3) Gly-Gly-Gly. The HILIC technique thus bears similarities with traditional NPLC, but with the important difference that HILIC employs semi-aqueous mobile phases. Consequently, with respect to analyte solubility in the eluent and matrix compatibility, HILIC is superior, as the mobile phase compositions used are comparable to RPLC separations. Typical eluents for HILIC consist of 40-97% acetonitrile in water or a volatile buffer. HILIC is thus a very mass spectrometry (MS) friendly technique, and by changing from RPLC to HILIC a 10-1000 fold increase in sensitivity is HILIC ICP-MS ESI-TOFMS
131. Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 Iron-citrate xylem transport Results and discussion
132. ICP-MS ESI-TOFMS © Copyright, 2005-2007 , SeQuant AB A PRACTICAL GUIDE TO HILIC A tutorial and application book Introduction This guide aims at introducing hydrophilic interaction liquid chromatography (HILIC), which is a technique suitable for separation of very polar and hydrophilic compounds. It deals with the basic theory of HILIC and the practical aspects of this separation mode. This booklet will also introduce the reader to the SeQuant zwitterionic ZIC®-HILIC and ZIC®-pHILIC stationary phases, see Figure 1, and contains a range of application examples for different types of hydrophilic compounds. You can read more about this and lots of other things in this guide. Figure 1: The functional group of the ZIC®-HILIC and ZIC®- pHILIC stationary phases. If your HILIC questions cannot be solved with this compilation SeQuant is at your service. We first recommend you to visit the SeQuant homepage (www.sequant.com), where you always find material update, applications and technical data on our products. If you need additional information the SeQuant staff will be happy to further assist you. Why HILIC? Despite the fact that reversed phase liquid chromatography (RPLC) is the overall most applied separation technique, and that it can be used for a variety of applications in junction with the most common detection principles, certain solutes, especially polar and hydrophilic compounds, are not retainable in a simple fashion. Over a long period normal phase liquid chromatography (NPLC) has been the technique of choice for this purpose, using non-aqueous mobile phases not very friendly to the environment. Yet, under such experimental conditions it is difficult to dissolve polar and hydrophilic compounds. In its place, HILIC is the alternative as the elution order is likewise inverted to RPLC, as illustrated in Figure 2. In other words, solutes that have little or no retention on RPLC columns generally experience strong retention on HILIC columns. Figure 2: Separation of peptides under HILIC and RPLC conditions. Eluents; (HILIC) 60:40 acetonitrile / 10 mM ammonium acetate, pH 7, (RPLC) 5:95 acetonitrile / 10 mM ammonium acetate, pH 7. Legend; (1) Phe-Gly-Gly-Phe, (2) Leu-Gly-Gly, (3) Gly-Gly-Gly. The HILIC technique thus bears similarities with traditional NPLC, but with the important difference that HILIC employs semi-aqueous mobile phases. Consequently, with respect to analyte solubility in the eluent and matrix compatibility, HILIC is superior, as the mobile phase compositions used are comparable to RPLC separations. Typical eluents for HILIC consist of 40-97% acetonitrile in water or a volatile buffer. HILIC is thus a very mass spectrometry (MS) friendly technique, and by changing from RPLC to HILIC a 10-1000 fold increase in sensitivity is often observed for hydrophilic analytes. Ion-pair reagents are also completely avoided, which is advantageous for preparative chromatography. natFe-Cit Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 Iron-citrate xylem transport Results and discussion
133. 54Fe-Cit ICP-MS ESI-TOFMS © Copyright, 2005-2007 , SeQuant AB A PRACTICAL GUIDE TO HILIC A tutorial and application book Introduction This guide aims at introducing hydrophilic interaction liquid chromatography (HILIC), which is a technique suitable for separation of very polar and hydrophilic compounds. It deals with the basic theory of HILIC and the practical aspects of this separation mode. This booklet will also introduce the reader to the SeQuant zwitterionic ZIC®-HILIC and ZIC®-pHILIC stationary phases, see Figure 1, and contains a range of application examples for different types of hydrophilic compounds. You can read more about this and lots of other things in this guide. Figure 1: The functional group of the ZIC®-HILIC and ZIC®- pHILIC stationary phases. If your HILIC questions cannot be solved with this compilation SeQuant is at your service. We first recommend you to visit the SeQuant homepage (www.sequant.com), where you always find material update, applications and technical data on our products. If you need additional information the SeQuant staff will be happy to further assist you. Why HILIC? Despite the fact that reversed phase liquid chromatography (RPLC) is the overall most applied separation technique, and that it can be used for a variety of applications in junction with the most common detection principles, certain solutes, especially polar and hydrophilic compounds, are not retainable in a simple fashion. Over a long period normal phase liquid chromatography (NPLC) has been the technique of choice for this purpose, using non-aqueous mobile phases not very friendly to the environment. Yet, under such experimental conditions it is difficult to dissolve polar and hydrophilic compounds. In its place, HILIC is the alternative as the elution order is likewise inverted to RPLC, as illustrated in Figure 2. In other words, solutes that have little or no retention on RPLC columns generally experience strong retention on HILIC columns. Figure 2: Separation of peptides under HILIC and RPLC conditions. Eluents; (HILIC) 60:40 acetonitrile / 10 mM ammonium acetate, pH 7, (RPLC) 5:95 acetonitrile / 10 mM ammonium acetate, pH 7. Legend; (1) Phe-Gly-Gly-Phe, (2) Leu-Gly-Gly, (3) Gly-Gly-Gly. The HILIC technique thus bears similarities with traditional NPLC, but with the important difference that HILIC employs semi-aqueous mobile phases. Consequently, with respect to analyte solubility in the eluent and matrix compatibility, HILIC is superior, as the mobile phase compositions used are comparable to RPLC separations. Typical eluents for HILIC consist of 40-97% acetonitrile in water or a volatile buffer. HILIC is thus a very mass spectrometry (MS) friendly technique, and by changing from RPLC to HILIC a 10-1000 fold increase in sensitivity is often observed for hydrophilic analytes. Ion-pair reagents are also completely avoided, which is advantageous for preparative chromatography. natFe-Cit Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 Iron-citrate xylem transport Results and discussion the whole process is done at xylem pH: 5.5 Fe:Cit 1:10
134. 54Fe-Cit ICP-MS ESI-TOFMS © Copyright, 2005-2007 , SeQuant AB A PRACTICAL GUIDE TO HILIC A tutorial and application book Introduction This guide aims at introducing hydrophilic interaction liquid chromatography (HILIC), which is a technique suitable for separation of very polar and hydrophilic compounds. It deals with the basic theory of HILIC and the practical aspects of this separation mode. This booklet will also introduce the reader to the SeQuant zwitterionic ZIC®-HILIC and ZIC®-pHILIC stationary phases, see Figure 1, and contains a range of application examples for different types of hydrophilic compounds. You can read more about this and lots of other things in this guide. Figure 1: The functional group of the ZIC®-HILIC and ZIC®- pHILIC stationary phases. If your HILIC questions cannot be solved with this compilation SeQuant is at your service. We first recommend you to visit the SeQuant homepage (www.sequant.com), where you always find material update, applications and technical data on our products. If you need additional information the SeQuant staff will be happy to further assist you. Why HILIC? Despite the fact that reversed phase liquid chromatography (RPLC) is the overall most applied separation technique, and that it can be used for a variety of applications in junction with the most common detection principles, certain solutes, especially polar and hydrophilic compounds, are not retainable in a simple fashion. Over a long period normal phase liquid chromatography (NPLC) has been the technique of choice for this purpose, using non-aqueous mobile phases not very friendly to the environment. Yet, under such experimental conditions it is difficult to dissolve polar and hydrophilic compounds. In its place, HILIC is the alternative as the elution order is likewise inverted to RPLC, as illustrated in Figure 2. In other words, solutes that have little or no retention on RPLC columns generally experience strong retention on HILIC columns. Figure 2: Separation of peptides under HILIC and RPLC conditions. Eluents; (HILIC) 60:40 acetonitrile / 10 mM ammonium acetate, pH 7, (RPLC) 5:95 acetonitrile / 10 mM ammonium acetate, pH 7. Legend; (1) Phe-Gly-Gly-Phe, (2) Leu-Gly-Gly, (3) Gly-Gly-Gly. The HILIC technique thus bears similarities with traditional NPLC, but with the important difference that HILIC employs semi-aqueous mobile phases. Consequently, with respect to analyte solubility in the eluent and matrix compatibility, HILIC is superior, as the mobile phase compositions used are comparable to RPLC separations. Typical eluents for HILIC consist of 40-97% acetonitrile in water or a volatile buffer. HILIC is thus a very mass spectrometry (MS) friendly technique, and by changing from RPLC to HILIC a 10-1000 fold increase in sensitivity is often observed for hydrophilic analytes. Ion-pair reagents are also completely avoided, which is advantageous for preparative chromatography. natFe-Cit Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 Iron-citrate xylem transport Results and discussion 2 Fe Peaks the whole process is done at xylem pH: 5.5 Fe:Cit 1:10
