A2 OCR Biology Unit F214 Communication, H C i ti Homeostasis and E t i d Energy

Module 3: Photosynthesis Module 4: Respiration

Phil Simpson ZigZag Education, 2009
Photocopiable/digital resources may only be copied by the purchasing institution on a single site and for their own use

Introduction
Photosynthesis and respiration are fundamental biological processes photosynthesis produces the carbohydrate and oxygen that plants animals and many microbes rely upon respiration releases the energy stored in organic molecules in a usable form Photosynthesis Respiration Click on the button to select the topic

Photosynthesis
Select topic by clicking on the button General introduction Photosynthesis and chloroplast structure LightLight-dependent and independent stages Factors affecting photosynthesis Measurement of photosynthesis Extension material Summary

Introduction
Photosynthesis is probably the most important biological process process. Without it, where would organisms get the carbohydrate and oxygen they need for respiration? With the exception of chemosynthetic bacteria, all other organisms ultimately depend upon plants and their photosynthetic activity. So, the ultimate source of the energy used by most , gy y organisms is the sun, and photosynthesis forms the basis of most food chains.

You may be familiar with the terminology already, but: Autotroph They are self-feeders, using simple selfinorganic molecules to build complex organic molecules. Uses light energy to fix CO2 into sugar sugar. Uses chemical energy to fix CO2 into gy sugar, e.g. nitrifying bacteria. nitrifying Other feeders that must have complex organic molecules like carbohydrates, lipid and p p protein in their food.

Photoautotroph Chemoautotroph p Heterotroph

The ultimate source of energy is the sun! Plants harvest solar energy and convert it i t Pl t h t l d t into chemical potential energy of photosynthetic products. These are a source of energy for the plants themselves and for the heterotrophs that feed off the plants. Respiration converts the chemical potential energy of organic matter into chemical p g potential energy of ATP. gy

Solar energy

Autotrophs trap light energy, p g gy converting it into chemical energy of organic molecules

Heterotrophs animals, fungi and bacteria g consume organic nutrients carbohydrate, lipid and protein

Respiration provides usable energy for cells ATP produced and stores energy in its bonds Heat energy lost/released because processes are inefficient Work uses the energy contained in the bonds of ATP

Photosynthesis and chloroplast structure

Photosynthesis is the process whereby green plants make sugar (glucose) from carbon dioxide and water water. Photosynthesis involves the capture of light energy, trapping of CO2 and its subsequent reduction into carbohydrate using hydrogen from water. 6CO2 + 6H2O + light -------> C6H12O6 + 6O2 -------> carbon dioxide + water + light ------> glucose + oxygen ------>

There are two sets of reactions in photosynthesis p y LightLight-dependent stage light energy is trapped by the photosynthetic pg pigments used to produce ATP and to split water into hydrogen and oxygen takes place in the membranes of th b f the chloroplasts LightLight-independent stage light is not necessary (hence used to be called the dark stages/reactions) energy trapped in the lightlight-dependent stages is used to reduce CO2 into sugar takes l t k place i th li id in the liquid component (stroma) of the chloroplast

Chloroplast structure
the chloroplast is an organelle found in photosynthetic eukaryotic cells it is where photosynthesis takes place light energy used to fix CO2 into sugar basic chloroplast structure was covered in Unit 1, so you should already be familiar with it

under the light microscope chloroplasts i hl l appear as biconvex discs (3(3-10 m diameter) little detail is evident

electron microscope has better resolution and magnification therefore more detail th f d t il can be seen
TEMofColeusblumei chloroplast.Starchgranulesand thylakoidsarrangedintogranaareclearlyvisible.Public domainimage:LouisaHowardandCharlesDaghlian

chloroplast are surrounded by a double membrane/envelope flattened sacs or thylakoids run through the internal solution or stroma the th sacs are stacked in t k di places. Here they are called grana

they are the site of photosynthesis. Grana trap light energy which is i used t fi CO2 i th d to fix in the stroma

Can you identify the key structures?

TEMofColeusblumei chloroplast.Publicdomainimage:LouisaHowardandCharlesDaghlian TEMofColeusblumei chloroplast.Publicdomainimage:LouisaHowardandCharlesDaghlian

Envelope. Double membrane that surrounds the chloroplast.

