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. Selective 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
ions/s
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

51. 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
others take up tetra- &
penta-peptides by H+ symport

52. 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
others take up tetra- &
penta-peptides by H+ symport
Also have ABC transporters
that import peptides

53. 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
others take up tetra- &
penta-peptides by H+ symport
Also have ABC transporters
that import peptides
N is vital!
NO3- & NH4+ are main forms

54. Nutrient uptake
Metals are taken up by ZIP proteins & by ABC transporters
same protein may import Fe, Zn & Mn!

55. Nutrient uptake
Much is coupled to
pH gradient

56. Nutrient transport in roots
Move from soil to endodermis in apoplast

57. Nutrient transport in roots
Move from soil to endodermis in apoplast
Move from endodermis to xylem in symplast

58. Nutrient transport in roots
Move from endodermis to xylem in symplast
Transported into xylem by H+ antiporters

59. Nutrient transport in roots
Move from endodermis to xylem in symplast
Transported into xylem by H+ antiporters, channels

60. Nutrient transport in roots
Transported into xylem by H+ antiporters, channels,pumps

61. Nutrient transport in roots
Transported into xylem by H+ antiporters, channels,pumps
Lowers xylem water
potential -> root pressure

62. Water Transport
Passes water & nutrients to xylem
Ys of xylem makes root pressure
Causes guttation: pumping water into shoot

63. Transport to shoot
Nutrients move up
plant in xylem sap

64. Nutrient transport in leaves
Xylem sap moves through apoplast
Leaf cells take up what
they want