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

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

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 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 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 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

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

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 D

Channels driving force : electrochemical D “non-saturable”

Channels driving force : electrochemical D “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 Closed Open 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- & NO3- enter 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- & tri- 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- & tri- peptides 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- & tri- peptides 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- & 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

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 Ys 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