Starch grain. Starch is insoluble and so it does not affect the water potential. Thylakoid. Flattened fluid-filled sac. The membranes hold pigments and carriers. Granum. A stack of thylakoids. Light-dependent stage takes place here. Stroma. Contains the enzymes that fix CO2. lightindependent stage.

TEMofColeusblumei chloroplast.Starchgranulesandthylakoids arrangedintogranaareclearlyvisible.Publicdomainimage: LouisaHowardandCharlesDaghlian

Additional points
In the stroma are starch grains and lipids It also has small 18nm/70S ribosomes a loop of DNA What is the significance of each of these?

Starch grains and lipid droplets are for storage of photosynthetic product
they are osmotically inactive and so can be stored without affecting the water potential

DNA loop codes for some chloroplast protein

made by the chloroplast ribosomes other protein is coded for by the genes on the DNA in the nucleus

Chloroplast ribosomes and DNA are like that of p prokaryotes

suggests symbiotic origin

LightLight-dependent and independent stages

Photosynthesis is a two-stage process twoLightLight-dependent stage
Light is Li ht i needed t d d to produce ATP and to split water into hydrogen and y g oxygen. Reactions only take place in the presence of suitable pigments. Takes place in the thylakoid membranes.

LightLight-independent stage
Light is t Li ht i not needed ( d d (used d to be called the dark stages/reactions). g ) Energy trapped in the lightlight-dependent stages is used to reduce carbon dioxide to sugar. Takes place in the stroma stroma.

Click to select topic on aspects of the light-dependent lightand independent stages of photosynthesis LightLight-dependent stage Absorption and action spectra Cyclic photophosphorylation Non- li h t h Non N -cyclic photophosphorylation h l ti Photolysis of water LightLight-independent stage Effect of photosynthesis on Calvin cycle intermediates Mechanism of ATP production Sequencing the Calvin cycle

LightLight-dependent stage
Light energy is trapped by photosynthetic pigments
organic molecules which absorb certain wavelengths of light, but t th li ht b t not others absorbed light is used to produce usable energy light that is not absorbed is transmitted or reflected

Most of the pigment in the chloroplast is chlorophyll, of which there are two basic types
chlorophyll a and b

Chlorophyll
absorbs blue and red light strongly transmits or reflects green light
hence the green colour of most leaves

Photosynthetic pigments can be categorised as

primary pigment or accessory pigment

The primary pigments are called P700 and P680

the P stands for pigment the number is the wavelength of light they absorb maximally

P700 and P680 are different forms of chlorophyll a

Accessory pigment includes various forms of chlorophyll a, chlorophyll b and others (carotene, xanthophyll, etc.). p y ) Pigments are arranged into light-harvesting clusters lightknown as photosystems. In photosystems, several hundred pigment molecules absorb and then funnel or feed the light they absorb into i t a reaction centre which contains primary ti t hi h t i i pigment.
sometimes the main light harvesting pigment is referred to as antenna pigment

P700 is at the reaction centre of PS I, whilst P680 is at the reaction centre of PS II.

A light-harvesting cluster or photosystem

Light energy Energy Accessory pigment Primary pigment

The light-dependent stages include the production of lightATP and splitting of water (photolysis) to produce H+ ions ions The H+ ions combine with NADP, reducing it. The Th production of ATP can be either cyclic or nond ti f b ith li noncyclic, depending upon the pattern of electron flow in the photosystems in cyclic, they end up where they started from. in non-cyclic they dont! non-cyclic don t!

Absorption and action spectra ...

