Today ’ s environmental factor folks Make Media Complete -N -S? -P? -K? -Fe? -micronutrients?

1. Today’s environmental factor folks • Make Media • Complete • -N • -S? • -P? • -K? • -Fe? • -micronutrients? • Varying [HCO3-]? • Varying [salt], [osmoticum] or pH? • Prepare flasks • decide how many • set them up? • Decide on other conditions • Count heterocysts • Measure cell number = OD • Measure chlorophyll? • Measure protein? • Save aliquot as baseline for later measures.

2. Today’s gene folks • Identify targets • Rationale for why they might affect internal environment • Alter nutrient uptake (including HCO3-) • Alter pH • Alter metabolism • Calvin cycle • Light reactions • Lipid metabolism • Carbohydrate metabolism • Nutrient assimilation • Figure out how to clone them • Must be from a bacterium or very short • Must be from an organism that we can obtain or short enough to have made • Design primers to obtain them by PCR • “external” primers to obtain gene + flanking sequence • “internal” primers to obtain gene from AUG to stop • Additional bases for cloning as described by Teresa

3. Nutrient uptake • Most nutrients are dissolved in water • Enter root through apoplast until hit endodermis

4. Crossing membranes • A) Diffusion through bilayer • B) Diffusion through protein pore • C) Facilitated diffusion • D) Active transport • E) Bulk transport • 1) Exocytosis • 2) Endocytosis Selective Active

5. Channels • Old model: S4 slides up/down • Paddle model: S4 rotates

6. Selective Transport • 1) Channels • 2) Facilitated Diffusion (carriers) • Carrier binds molecule

7. Selective Transport • Facilitated Diffusion (carriers) • Carrier binds molecule • carries it through membrane • & releases it inside

8. Selective Transport • Facilitated Diffusion (carriers) • Carrier binds molecule • carries it through membrane • & releases it inside • driving force = ∆ [ ]

9. Selective Transport • Facilitated Diffusion (carriers) • Carrier binds molecule • carries it through membrane • & releases it inside • driving force = ∆ [ ] • Important for sugar • transport

10. Selective Transport • Facilitated Diffusion (carriers) • Characteristics • 1) saturable • 2) specific • 3) passive: transports • down ∆ []

11. Selective Transport 1) Channels 2) Facilitated Diffusion (carriers) Passive transport should equalize [ ] Nothing in a plant cell is at equilibrium!

12. Selective Transport Passive transport should equalize [ ] Nothing in a plant cell is at equilibrium! Solution: use energy to transport specific ions against their ∆ [ ]

13. Active Transport • Integral membrane proteins • use energy to transport specific ions against their ∆ [ ] • allow cells to concentrate some chemicals, exclude others

14. Active Transport Characteristics 1) saturable 105-106 ions/s 102-104 molecules/s

15. Active Transport Characteristics 1) saturable 2) specific

16. Active Transport Characteristics 1) saturable 2) specific 3) active: transport up ∆ [ ] (or ∆ Em)

17. 4 classes of Active transport ATPase proteins • 1) P-type ATPases (P = “phosphorylation”) • Na/K pump • Ca pump in ER & PM • H+ pump in PM • pumps H+ out of cell

18. 4 classes of Active transport ATPase proteins • 1) P-type ATPases (P = “phosphorylation”) • 2) V-type ATPases (V = “vacuole”) • H+ pump in vacuoles

19. 4 classes of Active transport ATPase proteins • 1) P-type ATPases (P = “phosphorylation”) • 2) V-type ATPases (V=“vacuole”) • 3) F-type ATPases (F = “factor”) a.k.a. ATP synthases • mitochondrial ATP synthase • chloroplast ATP synthase

20. 4 classes of Active transport ATPase proteins • 1) P-type ATPases (P = “phosphorylation”) • 2) V-type ATPases (V = “vacuole”) • 3) F-type ATPases (F = “factor”) • 4) ABC ATPases (ABC = “ATP Binding Cassette”) • multidrug resistance proteins

21. 4 classes of Active transport ATPase proteins • 1) P-type ATPases (P = “phosphorylation”) • 2) V-type ATPases (V = “vacuole”) • 3) F-type ATPases (F = “factor”) • 4) ABC ATPases (ABC = “ATP Binding Cassette”) • multidrug resistance proteins • pump hydrophobic drugs out of cells • very broad specificity

