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.

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

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

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

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

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 ∆ [ ]

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