DISCUSSION AND ESSAY QUESTIONS SET 2 Ion transport 1. A certain barley root cell maintains a potential difference of 0.0885 V across its plasma membrane. The following concentrations of ions (molar) inside and outside the cell were measured: Ion Inside Outside K+ 0.1 0.003 Na+ 0.01 0.001 Cl- 0.06 0.002 2- SO 4 0.001 0.001 Ca 2+ 0.00001 0.001 a. Which of the ions would be considered to be at or near electrochemical equilibrium by the formula given in lecture, assuming a temperature of 25oC? Ion Inside Outside Observed ratio Expected Near Eq? (co/ci) (co/ci) K+ 0.1 0.003 0.03 0.0316 close Na+ 0.01 0.001 0.1 0.0316 exclusion Cl- 0.06 0.002 0.0333 31.6 Active up 2- SO 4 0.001 0.001 1.0 1000 Active up Ca 2+ 0.00001 0.001 100 0.001 active exp b. For each ion not in equilibrium, suggest a reason for its distribution (active uptake, active efflux, exclusion, magnetic effects, limited carrier capacity, no pressure for redistribution, etc.) c. Predict the concentrations of H+ inside and outside the cell. What do these have to do with the data given above? d. What will be the effect of adding the following chemicals: DNP (dinitrophenol, carries H+ ions across membranes along their electrochemical gradient); valinomycin (carries K+ ions across membranes along their electrochemical gradient); KCN (stops respiration and lowers the supply of ATP). Set ΔE to +88.5 mv OPPOSITE to voltage difference. for all but K+??? (a) The governing equation for equlibrium is the Nernst equation: ΔE= (2.3RT/zF)log10Co/Ci or ΔE = (59/z mv)log10co/ci or Co/Ci =10ΔEz/59. For the given voltage of -88.5 mv and z = +1, the equilibrium Co/Ci is 0.0316 (Ci/Co is 31.6). (b) K+ is close to equilibrium; Na+ is excluded; Cl- and SO42- are actively taken up; Ca2+ is excluded or actively exported. (c) Inside and outside the cell, the ph is 7 and 5; H+ concentration is 10-7 and 10-5 M, respectively. H+ is actively exported, and this contributes most to the membrane potential. The effect of H+ export is greater than appears from the concentrations, because of buffering. (d) DNP will set μ(h+) = 0 across the membrane; valinomycin will set μ(k+) = 0 across the membrane; KCN will inhibit the H+-ATPase pump. All three will tend to discharge the membrane potential. 2. A plant root that has been starved for Cl- is placed in a solution of 10 mm CaCl2. For a brief period, the ph around the root goes up. Discuss why. There is likely H+ / Cl- symport occurring. As Cl- is taken up by the root, H+ is taken up as well, leading to an increase in ph. 3. The electrochemical potential between roots and the medium in which the roots are immersed was measured to be -100 mv. If the medium has 50 μeq K+ per ml, what would you predict the K+ concentration to be in the root cells if the distribution between medium and cytoplasm followed the Nernst equation? If the actual K+ concentration in the cytoplasm was 500 μeq g-1 fresh weight of roots, what would you suspect about the mechanism of uptake? Note that 50 μeq K+ per ml = 50 mm (and 500 μeq g-1 FW is close
to 600 mm assuming root cells are 80% water). With ΔE = -100 mv, expect Ci/Co = 10^-ΔEz/59 = 50, and Ci = 50*50 = 2500 mm = 2.5 M. I would suspect that K+ is being excluded or actively exported. 4. Define the terms active and passive transport. Based on these definitions, describe the nature of the transport events that are thought to be involved in: (a) K+ uptake into the root symplast; K+ movement across the cells of the root to the xylem parenchyma; (c) efflux of K+ into the xylem transpiration stream. See chapter 6 (p. 88) in text. a) Active transport b) Active transport c) Passive transport 5. Explain how to test whether the transport of K+ from the soil solution into the epidermal cytoplasm is active or passive. Apply the test to the following data: K+ in epidermal cytoplasm = 80 mm K+ in soil solution = 0.05 mm Em (epidermal cytoplasm relative to soil solution) = -105 mv RT/F = 25.7 mv at 25oC JK net (soil solution to epidermal cytoplasm) has a positive value. Examine the difference between the observed concentration (in the epidermal cytoplasm) vs. the expected / predicted concentration (given the Nernst potential for that ion at equilibrium). If using Co/Ci, ΔE is the same sign as the membrane potential (negative). If the observed Co/Ci is greater than expected Co/Ci, that is interpreted as export. If observed Co/Ci is less than expected Co/Ci uptake Observed Co/Ci = 80/0.05 = 0.0006 Expected Co/Ci = 10^(ΔEz/(2.3*25.7) = 10^(105*1/(59)) = 60.2 Observed << expected, so ACTIVE uptake/ import. 