PMB 4412 Exam 2

two types of energy converting organelles

• chloroplasts

  • plastids, found only in plants and algae

• mitochondria

both are separated from the cytosol by a double membrane

characteristics of mitochondria and chloroplasts

semi-autonomous

• divide by fission

• contain circular chromosomal DNA, located in nucleoids within the stroma and matrix

• contain ribosomes, tRNAs

• depend on import of nuclear encoded proteins for many functions

• generate transmembrane hydrogen gradient and use gradient to make ATP

overall formula for photosynthesis

6 CO2 + 6 H2O → C6H12O6 + 6O2

what happens during the light reactions of photosynthesis?

energy from light is used to phosphorylate ADP (produce ATP) and to reduce NADP+ to NADPH

in chloroplasts, the light reactions drive _______ of the thylakoid lumen

acidification

where is ATP generated in chloroplasts? where is ATP generated in mitochondria?

in chloroplasts = stroma

in mitochondria = matrix 

A __________ can be formed when membranes allow selective permeation

diffusion potential

exists until chemical equilibrium is reached → then diffusion potential equals 0

diffusion is passive transport

what is the plant cell membrane potential inside relative to outside?

negative inside, positive outside

H+ ATPase pumps protons out

how does inhibiting ATP synthesis affect membrane potential?

membrane is depolarized

• H+ ATPase pumps out protons, makes the inside of the membrane negative and the outside positive

• turning off ATP synthesis mean means no more ATP hydrolysis, H+ ATPase becomes inactive

• inside of the membrane becomes more positive

what forms of transport across membranes are passive?

• diffusion

• channels

• uniporter: binds substrate on one side, changes conformation, releases substrate on other side

what forms of transport across membranes are active?

• pump

• symporter

• antiporters

• use energy to move something against its electrochemical gradient

molecular structure of H+ ATPase

• both N and C terminus in cytoplasm

• several transmembrane domains

  • transmembrane domains have occasional charged amino acids embedded in the membrane

  • these charged amino acids have special functions

• regulatory domains within the cytoplasm

  • have bindings sites for Mg2+

  • phosphorylation domain

structure of H+ ATPase in vacuole membrane

• domains in cytoplasm spin 

• channel allows proton flow from the cytoplasm to the lumen of the vacuole

• hydrolyze ATP to move protons

• process is reversible, based on stoich of protons to ATP

difference between H+ ATPase and ATP synthase

H+ ATPase hydrolyzes ATP to pump protons against their gradient -→ out of the cell or into the vacuole

ATP synthase makes ATP using the power of protons moving down their gradient

structure of ATP

• five carbon ribose sugar

• adenine nitrogenous base

• 3 phosphates

3 phosphate linkage is unstable because negative charges are next to each other → hydrolysis

what are standard conditions?

25 degrees Celsius

1 M products and reactants

• not cellular conditions

what is Keq?

concentration of products over concentration of reactants AT EQUILIBRIUM

• when Keq is big = more products than reactants at equilibrium

• when Keq is small = more reactants than products at equilibrium

ATP hydrolysis under standard conditions

ATP → ADP + Pi

• Keq is large = ATP is unstable = wants to lose P

• standard conditions are not cellular conditions

energy of ATP hydrolysis in the cell (no electrical component)

• dependent on concentration of ATP and hydrolysis products

how do we consider just the electrical component of membrane potential?

energy required to transport one mole of charge against a membrane potential 

how do we consider just the concentration component of membrane potential?

energy required to transport one mole of solute against a concentration gradient

what is equilibrium potential?

membrane potential at which the given ion concentrations are at equilibrium

how do we determine the equilibrium potential for a given ion?

• Nernst potential

• Walther Hermann Nernst

• takes into account electrical and concentration components

graphing of equilibrium potentials

• at zero, no net flux of the ion

• slope of the line gives the conductance of the channels mediating the current

• for cations

  • movement out of the cell = outward current = positive sign

  • movement into the cell = inward current = negative sign

• for anions

  • movement out of the cell = negative sign

  • movement into the cell = positive sign

how do voltage-gated channels sense changes in voltage?

charged amino acids within the transmembrane spans sense changes in voltage of the membrane

channels change conformation in response to voltage changes

how can we use equilibrium potential graphs to determine what kind of channels ions are moving through?