Photosynthetic pigments have particular lightlightabsorption characteristics
soso-called absorption spectra an absorption spectrum is essentially a graph of absorbance against wavelength of light

Chlorophyll absorbs light with wavelengths 400-700nm p y g g 400photosynthetically active radiation or PAR for short

Not all wavelengths are absorbed equally g q y

red and blue are absorbed strongly green is absorbed weakly most is reflected or transmitted

Re elative phot tosynthesis s

Relative absorbance a e

Chlorophyll a Chlorophyll b Carotene

400

500

600 Wavelength of light / nm

700

How are different wavelengths absorbed by the different photosynthetic pigments? Chlorophyll a and b have peaks of absorption in the blue and red regions of the spectrum with very little absorption in the green region. In contrast, carotene has a broad band of absorption from , p blue to green and extends the wavelengths that are available to the plant. It is likely that accessory pigments also protect the chlorophyll when light is very strong. What effect does wavelength (light quality) have on the rate ff ( ) of photosynthesis? Absorption spectra should not b confused with th action Ab ti t h ld t be f d ith the ti spectrum, which is a plot of photosynthesis against wavelength. wavelength Not surprisingly there are peaks of action surprisingly, (photosynthesis) where there are peaks of absorption!

Why do the leaves of trees turn yellow-orange in autumn? yellowThe Th magnesium i th chlorophyll i withdrawn so th i in the hl h ll is ithd the chlorophyll loses its green colour and the colour of the accessory pigment can be seen seen.

Cyclic photophosphorylation
Cyclic photophosphorylation only involves PSI Light is absorbed by pigments and passed to P700
This Thi excites an electron which moves t a hi h it l t hi h to higher energy state and are emitted from the chlorophyll a The electrons pass to an electron acceptor (FD) and then back to P700 via another electron acceptor (PQ) and a series of electron carriers which make up the electron transport chain or ETC As electrons travel along the ETC energy is lost, enough energy to generate ATP from ADP and inorganic P (Pi) i i

As ATP production is light driven, it is called photophosphorylation

FD

PQ E
e-

C P700 PSI

ADP+ Pi ATP

electrons transferred and ATP generated

light absorbed, electrons emitted

NonNon-cyclic photophosphorylation
This involves both PSI and PSII. The process has become known as the Z-scheme. ZLight is absorbed by both photosystems, electrons excited and emitted from the primary pigments and transmitted to the electron acceptors and transferred t itt d t th l t t dt f d along the ETC. P700 gets its electrons from PS II whilst P680 gets its electrons from the splitting of water.
PS II has a water-splitting enzyme water-

As in cyclic photophosphorylation
energy is released used to generate ATP from ADP and Pi

NADP reduced by e- and H+

FD

electrons transferred l f d photolysis of water

H2O 2e2e2H+ O2 ADP+ Pi P680 PSII ATP PQ E

NADP + 2H+ = reduced NADP

2eT C P700 PSI

light absorbed, electrons emitted

light absorbed, electrons emitted

Compare cyclic and non-cyclic photophosphorylation

FD NADP + 2H+ PQ H2O 2eADP ADP+ Pi 2H+ O2 P680 PSII ATP E 2eT C P700 PSI = reduced NADP

Cyclic Non-cyclic

Cyclic Photosystem used Requirements PSI

Non-cyclic PSI & PSII Light, ADP, Pi, water, NADP ATP, NADPH2, O2 Water splitting enzyme present associated with PSII

Light, ADP, Pi

Products

ATP

Other points

Photolysis of water
Photolysis of water is vital to photosynthesis. PSII includes a water-splitting enzyme: waterH2O ------> 2H+ + 2e- + O2 ------> The H+ ions combine with electrons from PSI and NADP to produce reduced NADP this is then used together with ATP in the light-independent stages to lightreduce CO2 into carbohydrate. carbohydrate

Photolysis of water and production of reducing power can be demonstrated by the Hill reaction DCPIP can reaction. substitute for NADP, receiving electrons and becoming reduced: g Oxidised O idi d DCPIP blue H2O Reduced DCPIP R d d colourless O2

LightLight-independent stage
The light-independent stage takes place in the lightstroma of the chloroplast
contains the necessary enzymes key enzyme is ribulose bisphosphate carboxylase (RUBISCO)

CO2 combines with ribulose bisphosphate to produce 2 molecules of glycerate phosphate (GP)
also known as phosphoglyceric acid/phosphoglycerate and PGA

GP is not a sugar, but is reduced into one using the energy and reduced NADP produced during the lightlightdependent stages

Calvin cycle:

CO2

RuBP

Unstable intermediate

GP

GP

redNADP redNADP ADP ATP Triose P NADP NADP ATP ADP+Pi Triose P

ATP ADP Pi ADP+Pi

Glucose, Glucose amino acids and lipids

Most of the triose produced is used to regenerate the CO2-acceptor ribulose bisphosphate. However
one turn of the Calvin cycle generates 1 spare fixed carbon two turns generate 2 spare fixed carbons (could be used to make acetyl CoA and then lipid or amino acids) three turns generate 3 spare fixed carbons or one spare triose six turns generate 6 spare fixed carbons or two spare triose enough for one hexose

Effect of photosynthesis on RuBP, GP and TP

To summarise:
during photosynthesis CO2 is added to ribulose bisphosphate by the enzyme ribulose bi h bi h h t b th ib l bisphosphate h t carboxylase (RUBISCO) produces an unstable C6 intermediate which splits into 2 molecules of glycerate phosphate GP then reduced to triose phosphate ( ) using ATP p p (TP) g and reduced NADP from the light-dependent stage lightsome of the TP used to regenerate the CO2-acceptor

Clearly levels of RuBP, GP and TP can fluctuate depending upon the prevailing conditions and the amount of photosynthesis occurring. occurring

Ribulose bisphosphate
The levels of RuBP are affected by a number of conditions
decrease when photosynthesis is rapid consumed as CO2 is added to it regenerated by th C l i cycle so l t d b the Calvin l levels reach a l h steady state

Ribulose bisphosphate carboxylase can also act as an oxygenase

hence RUBISCO
i.e. ribulose bisphosphate carboxylase oxygenase

adds O2 to RuBP instead of CO2

Oxygenase activity is wasteful of RuBP and energy

needs regenerating

Process is called photorespiration

can significantly reduce photosynthesis in C3 plants u de conditions under co d t o s where you would ot e se e pect ee ou d otherwise expect high rates of photosynthesis, namely:
high temperate high light intensity

Paradoxically, the high levels of photosynthesis under these conditions reduce the amount CO2 available and increase the amount of O2
makes it more likely that O2 occupies the active site of the enzyme
inhibits photosynthesis increases photorespiration

What type of enzyme inhibition is responsible for photorespiration? Explain. Competitive inhibition Oxygen and carbon dioxide compete for the active site What effect might photorespiration have upon crop p productivity? y Reduce it Photorespiration consumes RuBP, wasting the p , g energy that was used to make it. It also needs regenerating, this uses more energy Note: despite being called photorespiration, it is not respiration as it produces no ATP whatsoever.

Glycerate phosphate and triose phosphate

The concentrations of these two molecules are interlinked. When conditions are non-limiting the Calvin cycle nonoperates to produce glycerate phosphate which is then used to produce triose phosphate using ATP phosphate, and reduced NADP from the light-dependent stage. lightA steady state concentration of all Calvin cycle intermediates should be established until conditions change. g

Consider the example of a plant that has been in wellilluminated conditions and then the light is switched off.

Con ncentratio onofGPandTP

GP

TP

Inthelight

Inthedark

Inthelight

What happens to the concentration of GP and TP?

In the light:
concentrations of GP and TP reach a steady state.
Concentra ationofGP PandTP

GP TP

GP is made when CO2 is added to RuBP by the enzyme RUBISCO. TP is made from GP using ATP and reduced NADP from the light-dependent stage.

In the dark:
GP is still made (RUBISCO is still active) but it is not used to active), make TP
ATP and reduced NADP are not available. levels of GP increase and TP decrease. Note GP plateaus when all of the RuBP is consumed.