6. Analyze the following data to determine whether inorganic phosphate enters a root by a passive or an active mechanism: Em (membrane potential of the root epidermis relative to soil solution) = -95 mv [H2PO4-] in soil = 3.0 mm [H2PO4-] in cytoplasm = 0.5 mm Jinflux (phosphate) = 2.0 x 10-12 moles cm-2 sec-1 Jefflux(phosphate) = 1.0 x 10-12 moles cm-2 sec-1 Em = RT/zF ln (ao/ai) and RT/F = 25.7 mv Observed Co/Ci = 3/0.5 = 6 Expected Co/Ci = 10^(ΔEz/(2.3*25.7) = 10^(-95*1/(59)) = 1.99 Observed > expected, so ACTIVE efflux/ export. 7. Analyze the following theoretical situation involving K+ uptake into the root system of a maize plant: In one region of the soil, the concentration of K+ in the soil solution is 1 x 10-5 M, whereas in another region the concentration is 1 x 10-3 M. Although net uptake of K+ is occurring at both regions of the root, the membrane potential of the epidermal and cortical cells is different, being -180 mv and -130 mv in the regions of low and high soil K+ concentrations, respectively. However, the activity of K+ in the cytoplasm of the epidermal and cortical cells is the same in these two regions, being 0.1 M. Use the Nernst equation and the relation, RT/zF = 25.7 mv, to determine the thermodynamic nature of the K+ transport process that is occurring at each region of the root.
Low (region 1) High (region 2) Co 0.01 mm 1 mm Ci 100 mm 100 mm ΔE -180 mv -130 mv expected Ci/Co 1124 159 observed Ci/Co 10,000 100 conclusion active uptake; possibly co-transport with H+ exclusion; or active outflow to xylem 8. Use the Nernst equation to analyze the data presented below to identify the nature of the transport system for cation "X+". Use EX and JC net values to construct a current-voltage plot for this situation. Draw a schematic to describe the most likely transport system for "X+." X+ in the apoplasm = 0.5 mm X+ in the cytoplasm = 10 mm Em = -135 mv JX, net is an efflux RT/F = 25.7 mv Observed Co/Ci = 0.5/10= 0.05 Expected Co/Ci = 10^(ΔEz/(2.3*25.7) = 10^(-135*1/(59)) = 0.005 Observed > expected, so ACTIVE efflux/ export. Drawing components: - Membrane - Apoplast/ cytoplasm - Transporter - Direction of ion transport - Cartoon of relative concentrations of X+ on each side 9. The flow diagram below represents the pathway of ions from soil to leaf cell. For each step, indicate whether the process is "active" (crosses membranes, uses carriers and metabolic energy), "passive" (works by diffusion or by the non-metabolic flow of solution); or "symplastic" (flows through plasmodesmata, whether actively or passively). Soil--[passive]-->Intercellular space around cortex cells--[active]-->cortex cells--[symplastic]-->stelar cells--[active]-- >Intercellular space around stelar cells--[passive]-->root xylem--[passive]-->stem xylem--[passive]-->leaf xylem-- [passive]-->intercellular space around leaf cells--[active]-->leaf cell--[symplastic]-->other leaf cells 10. Does a plant depend on transpiration to obtain needed minerals? Does transpiration provide an advantage to a plant in terms of ion uptake? Does it provide any disadvantage? Advantage: bulk flow Disadvantage: dilution 11. Physiologists who discussed membrane transport in the 1970s spoke hypothetically of "carriers" and "channels." Both concerned the movement of specific ions, but carriers were thought to move ions one at a time in enzyme-like steps, while channels were thought to form pores that allowed the ions to flow rapidly. Discuss likely similarities and differences between carriers and channels. How, experimentally, could you determine which was involved in the transport of a specific ion? How would flow through each differ from simple diffusion?