135. 54Fe-Cit ICP-MS ESI-TOFMS © Copyright, 2005-2007 , SeQuant AB A PRACTICAL GUIDE TO HILIC A tutorial and application book Introduction This guide aims at introducing hydrophilic interaction liquid chromatography (HILIC), which is a technique suitable for separation of very polar and hydrophilic compounds. It deals with the basic theory of HILIC and the practical aspects of this separation mode. This booklet will also introduce the reader to the SeQuant zwitterionic ZIC®-HILIC and ZIC®-pHILIC stationary phases, see Figure 1, and contains a range of application examples for different types of hydrophilic compounds. You can read more about this and lots of other things in this guide. Figure 1: The functional group of the ZIC®-HILIC and ZIC®- pHILIC stationary phases. If your HILIC questions cannot be solved with this compilation SeQuant is at your service. We first recommend you to visit the SeQuant homepage (www.sequant.com), where you always find material update, applications and technical data on our products. If you need additional information the SeQuant staff will be happy to further assist you. Why HILIC? Despite the fact that reversed phase liquid chromatography (RPLC) is the overall most applied separation technique, and that it can be used for a variety of applications in junction with the most common detection principles, certain solutes, especially polar and hydrophilic compounds, are not retainable in a simple fashion. Over a long period normal phase liquid chromatography (NPLC) has been the technique of choice for this purpose, using non-aqueous mobile phases not very friendly to the environment. Yet, under such experimental conditions it is difficult to dissolve polar and hydrophilic compounds. In its place, HILIC is the alternative as the elution order is likewise inverted to RPLC, as illustrated in Figure 2. In other words, solutes that have little or no retention on RPLC columns generally experience strong retention on HILIC columns. Figure 2: Separation of peptides under HILIC and RPLC conditions. Eluents; (HILIC) 60:40 acetonitrile / 10 mM ammonium acetate, pH 7, (RPLC) 5:95 acetonitrile / 10 mM ammonium acetate, pH 7. Legend; (1) Phe-Gly-Gly-Phe, (2) Leu-Gly-Gly, (3) Gly-Gly-Gly. The HILIC technique thus bears similarities with traditional NPLC, but with the important difference that HILIC employs semi-aqueous mobile phases. Consequently, with respect to analyte solubility in the eluent and matrix compatibility, HILIC is superior, as the mobile phase compositions used are comparable to RPLC separations. Typical eluents for HILIC consist of 40-97% acetonitrile in water or a volatile buffer. HILIC is thus a very mass spectrometry (MS) friendly technique, and by changing from RPLC to HILIC a 10-1000 fold increase in sensitivity is often observed for hydrophilic analytes. Ion-pair reagents are also completely avoided, which is advantageous for preparative chromatography. natFe-Cit Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 Iron-citrate xylem transport Results and discussion 2 Fe Peaks 2 Fe-Complexes Peaks the whole process is done at xylem pH: 5.5 Fe:Cit 1:10
136. 54Fe-Cit ICP-MS ESI-TOFMS © Copyright, 2005-2007 , SeQuant AB A PRACTICAL GUIDE TO HILIC A tutorial and application book Introduction This guide aims at introducing hydrophilic interaction liquid chromatography (HILIC), which is a technique suitable for separation of very polar and hydrophilic compounds. It deals with the basic theory of HILIC and the practical aspects of this separation mode. This booklet will also introduce the reader to the SeQuant zwitterionic ZIC®-HILIC and ZIC®-pHILIC stationary phases, see Figure 1, and contains a range of application examples for different types of hydrophilic compounds. You can read more about this and lots of other things in this guide. Figure 1: The functional group of the ZIC®-HILIC and ZIC®- pHILIC stationary phases. If your HILIC questions cannot be solved with this compilation SeQuant is at your service. We first recommend you to visit the SeQuant homepage (www.sequant.com), where you always find material update, applications and technical data on our products. If you need additional information the SeQuant staff will be happy to further assist you. Why HILIC? Despite the fact that reversed phase liquid chromatography (RPLC) is the overall most applied separation technique, and that it can be used for a variety of applications in junction with the most common detection principles, certain solutes, especially polar and hydrophilic compounds, are not retainable in a simple fashion. Over a long period normal phase liquid chromatography (NPLC) has been the technique of choice for this purpose, using non-aqueous mobile phases not very friendly to the environment. Yet, under such experimental conditions it is difficult to dissolve polar and hydrophilic compounds. In its place, HILIC is the alternative as the elution order is likewise inverted to RPLC, as illustrated in Figure 2. In other words, solutes that have little or no retention on RPLC columns generally experience strong retention on HILIC columns. Figure 2: Separation of peptides under HILIC and RPLC conditions. Eluents; (HILIC) 60:40 acetonitrile / 10 mM ammonium acetate, pH 7, (RPLC) 5:95 acetonitrile / 10 mM ammonium acetate, pH 7. Legend; (1) Phe-Gly-Gly-Phe, (2) Leu-Gly-Gly, (3) Gly-Gly-Gly. The HILIC technique thus bears similarities with traditional NPLC, but with the important difference that HILIC employs semi-aqueous mobile phases. Consequently, with respect to analyte solubility in the eluent and matrix compatibility, HILIC is superior, as the mobile phase compositions used are comparable to RPLC separations. Typical eluents for HILIC consist of 40-97% acetonitrile in water or a volatile buffer. HILIC is thus a very mass spectrometry (MS) friendly technique, and by changing from RPLC to HILIC a 10-1000 fold increase in sensitivity is often observed for hydrophilic analytes. Ion-pair reagents are also completely avoided, which is advantageous for preparative chromatography. natFe-Cit Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 Iron-citrate xylem transport Results and discussion 2 Fe Peaks 2 Fe-Complexes Peaks the whole process is done at xylem pH: 5.5 Fe:Cit 1:10
137. 54Fe-Cit ICP-MS ESI-TOFMS © Copyright, 2005-2007 , SeQuant AB A PRACTICAL GUIDE TO HILIC A tutorial and application book Introduction This guide aims at introducing hydrophilic interaction liquid chromatography (HILIC), which is a technique suitable for separation of very polar and hydrophilic compounds. It deals with the basic theory of HILIC and the practical aspects of this separation mode. This booklet will also introduce the reader to the SeQuant zwitterionic ZIC®-HILIC and ZIC®-pHILIC stationary phases, see Figure 1, and contains a range of application examples for different types of hydrophilic compounds. You can read more about this and lots of other things in this guide. Figure 1: The functional group of the ZIC®-HILIC and ZIC®- pHILIC stationary phases. If your HILIC questions cannot be solved with this compilation SeQuant is at your service. We first recommend you to visit the SeQuant homepage (www.sequant.com), where you always find material update, applications and technical data on our products. If you need additional information the SeQuant staff will be happy to further assist you. Why HILIC? Despite the fact that reversed phase liquid chromatography (RPLC) is the overall most applied separation technique, and that it can be used for a variety of applications in junction with the most common detection principles, certain solutes, especially polar and hydrophilic compounds, are not retainable in a simple fashion. Over a long period normal phase liquid chromatography (NPLC) has been the technique of choice for this purpose, using non-aqueous mobile phases not very friendly to the environment. Yet, under such experimental conditions it is difficult to dissolve polar and hydrophilic compounds. In its place, HILIC is the alternative as the elution order is likewise inverted to RPLC, as illustrated in Figure 2. In other words, solutes that have little or no retention on RPLC columns generally experience strong retention on HILIC columns. Figure 2: Separation of peptides under HILIC and RPLC conditions. Eluents; (HILIC) 60:40 acetonitrile / 10 mM ammonium acetate, pH 7, (RPLC) 5:95 acetonitrile / 10 mM ammonium acetate, pH 7. Legend; (1) Phe-Gly-Gly-Phe, (2) Leu-Gly-Gly, (3) Gly-Gly-Gly. The HILIC technique thus bears similarities with traditional NPLC, but with the important difference that HILIC employs semi-aqueous mobile phases. Consequently, with respect to analyte solubility in the eluent and matrix compatibility, HILIC is superior, as the mobile phase compositions used are comparable to RPLC separations. Typical eluents for HILIC consist of 40-97% acetonitrile in water or a volatile buffer. HILIC is thus a very mass spectrometry (MS) friendly technique, and by changing from RPLC to HILIC a 10-1000 fold increase in sensitivity is often observed for hydrophilic analytes. Ion-pair reagents are also completely avoided, which is advantageous for preparative chromatography. natFe-Cit Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 Iron-citrate xylem transport Results and discussion 2 Fe Peaks 2 Fe-Complexes Peaks the whole process is done at xylem pH: 5.5 Fe:Cit 1:10
138. natFe-Cit Intensity m/z 54Fe-Cit Experimental Data Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
139. Experimental m/z Molecular
 formula 375.4047 natFe3C18H15O22 372.4119 54Fe3C18H15O22 natFe-Cit Intensity m/z 54Fe-Cit Experimental Data Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
140. Experimental m/z Molecular
 formula 375.4047 natFe3C18H15O22 372.4119 54Fe3C18H15O22 natFe-Cit Intensity m/z 54Fe-Cit Experimental Data Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
141. Experimental m/z Molecular
 formula 375.4047 natFe3C18H15O22 372.4119 54Fe3C18H15O22 natFe-Cit Intensity m/z 54Fe-Cit Experimental Data Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
142. Experimental m/z Molecular
 formula 375.4047 natFe3C18H15O22 372.4119 54Fe3C18H15O22 natFe-Cit Intensity m/z 54Fe-Cit Experimental Data Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
143. Experimental m/z Molecular
 formula 375.4047 natFe3C18H15O22 372.4119 54Fe3C18H15O22 natFe-Cit Intensity m/z 54Fe-Cit Experimental Data Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 Theoretical 
 m/z Error m/z (ppm) 375.4057 1.7 372.4127 2.3 Theoretical Data
144. Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
145. Fe3-Cit3 3D Modelling Jesús Orduna Uni. Zar. ICMA Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