Inthelight

Inthedark

Inthelight

Mechanism of ATP production

The original idea for ATP production was:
highhigh-energy electrons are produced when light energy is b b d by i i absorbed b primary pigments i t electrons pass along an electron transport chain, losing energy as they go lost energy is coupled to ATP production

This is not quite what happens!

highhigh-energy electrons are emitted they do travel down the electron transport chain However, the energy in the electrons is used to pump H+ ions (protons) across the thylakoid membranes to build a proton gradient

Protons (H+ ions) diffuse back down the gradient

into the stroma through an ATP synthase generating ATP

This mechanism is known as chemiosmosis

first proposed by Peter Mitchell in 1961

Key theory/concept
the basis of the mechanism for ATP production in chloroplasts also the mechanism for ATP production in respiration

NADP

NADPH + H+

H+ FD E T 2eC P700 PSI H+ H+

ADP + Pi

H+ ATP

PQ 2eP680 PSII 2eH 2O 2H+ +

H+ O2 H+

H+ H+ H+ H+

In a nutshell:
transfer of electrons through the electron transport chain releases energy h i l
this energy is used to pump protons (H+ ions) out of the stroma and into the thylakoid space generates a region with a high concentration of H+ ions

photolysis of water also generates H+ ions H+ ions then flow back into the stroma
through a membrane protein
contains an ATP synthase so generates ATP

Sequencing the Calvin cycle

Calvin used 14C to identify and sequence photosynthetic products. 14C is a radioactive isotope of carbon. It behaves as 12C would behave but, being radioactive, its presence can easily be detected detected. 14CO was fed to algal cultures which fixed the 14CO 2 2 during photosynthesis in a special lollipop vessel lollipop vessel. Samples of algae were taken after varying periods of photosynthesis and the photosynthetic products identified.

Side view of the special lollipop S f vessel Port f P t for injecting the 14CO (as 2 14CO ) NaH 3

Front view of the special lollipop f vessel Port for injecting the 14CO (as 2 NaH14CO3)

Culture of unicellular algae either Chlorella or Scenedesmus

Hot alcohol to stop metabolism and extract

Li ight source

Li ight source

Hot alcohol to stop metabolism and extract

Calvin reasoned that 14C label would appear first, and in the largest amount, in the first products of photosynthesis. h t th i Label would then be passed on to the subsequent products of photosynthesis in the order in which they were produced. The biochemical sequence he identified is now commonly referred to as the Calvin or C3 cycle.

He separated and identified products of photosynthesis paper chromatography to separate autoradiography to locate and identify (chromatograms placed on photographic ( h t l d h t hi film/paper. Radioactivity caused fogging when it was developed) He produced graphs that clearly show the sequence of 14C fixation during photosynthesis

First product appears

Radio oactivity/cps

Glycerate phosphate followed by triose phosphate followed by

Glycerate phosphate Triose phosphate

Hexose, Hexose amino acids, etc acids etc.

Time

Note: N t sometimes th C l i cycle i called th C3 cycle b ti the Calvin l is ll d the l because the first stable product is glycerate phosphate

Factors affecting photosynthesis ...

Photosynthesis is affected by a number of factors Overall rate will be governed by the factor in least or shortest supply law of limiting factors Key factors include: Light Intensity Quality Carbon dioxide Temperature Water Nutrients

Click on the button to select the limiting factor of photosynthesis: Light intensity and wavelength Carbon dioxide C b di id Temperature Water Nutrients Interaction of factors Controlling p g productivity y

Light: quantity (intensity) and quality () (

At low light intensities increasing light increases photosynthesis At high intensities further increases have little effect However, at very high light intensities damage can occur and cause a d d drop i photosynthetic rate ( t in h t th ti t (not evident in this graph). It is possible to identify:
light compensation point light saturation point

At LSP, some factor other than light will be limiting, e.g. e g temperature or CO2?

Light intensity
Rate +

Light saturation point

The light intensity at which further increases in light intensity bring about no y g further increase in the rate of photosynthesis.

_ Light intensity

Light compensation point

The light intensity at which photosynthesis is exactly matched by respiration. Net photosynthesis = zero.

Wavelength
Light is absorbed by the plant's photosynthetic pigments Chlorophyll absorbs light with wavelengths 400400700nm (so-called Photosynthetically Active Radiation (soor PAR for short) Not all wavelengths are absorbed equally red and blue are absorbed strongly green is absorbed weakly most is reflected or transmitted Why do plants appear green?

Re elative phot tosynthesis s

Relative absorbance a e

Chlorophyll a Chlorophyll b Carotene

400

500

600 Wavelength of light / nm

700

Carbon dioxide ...