If carriers are like most enzymes, then the initial binding step is faster than the transport (transformation) step; furthermore, the binding step has an association constant (Km), and the transport step has a maximum value (Vmax). If the Km and Vmax can be determined by measuring rate of transport vs concentration of substrate, this is evidence for a carrier-mediated transport. The substrate concentration kinetics of channels and simple diffusion might be difficult to distinguish, but channels may turn on and off (seen through patch-clamp studies) and show rectification, unlike diffusion 12. Compare and contrast apoplastic cell-to-cell transport with symplastic cell-to-cell transport. In terms of selectivity of transport, how do the two pathways compare? Why do they differ? What are the factors that would govern transport between cells by diffusion? Apoplastic: Movement through apoplast is passive only. No selectivity. Symplastic: Movement across membranes (selective, can be active) by pumps and pores. Passive movement through plasmodesmata. 13. Describe the mechanism by which a membrane can function as an osmotic barrier to the movement of solutes. SKIP. This concept is inadequately covered in lecture & the text. 14. Potassium ions have been implicated in the mechanism responsible for stomatal opening and closing. Speculate on a mechanism involving potassium ions that could account for the "sleep" movement of Albizzia julibrissin, as described by Taiz and Zeiger. The trick is to have similar cells (ventral and dorsal motor cells) that alternate in taking up, then releasing K+ and Cl-. One method would be by control of H+-ATPase pump (alternately on and off). Another method would be by control of Cl- flux, inward by turning on a H+- Cl- cotransporter, outward passively by opening Cl- channels. K+ transport would be inward passively through K+ channels; outward in response to the transient depolarization caused by Cl- efflux. 15. Discuss the symplast:apoplast concept in relation to the movement of solutes through the plant. Solutes can take a symplastic or apoplastic pathway in through the roots. However, upon reaching the root region where the Casparian strip has formed, all solutes are forced to follow the symplastic pathway. Further movement of solutes can occur symplastically (through the plasmodesmata or across membranes), or may be pumped into the apoplast. 16. Use the concepts presented in class and the text to discuss how Mg2+ might be acquired by the roots and subsequently transported to the young vegetative regions of the plant. Mg2+ channel?--probably Mg2+ transporter Mg2+ deficiency: necrosis of lower leaves easily remobilized to growing tissues passive transport? Photosynthetic light reactions 17. If a sample of algae, when illuminated, releases 3 x 10-5 mol O2/min, how rapidly will it incorporate CO2? How much starch will be formed per minute? How many photons of light will be needed per minute? (Equation 7.4) Assuming that all the NADPH synthesized by the electron transport chain is used to incorporate CO2 into carbohydrate, the rate of incorporation of CO2 will equal the rate of O2 synthesis.this will be true even if additional ATP is needed for the synthesis of starch from triose-p. The rate of CO2 incorporation may be lower if some of the NADPH is used for the reduction of NO3- to NH4+.
