1. Common themes
Nutrient deprivation
N and biodiesel
N and H2 production
S and biodiesel
Biotin and biodiesel
2. Hydrogen production
N deprivation
Knock down hydrogenases
Knock up hydrogen synthases, H+ pumps
Gene knockouts
FFA recycling
H2 metabolism
N metabolism
Cell walls
Abiotic stresses
Salinity
osmotic
Temperature

2. Common themes
Growth in different media
Differ in [N] or other nutrients
Growth in common medium, then change
Harvest, then resuspend in new media
Add something to medium
Salt
Biotin/avidin
Inducer
Inhibitor

3. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production

4. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate

5. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth

6. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?

7. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?
Growth?

8. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?
Growth?
Photosynthesis?

9. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?
Growth?
Photosynthesis?
Respiration?

10. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?
Growth?
Photosynthesis?
Respiration?
Protein synthesis?

11. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?
Growth?
Photosynthesis?
Respiration?
Protein synthesis?
DNA synthesis?

12. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?
Growth?
Photosynthesis?
Respiration?
Protein synthesis?
DNA synthesis?
Lipid synthesis?

13. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?
Growth?
Photosynthesis?
Respiration?
Protein synthesis?
DNA synthesis?
Lipid synthesis?
Hydrogen synthesis?

14. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?
Growth?
Photosynthesis?
Respiration?
Protein synthesis?
DNA synthesis?
Lipid synthesis?
Hydrogen synthesis?
How to test?

15. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?
Growth?
Photosynthesis?
Respiration?
Protein synthesis?
DNA synthesis?
Lipid synthesis?
Hydrogen synthesis?
How to test?
Alter external conditions

16. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?
Growth?
Photosynthesis?
Respiration?
Protein synthesis?
DNA synthesis?
Lipid synthesis?
Hydrogen synthesis?
How to test?
Alter external conditions
Alter internal conditions by bioengineering

17. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Will change allocation of photosynthate
Will invest their income in energy stores cf growth
Predictions?
Growth?
Photosynthesis?
Respiration?
Protein synthesis?
DNA synthesis?
Lipid synthesis?
Hydrogen synthesis?
How to test?
Alter external conditions
Alter internal conditions by bioengineering
Then measure growth, physiology and biofuels

18. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Alter external conditions
Nutrients
N, S, P, cofactors (including biotin)

19. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Alter external conditions
Nutrients
N, S, P, cofactors (including biotin)
Salinity/ water potential
NaCl vs KCl vs mannitol/sorbitol/PEG

20. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Alter external conditions
Nutrients
N, S, P, cofactors (including biotin)
Salinity/ water potential
NaCl vs KCl vs mannitol/sorbitol/PEG
pCO2: pCO2 in air and [HCO3-] in medium

21. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Alter external conditions
Nutrients
N, S, P, cofactors (including biotin)
Salinity/ water potential
NaCl vs KCl vs mannitol/sorbitol/PEG
pCO2: pCO2 in air and [HCO3-] in medium
Temperature

22. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Alter external conditions
Nutrients
N, S, P, cofactors (including biotin)
Salinity/ water potential
NaCl vs KCl vs mannitol/sorbitol/PEG
pCO2: pCO2 in air and [HCO3-] in medium
Temperature
Light: intensity & duration

23. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Alter external conditions
Nutrients
N, S, P, cofactors (including biotin)
Salinity/ water potential
NaCl vs KCl vs mannitol/sorbitol/PEG
pCO2: pCO2 in air and [HCO3-] in medium
Temperature
Light: intensity & duration

24. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Alter external conditions
Nutrients
N, S, P, cofactors (including biotin)
Salinity/ water potential
NaCl vs KCl vs mannitol/sorbitol/PEG
pCO2: pCO2 in air and [HCO3-] in medium
Temperature
Light: intensity & duration
Turn down light reactions with atrazine

25. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Alter external conditions
Nutrients
N, S, P, cofactors (including biotin)
Salinity/ water potential
NaCl vs KCl vs mannitol/sorbitol/PEG
pCO2: pCO2 in air and [HCO3-] in medium
Temperature
Light: intensity & duration
Turn down light reactions with atrazine
Bioengineer internal changes
Nutrients (including HCO3-) by altering transporters

26. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Alter external conditions
Nutrients
N, S, P, cofactors (including biotin)
Salinity/ water potential
NaCl vs KCl vs mannitol/sorbitol/PEG
pCO2: pCO2 in air and [HCO3-] in medium
Temperature
Light: intensity & duration
Turn down light reactions with atrazine
Bioengineer internal changes
Nutrients (including HCO3-) by altering transporters
Light reactions

27. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Alter external conditions
Nutrients
N, S, P, cofactors (including biotin)
Salinity/ water potential
NaCl vs KCl vs mannitol/sorbitol/PEG
pCO2: pCO2 in air and [HCO3-] in medium
Temperature
Light: intensity & duration
Turn down light reactions with atrazine
Bioengineer internal changes
Nutrients (including HCO3-) by altering transporters
Light reactions
H2 production via N2ases, H2ases, H+ pumps, etc
Redirect photosynthate
K/O FFA recycling

28. Hypothesis: manipulating the internal environment of
cyanobacteria will affect biofuel production
Alter external conditions
Nutrients
N, S, P, cofactors (including biotin)
Salinity/ water potential
NaCl vs KCl vs mannitol/sorbitol/PEG
pCO2: pCO2 in air and [HCO3-] in medium
Temperature
Light: intensity & duration
Turn down light reactions with atrazine
Bioengineer internal changes
Nutrients (including HCO3-) by altering transporters
Light reactions
H2 production via N2ases, H2ases, H+ pumps, etc
Redirect photosynthate
K/O FFA recycling
????

29. Suggested Game Plan
Run everything in parallel in Synechococcus elongatus and Anabaena
We have experience growing S. elongatus + expertise & materials to engineer its genome
Anabaena will be the exptl organism

30. Suggested Game Plan
Run everything in parallel in Synechococcus elongatus and Anabaena
We have experience growing S. elongatus + expertise & materials to engineer its genome
Anabaena will be the exptl organism
2. For environmental folks:
Grow large batches of S. elongatus and Anabaena, then subdivide into different media/ conditions

31. Suggested Game Plan
Run everything in parallel in Synechococcus elongatus and Anabaena
We have experience growing S. elongatus + expertise & materials to engineer its genome
Anabaena will be the exptl organism
2. For environmental folks:
Grow large batches of S. elongatus and Anabaena, then subdivide into different media/ conditions
At suitable intervals measure
Growth
Heterocysts
H2 production
Photosynthesis
Respiration
DNA, RNA, gene expression in general
Lipids

32. Suggested Game Plan
2. For environmental folks:
Grow large batches of S. elongatus and Anabaena, then subdivide into different media/ conditions
At suitable intervals measure
Growth
Heterocysts
H2 production
Photosynthesis
Respiration
DNA, RNA, gene expression in general
Lipids
3. For gene folks
Identify genes predicted to affect biofuel production

33. Suggested Game Plan
2. For environmental folks:
Grow large batches of S. elongatus and Anabaena, then subdivide into different media/ conditions
At suitable intervals measure
Growth
Heterocysts
H2 production
Photosynthesis
Respiration
DNA, RNA, gene expression in general
Lipids
3. For gene folks
Identify genes predicted to affect biofuel production
Nutrient uptake or metabolism

34. Suggested Game Plan
2. For environmental folks:
Grow large batches of S. elongatus and Anabaena, then subdivide into different media/ conditions
At suitable intervals measure
Growth
Heterocysts
H2 production
Photosynthesis
Respiration
DNA, RNA, gene expression in general
Lipids
3. For gene folks
Identify genes predicted to affect biofuel production
Nutrient uptake or metabolism
Lipid unsaturation

35. Suggested Game Plan
2. For environmental folks:
Grow large batches of S. elongatus and Anabaena, then subdivide into different media/ conditions
At suitable intervals measure
Growth
Heterocysts
H2 production
Photosynthesis
Respiration
DNA, RNA, gene expression in general
Lipids
3. For gene folks
Identify genes predicted to affect biofuel production
Nutrient uptake or metabolism
Lipid unsaturation
Alternate biofuels

36. Suggested Game Plan
3. For gene folks
Identify genes predicted to affect biofuel production
Nutrient uptake or metabolism
Lipid unsaturation
Alternate biofuels
4. Clone and transform genes into host

