1. Ion and Solute Transport across Plant Cell Membranes HORT 301 – Plant Physiology October 10, 2007 Taiz and Zeiger, Chapter 6, Web Chapter 2 (p 1-10), Web Topic 6.3 paul.m.hasegawa.1@purdue.edu Plant Mineral Nutrition Lectures Lecture 1 – mineral nutrients/essential elements, plant nutrient status, acquisition by plant roots Lecture 2 – transport across cell membranes, transport proteins Lecture 3 – mineral nutrient absorption, assimilation and translocation

2. Mineral Nutrient (Ion) and Solute Transport across Cellular Membranes Transport into or out of plant cells is across the plasma membrane lipid bilayer, composed of phospholipids Phospholipids – fatty acids are linked to two carbons in glycerol (ester linkages) and a head group (phosphate, phosphate-linked molecule) is attached to the third carbon Fatty acids - hydrophobic membrane region (low or no affinity for water) and head groups - hydrophilic membrane region (affinity for water)

3. Plasma membrane - lipid molecules are joined end-to-end in the hydrophobic regions, with the hydrophilic phospho head groups on the outside of the membrane, i.e. cytoplasm and apoplast (outside) Hydrophobic region - restricts diffusion of ions and other water soluble molecules directly across the membrane bilayer solution Transport proteins - embedded across the membrane and have specific affinities for ions and other solutes, i.e. Na +, sucrose, etc.

4. Chemical potential (j mol -1 ) is the driving force (energy) for ion and solute transport across membranes – chemical potential components are: Solute potential Hydrostatic/pressure And electrical potential Gravity effect is negligible Passive transport is down (with/downhill) the chemical (electrochemical) potential gradient and active transport is up (against/uphill) the chemical potential gradient

5. Water (H 2 O) - chemical potential = water potential (  w )  w = solute/osmotic potential (  s ) + hydrostatic pressure/pressure potential (  p ) H 2 O - no net charge (localized positive and negative charges) = no electrical potential Ions and solutes - negligible hydrostatic pressure/pressure potential 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) Mineral nutrients are absorbed by plants as ions, electrical potential

6. Membrane potential – electrical gradients that buildup across a membrane, differential accumulation of ions on sides of the membrane Chemical potential (electrochemical potential) of ions: Δµ (electrochemical potential) = RT ln C i /C o (concentration activity) + zF∆E (electrical potential) C i and C o – concentration inside and outside, respectively, R – gas constant, T – temperature (°K), 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, and anions (negatively charged) must be actively transported into the cell

7. 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 Univalent cation (+, e.g. Na + ) - membrane potential of about -59 mV (inside negative) = the energy to drive a 10-fold concentration gradient ∆E = 59 mV log C o /C i, membrane potential of 59 mV (C o /C i = 10, log 10 = 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 For a univalent anion (-, e.g. Cl - ) a membrane potential of -120 mV (inside negative) requires that a Cl - apoplast must be >100X relative to Cl - symplast for passive transport

8. Divalent (Ca 2+ or SO 4 2- ) ions have 2X the electrical potential Each ion has its own electrochemical potential Differential membrane permeability for an ion causes a membrane potential Movement of Ions with the electrochemical potential gradient

9. Major cellular ion (solute) compartments of a cell - apoplast, cytosol and vacuole Apoplast - variable in size relative to the symplast Symplast - cytosol = 5-10% and vacuole = 90-95%, fully expanded cell

10. Active proton (H + ) transport generates membrane potential (electrical) and pH (H + ) gradients that facilitate passive and active transport of ions and solutes - H + electrochemical potential gradient Transport across these membranes is mediated by electrongenic H + - ATPases and pyrophophatases, 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) 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

11. Membrane potential and pH gradients drive transport of nearly all ions and solutes in plants, via transport proteins Exceptions: Ca 2+ -ATPases and ATP–binding cassette (ABC) transporters that mediate active transport of Ca 2+ and macromolecules, respectively

12. Distribution of some essential mineral nutrients within the cell, e.g. transport across the plasma membrane for cations (Na +, K +, Ca 2+ ) is passive (dashed) and for anions (Cl -, NO 3 -, H 2 PO 4 - ) is active (solid)

13. Transport Proteins – individual proteins or multi-subunit structures (quaternary structure) that are embedded in the membrane Facilitate passive and active transport across membranes Transport proteins are usually highly specific – transport a particular ion or solute with high specificity, tightly control active or passive transport of ions and solutes ~450 Arabidopsis genes encode transport proteins

14. Transport protein categories – channels, carriers and pumps

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

16. Carrier – specific substrate binding site on one side of the membrane, protein undergoes a conformational change that exposes the substrate to the opposite side Substrate binding site confers high specificity (affinity) for transport Transport rate of carriers is between 100 to 1000 ions or molecules per second, about 10 6 times slower than transport through channels Carrier mediated transport is passive diffusion (uniport) or Secondary active (discussed after pumps) e.g. symport or antiport

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. Transport protein categories – channels, carriers and pumps

19. H + -ATPases transducers of ATP hydrolysis → ADP + Pi - unidirectional electrogenic H + transport, pH gradients and membrane potential across the plasma membrane (apopolast) and tonoplast membrane (vacuole) Tonoplast pyrophosphatase - H + pump hydrolyzes PPi to 2Pi, energy is used electrogenic H + transport to the inside of the vacuole H + pumps generate proton (H + ) electrochemical gradients (ΔpH and membrane potential) across membranes that facilitate secondary active ion and solute transport in plants

20. 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

21. Primary and Secondary Active Transport of Ions and Solutes – active transport mechanisms in plants H + pumps - primary active transport to generate H + electrochemical gradients (ΔpH and membrane potential) across the plasma membrane and tonoplast

22. 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)

23. Electrophoretic flux – passive transport of an ion that at equilibrium is defined by the Nernst equation, -120 mV (inside negative) - K + can accumulate to 100-fold in the cytosol relative to the apoplast Secondary active transport (carrier mediated) – active transport of an ion or solute (against the electrochemical gradient) by coupling to passive transport of H + s (down the H + electrochemical gradient)

24. Antiporter - H + and ion/solute transport is in the opposite direction, H + electrochemical gradient is greater than the electrochemical gradient of the substrate Red arrow denotes the direction of the electrochemical gradient

25. Symporter – H + and ion/solute transport is in the same direction

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