1. Solute Transport HORT 301 – Plant Physiology September 15, 2008 Taiz and Zeiger, Chapter 6, Web Chapter 2 (p 1-10), Web Topic 6.3 paul.m.hasegawa.1@purdue.edu Transport of ions and other solutes across membranes is facilitated by proteins Membranes are composed primarily of phospholipids joined end-to-end in a lipid bilayer Proteins and carbohydrates also comprise the membrane Proteins – integral proteins often function in transport, peripheral proteins may be sensors or receptors

2. Membrane phospholipids Glycerol is the central backbone of the molecule Hydrophobic region - two fatty are linked to carbons in glycerol (ester linkages) Hydrophilic head group – third carbon is usually linked to a phosphorylated molecule e.g. choline, or another hydrophilic molecule, e.g. galactose Hydrophilic region has affinity for water and hydrophobic region has little or no affinity for water

3. Plasma membrane – head groups on the apoplastic and symplastic (cytoplasmic) sides Transport proteins – facilitate transport across membranes and each has a specific affinity for an ions or solute, i.e. Na +, sucrose, etc.

4. Chemical potential (j mol -1 ) is the driving force (energy) for solute transport across membranes – sum of concentration gradient and electrical potential Passive transport is with the chemical (electrochemical) potential gradient (higher to lower) Passive Active Active transport (requires energy) is against the chemical potential gradient (lower to higher)

5. Membrane potential – electrical gradient that builds across a semipermeable membrane, sum of ion distribution, measured in voltage Uncharged solutes have no electrical potential like water, e.g. sucrose, starch Charged solutes – electrical gradient contributes to the chemical potential to drive transport of ions (charged atoms or molecules) Plant plasma membranes – inside negative membrane potential, -120 mV

6. Existence of a membrane potential indicates that ions accumulated differentially on sides of the membrane Membrane is “selectively” permeable, i.e. transport protein selectivity

7. Chemical potential (electrochemical potential) gradient for an ion: Δµ (electrochemical potential gradient) = RT ln C i /C o (concentration activity) + zF∆E (electrical potential) Concentration activity - C i and C o – concentration inside and outside of membrane, respectively, R – gas constant, T – temperature (°K) Electrical potential - z = electrostatic charge of the ion (+ or -), F = Faraday’s constant, ∆E = membrane potential Electrical potential across the plasma membrane of plant cells (steady- state) is inside negative, about -120 mV Therefore, cations (positively charged ions) move passively into the cell, even against a concentration gradient Anions (negatively charged) must be actively transported into the cell

8. Transformation of an electrical gradient (membrane potential) into a concentration gradient – at equilibrium defined by the Nernst equation, ∆E = -2.3RT/zF log C o /C i Plasma membrane potential of -59 mV (inside negative) corresponds to an energy that will drive a 10-fold concentration of a monovalent cation (+, e.g. Na + ) into the cytosol; Na + transports passively to 10-fold greater concentration inside the cell relative to the outside ∆E = 59 mV log C o /C i if C o /C i = 10, log 10 = 1 then ∆E = 59 mV x 1 Membrane potential across the plasma membrane – usually about -120 mV, Na + accumulates ~10 2 (100-fold) greater concentration in the symplast relative to the apoplast based on the electrical potential

9. For a univalent anion (-, e.g. Cl - ) a membrane potential of -120 mV (inside negative) requires that Cl - apoplast must be >100X relative to Cl - symplast for passive transport Divalent (Ca 2+ or SO 4 2- ) ions have 2X the electrical potential Each ion has its own electrochemical potential K + and Cl - each diffuse to net chemical steady-state Specificity is due to unique concentration activity

10. Membranes separate major cellular compartments – focus is on the apoplast, cytosol and vacuole Apoplast - variable in size relative to the symplast Symplast - cytosol = 5-10% and vacuole = 90-95%, fully expanded cell Tonoplast – vacuolar membrane