146. Fe3-Cit3 3D Modelling Jesús Orduna Uni. Zar. ICMA Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
147. Oxo-bridged tri-Fe core center Fe3-Cit3 3D Modelling Jesús Orduna Uni. Zar. ICMA Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
148. Oxo-bridged tri-Fe core center Distal carboxylate groups Fe3-Cit3 3D Modelling Jesús Orduna Uni. Zar. ICMA Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
149. Oxo-bridged tri-Fe core center Distal carboxylate groups Central carboxylate group Fe3-Cit3 3D Modelling Jesús Orduna Uni. Zar. ICMA Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
150. Oxo-bridged tri-Fe core center Distal carboxylate groups Central carboxylate group Octahedral configuration Fe3-Cit3 3D Modelling Jesús Orduna Uni. Zar. ICMA Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
151. Oxo-bridged tri-Fe core center H-bonds Distal carboxylate groups Central carboxylate group Octahedral configuration Fe3-Cit3 3D Modelling Jesús Orduna Uni. Zar. ICMA Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
152. Fe2-Cit2 3D Modelling Iron-citrate xylem transport Results and discussion
153. Fe2-Cit2 3D Modelling Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
154. Iron-citrate xylem transport Results and discussion
155. Iron-citrate xylem transport Results and discussion López-Millán et al. 2000 Plant Physiol 124: 885 Larbi et al. 2003 J Plant Physiol 160: 1473 Fe:Cit ratios change with Fe-deficiency Sugar beet Peach +Fe 1:36 1:8 -Fe 1:2474 1:125 Fact
156. Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 López-Millán et al. 2000 Plant Physiol 124: 885 Larbi et al. 2003 J Plant Physiol 160: 1473 Fe:Cit ratios change with Fe-deficiency Sugar beet Peach +Fe 1:36 1:8 -Fe 1:2474 1:125 Fact Fe2Cit2 Fe3Cit3 ICP-MS Fe:Cit 1:1 1:10 1:10 1:50 0.0 0.2 0.4 0.6 0.8 1.0 1.2 54Fe nmol Fe:Cit Ratios drive the formation of Fe-Cit Complexes
157. ESI-TOFMS Fe2Cit2 Fe3Cit3 Fe-Cit ESI-MS Intensity x 104 Fe:Cit 1:1 1:10 1:10 1:50 0 1 2 3 4 Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 López-Millán et al. 2000 Plant Physiol 124: 885 Larbi et al. 2003 J Plant Physiol 160: 1473 Fe:Cit ratios change with Fe-deficiency Sugar beet Peach +Fe 1:36 1:8 -Fe 1:2474 1:125 Fact Fe2Cit2 Fe3Cit3 ICP-MS Fe:Cit 1:1 1:10 1:10 1:50 0.0 0.2 0.4 0.6 0.8 1.0 1.2 54Fe nmol Fe:Cit Ratios drive the formation of Fe-Cit Complexes
158. ESI-TOFMS Fe2Cit2 Fe3Cit3 Fe-Cit ESI-MS Intensity x 104 Fe:Cit 1:1 1:10 1:10 1:50 0 1 2 3 4 Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 López-Millán et al. 2000 Plant Physiol 124: 885 Larbi et al. 2003 J Plant Physiol 160: 1473 Fe:Cit ratios change with Fe-deficiency Sugar beet Peach +Fe 1:36 1:8 -Fe 1:2474 1:125 Fact Fe2Cit2 Fe3Cit3 ICP-MS Fe:Cit 1:1 1:10 1:10 1:50 0.0 0.2 0.4 0.6 0.8 1.0 1.2 54Fe nmol Fe:Cit Ratios drive the formation of Fe-Cit Complexes
159. ESI-TOFMS Fe2Cit2 Fe3Cit3 Fe-Cit ESI-MS Intensity x 104 Fe:Cit 1:1 1:10 1:10 1:50 0 1 2 3 4 Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 López-Millán et al. 2000 Plant Physiol 124: 885 Larbi et al. 2003 J Plant Physiol 160: 1473 Fe:Cit ratios change with Fe-deficiency Sugar beet Peach +Fe 1:36 1:8 -Fe 1:2474 1:125 Fact Fe2Cit2 Fe3Cit3 ICP-MS Fe:Cit 1:1 1:10 1:10 1:50 0.0 0.2 0.4 0.6 0.8 1.0 1.2 54Fe nmol Fe:Cit Ratios drive the formation of Fe-Cit Complexes
160. Tomato Xylem Sap Control Fe-Deficient Fe-Resupplied Sampling Time natFe 54Fe natFe 54Fe natFe 54Fe 11:00 X X X X 17:00 X X X X 23: 00 X X X X 05:00 X X X X 11:00 X X X X Iron-citrate xylem transport Materials and Methods
161. Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
162. [Fe] µM 0 75 150 225 300 Time after Fe-resupply (h) 0 6 12 18 24 -Fe Resupplied +Fe -Fe Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
163. [Fe] µM 0 75 150 225 300 Time after Fe-resupply (h) 0 6 12 18 24 -Fe Resupplied +Fe -Fe Cit:172 µM Fe:Cit 1:1.4 Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
164. [Fe] µM 0 75 150 225 300 Time after Fe-resupply (h) 0 6 12 18 24 -Fe Resupplied +Fe -Fe Cit:172 µM Fe:Cit 1:1.4 Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91
165. [Fe] µM 0 75 150 225 300 Time after Fe-resupply (h) 0 6 12 18 24 -Fe Resupplied +Fe -Fe Cit:172 µM Fe:Cit 1:1.4 Iron-citrate xylem transport Results and discussion Rellán-Álvarez et al. 2010 Plant Cell Physiol 51: 91 Only one Fe-Cit Complex: Fe3OCit3 54Fe natFe
166. Iron-citrate xylem transport
167. Iron-citrate xylem transport Concluding Remarks
168. -An HPLC-ESI-MS method was designed for the determination of natural Fe complexes in plant fluids Iron-citrate xylem transport Concluding Remarks
169. -Using this method, the first unequivocal identification of a natural Fe complex in xylem sap was achieved: Fe3OCit3 -An HPLC-ESI-MS method was designed for the determination of natural Fe complexes in plant fluids Iron-citrate xylem transport Concluding Remarks
170. -Using this method, the first unequivocal identification of a natural Fe complex in xylem sap was achieved: Fe3OCit3 -An HPLC-ESI-MS method was designed for the determination of natural Fe complexes in plant fluids -Another Fe-Cit complex, Fe2Cit2, was also found in Fe-Cit standards Iron-citrate xylem transport Concluding Remarks
171. -Using this method, the first unequivocal identification of a natural Fe complex in xylem sap was achieved: Fe3OCit3 -An HPLC-ESI-MS method was designed for the determination of natural Fe complexes in plant fluids -Another Fe-Cit complex, Fe2Cit2, was also found in Fe-Cit standards -The Fe:Cit ratio drives the formation of these two compounds Iron-citrate xylem transport Concluding Remarks
172. -Using this method, the first unequivocal identification of a natural Fe complex in xylem sap was achieved: Fe3OCit3 -An HPLC-ESI-MS method was designed for the determination of natural Fe complexes in plant fluids -Another Fe-Cit complex, Fe2Cit2, was also found in Fe-Cit standards -The Fe:Cit ratio drives the formation of these two compounds -Keep the original plant fluid pH constant Iron-citrate xylem transport Concluding Remarks
173. 3. To develop an HPLC-ESI-TOFMS method for the determination of organic acids in plant tissues Objective HPLC-MS determination of organic acids
174. HPLC-MS determination of organic acids Separation Identification HPLC ESI-TOFMS Quantification Isotope labelled Internal Standards Sara López Estación Experimental Aula Dei Materials and Methods
175. Internal Standards (IS) Matrices Organic Acids 13C-Malic 13C-Succinic Sugar Beet Leaves Tomato Xylem Sap Orange Juice Oxalic X X X X Cis-aconitic X X X X Oxo-glutaric X X X X Citric X X X X Malic X X X X Quinic X X X X Ascorbic X X X X Shikimic X X X X Succinic X X X X Fumaric X X X X HPLC-MS determination of organic acids Materials and Methods
176. HPLC Separation + ESI-TOFMS Detection Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Results and Discussion
177. HPLC Separation + ESI-TOFMS Detection Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Results and Discussion
178. Validation Data Repeatability - RSD % Interday Intraday LODs in pmol LOQs in pmol Linearity Ranges in µM R.T. Peak Area R.T. Peak Area 0.05-255 3.15-337 0.2-17 to 70-1000 1.63 4.37 0.06 0.14 Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Results and Discussion
179. Recoveries Recoveries (%) obtained for the different organic acids in, sugar beet le em sap and commercial orange juice. Results are means ± SE. (n=5). Organic acid Sugar beet leaf Tomato xylem Orange juice Oxalic 107.8 ± 3.6 46.5 ± 1.0 38.9 ± 1.0 Cis-aconitic 99.2 ± 1.7 102.8 ± 2.3 95.8 ± 2.0 2-oxoglutaric 66.2 ± 0.9 68.8 ± 1.8 43.9 ± 0.7 Citric 100.0 ± 1.8 109.6 ± 2.2 96.7 ± 1.5 Malic 104.0 ± 1.4 100.6 ± 1.0 101.0 ± 0.5 Quinic 98.0 ± 0.4 96.7 ± 2.4 98.2 ± 1.4 Ascorbic 21.5 ± 0.4 85.5 ± 1.9 76.4 ± 0.5 Shikimic 103.2 ± 0.8 97.4 ± 2.5 91.9 ± 1.9 Succinic 100.4 ± 0.6 96.8 ± 2.5 96.4 ± 2.5 Fumaric 93.2 ± 0.5 94.8 ± 3.1 93.5 ± 2.7 Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Results and Discussion
180. Recoveries Recoveries (%) obtained for the different organic acids in, sugar beet le em sap and commercial orange juice. Results are means ± SE. (n=5). Organic acid Sugar beet leaf Tomato xylem Orange juice Oxalic 107.8 ± 3.6 46.5 ± 1.0 38.9 ± 1.0 Cis-aconitic 99.2 ± 1.7 102.8 ± 2.3 95.8 ± 2.0 2-oxoglutaric 66.2 ± 0.9 68.8 ± 1.8 43.9 ± 0.7 Citric 100.0 ± 1.8 109.6 ± 2.2 96.7 ± 1.5 Malic 104.0 ± 1.4 100.6 ± 1.0 101.0 ± 0.5 Quinic 98.0 ± 0.4 96.7 ± 2.4 98.2 ± 1.4 Ascorbic 21.5 ± 0.4 85.5 ± 1.9 76.4 ± 0.5 Shikimic 103.2 ± 0.8 97.4 ± 2.5 91.9 ± 1.9 Succinic 100.4 ± 0.6 96.8 ± 2.5 96.4 ± 2.5 Fumaric 93.2 ± 0.5 94.8 ± 3.1 93.5 ± 2.7 Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Results and Discussion
181. Sugar beet leaves - HPLC-ESI-TOFMS Analysis Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Results and Discussion
182. Sugar beet leaves - HPLC-ESI-TOFMS Analysis Major Carboxylates Intensity time (min) Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Results and Discussion
183. Sugar beet leaves - HPLC-ESI-TOFMS Analysis Major Carboxylates Intensity time (min) Minor Carboxylates time (min) Intensity Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Results and Discussion
184. Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids
185. Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Concluding Remarks
186. Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Concluding Remarks -Up to nine different carboxylates can be directly determined without derivatization
187. -Quantification was done using C-labelled IS, avoiding matrix effects Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Concluding Remarks -Up to nine different carboxylates can be directly determined without derivatization
188. -Good LODs, in line or better than those already published -Quantification was done using C-labelled IS, avoiding matrix effects Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Concluding Remarks -Up to nine different carboxylates can be directly determined without derivatization
189. -Good LODs, in line or better than those already published -Quantification was done using C-labelled IS, avoiding matrix effects -The method was validated in three different plant tissues Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Concluding Remarks -Up to nine different carboxylates can be directly determined without derivatization
