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(i)1: Screen to identify new key players linking iron homeostasis and transport

(i)1: Screen to identify new key players linking iron homeostasis and transport. Fe homeostasis gene families from peach.

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(i)1: Screen to identify new key players linking iron homeostasis and transport

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  1. (i)1: Screen to identify new key players linking iron homeostasis and transport • Fe homeostasis gene families from peach Jiménez S, López-Millán AF, Duy D, Philippar K, Abadía J, Abadía A, Gogorcena Y. Phylogenetic analysis of the iron homeostasis gene families in peach (Prunus persica). In preparation 8 families ZIP, OPT, YSL, NRAMP, FRO, FER, NAS, IREG

  2. (i)4: Identification of factors limiting iron homeostasis and transport • Fe tracing using stable isotopes Orera et al. (2010) Metallomics 2, 646-657

  3. (i)4: Identification of factors limiting iron homeostasis and transport Fe tracing using stable isotopes Orera et al. (2010) Metallomics 2, 646-657

  4. (i)4: Identification of factors limiting iron homeostasis and transport • Determination of carboxylates in plant materials Rellán-Álvarez et al. (2011) J Agric Food Chem, 59, 6864-6870

  5. Determination of carboxylates in plant materials (i)4: Identification of factors limiting iron homeostasis and transport Rellán-Álvarez et al. (2011) J Agric Food Chem, 59, 6864-6870

  6. Determination of carboxylates in plant materials (i)4: Identification of factors limiting iron homeostasis and transport Schuler M, Rellán-Álvarez R, Fink-Straube C, Abadía J, Bauer P (2012) New functions of nicotianamine in the phloem-based transport of iron to sink organs, in pollen development and in pollen tube growth. Plant Cell, accepted.

  7. (i)4: Identification of factors limiting iron homeostasis and transport • Metabolic profiles in xylem sap and leaf extracts Search for consistent changes Rellán-Álvarez et al. (2011) Frontiers Plant Sci 2, 66

  8. Metabolomics analysis "untargeted" and "high throughput" (GC-MS) Metabolites "Fishing" unknown changes Metabolomics, Fiehn’s lab, UC Davis

  9. Metabolomics analysis "untargeted" and "high throughput" (GC-MS) Metabolites Wealth of data: bioinformatics Metabolomics, Fiehn’s lab, UC Davis GC-MS

  10. >200.000 metabolites (10.000 primaries) Metabolomics analysis "untargeted" and "high throughput" (GC-MS) Pichersky and Lewinsohn, Ann Rev Plant Biol 2011 Metabolites One must be aware of the limitations Metabolomics, Fiehn’s lab, UC Davis GC-MS 1.013 metabolites Fiehn's database <10%

  11. Changes in metabolite profiles in xylem sap and leaf extracts (i)4: Identification of factors limiting iron homeostasis and transport Principal components: control and Fe-deficient clusters Rellán-Álvarez et al. (2011) Frontiers Plant Sci 2, 66

  12. Changes in metabolite profiles in xylem sap and leaf extracts (i)4: Identification of factors limiting iron homeostasis and transport Xylem tomato/lupine/peach 253/233/251 resolved 77/83/77 identified 11/3/1 increase 32/16/5 decrease Leaf tomato/sugar beet/peach 254/113/239 resolved 65/82/92 identified 12/29/4 increase 8/4/3 decrease <3% Rellán-Álvarez et al. (2011) Frontiers Plant Sci 2, 66

  13. Changes in metabolite profiles in xylem sap and leaf extracts (i)4: Identification of factors limiting iron homeostasis and transport Metabolite relationships change Rellán-Álvarez et al. (2011) Frontiers Plant Sci 2, 66

  14. Changes in metabolite profiles in xylem sap and leaf extracts (i)4: Identification of factors limiting iron homeostasis and transport Clustering of +Fe, -Fe and Fe resupplied at different times Rellán-Álvarez et al. (2011) Frontiers Plant Sci 2, 66

  15. Changes in metabolite profiles in xylem sap and leaf extracts (i)4: Identification of factors limiting iron homeostasis and transport New information on N recycling in Fe deficiency Rellán-Álvarez et al. (2011) Frontiers Plant Sci 2, 66

  16. Changes in metabolite profiles in xylem sap and leaf extracts (i)4: Identification of factors limiting iron homeostasis and transport Pathway activation Pathway deactivation Consistent changes in three species Rellán-Álvarez et al. (2011) Frontiers Plant Sci 2, 66