11. Proton (H + ) electochemical gradient generates membrane potential (electrical gradient) and pH (H + ) gradients that facilitate passive and active transport of ions and solutes in plants H + electrochemical potential gradient H + transport across these membranes is mediated by electrongenic H + - ATPases and pyrophosphatases, pumps Electrogenic transport - transfer of charged atoms/molecules unequally across a membrane, causing a membrane potential and a concentration gradient for that ion H + -ATPases and pyrophosphatases - hydrolyze high energy phosphoester bonds, ATP or pyrophosphate (PPi), respectively Energy from hydrolysis is used for active transport of H + s (against the H + electrochemical potential gradient, uphill) to establish membrane potential and pH gradients

12. Membrane potential and pH gradients drive transport of ions and solutes in plants, via transport proteins Based on steady-state membrane potentials and ion concentration gradients, intracellular distribution of essential elements is due to active (solid line) or passive (dashed) transport Inward transport across the plasma membrane of cations (Na + and Ca 2+ ) is passive and for anions (Cl -, NO 3 -, H 2 PO 4 - ) is active

13. Transport Proteins – individual proteins or multi-subunit complexes (quaternary structure) that are embedded in the membrane Facilitate passive and active transport across membranes Transport proteins are highly specific – transport a particular solute with high specificity, tightly control active or passive transport of ions and solutes However, similar solutes are transported at lower affinity, e.g. K + transport proteins also transport Na + but not Cl - ~450 Arabidopsis genes encode transport proteins

14. Transport protein categories – channels, carriers and pumps

15. Channel – selective pore that transports a solute by diffusion (passive), regulation is based on channel pore opening and closing (gating) Channel transport is usually restricted to ions or small molecules The “gate” is a component of protein structure – gating is regulated by stimuli, such as voltage (membrane potential changes), osmotic, hormones, Ca 2+, light Specific channels may transport solutes (or water) inwards (inward rectifying) or outwards (outward rectifying)

16. Carrier – substrate binding site on one side of the membrane, protein conformational change facilitates substrate movement to the opposite side Substrate binding site confers high specificity (affinity) for transport Transport rate of carriers is between 100 to 1000 molecules per second, about 10 6 times slower than transport through channels Carrier mediated transport is passive diffusion via a uniporter (like a channel) Or secondary active transport via a symporter or an antiporter, driven by the H + electrochemical gradient

17. Pump – transport protein that couples energy production to the movement of a solute against the chemical (electrochemical) potential, primary active transport Proton (H + )-ATPases (plasma membrane and tonoplast) - most common pumps in plants, plasma membrane and tonoplast membrane

18. ATP binding cassette (ABC) transport proteins – active transport of large molecules (secondary products, flavonoids, anthocyanins, xenobiotics) by the transduction of energy from ATP hydrolysis Ca 2+ -ATPases - localized in the plasma membrane, tonoplast membrane and endomembranes, couple ATP hydrolysis to active transport of Ca 2+ from the cytosol Tonoplast pyrophosphatase - H + pump hydrolyzes PPi to 2Pi, energy is used electrogenic H + transport to the inside of the vacuole

19. Primary and Secondary Active Transport of Solutes – active transport mechanisms in plants Primary active transport – pumps mediate primary active transport, couple ATP (or pyrophosphate) hydrolysis to unidirectional solute transport Secondary active transport - H + pumps generate H + electrochemical gradients across the plasma membrane (ΔpH and membrane potential) and tonoplast (primarily ΔpH) to facilitate active transport of solutes

20. Plasma membrane – ΔpH ~2 units (apoplast - pH 5.5 and cytosol - pH 7.2, membrane potential ~-120 mV (cytosol negative relative to apoplast) Tonoplast - ΔpH ~2 units (vacuole - pH 5.5 and cytosol - pH 7.2 – cytosol), membrane potential ~+30 mV (vacuole positive relative to cytosol)

21. Electrophoretic flux – passive transport of an ion that at steady state is driven by electrochemical potential gradient -120 mV (inside negative) - K + can accumulate to 100-fold in the cytosol relative to the apoplast

22. Secondary active transport is carrier mediated – transport of a solute against its the electrochemical gradient by coupling to passive transport of H + s down the H + electrochemical gradient, antiporter or symporter Antiporter - H + and ion/solute transport is in the opposite direction, H+ electrochemical gradient is greater than the electrochemical gradient of the substrate Symporter – H + and ion/solute transport is in the same direction, H + electrochemical gradient is greater than S

24. Transport proteins in planta, expression in heterologous systems or loss-or gain-of-function genetics