190. -Good LODs, in line or better than those already published -Quantification was done using C-labelled IS, avoiding matrix effects -The method was validated in three different plant tissues -It allows us to measure a group of metabolites very important in the plant Fe deficiency response Rellán-Álvarez et al. 2011 Submitted to JAFC HPLC-MS determination of organic acids Concluding Remarks -Up to nine different carboxylates can be directly determined without derivatization
191. Iron-deficient Beta vulgaris root tips metabolomics Metabolomics of xylem sap and leaves of Fe-deficient plants Objective 2: Study the metabolite profile of Fe-deficient plants Objectives
192. 4. To characterize the changes induced in the root metabolite profile in response to Fe deficiency and resupply, using gas chromatography coupled to mass spectrometry (GC-MS) Objective Iron-deficient Beta vulgaris root tips metabolomics 1 2 3 4 5 6 7 8 9 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2
193. Picture by C. Ortega-Villasante +Fe -Fe Sugar beet plants Iron-deficient Beta vulgaris root tips metabolomics Materials and Methods +Fe
194. +Fe López-Millán et al. 2001 Aust. J Plant Physiol 28:171 Iron-deficient Beta vulgaris root tips metabolomics Materials and Methods White Zone Yellow Zone -Fe Yellow Zone 24h Fe-resupply White Zone Yellow Zone 72h
195. +Fe López-Millán et al. 2001 Aust. J Plant Physiol 28:171 Iron-deficient Beta vulgaris root tips metabolomics Materials and Methods White Zone 10 mm Yellow Zone -Fe 10 mm Yellow Zone 24h 10 mm Fe-resupply White Zone 10 mm 10 mm Yellow Zone 72h
196. Metabolites GC-MS Metabolomics Oliver Fiehn Proteins 2D-Gels Ana Flor López Sofía Andaluz Jorge Rodríguez Flavins HPLC-PDA RFOs HPLC-ESI-TOFMS Materials and Methods Iron-deficient Beta vulgaris root tips metabolomics
197. -Fe ↑ +Fe ↓ Protein changes and Identification 29 Increased 13 Only in Fe-deficient 13 Decreased 6 only in Fe-sufficient Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120 Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion IEF pI 5-8 12% SDS-PAGE
198. Spot Homology Species Increased proteins in Fe-deficiency a fructose 1,6-bisphosphate aldolase A. Thaliana b triose-phosphate isomerase C. japonica c cytosolic 3-phosphoglycerate kinase T. Aestivum d enolase M. crystallinum e enolase L. sativa f malate dehydrogenase B. vulgaris g malate dehydrogenase B. vulgaris h malate dehydrogenase N. tabacum i F1 ATPase α subunit B. vulgaris j F1 ATPase ß subunit S. bicolor k fructokinase B. vulgaris l formate dehydrogenase Q. robur m At1g79210/YUP8H12R_1 A. thaliana n glycine rich protein A. thaliana New spots in Fe-deficiency o glyceraldehyde 3-phosphate DH M. quinquepeta p DMRL synthase S. oleracea -Fe ↑ Increased and New Spots in Fe deficiency Glycolisis and TCA Cycle Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
199. Spot Homology Species Increased proteins in Fe-deficiency a fructose 1,6-bisphosphate aldolase A. Thaliana b triose-phosphate isomerase C. japonica c cytosolic 3-phosphoglycerate kinase T. Aestivum d enolase M. crystallinum e enolase L. sativa f malate dehydrogenase B. vulgaris g malate dehydrogenase B. vulgaris h malate dehydrogenase N. tabacum i F1 ATPase α subunit B. vulgaris j F1 ATPase ß subunit S. bicolor k fructokinase B. vulgaris l formate dehydrogenase Q. robur m At1g79210/YUP8H12R_1 A. thaliana n glycine rich protein A. thaliana New spots in Fe-deficiency o glyceraldehyde 3-phosphate DH M. quinquepeta p DMRL synthase S. oleracea -Fe ↑ Increased and New Spots in Fe deficiency Glycolisis and TCA Cycle Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
200. DMRL RNA, protein and flavin analysis RNA Protein Flavins Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
201. -Fe/+Fe metabolite response ratios Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
202. -Fe/+Fe metabolite response ratios 328 metabolites were detected Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
203. -Fe/+Fe metabolite response ratios 328 metabolites were detected 77 were identified Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
204. -Fe/+Fe metabolite response ratios 328 metabolites were detected 77 were identified 66 changed (p <0.05) Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
205. metabolite + metabolite - galactinol 38.1 oxalic a. -16.6 citric a. 23.2 PGA -2 raffinose 18.2 myo-inositol 5.7 cellobiose 5.5 sucrose 5.4 aconitic a. 5.1 threonic a. 3.9 citrulline 3.9 glyceric a. 3.7 malic a. 3.7 -Fe/+Fe metabolite response ratios 328 metabolites were detected 77 were identified 66 changed (p <0.05) Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
206. metabolite + metabolite - galactinol 38.1 oxalic a. -16.6 citric a. 23.2 PGA -2 raffinose 18.2 myo-inositol 5.7 cellobiose 5.5 sucrose 5.4 aconitic a. 5.1 threonic a. 3.9 citrulline 3.9 glyceric a. 3.7 malic a. 3.7 -Fe/+Fe metabolite response ratios 328 metabolites were detected 77 were identified 66 changed (p <0.05) Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
207. metabolite + metabolite - galactinol 38.1 oxalic a. -16.6 citric a. 23.2 PGA -2 raffinose 18.2 myo-inositol 5.7 cellobiose 5.5 sucrose 5.4 aconitic a. 5.1 threonic a. 3.9 citrulline 3.9 glyceric a. 3.7 malic a. 3.7 -Fe/+Fe metabolite response ratios 328 metabolites were detected 77 were identified 66 changed (p <0.05) Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
208. Metabolite PLS Analysis 30 20 10 0 -10 -20 -30 30 20 10 0 -10 -20 -30 v1 (35.3 %) v2 (20.1 %) +Fe -Fe Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
209. Metabolite PLS Analysis 30 20 10 0 -10 -20 -30 30 20 10 0 -10 -20 -30 v1 (35.3 %) v2 (20.1 %) +Fe -Fe Metabolite Contribution (X-weights) to PLS vector 1 Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
210. Metabolite PLS Analysis 30 20 10 0 -10 -20 -30 30 20 10 0 -10 -20 -30 v1 (35.3 %) v2 (20.1 %) +Fe -Fe metabolite + citric a. 0.21505 galactinol 0.200043 aconitic a. 0.195182 myo-inositol 0.194711 glyceric a. 0.194598 malate 0.193229 raffinose 0.191252 citrulline 0.190349 sucrose 0.181604 threonic a. 0.1773 Metabolite Contribution (X-weights) to PLS vector 1 Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
211. Metabolite PLS Analysis 30 20 10 0 -10 -20 -30 30 20 10 0 -10 -20 -30 v1 (35.3 %) v2 (20.1 %) +Fe -Fe metabolite - oxalic a. -0.202023 PGA -0.12164 alanine -0.091395 glutamine -0.074066 fructose -0.071387 adenosine-5-P -0.070121 oxoproline -0.048596 mannitol -0.043351 inositol-P -0.041337 linoleic acid -0.037509 metabolite + citric a. 0.21505 galactinol 0.200043 aconitic a. 0.195182 myo-inositol 0.194711 glyceric a. 0.194598 malate 0.193229 raffinose 0.191252 citrulline 0.190349 sucrose 0.181604 threonic a. 0.1773 Metabolite Contribution (X-weights) to PLS vector 1 Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
212. Metabolite PLS Analysis 30 20 10 0 -10 -20 -30 30 20 10 0 -10 -20 -30 v1 (35.3 %) v2 (20.1 %) +Fe -Fe metabolite - oxalic a. -0.202023 PGA -0.12164 alanine -0.091395 glutamine -0.074066 fructose -0.071387 adenosine-5-P -0.070121 oxoproline -0.048596 mannitol -0.043351 inositol-P -0.041337 linoleic acid -0.037509 metabolite + citric a. 0.21505 galactinol 0.200043 aconitic a. 0.195182 myo-inositol 0.194711 glyceric a. 0.194598 malate 0.193229 raffinose 0.191252 citrulline 0.190349 sucrose 0.181604 threonic a. 0.1773 Metabolite Contribution (X-weights) to PLS vector 1 TCA Cycle Carbohydrates AAs - N Metabolism Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
213. RFOs HPLC-ESI-MS Analysis Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
214. sucrose + galactinol raffinose + myo-inositol raffinose + galactinol stachyose + myo-inositol stachyose + galactinol verbascose + myo-inositol RFOs HPLC-ESI-MS Analysis Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
215. sucrose + galactinol raffinose + myo-inositol raffinose + galactinol stachyose + myo-inositol stachyose + galactinol verbascose + myo-inositol RFOs HPLC-ESI-MS Analysis Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120 Intensity x104 time (min) 10.8 11.8 12.8 6 8 10 12 14 16 18 0 2 4 6 Sucrose +Fe Raffinose Galactinol Sucrose Raffinose Galactinol -Fe 10.9 11.812.8 8 10 12 14 16 6
216. sucrose + galactinol raffinose + myo-inositol raffinose + galactinol stachyose + myo-inositol stachyose + galactinol verbascose + myo-inositol RFOs HPLC-ESI-MS Analysis % Sucrose of Raf + Gal 13.9 0.6 Iron-deficient Beta vulgaris root tips metabolomics Results and Discussion Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120 Intensity x104 time (min) 10.8 11.8 12.8 6 8 10 12 14 16 18 0 2 4 6 Sucrose +Fe Raffinose Galactinol Sucrose Raffinose Galactinol -Fe 10.9 11.812.8 8 10 12 14 16 6
217. Pentose Phosphate Carbohydrate Metabolism AAs and N Metabolism Glycolysis TCA Cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 24 23 25 Others a b c d e k o m s v i j n q v f g h Metabolite Protei n Iron-deficient Beta vulgaris root tips metabolomics Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120
218. Pentose Phosphate Carbohydrate Metabolism AAs and N Metabolism Glycolysis TCA Cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 24 23 25 Others a b c d e k o m s v i j n q v f g h Metabolite Protei n Iron-deficient Beta vulgaris root tips metabolomics Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120 Concluding Remarks
219. Pentose Phosphate Carbohydrate Metabolism AAs and N Metabolism Glycolysis TCA Cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 24 23 25 Others a b c d e k o m s v i j n q v f g h -Increases in glycolysis and TCA cycle were found at the metabolite and protein level confirming previous results Metabolite Protei n Iron-deficient Beta vulgaris root tips metabolomics Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120 Concluding Remarks
220. Pentose Phosphate Carbohydrate Metabolism AAs and N Metabolism Glycolysis TCA Cycle 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 24 23 25 Others a b c d e k o m s v i j n q v f g h -Increases in glycolysis and TCA cycle were found at the metabolite and protein level confirming previous results Metabolite Protei n -Two major findings, not previously described were found for: -Proteins: DMRL -Metabolites: RFOs Iron-deficient Beta vulgaris root tips metabolomics Rellán-Álvarez et al. 2010 BMC Plant Biol 10:120 Concluding Remarks
221. 5. To characterize the changes induced in the metabolite profile of xylem sap and leaves of several plant species in response to Fe deficiency and resupply, using GC-MS Objective Metabolomics of xylem sap and leaves of Fe-deficient plants +Fe -Fe