  17. HPLC-MS techniques Proteins Other techniques 2-DE Techniques "Fishing" unknown changes Protein analysis: proteomics IEF-SDS/PAGE Blue native (PAGE-SDS/PAGE) DIGE iTraq ICAT Label free shotgun proteomics Image analysis

  18. 2-DE: strips IEF and gels 7 cm, colloidal Coomasie MSn (ion trap) nHPLC MALDI-TOF UPM/INIA J. Kehr Tripsin digestion 2-DE techniques Proteins Spot identification IEF-SDS/PAGE EEAD-CSIC, Zaragoza Tripsin digestion 2-DE Spot cutter

  19. 2-DE techniques Proteins Experimental design for differential proteomics 2-DE IEF-SDS/PAGE IEF-SDS/PAGE Consistent spots: in 80% gels in at least one tratment Significant differences: t-test p<0,10; changes >2 fold EEAD-CSIC, Zaragoza 2-DE • 5 batches (biological replicates) with all treatments • 1 gel (7 cm BioRad) per biological replicate (5 biol. rep. x 3 treatments, 15 geles in total)

  20. (i)4: Identification of factors limiting iron homeostasis and transport • Changes in root protein profiles with Fe deficiency 0 µM Fe + CaCO3, pH 7,7 Control 0 µM Fe, pH 5,5 Medicago truncatula Proteins Hydroponics Rodríguez-Celma et al. (2011) J Proteome Res roots 2-DE

  21. Control 323 spots 13 appear 8 appear Changes in protein profiles in roots (i)4: Identification of factors limiting iron homeostasis and transport 0 µM Fe, pH 5,5 341 spots 345 spots 0 µM Fe + CaCO3, pH 7.7 31 change 21 identified (68%) 69 change 51 identified (74%) 19 increase 12 increase Medicago truncatula 26 decrease 8 decrease IEF pI 11 dissapear 3 disappear roots SDS-PAGE kDa Rodríguez-Celma et al. (2011) J Proteome Res

  22. appear Changes in protein profiles in roots (i)4: Identification of factors limiting iron homeostasis and transport 0 µM Fe + CaCO3, pH 7,7 0 µM Fe, pH 5,5 Pathway inactivation Pathway activation increase Medicago truncatula decrease disappear roots Rodríguez-Celma et al. (2011) J Proteome Res

  23. Changes in protein profiles in shoots (i)4: Identification of factors limiting iron homeostasis and transport Shoot sampling Medicago truncatula Hydroponics shoots Rodríguez-Celma et al., in preparation

  24. 5 appear 0 appear 268 spots Changes in protein profiles in shoots (i)4: Identification of factors limiting iron homeostasis and transport 0 µM Fe, pH 5,5 270 spots 274 spots Control 0 µM Fe + CaCO3, pH 7.7 18 change 16 identified (89%) 43 change 39 identified (91%) 24 increase 7 increase Medicago truncatula 9 decrease 11 decrease IEF shoots pI 2 disappear 3 disappear SDS-PAGE kDa Rodríguez-Celma et al., in preparation

  25. Changes in protein profiles in shoots (i)4: Identification of factors limiting iron homeostasis and transport 0 µM Fe, pH 5.5 0 µM Fe + CaCO3, pH 7.7 Medicago truncatula 6 3 shoots 4 6 0 4 1 1 2 1 0 3 3 4 1 4 5 1 Rodríguez-Celma et al., in preparation

  26. (i)4: Identification of factors limiting iron homeostasis and transport • Flavins in roots of Fe-deficient plants Rodríguez-Celma et al. (2011) Plant Cell Physiol

  27. (i)4: Identification of factors limiting iron homeostasis and transport Flavins in roots Susín et al. (1993) JBC Rodríguez-Celma et al. (2011) Plant Cell Physiol

  28. (i)4: Identification of factors limiting iron homeostasis and transport Flavins in roots Rodríguez-Celma et al. (2011) Plant Cell Physiol

  29. (i)4: Identification of factors limiting iron homeostasis and transport Flavins in roots Rodríguez-Celma et al. (2011) Plant Cell Physiol

  30. (i)4: Identification of factors limiting iron homeostasis and transport • Protein profiles in phloem sap Lupinus texensis IEF Phloem SDS-PAGE Lattanzio G, Andaluz S, Abadía A, Abadía J, López-Millán AF. Protein profile of Lupinus texensis phloem sap exudates with a special enphasis on Fe and Zn containing proteins. Submitted for publication 249 spots, 54 unambiguous id (+44)