37. Suggested Game Plan
3. For gene folks
Identify genes predicted to affect biofuel production
Nutrient uptake or metabolism
Lipid unsaturation
Alternate biofuels
4. Clone and transform genes into host
5. Measure effects of transgenes on physiology and biofuel production

38. Suggested Game Plan
3. For gene folks
Identify genes predicted to affect biofuel production
Nutrient uptake or metabolism
Lipid unsaturation
Alternate biofuels
4. Clone and transform genes into host
5. Measure effects of transgenes on physiology and biofuel production
Monday
Environmental folks make the various media and start growing cells

39. Suggested Game Plan
3. For gene folks
Identify genes predicted to affect biofuel production
Nutrient uptake or metabolism
Lipid unsaturation
Alternate biofuels
4. Clone and transform genes into host
5. Measure effects of transgenes on physiology and biofuel production
Monday
Environmental folks make the various media and start growing cells
Gene folks work on identifying suitable targets and devising strategies to clone them.

40. Mineral Nutrition
Soil nutrients
Amounts & availability vary
Many are immobile, eg P, Fe

41. Mineral Nutrition
Immobile nutrients must be mined
Root hairs get close
Mycorrhizae get closer

42. Rhizosphere
Endomycorrhizae invade root cells: Vesicular/Arbuscular
Most angiosperms, especially in nutrient-poor soils
Deliver nutrients into symplast or release them when arbuscule dies
Also find bacteria, actinomycetes, protozoa associated with root surface = rhizosphere

43. Rhizosphere
Also find bacteria, actinomycetes, protozoa associated with root surface = rhizosphere
Plants feed them lots of C!
They help make nutrients available
N-fixing bacteria supply N to many plant spp

44. Nutrient uptake
Most nutrients are dissolved in water

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

46. Nutrient uptake
Most nutrients are dissolved in water
Enter root through apoplast until hit endodermis
Then must cross plasma membrane

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

48. Nutrient uptake
Then must cross plasma membrane
Gases, small uncharged & non-polar molecules diffuse

49. Nutrient uptake
Then must cross plasma membrane
Gases, small uncharged & non-polar molecules diffuse
down their ∆ [ ]
Important for CO2, auxin & NH3 transport

50. Nutrient uptake
Then must cross plasma membrane
Gases, small uncharged & non-polar molecules diffuse
down their ∆ [ ]
Polar chems must go through proteins!

51. Selective Transport
1) Channels
integral membrane proteins with pore that specific ions diffuse through

52. Selective Transport
1) Channels
integral membrane proteins with pore that specific ions diffuse through
depends on size
& charge

53. Channels
integral membrane proteins with pore
that specific ions diffuse through
depends on size & charge
O in selectivity filter bind
ion (replace H2O)

54. Channels
integral membrane proteins with pore
that specific ions diffuse through
depends on size & charge
O in selectivity filter bind
ion (replace H2O)
only right one fits

55. Channels
O in selectivity filter bind
ion (replace H2O)
only right one fits
driving force?
electrochemical D

56. Channels
driving force : electrochemical D
“non-saturable”

57. Channels
driving force : electrochemical D
“non-saturable”
regulate by opening & closing

58. Channels
regulate by opening & closing
ligand-gated channels open/close when bind specific chemicals

59. Channels
ligand-gated channels open/close when bind specific chemicals
Stress-activated channels open/close in response to mechanical stimulation

60. Channels
Stress-activated channels open/close in response to mechanical stimulation
voltage-gated channels open/close in response to changes in electrical potential

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

62. Channels
Old model: S4 slides up/down
Paddle model: S4 rotates
3 states
Closed
Open
Inactivated

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

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

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

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

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

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

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

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

71. Active Transport
Characteristics
1) saturable
ions/s
molecules/s

72. Active Transport
Characteristics
1) saturable
2) specific

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

74. 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

75. 4 classes of Active transport ATPase proteins
1) P-type ATPases (P = “phosphorylation”)
2) V-type ATPases (V = “vacuole”)
H+ pump in vacuoles

76. 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

77. 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

78. 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

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

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

81. 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

82. 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

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

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

85. 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

86. 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)!

87. 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!

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

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

90. 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

91. 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

92. 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+

93. 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?

94. 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

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

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

97. 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

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

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

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

101. 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

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

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

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

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

106. 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

107. 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

108. 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

109. 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

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

111. Nutrient uptake
Much is coupled to
pH gradient

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

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

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

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

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

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

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

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

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