The number of light photons will be at least 8 for every O2 released (2 per electron, 4 per O, 8 per O2), but additional photons may be needed depending on the efficiency of coupling electron transport to ATP synthesis (on the need for cyclic photophosphorylation) and on the need for ATP in other metabolic reactions. So 24 x 10-5 mol photons/ min 18. Diagram a chloroplast. Draw and explain the protein complexes of the thylakoid membrane. Explain what happens at each complex in the course of non-cyclic and cyclic photosynthetic electron flow. See Fig. 7.16 and Fig. 7.18 and Fig. 7.22 19. Draw the Z scheme. Explain how this diagram differs from, and contributes to, your description of non-cyclic electron flow in question 18. (*previous typo) See Fig. 7.21 Higher energy state = lower redox potential (the larger negative values on y axis) measured in voltage Chain of redox reactions as lower potential reactants reduce next step s substrate 20. What is happening in the fall when the leaves change colors? Degradation and reallocation of chlorophyll while carotenoids and other pigments remain. Chlorophyll absorbs in red wavelengths Other pigments absorb in greener wavelengths Less chlorophyll lose red absorption more reds reflected leaf color appears red/orange/yellow 21. What is quantum yield? What is the maximum quantum yield for NADPH? For O2? For triose phosphate? Quantum yield is the moles of product formed per moles of quanta (photons) absorbed. O2: (Lecture 14 slide 19 // Figure 7.11) (p.178 // Lecture 16 slide 16) 8 photons activate 4 electrons through ETC, 4 electrons released when 2 molecules H2O oxidized to form 2 O atoms, 2 O atoms per O2 molecule. Plus, cyclic phosphorylation requires 1 photon. It takes about 10 photons to produce 1 molecule of O2, so its quantum yield is about 0.1 NADPH: (Lecture 16 slide 8) 4 photons activate 2 electrons Z scheme 1 H+ bound in 1 NADPH So quantum yield NADPH = ¼ = 0.25 Triose phosphate: (Lecture 17 slide 5) 6 ATP to produce 6 triose phosphate. = 3 ATP to produce 3 triose phosphate 14 H+ per 3 ATP 14 H+ from 2 NADPH plus 2 from cyclic phosphorylation From above, 4 photons per NADPH = 8 photons for 2 NADPH, 14 H+ So quantum yield triose phosphate = 3 / 8 = 0.375 22. Explain why the most efficient photosynthetic reaction in higher plants requires the absorption of 9-10 photons per O2 molecule formed. The number of light photons will be at least 8 for every O2 released (2 per electron, 4 per O, 8 per O2) from noncyclic electron transport, but additional photons may be needed depending on the efficiency of coupling electron transport to ATP synthesis (on the need for cyclic photophosphorylation) and on the need for ATP in other metabolic reactions.
23. Compare the energy of a photon of light at 700 nm and the energy needed to move an electron from H2O to NADP+. Suggest reasons why two photons are needed for that reaction. Energy of a photon, E = hc/λ, using = 700 nm, E = (4.14 x 10-15 ev-s)(3.00 x 108 m/s)/(700 x 10-9) m (Lecture 15 p 5) E = 1.76 electron volts per photon. (Lecture 15 p 5) Energy to move an electron from H2O to NADP+ is [0.82 (-0.32) = 1.14 electron volts] (Lecture 15 p 5) Refer to Z scheme in Figure 7.21 of text: energy lost in indirect transfer of electron from H2O to NADPH 24. Explain how plants absorb light at many wavelengths of the spectrum. Calculate the energy carried in a mmol of light photons at 528, at 620, and at 700 nm. Describe the probable path by which that energy could be used to power photosynthetic electron flow. The absorption of different wavelengths occurs because of (a) alterations in the environments of chlorophyll molecules (the electronic transitions are affected by environment); (b) the inclusion of chlorophylls a and b, which have different absorption peaks, in the antennas; (c) the presence of various carotenoids in the antennas. The energy in a mmol of photons of 528 nm is 225 J/mmol; 620, 192 J/mmol; 700, 170 J/mmol. Energy absorbed in the blue, green, or orange regions of the spectrum will be transferred by fluorescence or, preferably, by resonance energy transfer to the reaction center of P680 (PS II) or P700 (PSI), where it excites an electron to move to an acceptor. 