22. Secondary active transport Uses ∆ [ ] created by active transport to pump something else across a membrane against its ∆ [ ]

23. Secondary active transport Uses ∆ [ ] created by active transport to pump something else across a membrane against its ∆ [ ] Symport:both substances pumped same way

24. Secondary active transport Uses ∆ [ ] created by active transport to pump something else across a membrane against its ∆ [ ] Symport:both substances pumped same way Antiport: substances pumped opposite ways

25. Secondary active transport Uses ∆ [ ] created by active transport to pump something else across a membrane against its ∆ [ ] Symport:both substances pumped same way Antiport: substances pumped opposite ways

26. Nutrient uptake Gases enter/exit by diffusion down their ∆ [ ]

27. Nutrient uptake Gases enter/exit by diffusion down their ∆ [ ] Ions vary dramatically!

28. Nutrient uptake Ions vary dramatically! H+ is actively pumped out of cell by P-type H+ -ATPase

29. Nutrient uptake Ions vary dramatically H+ is actively pumped out of cell by P-type H+ -ATPase and into vacuole by V-type ATPase & PPase

30. Nutrient uptake • H+ is actively pumped out of cell by P-type H+ -ATPase • and into vacuole by V-type ATPase & PPase • Main way plants make membrane potential (∆Em)!

31. Nutrient uptake • H+ is actively pumped out of cell by P-type H+ -ATPase • and into vacuole by V-type ATPase & PPase • Main way plants make membrane potential (∆Em)! • Used for many kinds of transport!

32. Nutrient uptake Many ions are imported by multiple transporters with varying affinities

33. Nutrient uptake • Many ions are imported by multiple transporters with varying affinities • K+ diffuses through channels down ∆Em: low affinity

34. Nutrient uptake • Many ions are imported by multiple transporters with varying affinities • K+ diffuses through channels down ∆Em: low affinity • Also taken up by H+ symporters : high affinity

35. Nutrient uptake • Many ions are imported by multiple transporters with varying affinities • K+ diffuses through channels down ∆Em: low affinity • Also taken up by H+ symporters : high affinity • Low affinity is cheaper • but less effective

36. Nutrient uptake K+ diffuses through channels down ∆Em: low affinity Also taken up by H+ symporters : high affinity Low affinity is cheaper but less effective some channels also transport Na+

37. Nutrient uptake K+ diffuses through channels down ∆Em: low affinity Also taken up by H+ symporters : high affinity Low affinity is cheaper but less effective some channels also transport Na+ why Na+ slows K+ uptake?

38. Nutrient uptake K+ diffuses through channels down ∆Em: low affinity Also taken up by H+ symporters : high affinity Low affinity is cheaper but less effective some channels also transport Na+ why Na+ slows K+ uptake? Na+ is also expelled by H+ antiport

39. Nutrient uptake Ca2+ is expelled by P-type ATPases in PM

40. Nutrient uptake • Ca2+ is expelled by P-type ATPases in PM • pumped into vacuole & ER by H+ antiport & P-type

41. Nutrient uptake • Ca2+ is expelled by P-type ATPases in PM • pumped into vacuole & ER by H+ antiport & P-type • enters cytosol via gated channels

42. Nutrient uptake • PO43-, SO42-, Cl- & NO3- • enter by H+ symport

43. Nutrient uptake • PO43-, SO42-, Cl- & NO3- enter by H+ symport • also have anion transporters of ABC type

44. Nutrient uptake • PO43-, SO42-, Cl- & NO3- enter by H+ symport • also have anion transporters of ABC type • and anion channels

45. Nutrient uptake • PO43-, SO42-, Cl- & NO3- enter by H+ symport • also have anion transporters of ABC type • and anion channels • Plants take up N many ways

46. Nutrient uptake • Plants take up N many ways: NO3- & NH4+ are main forms

47. Nutrient uptake • Plants take up N many other ways • NO3- also by channels • NH3 by diffusion • NH4+ by carriers

48. Nutrient uptake • Plants take up N many other ways • NO3- by channels • NH3 by diffusion • NH4+ by carriers • NH4+ by channels

49. Nutrient uptake • Plants take up N many other ways • 3 families of H+ symporters take up amino acids

50. Nutrient uptake • Plants take up N many other ways • 3 families of H+ symporters take up amino acids • Also have many peptide transporters • some take up di- & tri- • peptides by H+ symport