• outward channels open when membrane potential exceeds equilibrium potential for potassium → potassium moves out

• inward channels open when membrane potential is below equilibrium potential for potassium → potassium moves in

“goal” = get the membrane potential as close to the ion equilibrium potential as possible

what is channel gating?

opening and closing of ion or solute channels

involves protein conformation change

• once open, ions or solutes are conducted at very fast rates

what is voltage dependence?

regulation of the channel by membrane potential

open/close in response to voltage

how can we measure ion channels?

• patch clamping

  • discovered by Erwin Neher and Bert Sakmann

• electrophysiology methods

what is the first key reaction of photosynthesis?

splitting water

• 2 H2O → 4 e + 4 H+ + O2

• done by O2-evolving complex that contains Mn

• O2 is a waste product

• negative redox potential

what is redox potential mean?

• negative redox potential = more willing to give up electrons

• positive redox potential = more willing to take up electrons

electrons flow spontaneously from negative redox potentials to positive redox potentials

in the light reactions of photosynthesis, hydrogen is stockpiled into the __________, flows out the ATP synthase channel into the ________, and makes ATP in the __________

thylakoid lumen

stroma

stroma

PSII is located primarily in the ________________

stacked grana

PSII

1. light oxidizes P680

2. P680 is reduced by the electrons from splitting H2O, protons from water are left in lumen

3. Reduced P680 passes electrons to a pheophytin (chlorophyll)

4. Pheophytin passes electrons to plastoquinones QA and QB

5. QB is reduced by 2 electrons and 2 protons → protons are taken from the stroma

6. Cytochrome b6f takes electrons from QB, the protons from QB are pumped into the lumen

• 4 H+ pumped in for each 2 electrons that go through cyt b6f

7. cytochrome b6f passes electrons to plastocyanin (final electron acceptor of PSII

4 ways protons are moved into thylakoid lumen during PSII

1. splitting of water by oxygen-evolving complex → protons in the lumen

2. plastoquinones take up protons from the stroma → protons removed from stroma

3. protons are pumped into the lumen when plastoquinones pass electrons to cyt b6f → protons in the lumen

4. protons are used to reduce NADP+ in the stroma → protons removed from the stroma

final electron acceptor of PSII

plastocyanin

initial electron donor of PSII

H2O

initial electron donor of PSI

plastocyanin

where is PSI and ATP synthase?

stromal thylakoids

• unstacked regions

PSI

1. light oxidizes P700

2. plastocyanin reduces P700

3. P700 reduces A0 quinone

4. A0 quinones transfers electrons through a series of FeS proteins

5. FeSB reduces soluble ferredoxin

6. reduced ferredoxin transfers electrons to FNR

7. FNR reduces NADP+ to NADPH → removes protons from the stroma

what are the products of the light reactions?

O2

NADPH → used in the Calvin cycle to reduce CO2

ATP

ultimate electron donor for the light reactions?

H2O

ultimate electron acceptor for the light reactions?

NADP+

how does the ATP synthase use the proton gradient to make ATP?

proton concentration is much higher in the thylakoid lumen than in the stroma

protons move down their gradient from thylakoid lumen to the stroma through a channel in the ATP synthase

proton motive force powers the phosphorylation of ADP into ATP in the stroma

important features of thylakoid structure

• continuous thylakoid lumen

• extensive contact and continuity between stromal and thylakoid membranes

what ion is required for the splitting of H2O in PSII?

need 4 Mn ions

what does FNR stand for?

ferredoxin NADP+ reductase

FNR reduces NADP+ to NADPH → removes proton from stroma and gives NADP+ electrons

what is cyclic electron transport?

chloroplast can redirect electrons from ferredoxin to cyt b6f to make more ATP rather than making more NADPH

function of the light reactions of photosynthesis

generate ATP and NADPH

function of the carbon reactions of photosynthesis

fix CO2 and regenerate ribulose-1,5-bisphosphate

requires NADPH and ATP

light reactions occur on ______________

thylakoid membrane

carbon reactions occur in the _________

stroma

NADP+ is reduced to NADPH during __________ in the ___________

PSI
stroma

what is FNR?

protein in the stroma 

exists as either a soluble monomer (inactive) or a thylakoid bound dimer (active)

NOT A TRANSMEMBRANE PROTEIN

contains a FAD cofactor → uses reducing power of two ferredoxin to reduce NADP+ → NADPH

how did Melvin Calvin discover the carbon reactions?