25. Some herbicides block electron transport in the light reactions. Explain how and why these are effective herbicides. DCMU is an example described in class. Will DCMU inhibit the production of NADPH, ATP, or both? Explain. DCMU blocks the transfer of electrons from PSII to PSI, so it will inhibit NADPH production AND H+ transfer to the lumen, thus ATP production. 26. Some herbicides block electron transport in the light reactions. Paraquat is an example described in class. Will Paraquat inhibit the production of NADPH, ATP, or both? Explain. Paraquat blocks the transfer of electrons from PSI to reduce NADP+ to NADPH. So it will inhibit NADPH production. PSI does not pump protons across the membrane, so ATP production can continue. 27. Explain the Mitchell theory and how it is related to photophosphorylation. Aka the chemiosmotic hypothesis. See Ch.11 (p. 238) 28. Using the concepts described in lecture, explain how the addition of ATP to vesicles containing ATP synthase can form a ph gradient across the vesicle membrane. Will the ph inside the vesicle be higher or lower than outside? Adding ATP to vesicles will drive the ATP synthase backwards. If the ATP is inside the vesicles, the ATP synthase will pump H+ out of the vesicles, creating a gradient with higher ph inside the vesicles. 29. Do you think chloroplasts lacking the outer envelopes, when prepared for in vitro studies, would make ATP from ADP and inorganic phosphate better than chloroplasts with intact outer envelopes and stroma? Would chloroplasts lacking outer envelopes and stroma fix CO2?
Chloroplasts lacking outer envelopes may make ATP more inefficiently due to difficulty forming a sufficiently strong H+ concentration gradient in a large volume of solution. Chloroplasts lacking outer envelopes would likely be unable to fix CO2, as light-dependent stromal changes to ph and Mg2+ facilitate Rubisco activation. 30. Explain why cyclic electron flow is thought to be necessary for photosynthesis. If it can be shown that under optimum conditions the ratio of ATP synthesis to NADPH synthesis is 3:2, is cyclic electron flow necessary? Can you think of experiments to demonstrate cyclic electron flow in vitro and in vivo? 31. If the ratio of NADPH to NADP+ in chloroplasts in the light is 10, compute the actual oxidation-reduction potential. Eo for the NADPH/NADP+ redox couple is -0.32 mv. SKIP. This concept is inadequately covered in lecture & the text. C3 carbon fixation 32. Describe the Calvin cycle and explain how it is connected to the light reactions. See chapter 8 of text (p. 146) The connection is the ATP and NADPH needed for the Calvin cycle reactions. 33. Suggest two mechanisms by which an increased light intensity could increase the rate of photosynthesis (1) Increased rate of synthesis of ATP and NADPH, stimulating Calvin Cycle reactions (2) Activation of rubisco by rubisco activase (stimulated by ATP and Mg2+ uptake) (3) Reduction of photosynthetic enzymes by ferridoxin. 34. In comparisons of photosynthesis with respiration, the process of photosynthesis is usually described in terms of the synthesis of glucose: 6 CO2 + 6 H2O --> C6H12O6 + 6 O2. How accurate is this characterization? How would you change it? Glucose is almost never the product of photosynthesis in plants. The description in the textbook of the Calvin cycle describes the product and triose-p, and this too is never a final product. Under most conditions of active photosynthesis, triose-p will be converted to glucose-p, and the glucose-p will be polymerized to form starch, visible as large starch grains in chloroplasts. Alternatively, the glucose can be exported from the chloroplast and used to synthesize sucrose, which in turn can be exported from the cell to phloem. 35. In many plants, a large fraction of the protein is RuBisCO. For instance, in bean leaves about half the protein is RuBisCO. Suggest two reasons why plants might have so much resource invested in this protein. a) RuBisCO is essential for energy and growth, but inefficient b) RuBisCO has relatively low affinity for CO2, so high concentrations assure it is never limiting c) It is used as a form of storage of nitrogen. (Evidence: Some plants, like beans, have much more rubisco than other plants that grow as well. In many biennial/perennial plants, rubisco is metabolized and the products are transported to storage in the roots.)