222. Metabolites GC-MS Metabolomics Oliver Fiehn Metabolomics of xylem sap and leaves of Fe-deficient plants Materials and Methods
223. +Fe -Fe Sugar beet Metabolomics of xylem sap and leaves of Fe-deficient plants Materials and Methods
224. +Fe -Fe Tomato Metabolomics of xylem sap and leaves of Fe-deficient plants Materials and Methods
225. +Fe -Fe Lupinus Metabolomics of xylem sap and leaves of Fe-deficient plants Materials and Methods
226. +Fe -Fe Peach Metabolomics of xylem sap and leaves of Fe-deficient plants Materials and Methods
227. +Fe -Fe Peach Hamdi El-Jendoubi Monona Abadía Metabolomics of xylem sap and leaves of Fe-deficient plants Materials and Methods
228. Tomato Sugar beet Lupin Peach Xylem Sap Leaves Xylem Sap Leaves Xylem Sap Leaves Xylem Sap Leaves X X X X X X 11:00 X 17:00 X 23: 00 X 05:00 X 11:00 X Fe Resupply Metabolomics of xylem sap and leaves of Fe-deficient plants Materials and Methods +Fe -Fe
229. -Fe/+Fe metabolite response ratios Tomato Lupine Peach Detected 233 233 251 Identified 77 83 77 Changed 41 19 6 Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Xylem Sap + Tomato Lupine Peach oxoglutaric 91 aconitic. 2.6 NA 2.4 suberyl glycine 7.1 maleic 2.5 tryptophan 6.4 succinic 2.4 malic 6.2 citric 5.7 succinic 4.7 aconitic 2.8 asparagine 2.5
230. -Fe/+Fe metabolite response ratios Tomato Lupine Peach Detected 233 233 251 Identified 77 83 77 Changed 41 19 6 Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Xylem Sap + Tomato Lupine Peach oxoglutaric 91 aconitic. 2.6 NA 2.4 suberyl glycine 7.1 maleic 2.5 tryptophan 6.4 succinic 2.4 malic 6.2 citric 5.7 succinic 4.7 aconitic 2.8 asparagine 2.5
231. -Fe/+Fe metabolite response ratios Tomato Lupine Peach Detected 233 233 251 Identified 77 83 77 Changed 41 19 6 Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Xylem Sap + AA decreases - Tomato Lupine Peach oxoglutaric 91 aconitic. 2.6 NA 2.4 suberyl glycine 7.1 maleic 2.5 tryptophan 6.4 succinic 2.4 malic 6.2 citric 5.7 succinic 4.7 aconitic 2.8 asparagine 2.5
232. 11:00 17:00 23:00 05:00 11:00 30 20 10 0 -10 -20 -30 30 20 10 0 -10 -20 v1 v2 Fe ressupplied Tomato Xylem Sap PLS Analysis Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation
233. 11:00 17:00 23:00 05:00 11:00 30 20 10 0 -10 -20 -30 30 20 10 0 -10 -20 v1 v2 Fe ressupplied Tomato Xylem Sap PLS Analysis +Fe -Fe R-6 R-12 R-18 R-24 Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation
234. -Fe/+Fe metabolite response ratios Tomato S. beet Peach Detected 238 128 375 Identified 65 82 92 Changed 21 34 4 Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Leaves + Tomato S. beet Peach threitol 6.5 glutamine 32.8 sub-glycine 5.9 tyrosine 4.2 serine 19.6 asparagine 4.3 ribonic 4.1 g-tocopherol 17.3 isoleucine 2 ethanolamine 3.8 citric 13.7 D-hexosamine 3.8 isoleucine 13.7 succinic 2.8 succinic 12.2 galactinol 2.5 valine 8.9 maleic 2.1 oxoproline 8
235. -Fe/+Fe metabolite response ratios Tomato S. beet Peach Detected 238 128 375 Identified 65 82 92 Changed 21 34 4 Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Leaves + Tomato S. beet Peach threitol 6.5 glutamine 32.8 sub-glycine 5.9 tyrosine 4.2 serine 19.6 asparagine 4.3 ribonic 4.1 g-tocopherol 17.3 isoleucine 2 ethanolamine 3.8 citric 13.7 D-hexosamine 3.8 isoleucine 13.7 succinic 2.8 succinic 12.2 galactinol 2.5 valine 8.9 maleic 2.1 oxoproline 8
236. -Fe/+Fe metabolite response ratios Tomato S. beet Peach Detected 238 128 375 Identified 65 82 92 Changed 21 34 4 Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Leaves + Tomato S. beet Peach threitol 6.5 glutamine 32.8 sub-glycine 5.9 tyrosine 4.2 serine 19.6 asparagine 4.3 ribonic 4.1 g-tocopherol 17.3 isoleucine 2 ethanolamine 3.8 citric 13.7 D-hexosamine 3.8 isoleucine 13.7 succinic 2.8 succinic 12.2 galactinol 2.5 valine 8.9 maleic 2.1 oxoproline 8
237. Beta vulgaris. +Fe -Fe 30 20 10 0 -10 -20 -30 30 20 10 0 -10 -20 v1 (32.8%) v2 (8.6%) metabolite + succinic a. 0.175703 lactobionic a. 0.172614 xylitol 0.172469 oxoproline 0.166707 serine 0.165424 alpha tocopherol 0.165124 citric a. 0.164217 threonine 0.16214 N-acetyl-D-hexosamine 0.158702 N-acetyl-D-mannosamine 0.156364 metabolite - phytol -0.167686 oxalic a. -0.146914 PGA -0.138575 fructose-6-P -0.125472 beta-sitosterol -0.099845 glucose-6-P -0.0984 mannitol -0.090316 putrescine -0.061401 aspartate -0.057571 ribonic a. -0.0432 Metabolite Contribution (X-weights) to PLS vector 1 Beta vulgaris PLS Analysis Metabolomics of xylem sap and leaves of Fe-deficient plants Results and Discussion Rellán-Álvarez et al. In Preparation
238. Beta vulgaris. +Fe -Fe 30 20 10 0 -10 -20 -30 30 20 10 0 -10 -20 v1 (32.8%) v2 (8.6%) metabolite + succinic a. 0.175703 lactobionic a. 0.172614 xylitol 0.172469 oxoproline 0.166707 serine 0.165424 alpha tocopherol 0.165124 citric a. 0.164217 threonine 0.16214 N-acetyl-D-hexosamine 0.158702 N-acetyl-D-mannosamine 0.156364 metabolite - phytol -0.167686 oxalic a. -0.146914 PGA -0.138575 fructose-6-P -0.125472 beta-sitosterol -0.099845 glucose-6-P -0.0984 mannitol -0.090316 putrescine -0.061401 aspartate -0.057571 ribonic a. -0.0432 Metabolite Contribution (X-weights) to PLS vector 1 TCA Cycle AAs - N Metab Glycolisis P-P Carbohydrates Beta vulgaris PLS Analysis Metabolomics of xylem sap and leaves of Fe-deficient plants Results and Discussion Rellán-Álvarez et al. In Preparation
239. PEPC TCA cycle Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Other Anaplerotic Reactions Occurring in Leaves?
240. PEPC TCA cycle Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Other Anaplerotic Reactions Occurring in Leaves? Asparagine Aspartate Tyrosine Glutamine Glutamate
241. Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Other Anaplerotic Reactions Occurring in Leaves?
242. Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Other Anaplerotic Reactions Occurring in Leaves?
243. Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Other Anaplerotic Reactions Occurring in Leaves?
244. Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Other Anaplerotic Reactions Occurring in Leaves?
245. Results and Discussion Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Other Anaplerotic Reactions Occurring in Leaves?
246. Glycolysis TCA Cycle Carbohydrates AAs and N Metabolism Pentose-P TCA Carbohydrates AAs and N Metabolism Glycolysis Pentose-P Xylem Sap Leaves Concluding Remarks Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation
247. Glycolysis TCA Cycle Carbohydrates AAs and N Metabolism Pentose-P TCA Carbohydrates AAs and N Metabolism Glycolysis Pentose-P Xylem Sap Leaves Concluding Remarks Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation Concluding Remarks
248. Glycolysis TCA Cycle Carbohydrates AAs and N Metabolism Pentose-P TCA Carbohydrates AAs and N Metabolism Glycolysis Pentose-P Xylem Sap Leaves Concluding Remarks Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation -Increases in TCA cycle metabolites and AAs Concluding Remarks
249. Glycolysis TCA Cycle Carbohydrates AAs and N Metabolism Pentose-P TCA Carbohydrates AAs and N Metabolism Glycolysis Pentose-P Xylem Sap Leaves Concluding Remarks Metabolomics of xylem sap and leaves of Fe-deficient plants Rellán-Álvarez et al. In Preparation -Increases in TCA cycle metabolites and AAs -Anaplerotic reactions using AAs as C source might be occurring in sugar beet leaves Concluding Remarks
250. Glycolysis TCA Cycle Carbohydrates AAs and N Metabolism Pentose-P TCA Carbohydrates AAs and N Metabolism Glycolysis Pentose-P Xylem Sap Leaves Concluding Remarks Metabolomics of xylem sap and leaves of Fe-deficient plants -Increases in TCA cycle Metabolites Rellán-Álvarez et al. In Preparation -Increases in TCA cycle metabolites and AAs -Anaplerotic reactions using AAs as C source might be occurring in sugar beet leaves Concluding Remarks
251. Glycolysis TCA Cycle Carbohydrates AAs and N Metabolism Pentose-P TCA Carbohydrates AAs and N Metabolism Glycolysis Pentose-P Xylem Sap Leaves Concluding Remarks Metabolomics of xylem sap and leaves of Fe-deficient plants -Increases in TCA cycle Metabolites Rellán-Álvarez et al. In Preparation -Decreases in AAs and carbohydrates -Increases in TCA cycle metabolites and AAs -Anaplerotic reactions using AAs as C source might be occurring in sugar beet leaves Concluding Remarks
252. Glycolysis TCA Cycle Carbohydrates AAs and N Metabolism Pentose-P TCA Carbohydrates AAs and N Metabolism Glycolysis Pentose-P Xylem Sap Leaves Concluding Remarks Metabolomics of xylem sap and leaves of Fe-deficient plants -Increases in TCA cycle Metabolites Rellán-Álvarez et al. In Preparation -Decreases in AAs and carbohydrates -Increases in TCA cycle metabolites and AAs -Very rapid changes in tomato xylem sap metabolites after Fe Resupply -Anaplerotic reactions using AAs as C source might be occurring in sugar beet leaves Concluding Remarks
253. Summary
254. Summary Previously on Fe deficiency This PhD
255. Summary Previously on Fe deficiency This PhD
256. Summary Previously on Fe deficiency This PhD
257. Flavins Glycolysis TCA cycle PEPC DMRL and flavins Confirmed by Metabolomics/ Proteomics RFOs Summary Previously on Fe deficiency This PhD
258. Flavins Glycolysis TCA cycle PEPC DMRL and flavins Confirmed by Metabolomics/ Proteomics RFOs Summary Previously on Fe deficiency This PhD
259. Flavins Glycolysis TCA cycle PEPC DMRL and flavins Confirmed by Metabolomics/ Proteomics RFOs Carboxylates C transport Fe transport Fe is transported as Fe3OCit3 in tomato xylem sap C transport by Metabol NA might chelate Fe depending on pH N transport is reduced Summary Previously on Fe deficiency This PhD
260. Flavins Glycolysis TCA cycle PEPC DMRL and flavins Confirmed by Metabolomics/ Proteomics RFOs Carboxylates C transport Fe transport Fe is transported as Fe3OCit3 in tomato xylem sap C transport by Metabol NA might chelate Fe depending on pH N transport is reduced C Fixation Carboxylates Free Aminoacids Large increases in Free Aminoacids Carboxylates Other Anaplerotic reactions might be ocurring in order to provide more C Summary Previously on Fe deficiency This PhD
261. Conclusions
262. Conclusions 1. Complexes of nicotianamine (NA) with Fe(II) and Fe(III) can be determined using electrospray time-of-flight mass spectrometry (ESI- TOFMS).