• used radioactive carbon dioxide C14

• give to algae

• rapid sampling

• 2D chromatograph to identify metabolic intermediates

what is the first stable product of carboxylation in the carbon cycle?

3PGA

ribulose-1,5-bisphosphate + CO2 → Rubisco → 3PGA

where do the carbon reactions (Calvin cycle) of photosynthesis happen?

stroma

where is Rubisco found?

chloroplast stroma

what are the substrates and products of Rubisco?

ribulose-1,5-bisphosphate + CO2 → 3PGA

what are the three stages of the Calvin cycle?

1. Carboxylation of ribulose-1,5-bisphosphate with CO2 to 3PGA

2. reduction using ATP and NADPH to G3P and DHAP → can be used to regenerate ribulose-1,5-bisphosphate or to make sucrose, starch

3. regeneration of ribulose-1,5-bisphosphate using ATP

what are the triose phosphates?

DHAP and G3P

what are the two uses of G3P?

1. used to make sucrose and starch

2. combined with DHAP to regenerate ribulose-1,5-bisphosphate

where is starch synthesized?

stroma

where is sucrose synthesized?

cytoplasm

DHAP and G3P transported to the cytoplasm in exchange for phosphate

then made into sucrose

what does Rubisco mean?

ribulose-1,5-bisphosphate carboxylase/oxygenase

what activates Rubisco?

• increasing stromal pH

• increasing concentration of Mg2+ in the stroma

• four light regulated enzymes in the Calvin cycle are activated by the ferredoxin-thioredoxin pathway → ensures that Calvin cycle happens when light reactions are happening

why does increasing Mg2+ concentration activate Rubisco?

• light reactions create a membrane potential across the thylakoid

• membrane potential is negative on stromal side

• Mg2+ is driven from thylakoid lumen to negative stroma

what are the substrates and products of Rubisco when it is acting as an oxygenase?

ribulose-1,5-bisphosphate + O2 → Rubisco → Phosphoglycolate + 3PGA

PHOTORESPIRATION

when is photorespiration more dominant?

high temperatures and dry conditions

more O2 than CO2 in solution at high temperatures

stomatal pores close in drought to conserve water → CO2 in leaves is low

why is photorespiration wasteful?

because we need to convert glycolate to a useful form and carbon is lost in the process →turns glycolate into 3PGA

requires 2.5 ATP and NADPH per glycolate because a CO2 is lost in the process and 1 ATP is required to make 3PGA

what is the primary carboxylation for C3 photosynthesis?

ribulose-1,5-bisphosphate + CO2 → Rubisco → 3PGA

• happens in the stroma

what is the goal of C4?

avoid photorespiration by separating carboxylation from Rubisco

what is the primary carboxylation for C4 photosynthesis?

HCO3- + PEP → PEP carboxylase → oxaloacetate

• occurs in mesophyll cells

• oxaloacetate converted to C4 acid and is exported to bundle sheath cells

where does the Calvin cycle happen in C4 photosynthesis?

oxaloacetate made in the mesophyll cells are exported to bundle sheath cells

• C4 acid is decarboxylated

• decarboxylated product is combined with CO2 and carbon cycle starts

what is the cost of C4 photosynthesis?

• you have to accumulate CO2 in bundle sheath cells → 2 ATP per CO2

why do bundle sheath chloroplasts have little PSII?

PSII begins with splitting of H2O

produces O2

high levels of O2 = photorespiration = defeats the purpose of C4 photosynthesis

how is PEP carboxylase regulated?

light regulated

in the light → phosphorylated by PEP carboxylase kinase→ activated

how is pyruvate dikinase regulated?

pyruvate dikinase

• used to regenerate PEP

regulated by light through phosphorylation

• phosphorylated in the dark = inactivated

• ADP is the phosphate donor = generates AMP

• when photosynthesis is low, high levels of ADP = pyruvate dikinase inactivated in the dark

what is Kranz anatomy in plants?