  31. (i)4: Identification of factors limiting iron homeostasis and transport metal affinity chromatography Protein profile: phloem sap Lattanzio G et al. Submitted for publication Fe Zn

  32. June 1-3 • -3 dates, +Fe, -Fe (i)4: Identification of factors limiting iron homeostasis and transport • Changes in xylem sap protein profiles with Fe deficiency in peach July 14-16 Ago 30-31 Ago 31 May 15 June 14 July 24 El-Jendoubi (2012) PhD Thesis • Resupply

  33. (i)4: Identification of factors limiting iron homeostasis and transport Changes in xylem sap protein profiles

  34. (i)4: Identification of factors limiting iron homeostasis and transport Changes in xylem sap protein profiles IEF SDS-PAGE Gutierrez-Carbonell et al. In preparation

  35. appear • Metabolite and protein profiles in flowers of the atfer1-3-4 mutant (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players increase decrease disappear search

  36. appear (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players Metabolite and protein profiles in flowers increase decrease disappear

  37. (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players Metabolite and protein profiles in flowers 63 spots 62 identified

  38. appear (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players Metabolite and protein profiles in flowers increase decrease disappear

  39. (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players Metabolite and protein profiles in flowers 252 metabolites consistently present (in 80% of the samples -in 5 samples out of 6- in at least one class)

  40. 25 unknowns (15 increase, 10 decrease) (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players Metabolite and protein profiles in flowers for unknowns, go to http://eros.fiehnlab.ucdavis.edu:8080 10 known metabolites (5 increase, 5 decrease)

  41. (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players Metabolite and protein profiles in flowers

  42. (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players Metabolite profiles in leaves

  43. 160 spots 89 identified (56%) • Protein profiles of Pisum sativum inner and outer chloroplast envelopes (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players Inner chloroplast envelope Outer chloroplast envelope 181 spots 54 identified (30%)

  44. Complex mixture Tripsin digestion nano rp-LC-MS/MS Proteins "Shotgun proteomics" Tripsin digestion Protein fractionation HPLC-MS Protein identification and quantification SDS-PAGE, IEF, rp-HPLC, FFE Peptide fractionation IEF, SCX, rp-HPLC, FFE, m/z in MS

  45. Phenolics in Arabidopsis thaliana roots (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players A root phenolic compound increasing with Fe deficiency in WT Root extracts m/z 193.051 WT UV, 320 nm Col0 -Fe Scopoletin PDR9 +Fe C10H9O4 PDR9 -Fe Col0 +Fe Root extracts PDR9 UV, 320 nm RT 14.7

  46. Identification of phenolics accumulated and exported in Arabidopsis, peach and tomato (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players Root extracts UV, 320 nm - Fe, 7 days + Fe, 7 days Root phenolic compounds increasing with Fe deficiency PDR3 ortholog in P. persica: ppa000248m PDR9 orthologs in P. persica ppa000249m and ppa023381m (ppa017338m, ppa000229m with homologies)

  47. Spots with -statistically significant changes in relative abundance -present in 80% of the gels (4/5) -increases >2-fold or decreases >50% • Changes in root protein profiles in Prunus x amygdalopersica (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players (see Rodríguez-Celma et al., 2011, Journal of Proteomics Research 10, 2590-2601) Jiménez et al. 2009 Plant Soil 315, 93-106 • 1 gel (7 cm BioRad) per biological replicate (2 batches x 3 reps x 3 treatments, 18 gels in total)

  48. Control 90 µM Fe (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players 339±1 spots 0 µM Fe 336±5 spots +180 µM Fe, 24 h 338±5 spots Changes in root protein profiles in Prunus 1 appear 1 appear 10 increase 15 increase IEF pI 14 decrease 6 decrease 0 dissapear 0 dissapear SDS-PAGE kDa Rodríguez-Celma et al., in preparation

  49. (iii)2: Changes at the transcriptional, protein and metabolite levels of major homeostasis players 0 µM Fe 0 µM Fe +180 µM Fe, 24 h 42 changes 39 ids (92%) Changes in root protein profiles in Prunus Control Control +180 µM Fe, 24 h increase 2 2 1 8 5 5 1 1 4 2 11 decrease Rodríguez-Celma et al., in preparation

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