263. Conclusions 1. Complexes of nicotianamine (NA) with Fe(II) and Fe(III) can be determined using electrospray time-of-flight mass spectrometry (ESI- TOFMS). 2. Changes in pH and the concentrations of citrate and metals can have significant effects in NA speciation in plant fluids.
264. Conclusions 1. Complexes of nicotianamine (NA) with Fe(II) and Fe(III) can be determined using electrospray time-of-flight mass spectrometry (ESI- TOFMS). 2. Changes in pH and the concentrations of citrate and metals can have significant effects in NA speciation in plant fluids. 3. Nicotianamine is a candidate for chelating Fe at the pH usually found in the phloem sap, whereas NA is not likely to be involved in xylem Fe transport, conversely to what occurs with other metals such as Cu and Ni.
265. Conclusions 1. Complexes of nicotianamine (NA) with Fe(II) and Fe(III) can be determined using electrospray time-of-flight mass spectrometry (ESI- TOFMS). 2. Changes in pH and the concentrations of citrate and metals can have significant effects in NA speciation in plant fluids. 3. Nicotianamine is a candidate for chelating Fe at the pH usually found in the phloem sap, whereas NA is not likely to be involved in xylem Fe transport, conversely to what occurs with other metals such as Cu and Ni. 4. An oxo-bridged tri-Fe(III), tri-citrate complex (Fe3OCit3) has been found in the xylem sap of Fe-deficient Solanum lycopersicum Mill. plants resupplied with Fe, by using an integrated mass spectrometry approach.
266. Conclusions
267. Conclusions 5. High Fe-to-citrate ratios favour the formation of Fe-citrate polymeric forms and low ratios (such as those found in Fe deficiency) favour dimeric or monomeric species.
268. Conclusions 5. High Fe-to-citrate ratios favour the formation of Fe-citrate polymeric forms and low ratios (such as those found in Fe deficiency) favour dimeric or monomeric species. 6. An HPLC-ESI-TOFMS method has been developed to determine organic acids in different plant tissues (xylem sap, leaves and fruit juice) with high selectivity, sensitivity and reproducibility.
269. Conclusions 5. High Fe-to-citrate ratios favour the formation of Fe-citrate polymeric forms and low ratios (such as those found in Fe deficiency) favour dimeric or monomeric species. 6. An HPLC-ESI-TOFMS method has been developed to determine organic acids in different plant tissues (xylem sap, leaves and fruit juice) with high selectivity, sensitivity and reproducibility. 7. Major increases in the raffinose family of oligosaccharide (RFOs) are elicited by Fe deficiency and resupply in root tips of Beta vulgaris plants.
270. Conclusions 5. High Fe-to-citrate ratios favour the formation of Fe-citrate polymeric forms and low ratios (such as those found in Fe deficiency) favour dimeric or monomeric species. 6. An HPLC-ESI-TOFMS method has been developed to determine organic acids in different plant tissues (xylem sap, leaves and fruit juice) with high selectivity, sensitivity and reproducibility. 8. Increases in proteins and metabolites related to carbohydrate, TCA cycle and flavin synthesis have been confirmed to occur in roots of Beta vulgaris plants grown under Fe deficiency. 7. Major increases in the raffinose family of oligosaccharide (RFOs) are elicited by Fe deficiency and resupply in root tips of Beta vulgaris plants.
271. Conclusions
272. Conclusions 9. The major changes in the metabolite profile of xylem sap from Solanum lycopersicum and Lupinus albus in response to Fe deficiency are an increase in TCA cycle metabolites and a decrease in aminoacids and carbohydrates.
273. Conclusions 9. The major changes in the metabolite profile of xylem sap from Solanum lycopersicum and Lupinus albus in response to Fe deficiency are an increase in TCA cycle metabolites and a decrease in aminoacids and carbohydrates. 10. The xylem sap metabolite profile of Fe-deficient Solanum lycopersicum plants becomes similar to that of Fe-sufficient controls one day after Fe resupply.
274. Conclusions 9. The major changes in the metabolite profile of xylem sap from Solanum lycopersicum and Lupinus albus in response to Fe deficiency are an increase in TCA cycle metabolites and a decrease in aminoacids and carbohydrates. 10. The xylem sap metabolite profile of Fe-deficient Solanum lycopersicum plants becomes similar to that of Fe-sufficient controls one day after Fe resupply. 11. The main changes in the metabolite profile of leaves from Solanum lycopersicum and Beta vulgaris in response to Fe deficiency are increases in TCA cycle metabolites, aminoacids and carbohydrates.
275. Conclusions 9. The major changes in the metabolite profile of xylem sap from Solanum lycopersicum and Lupinus albus in response to Fe deficiency are an increase in TCA cycle metabolites and a decrease in aminoacids and carbohydrates. 10. The xylem sap metabolite profile of Fe-deficient Solanum lycopersicum plants becomes similar to that of Fe-sufficient controls one day after Fe resupply. 11. The main changes in the metabolite profile of leaves from Solanum lycopersicum and Beta vulgaris in response to Fe deficiency are increases in TCA cycle metabolites, aminoacids and carbohydrates. 12. High correlations between aminoacids and TCA cycle metabolites levels suggest that some anaplerotic reactions using aminoacids as a C source may occur in Beta vulgaris leaves in response to Fe deficiency.
276. Questions
277. Fe status Fe in solution (µM) pH Fe (µM) Cit (µM) Fe:Cit Fe3Cit3 (%) Fe2Cit2 (%) +Fe 45 5.8 19.9 11.6 1:0.6 bld bld -Fe 0 + HCO3 - 5.8 5.4 165 1:31 bld bld Fe- resupplied 0 + HCO3 - + 45 5.5 121 172.2 1:1.4 71 bld Problems and Future Research Results and Discussion -Rellán-Álvarez et al. (2010) Plant Cell Physiol 51: 91
278. Fe status Fe in solution (µM) pH Fe (µM) Cit (µM) Fe:Cit Fe3Cit3 (%) Fe2Cit2 (%) +Fe 45 5.8 19.9 11.6 1:0.6 bld bld -Fe 0 + HCO3 - 5.8 5.4 165 1:31 bld bld Fe- resupplied 0 + HCO3 - + 45 5.5 121 172.2 1:1.4 71 bld Only 25% of total Fe eluted from column: Fe3Cit3 represents 16% of total Fe Problems and Future Research Results and Discussion -Rellán-Álvarez et al. (2010) Plant Cell Physiol 51: 91
279. Fe:Cit ratios drive the formation of Fe-Cit species…. also in humans
280. Clinically observed range: Oligomeric Iron 1:0 1:2 1:3 1:5 1:10 1:100 1:1,000 Iron-Citrate Ratio Polymeric Iron Dimeric Iron Monomeric Iron Iron(III) Citrate speciation at pH 7.4 1:10,000 1:1 citrate speciation as a function of iron-to-citrate ratio ution at pH 7.4. The proposed speciation is derived wn chemistry of iron citrate complexes and the on citrate solutions of various iron-to-citrate ratios to chelator access and ultrafiltration described he indicates the expected iron-to-citrate ratio range in where the NTBI concentration ranges from 1 to 10 present at 100 lM em (2008) 13:57–74 Evans, JBIC, 2008 Fe:Cit ratios drive the formation of Fe-Cit species…. also in humans
281. Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
282. Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Beta vulgaris xylem sap Abadía J. et al. ISINIP Budapest 2010
283. Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Beta vulgaris xylem sap high concentrations of citrate (mM) Abadía J. et al. ISINIP Budapest 2010
284. Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Larbi et al. (2009) J Plant Physiol Beta vulgaris xylem sap high concentrations of citrate (mM) Abadía J. et al. ISINIP Budapest 2010
285. Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Larbi et al. (2009) J Plant Physiol Beta vulgaris xylem sap high concentrations of citrate (mM) Abadía J. et al. ISINIP Budapest 2010
286. Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Larbi et al. (2009) J Plant Physiol Fe increase after resupply Beta vulgaris xylem sap high concentrations of citrate (mM) Abadía J. et al. ISINIP Budapest 2010
287. Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Larbi et al. (2009) J Plant Physiol Fe increase after resupply Beta vulgaris xylem sap high concentrations of citrate (mM) Abadía J. et al. ISINIP Budapest 2010
288. Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Larbi et al. (2009) J Plant Physiol decrease in the Cit:Fe ratio after resupply Fe increase after resupply Beta vulgaris xylem sap high concentrations of citrate (mM) Abadía J. et al. ISINIP Budapest 2010
289. Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Larbi et al. (2009) J Plant Physiol decrease in the Cit:Fe ratio after resupply Fe increase after resupply Beta vulgaris xylem sap high concentrations of citrate (mM) Fe-citrate complexes Abadía J. et al. ISINIP Budapest 2010
290. Fe long distance transport Abadía J. et al. ISINIP Budapest 2010
291. Fe long distance transport Reduction by leaf mesophyll Abadía J. et al. ISINIP Budapest 2010
292. Fe long distance transport protoplasts Beta vulgaris Reduction by leaf mesophyll Abadía J. et al. ISINIP Budapest 2010
293. Fe long distance transport protoplasts Beta vulgaris Reduction by leaf mesophyll Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
294. Fe long distance transport protoplasts Beta vulgaris Reduction by leaf mesophyll - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
295. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
296. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll BPDS Fe(II)-BPDS - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
297. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll BPDS Fe(II)-BPDS Vázquez, Rellán-Álvarez, unpublished Fe(III)2 -Cit2 Fe(III)3 -Cit3 Fe(III)-EDTA 0 5 10 15 20 25 30 0 50 100 150 200 250 nmol µm-2 s-1 (x10-12 ) µM - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
298. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll BPDS Fe(II)-BPDS Vázquez, Rellán-Álvarez, unpublished Fe(III)2 -Cit2 Fe(III)3 -Cit3 Fe(III)-EDTA 0 5 10 15 20 25 30 0 50 100 150 200 250 nmol µm-2 s-1 (x10-12 ) µM good reduction with Fe-citrate - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
299. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll BPDS Fe(II)-BPDS Vázquez, Rellán-Álvarez, unpublished Fe(III)2 -Cit2 Fe(III)3 -Cit3 Fe(III)-EDTA 0 5 10 15 20 25 30 0 50 100 150 200 250 nmol µm-2 s-1 (x10-12 ) µM good reduction with Fe-citrate - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
300. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll BPDS Fe(II)-BPDS Vázquez, Rellán-Álvarez, unpublished Fe(III)2 -Cit2 Fe(III)3 -Cit3 Fe(III)-EDTA 0 5 10 15 20 25 30 0 50 100 150 200 250 nmol µm-2 s-1 (x10-12 ) µM good reduction with Fe-citrate - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
301. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll BPDS Fe(II)-BPDS Vázquez, Rellán-Álvarez, unpublished Fe(III)2 -Cit2 Fe(III)3 -Cit3 Fe(III)-EDTA 0 5 10 15 20 25 30 0 50 100 150 200 250 nmol µm-2 s-1 (x10-12 ) µM good reduction with Fe-citrate - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA low Km values Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
302. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll BPDS Fe(II)-BPDS Vázquez, Rellán-Álvarez, unpublished Fe(III)2 -Cit2 Fe(III)3 -Cit3 Fe(III)-EDTA 0 5 10 15 20 25 30 0 50 100 150 200 250 nmol µm-2 s-1 (x10-12 ) µM good reduction with Fe-citrate - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA low Km values Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
303. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll BPDS Fe(II)-BPDS Vázquez, Rellán-Álvarez, unpublished Fe(III)2 -Cit2 Fe(III)3 -Cit3 Fe(III)-EDTA 0 5 10 15 20 25 30 0 50 100 150 200 250 nmol µm-2 s-1 (x10-12 ) µM good reduction with Fe-citrate - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA low Km values FCR decrease in -Fe on a surface basis Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
304. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll BPDS Fe(II)-BPDS Vázquez, Rellán-Álvarez, unpublished Fe(III)2 -Cit2 Fe(III)3 -Cit3 Fe(III)-EDTA 0 5 10 15 20 25 30 0 50 100 150 200 250 nmol µm-2 s-1 (x10-12 ) µM good reduction with Fe-citrate - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA low Km values FCR decrease in -Fe on a surface basis Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
305. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll BPDS Fe(II)-BPDS Vázquez, Rellán-Álvarez, unpublished Fe(III)2 -Cit2 Fe(III)3 -Cit3 Fe(III)-EDTA 0 5 10 15 20 25 30 0 50 100 150 200 250 nmol µm-2 s-1 (x10-12 ) µM good reduction with Fe-citrate - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA low Km values FCR decrease in -Fe on a surface basis FCR increase in -Fe on a Chl basis Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
306. Fe long distance transport protoplasts Beta vulgaris FRO Fe(II) Reduction by leaf mesophyll BPDS Fe(II)-BPDS Vázquez, Rellán-Álvarez, unpublished Fe(III)2 -Cit2 Fe(III)3 -Cit3 Fe(III)-EDTA 0 5 10 15 20 25 30 0 50 100 150 200 250 nmol µm-2 s-1 (x10-12 ) µM good reduction with Fe-citrate - Fe(III)3 -Cit3 -2 Fe(III)2 -Cit2 -2,-1 Fe(III)-EDTA low Km values FCR decrease in -Fe on a surface basis FCR increase in -Fe on a Chl basis not too specific? Fe(II)-NA Fe(III)-Cit Fe(III)-NA Fe species Abadía J. et al. ISINIP Budapest 2010
307. Fe long distance transport Abadía J. et al. ISINIP Budapest 2010
308. Evidence for Fe immobilization in leaves Fe long distance transport Abadía J. et al. ISINIP Budapest 2010
309. Evidence for Fe immobilization in leaves Fe long distance transport Beta vulgaris Abadía J. et al. ISINIP Budapest 2010
310. Vázquez, unpublished Evidence for Fe immobilization in leaves Fe long distance transport Beta vulgaris Abadía J. et al. ISINIP Budapest 2010
311. Vázquez, unpublished Evidence for Fe immobilization in leaves Fe long distance transport Beta vulgaris Abadía J. et al. ISINIP Budapest 2010
312. Vázquez, unpublished Evidence for Fe immobilization in leaves Fe long distance transport Beta vulgaris accumulation in vessels Abadía J. et al. ISINIP Budapest 2010
313. Curie et al. Ann Bot (2009) Mutants Chlorosis young leaves, disappears with age (phloem fed) Overexpression NAAT in tobaco mimics Chln, Fe accumulated in veins (Takahashi 03) Protoplasts Chln =FCR, +Fe uptake, = with exogenous NA (Pich and Scholz, 1991) In root Chln tips, NA in vacuole, cytosol (Pich et al. 1997) In controls NA in vacuole in excess Fe (Pich et al. 1991) In dgl and brz NA in vacuole, 10x NA In Chln Fe in chloroplast stroma and phloem. P depositions? In root cells cytosol and vacuole (Becker et al., 1995) YSLs (OPT) 4 subfamilies. 18 YSL in rice, 8 Arabidopsis (Curie et al. 2001), 3 Thlaspi ZmYSL1 and HvYS1 (and OsYSL15) upregulated in root with -Fe (Curie et al 2001, Roberts et al 2004, Koike at el. 2004, Murata et al 2006) Domain swapping in HvYS1: specificity between loops 6 and 7 (Harada et al. 2007) AtYSL1-8 and TcYSL3,5,7 OsYSL except2 not regulated by Fe (DiDonato et al. 2004, LeJean et al. 2005, Schaaf et al. 2005, Waters 2006) OsYSL2 upregulated in leaves -Fe (Koike et al. 2004) AtYSL2 downregulated by -Zn and +Cu (Schaaf et al. 2005) YSLs Abadía J. et al. ISINIP Budapest 2010
314. Curie et al. Ann Bot (2009) HvYS1 (and OsYSL15) in PM of root epidermal cells by anti- and transient GFP (Murata et al. 2006) OsYSL2,13,14 in aereal parts (Koike et al. 2004, Murata et al. 2006, Nishizawa et al. 2006). OsYSL2 in phloem companion cells (??) ZmYSL1 also in aereal parts, perhaps transporting Fe-NA or Fe(III)-PS (Curie et al. 2001) Localization ZmYSL1 and HvYS1 (and OsYSL15) in root epidermis (Curie et al 2001, Roberts et al 2004, Koike at el. 2004, Murata et al 2006) Other YSLs in vascular tissues, pollen grains and seeds. AtYSL1, AtYSL3 in senescent leaves (5/8 AtYSLs microarrays). AtYSL2 in lateral PM of pericycle (xylem parenchima) cells, suggesting loading/unloading of root vessels (DiDonato et al. 2004) TcYSL3,5 in PM (Gendre et al. 2007) AtYSL4,6 in tonoplast, suggesting influx from vacuole or efflux to neutral vacuoles (Jaquinod et al. 2007) (OsYSL5,6?) AtYSL1 in pollen of flower buds and vascular tissue of the anther (LeJean et al. 2005). AtYSL3 in pollen of open flowers (Waters et al. 2006). ysl1ysl3 plants little pollen bur normal Fe as ysl1 (Waters et al. 2006). ysl5ysl7 few pollen grains, female gametophyte also affected (Curie et al., 2009) AtYSL2,5,6,7 expression enhanced in stamen (https://www.geneinvestigator.ethz.ch). AtYSL1 in seed chalazal endosperm, silique vascular tissue, peduncle of the silique (Curie et al. 2001) OsYSL2 strong promoter activity developing seeds (Koike et al. 2004) AtYSL1 in xylem parenchima (Waters et al. 2006?) TcYSL3, AtYSL3,5 phloem/xylem (Curie et al. 2009) Abadía J. et al. ISINIP Budapest 2010
315. Curie et al. Ann Bot (2009) ZmYSL1 transports (in oocytes) Fe(III)-PS 5-10 µM Km, H+ co-transport, membrane potential. Also Fe(II), Ni(II), Zn(II), Cu(II), Mn(II), Cd(II) (Schaaf et al 2004). Also trasports Fe(II)-NA and Ni(II)-NA, does not transport DMA alone HvYS1 transports (in oocytes) only Fe(III)-PS (Murata et al. 2006) ZmYS1 complements fet3fet4 with Fe(III)-DMA, Fe(III)-NA and Fe(II)-NA, not Fe(III)-Cit (Curie et al. 2001, Schaaf et al. 2004) HvYS1 and OsYSL15 complement fet3fet4 with Fe(III)-DMA (Murata et al. 2006, Nishizawa et al. 2006) TcYSL3 complements fet3fet4 with Fe(II)-NA and Ni(II)-NA (Gendre et al. 2007) AtYSL1 does not complement fet3fet4 with any Fe source (Le Jean 2005). The same for other AtYSLs (Curie et al. 2009) AtYSL2 complements fet3fet4 with Fe(II)-NA (DiDonato et al. 2004), results not confirmed in Schaaf et al. (2005) AtYSL2 and ZmYSL1 coexpression in fet3fet4: no efflux of Ni (Schaaf et al. 2005) AtYSLs does not complement ztr1zrt2 or ctr1 (Curie et al. 2009) Expression in yeast Expression in oocites OsYSL2 transports (in oocytes) Fe(II)-NA and Mn(II)-NA (Koike et al. 2004) AtYSL2 does not transport Fe(II)-NA, Fe(III)-NA and Ni(II)-NA (Schaaf et al. 2005)
