13. Transport can be active or passive.•Passive transport is movement downan electrochemical gradient.•Active transport is movement against an electrochemical gradient.What is an electrochemical gradient?How is it formed?Passive and active transport of ions result in electric potential difference across membranes.•Movement of an uncharged mol Is dependent on conc. gradient alone. •Movement of an ion depends on the electric gradient and the conc. gradient. •Diffusion potential-Pump potential-How do you know if an ion is moving uphill or downhill? Nernst EqWhat is the driving force for uphill movement?A) ATP ; b) H+ gradientF 6-3 Taiz. Microelectrodes are used to measure membrane potentials across cell membrane6-5. Pump potential and diffusion potential.Nernst equation states that at equilibrium the difference in concentration of an ion between two compartments is balanced by the voltage difference. Thus it can predict the ion conc at equilibrium at a certain ΔE.Very useful to predict active or passive transport of an ion. How can we determine whether an ion moves in or out by active or passive transport?----------------------------------------------------------------------------------------Ext Conc. Ion Internal concentration (mM) observed Nernst (Predicted)----------------------------------------------------------------------------------------1 mM K+75 mM 741 mM Na+8 mM 741 mM Ca2+2 mM 5,0000.2 mM Mg2+3 1,3402 mM NO3 -5 mM 0.021 Cl-10 mM 0.011H2PO4-21 0.01----------------------------------------------------------------------------------------Tab 6-1, Taiz . Using the Nernst equation to predict ion conc. at equilibrium when the Cell electrical potential, Δψ = -110 mVSummary: In generalCation uptake: passiveCation efflux: activeAnion uptake: activeAnion release: passiveFig. 6-4, Taiz. Passive and active transporters.26-7. Proteins catalyze transport. Channel, carrier pump6-10. Secondary active transport15-3 Lodish. Three types of transport proteinsOutline: Major Transport Proteins in Plants1. Primary Pumps: H+-pumping ATPases are the major ion pumps in plants Ca-pumps. Cu, Zn. Few ions moved by pumps.2. Secondary active transport: H+-coupled cotransportEnergy from H+ gradient is used to drive uphill movement of other nutrients. E.g. ions, sugars, amino-acids, 3. Channels allow rapid, passive transport of ions and metabolites. 4. Water Channels or AQUAPORINS in membranes that conduct large volumes of water rapidly.5. ABC transporters pump organic molecules. E.g. auxins, Cd-X Genome of Arabidopsis was completely sequenced in 2000. ~ 30 k genes~5% of genes encode transporters. E.g.ChannelsSecondary transportPrimary transport TAGI 2000. Nature 408, 7963 H+pumps:PM H+-ATPaseVac H+-ATPaseVac H+-PPaseH+H+pH 7.3pH 5.5pH 5.5H+pumps generate a proton electrochemical gradientWhy are H+pumps important?345 P-type ion ATPases in ArabidopsisAxelsen & Palmgren 2001 Plant PhysiolHeavy MetalPM H+ATPaseCa2+-ATPaseAmino-phospholipid6-17. Taiz. PM-H+-ATPase acidify the cell exterior10 Transmembrane100 kDaArabidopsis: 12 genesActivity is Regulated by hormone, light, pathogenBinds ATPModel of a pump at work6-16 Taiz. Vacuolar H+-ATPase acidify endomembrane compartments:vacuole, Golgi, vesicles.From Wilkens et al. 1999. JBC; Sagerman et al 2001. PNASEvidence for H+pumps• Cell- vac pH is acidic• Isolated membrane vesicles-ATP generate pH gradient and electrical gradient• Purify protein & reconstitute activity in liposomes• ----------------• Clone gene• Express & show transport in yeastH+H+H+ABAntiportSymportExpt. Evidence for H+pumps. Importance of H+pumps.How will uptake of A affect the membrane potential?pH gradient?H+4Active transport of many ions and metabolites is dependent on Secondary active transport: H+-coupled cotransport (Fig. 6-11)H+H+Model of an antiporterTaiz. Evidence that Glucose uptake depends on H+-coupled glucose symportChannels allow rapid passive diffusion of ions. Fig. 6-15. K+enters cells via a gated channel.K+Channels are gated by -voltage, -Ligands: Ca, hormone, ATP, GlutamateHyperpolarization-activatedDepolarization-actWeb topic. Box 6-1 Taiz. Measuring ion fluxes via channels with patch clamp method. Measures current (movement of a + charge)Voltage-gated current. Evidence for K+ conductance5Aquaporin: water channelProteins that conduct fast diffusion of water down its water potential gradient.Experimental evidence. How do you prove this? 1. Express a candidate gene in an animal cell.2. Put cell in dilute solution.3. Measure swelling……. Until boom!Cells control their osmotic conc and cell vol.15-32. Evidence for a water channel protein (aquaporin)Microinject CHIP 28 mRNA into oocyteTransfer cell from 0.2 M --> 0.035 MControl oocyte has low permeability to water.controlNobel Laureates in Chemistry 2003"for discoveries concerning channels in cell membranes“Agre"for the discovery of water channels“McKinnon "for structural and mechanistic studies of ion channels"Putting all the info. togetherHow is a nutrient transported into roots?How do guard cells open and close?How does a mineral nutrient move from root to leaf?a. Movement into root. Consider the mode of transport at each stage.Passive, active, and type of transport protein?b. Up the xylem in the stem to the leafc. Movement into mesophyll.Solutes move through apoplast and symplastIon transport into rootsXylem loading by xylem parenchyma cells.618-8. Stomata. Open and closed stateWhat controls opening? How? Increase in turgor pressureWhat controls closing?18.9 Stomatal opening tracks photosynthetic active radiation at the leaf surface18.16 Daily course of changes in stomatal aperture18.16 Daily course of changes in stomatal apertureLight stimulates stomatal openingHow?Draw on board18.13 Acidification of a suspension medium of guard cell protoplasts of V. faba• Membrane potential hyperpolarize.• K+ conc. Increase 100 mM Æ 400 mM• Anions: Cl- and malate increase• Water potential drops, water enters• Guard cells increase their turgor pressure• Stomata open.7Closure•How?From Schroeder and Allen. 2001. NatureClosing stomatal aperture: the long-term efflux of both anions and K+ from guard cells contributes to the loss of guard cell turgor, leading to stomatal closing.Lab. - guard cell movement1. Test effect of light versus dark on stomatalaperture2. Determine which ion or solute is needed for