Common themes 1.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 3.Gene knockouts FFA recycling H2 metabolism N metabolism Cell walls
Abiotic stresses Salinity osmotic Temperature Common themes 1.Growth in different media Differ in [N] or other nutrients
2.Growth in common medium, then change Harvest, then resuspend in new media Add something to medium Salt Biotin/avidin Inducer Inhibitor
Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production Will change allocation of photosynthate 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 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? 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?
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?
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? 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? 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? 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? 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?
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? 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 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 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 Hypothesis: manipulating the internal environment of cyanobacteria will affect biofuel production
Alter external conditions Nutrients N, S, P, cofactors (including biotin) 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 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 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 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 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 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
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 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 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
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 Suggested Game Plan 1.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
Suggested Game Plan 1.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 Suggested Game Plan 1.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 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 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 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 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 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
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 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
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.
Mineral Nutrition Soil nutrients Amounts & availability vary Many are immobile, eg P, Fe Mineral Nutrition Immobile nutrients must be mined
Root hairs get close Mycorrhizae get closer 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 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 Nutrient uptake Most nutrients are dissolved in water Nutrient uptake Most nutrients are dissolved in water Enter root through apoplast until hit endodermis
Nutrient uptake Most nutrients are dissolved in water Enter root through apoplast until hit endodermis Then must cross plasma membrane Crossing membranes A) Diffusion through bilayer
B) Difusion through protein pore Selective C) Facilitated diffusion D) Active transport E) Bulk transport Active 1) Exocytosis 2) Endocytosis
Nutrient uptake Then must cross plasma membrane Gases, small uncharged & non-polar molecules diffuse Nutrient uptake Then must cross plasma membrane Gases, small uncharged & non-polar molecules diffuse
down their [ ] Important for CO2, auxin & NH3 transport Nutrient uptake Then must cross plasma membrane Gases, small uncharged & non-polar molecules diffuse down their [ ] Polar chems must go through proteins!
Selective Transport 1) Channels integral membrane proteins with pore that specific ions diffuse through Selective Transport 1) Channels
integral membrane proteins with pore that specific ions diffuse through depends on size & charge Channels integral membrane proteins with pore that specific ions diffuse through
depends on size & charge O in selectivity filter bind ion (replace H2O) 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 Channels O in selectivity filter bind ion (replace H2O) only right one fits
driving force? electrochemical Channels driving force : electrochemical non-saturable Channels
driving force : electrochemical non-saturable regulate by opening & closing Channels regulate by opening & closing ligand-gated channels open/close when bind specific chemicals
Channels ligand-gated channels open/close when bind specific chemicals Stress-activated channels open/close in response to mechanical stimulation Channels
Stress-activated channels open/close in response to mechanical stimulation voltage-gated channels open/close in response to changes in electrical potential Channels
Old model: S4 slides up/down Paddle model: S4 rotates Channels Old model: S4 slides up/down Paddle model: S4 rotates 3 states 1.Closed
2.Open 3. Inactivated Selective Transport 1) Channels 2) Facilitated Diffusion (carriers) Carrier binds molecule
Selective Transport Facilitated Diffusion (carriers) Carrier binds molecule carries it through membrane & releases it inside Selective Transport Facilitated Diffusion (carriers)
Carrier binds molecule carries it through membrane & releases it inside driving force = [ ] Selective Transport Facilitated Diffusion (carriers) Carrier binds molecule
carries it through membrane & releases it inside driving force = [ ] Important for sugar transport Selective Transport Facilitated Diffusion (carriers)
Characteristics 1) saturable 2) specific 3) passive: transports down [] Selective Transport 1) Channels
2) Facilitated Diffusion (carriers) Passive transport should equalize [ ] Nothing in a plant cell is at equilibrium! Selective Transport Passive transport should equalize [ ] Nothing in a plant cell is at equilibrium! Solution: use energy to transport specific ions against
their [ ] Active Transport Integral membrane proteins use energy to transport specific ions against their [ ] allow cells to concentrate some chemicals, exclude others Active Transport
Characteristics 1) saturable 105-106 ions/s 102-104 molecules/s Active Transport
Characteristics 1) saturable 2) specific Active Transport Characteristics 1) saturable 2) specific
3) active: transport up [ ] (or Em) 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
4 classes of Active transport ATPase proteins 1) P-type ATPases (P = phosphorylation) 2) V-type ATPases (V = vacuole) H+ pump in vacuoles 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 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 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 Secondary active transport Uses [ ] created by active transport to pump something else across a membrane against its [ ]
Secondary active transport Uses [ ] created by active transport to pump something else across a membrane against its [ ] Symport: both substances pumped same way 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 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 Nutrient uptake Gases enter/exit by diffusion down their [ ] Ions vary dramatically!
Nutrient uptake Ions vary dramatically! H+ is actively pumped out of cell by P-type H+ -ATPase 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 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)! 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! Nutrient uptake Many ions are imported by multiple transporters with varying affinities
Nutrient uptake Many ions are imported by multiple transporters with varying affinities K+ diffuses through channels down Em: low affinity 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 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 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+ 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?
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 Nutrient uptake Ca2+ is expelled by P-type ATPases in PM Nutrient uptake Ca2+ is expelled by P-type ATPases in PM pumped into vacuole & ER by H+ antiport & P-type
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 Nutrient uptake PO43-, SO42-, Cl- & NO3enter by H+ symport
Nutrient uptake PO43-, SO42-, Cl- & NO3- enter by H+ symport also have anion transporters of ABC type Nutrient uptake PO43-, SO42-, Cl- & NO3- enter by H+ symport also have anion transporters of ABC type
and anion channels 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
Nutrient uptake Plants take up N many ways: NO3- & NH4+ are main forms Nutrient uptake Plants take up N many other ways NO3- also by channels NH3 by diffusion
NH4+ by carriers Nutrient uptake Plants take up N many other ways NO3- by channels NH3 by diffusion NH4+ by carriers NH4+ by channels
Nutrient uptake Plants take up N many other ways 3 families of H+ symporters take up amino acids 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- & tripeptides by H+ symport 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- & tripeptides by H+ symport
others take up tetra- & penta-peptides by H+ symport 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- & tripeptides by H+ symport
others take up tetra- & penta-peptides by H+ symport Also have ABC transporters that import peptides 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- & tripeptides 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
Nutrient uptake Metals are taken up by ZIP proteins & by ABC transporters same protein may import Fe, Zn & Mn! Nutrient uptake Much is coupled to
pH gradient Nutrient transport in roots Move from soil to endodermis in apoplast Nutrient transport in roots Move from soil to endodermis in apoplast Move from endodermis to xylem in symplast
Nutrient transport in roots Move from endodermis to xylem in symplast Transported into xylem by H+ antiporters Nutrient transport in roots Move from endodermis to xylem in symplast Transported into xylem by H+ antiporters, channels
Nutrient transport in roots Transported into xylem by H+ antiporters, channels,pumps Nutrient transport in roots Transported into xylem by H+ antiporters, channels,pumps Lowers xylem water potential -> root pressure
Water Transport Passes water & nutrients to xylem s of xylem makes root pressure Causes guttation: pumping water into shoot Transport to shoot Nutrients move up
plant in xylem sap Nutrient transport in leaves Xylem sap moves through apoplast Leaf cells take up what they want