316. Flavin functions cently characterized in Arabidopsis (Robinson et al., 1999). The white parts of the iron-deficient roots, which do not contain flavin sulfates and do not have in- creased Fe(III)-reducing activities, had large pools of organic anions, similar to those of the yellow distal root parts. However, the respiration rates of these root zones were similar to the controls and increases Figure 5. Proposed electron transport pathways in iron-deficient sugar beet roots. Reduced pyridine nucleotides would reduce flavins (Flv), which are oxidized and in large amounts in the cytosol (up to 700 ␮M). Flavins would finally provide electrons to the FC-R enzyme of the PM. This enzyme would be able to reduce not only Fe(III)- chelates, but also oxygen when Fe(III)-chelates are absent. Plant Physiol. Vol. 124, 2000 Redox Bridge and are thus more difficult to detect (Figure S1 and (Hagerha ¨ll et al. 1995)). The long expression times used in our study (compare Figure S1, 16 h and Figure S2, 4 days), necessary to express the plant polypeptides in E. coli, most probably contributed to an even more pronounced degradation of both folded and misfolded protein, resulting in multiple bands on the gel. Similar difficulties were encountered when determining the transmembrane topology of a potassium channel from A. thaliana with the PhoA fusion protein technique (Uozumi et al. 1998). Thus, as a control experiment to rule out false negatives caused by poor expression levels rather than production of inactive fusion protein, we also expressed the fusion-proteins from the same plasmid constructs in E. coli Rosetta-Gami (DE3), which lacks a chromosomally encoded PhoA, but contains muta- tions in thioredoxin reductase that enables disulfide bond formation in the cytoplasm. In this case the oxi- dizing environment caused by the trxB/gor mutations will result in production of a properly folded, active PhoA enzyme also on the cytoplasmic side of the membrane. Fusion proteins FP2, FP6, FP7, FP9, FP10 and FP12 exhibit a PhoA activity of 5 units or less when expressed in E. coli CC118 and were subse- quently assigned as inactive/located on the inside. The Based on these data, a new topology model of FRO2 could be constructed (Fig. 2). The model contains a total of 8 transmembrane helices and a large water soluble domain, which is located on the cytoplasmic side of the membrane. The high activity of FP3 and FP4 clearly support an outside location of helix IV, whereas both FP6 and FP7, which flank helix VI, predicted to be Fig. 2 Topology model of FRO2 based on the results from the PhoA fusion protein analysis. Arrows indicate the locations of the different fusion points. The position of the C-terminus could not be determined experimentally, but the most probable location is inside 123 López-Millán et al. 2000 Plant Phys 124: 885 Schagerlöf et al. 2006 Plant Mol Biol 62: 115 Excretion induced by bHLH TFs
317. Riboflavin Synthesis Pathway
318. Riboflavin Synthesis Pathway
319. Figure 1. Scanning electron micrographs showing the root distal segments of iron-deficient plants 5 to 10 mm from the apex (A), 0 to 5 mm from the apex (B), and iron-sufficient sugar beet plants 5 to 10 mm from the apex (C), and 0 to 5 mm from the apex (D). Plant Physiol. Vol. 12 Fe-sufficient Root Tips López-Millán et al. 2001 Aust. J Plant Physiol 28:171
320. Fe-deficient Root Tips YZ WZ López-Millán et al. 2001 Aust. J Plant Physiol 28:171
321. López-Millán et al. 2001 Aust. J Plant Physiol 28:171 Fe-resupplied (24 h) Root Tips YZ WZ
322. Fe-resupplied (72 h) Root Tips YZ New WZ
323. RFOs can protect plants from oxidative damage 50 µM MV treatment KD-GolS2, and KD-GolS3 mutants were isolated and characterized. There was no significant difference in the expression of GolS genes between the wild type and the KO-GolS1, KD-GolS2, and KD-GolS3 mutants under growth or stressful conditions (Supplemental Fig. S2). However, the reduced expression of GolS1, -2, or -3 had no effect on the levels of galactinol and raffinose and the total activities of GolS under stressful conditions (Supplemental Fig. S3). Accordingly, it is likely that the double or triple knockout GolS plants might be necessary to produce the mutants with reduced levels of galactinol and/or raffinose. We assessed the stress tolerance of GolS1- or GolS2- Ox-GolS2-8, and O wild-type plants un (Fig. 7C). These incre of AsA and GSH in t Ox-GolS2-29 plants u (Fig. 8). At 6 h after MV conditions (100 mE developed visible lea did not (Fig. 9A). Al wild-type, Ox-GolS1- plants decreased sig ment under control Effects of the stress on PSII activity in wild-type, Ox-GolS1-11, Ox-GolS2- 8, and Ox-GolS2-29 plants. The PSII activity (Fv /Fm ) in the rosette leaves of Arabidopsis plants was determined at 25°C after adaptation to the dark for 30 min. C, CO2 fixation in wild- type, Ox-GolS1-11, Ox-GolS2-8, and Ox-GolS2-29 plants. D, Lipid peroxidation expressed as MDA con- tent in wild-type, Ox-GolS1-11, Ox- GolS2-8, and Ox-GolS2-29 plants. Mean 6 SD values from three exper- iments are shown. Asterisks indicate that the values are significantly dif- ferent from those in the wild-type plants (P , 0.05). under control growth conditions on phenotype, PSII activity, CO2 fixa- tion, and lipid hydroperoxide levels in the leaves of wild-type, Ox- GolS1, and Ox-GolS2 plants. Four- week-old wild-type, Ox-GolS1-11, Ox-GolS2-8, and Ox-GolS2-29 plants grown in soil under control growth conditions were sprayed with MV (50 mM) in 0.1% Tween 20 (5 mL) and then transferred to control growth conditions (100 mE m22 s21) for 6 h. A, Phenotypes of plants exposed to oxidative stress. B, Effects of the stress on PSII activity in wild-type, Ox-GolS1-11, Ox-GolS2- 8, and Ox-GolS2-29 plants. The PSII activity (Fv /Fm ) in the rosette leaves of Arabidopsis plants was determined at 25°C after adaptation to the dark for 30 min. C, CO2 fixation in wild- type, Ox-GolS1-11, Ox-GolS2-8, and Ox-GolS2-29 plants. D, Lipid peroxidation expressed as MDA con- tent in wild-type, Ox-GolS1-11, Ox- GolS2-8, and Ox-GolS2-29 plants. Mean 6 SD values from three exper- iments are shown. Asterisks indicate that the values are significantly dif- ferent from those in the wild-type plants (P , 0.05). Figure 9. Effects of MV treatment under control growth conditions on phenotype, PSII activity, CO2 fixa- tion, and lipid hydroperoxide levels in the leaves of wild-type, Ox- GolS1, and Ox-GolS2 plants. Four- week-old wild-type, Ox-GolS1-11, Ox-GolS2-8, and Ox-GolS2-29 plants grown in soil under control growth conditions were sprayed with MV (50 mM) in 0.1% Tween 20 (5 mL) and then transferred to control growth conditions (100 mE m22 s21) for 6 h. A, Phenotypes of plants exposed to oxidative stress. B, Effects of the stress on PSII activity in wild-type, Ox-GolS1-11, Ox-GolS2- 8, and Ox-GolS2-29 plants. The PSII activity (Fv /Fm ) in the rosette leaves of Arabidopsis plants was determined at 25°C after adaptation to the dark for 30 min. C, CO2 fixation in wild- type, Ox-GolS1-11, Ox-GolS2-8, and Ox-GolS2-29 plants. D, Lipid peroxidation expressed as MDA con- tent in wild-type, Ox-GolS1-11, Ox- GolS2-8, and Ox-GolS2-29 plants. Mean 6 SD values from three exper- iments are shown. Asterisks indicate that the values are significantly dif- ferent from those in the wild-type plants (P , 0.05). Lines Overexpressing Galactinol Synthase
324. Metabolic changes López-Millán et al. 2000 Plant Phys 124: 885
325. root Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Root Uptake
326. root Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Fe(III)-X AtFRO H+ AHA2 Fe(II) AtIRT1 Fe(II) Fe(III)-X Phe, OA, Flv PDR9 Phenolics PEZ Reduction Root Uptake
327. root Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Fe(III)-X AtFRO H+ AHA2 Fe(II) AtIRT1 Fe(II) Fe(III)-X Phe, OA, Flv PDR9 Phenolics PEZ Fe(III)-Phx AtYSL3 Fe(III)-Phx Fe(III)-PS OsYSL15 Fe(III)-PS PS TOM Fe(III)-PS HvYS1 Fe(III)-PS Reduction Chelation Root Uptake
328. root Fe-NA ? Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Fe-NA Fe(III)-X AtFRO H+ AHA2 Fe(II) AtIRT1 Fe(II) Fe(III)-X Phe, OA, Flv PDR9 Phenolics PEZ Fe(III)-Phx AtYSL3 Fe(III)-Phx Fe(III)-PS OsYSL15 Fe(III)-PS PS TOM Fe(III)-PS HvYS1 Fe(III)-PS Reduction Chelation Root Uptake
329. xylem root Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Fe-NA Transport Xylem Loading Phenolics PEZ
330. xylem root Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Fe-NA Fe-NA AtYSL3 Fe-NA Citrate OsFRDL1 Citrate AtFRD3 AtIREG1 Fe Fe Transport Xylem Loading Phenolics PEZ
331. xylem root Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Fe-NA Fe-Cit ? Fe-NA AtYSL3 Fe-NA Citrate OsFRDL1 Citrate AtFRD3 AtIREG1 Fe Fe Transport Xylem Loading Phenolics PEZ
332. Fe-Cit ? leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Introduction Xylem Unloading
333. Fe-Cit ? xylem vessel xylem parenchima cell apoplastic space mesophyll cell leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez Introduction Xylem Unloading
334. Fe-Cit ? xylem vessel xylem parenchima cell apoplastic space mesophyll cell leaf Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez Introduction Bottleneck Xylem Unloading
335. Fe-Cit ? xylem vessel xylem parenchima cell apoplastic space mesophyll cell ZIP8 leaf hν Plant Fe uptake and long distance transport Abadía et al, 2011 Plant Physiol Biochem, In Press Curie et al, 2009 Ann Bot 103: 1 Palmer & Guerinot 2009 Nat Chem Biol 5: 333 Picture by Saúl Vazquez Introduction Fe(III)-X AtFRO Fe(II) IRT Fe(II) YSL2 Bottleneck Xylem Unloading