in C4 plants

• enlarged bundle sheath cells surround vascular bundles → high concentration of CO2 in the bundle sheath cells → very little PSII, very little stacking

• mesophyll cells surround the enlarged bundle sheath cells

• in C3 plants, mesophyll cells are adjacent to the vascular bundles

function of CAM

• separates Calvin cycle and carboxylation by time

• prevent water loss in arid environments

• open stomata during cool night and close stomata during hot day

night in CAM plants

• stomata are open

• PEP is made from broken down starch, exported from chloroplast to cytoplasm

• CO2 comes in → PEP + HCO3- decarboxylated by PEP carboxylase→ C4 acid stored in vacuole

• pH is low

day in CAM plants

• stomata are closed

• C4 acid is exported from vacuole to chloroplast

• C4 acid is decarboxylated and Calvin cycle happens

• pH is high

cost of CAM photosynthesis

• ATP is required to transport C4 acid in and out of the vacuole

• ATP is required to regenerate PEP

• CO2 uptake is limited by vacuole storage space

why is excess light dangerous to plants?

excess light causes the creation of reactive oxygen radicals

over-excitation of PSII can transfer excess energy to O2 to create a free radical

can cause cellular damage

how do carotenoids protect against excess light?

• carotenoids are in LHCII

• carotenoids quench the excited state of chlorophyll and dissipate energy as heat

• violaxanthin is converted to zeaxanthin, releases energy as heat

• protects against formation of free radicals from light damage

why is carotenoid protection from excess light important during water stress conditions?

• in water stress, stomata is closed and CO2 photosynthesis is limited

• good way to dissipate heat

what is non-photochemical quenching?

quenching of chlorophyll fluorescence by processes other than photochemistry

what is the xanthophyll cycle?

in high light

violaxanthin converted to zeaxanthin

allows for dissipation of heat energy

quenched state of PSII  = zeazanthin

unquenched state of PSII = violaxanthin

what is photoinhibition?

inhibition of photosynthesis by excess light

D1 membrane protein in PSII is a primary target for photoinhibition

• D1 has a high turnover

• susceptible to oxidative damage

how does LHCII movement prevent high light damage

• when tons of light energy is going towards PSII

• reduced plastoquinone accumulates

• kinase phosphorylates LHCII

• phosphorylation causes LHCII to move out of stacked grana (PSII) to unstacked regions (PSI)

• directs absorbed light energy to PSI

• prevents creation of oxygen free radicals

how is heat dissipated from leaves?

• evaporative heat loss: H2O evaporation through stomata

• sensible heat loss: directs heat loss to the air

how does isoprene production dissipate excess heat?

• high temperature causes isoprene to be made from terpenes

• isoprenes stabilize photosynthetic membranes and are released into the atmosphere

examples of sources

• mature leaves

• starch

examples of sinks

• fruit

• flowers

• new leaves

• roots

characteristics of sieve cells

• no nucleus

• no ribosomes

• no vacuole

• no microfilaments

• no golgi

• modified endoplasmic reticulum

• separated by sieve plates with pores

characteristics of companion cells

• numerous mitochondria

• supply sieve elements with ATP, proteins, and RNA

• connected to sieve elements by plasmodesmata

mechanisms for phloem loading

• passive/symplastic: cells of mesophyll are connected to phloem and companion cells through plasmodesmata → sugar concentrations are equal in mesophyll and phloem in leaves

• polymer trapping: all cells connected by plasmodesmata but not all plasmodesmata have same size exclusion limit, higher order oligosaccharides are made in companion cells → higher order oligosaccharides are trapped, can only move forward into the phloem

• active/apoplastic: sieve cells and companion cells are connected by plasmodesmata but not connected to the rest of the mesophyll → requires transmembrane loading, SWEETs and SUCs/SUTs

what is polymer trapping?

• all cells connected by plasmodesmata

• not all plasmodesmata have the same size exclusion limit

• higher order oligosaccharides are synthesized in companion cells

• forces sugars to move forward into the phloem, cannot go backward

• sucrose + galactose → raffinose

• raffinose + galactose → stachyose

• sucrose, raffinose, and stachyose are transported into the phloem

what are SWEETs?

• sucrose uniporters

• passive transport

• usually exporters because concentration of sucrose is higher inside the cell than outside