CO2 is a substrate for photosynthesis, so
increasing CO2 concentration increases photosynthesis.

Only happens up to a point

eventually increasing CO2 no longer brings about an increase i photosynthesis. i in h t th i

In the plot of CO2 concentration against photosynthesis it is possible to recognise:

CO2 compensation point CO2 saturation point

At CO2 saturation point some other factor is limiting

e.g. temperature or light? eg

photosynthesis

CO2 saturation point p

Photosynthesis is proceeding at maximum rate. Some factor other than CO2 is limiting g photosynthesis.

CO2 concentration

CO2 compensation point

As much CO2 is being produced by respiration as is being consumed by photosynthesis. Net photosynthesis = zero.

CO2 enters leaves through stomata (singular stoma)

stomata are usually found on the lower side of the leaf stomata are open during the day and closed at night

Under most circumstances CO2 limits photosynthesis Note: when plants are water stressed they close stomata
to li it t limit water loss t l but it also limits CO2 uptake therefore affects photosynthesis

Temperature
Photosynthesis is enzyme controlled. There are two important enzymes involved in photosynthesis:
ribulose bisphosphate carboxylase (RUBISCO) waterwater-splitting enzyme of PSII

Enzymes are affected by temperature

increasing temperature increases the rate of p photosynthesis up to an optimum y p p exact optimum depends upon the species
it is often around 25oC

There is a doubling of rate for every 10oC rise Q10 = 2

rate at t + 10C Q10 = rate at t C

Above the temperature optimum
photosynthesis declines
enzyme denaturation irreversible

Interestingly, temperature has little effect on photosynthesis at low light

Photosynthesis Doubling of rate per 10oC rise in temperature up to the optimum. Q10 of 2.

Optimum temperature
Photosynthesis proceeds at fastest rate.

Low t L temperature inhibits t i hibit the enzymes of photosynthesis.

Denaturation
Irreversible damage to enzymes means rate falls rapidly to zero.

Temperature / oC

Water
An adequate supply of water is needed:
water is a substrate in photosynthesis
however, most of the plants water requirements are to replace water lost by transpiration.

if water is in short supply the stomata close

this reduces photosynthesis.

In salt marshes there is plenty of water but plants suffer physiological drought:
plants cannot generate a sufficiently low water potential to absorb all of the water needed. they have xerophytic adaptations.

Nutrients
Plants need mineral nutrients for photosynthesis. May be needed:
to make chlorophyll for photosynthetic enzymes

Key minerals are: y Magnesium in chlorophyll i hl h ll Iron for chlorophyll ETC components

Interaction of factors
Photosynthesis is affected by a number of factors, the one in least supply limits the overall process Consider interaction of light intensity temperature CO2 concentration

photosynthesis

High CO2, 25oC

High CO2, 15oC Low CO2, 15oC Relative light intensity g y

Increasing light intensity increases photosynthesis until saturation is reached and some other factor becomes limiting Raising the temperature raises the rate of photosynthesis h t th i doubling it? Q10 = 2 Raising CO2 raises the rate of photosynthesis f it is the substrate

At low light intensities, increasing the temperature has no effect on photosynthesis gradient is the same and so the Q10 must be 1 Photochemical reactions are not affected by temperature (Q10 = 1) t t EnzymeEnzyme-controlled reactions are affected by temperature (Q10 = 2) this observation led early plant physiologists to suggest that photosynthesis consisted of lightlightdependent and light-independent stages/processes light-

Controlling productivity ...

With knowledge of limiting factors can increase crop yield by controlling limiting factors
i.e. remove the limitation by enhancing the factor.

Not practicable in the field

possible if crops under glass or plastic
in a greenhouse or a polythene tunnel.

Glasshouse cultivation has advantages

it produces a better yield; crops out of season so they warrant a premium price; crops can be grown in regions where they would not normally grow. yg

Light intensity and duration

Supplementary light
on dull days at either end of growing g g season

Carbon dioxide
Providing extra CO2 should boost photosynthesis If CO2 concentration > 0.5%, ,
can cause stomata to close have the opposite of the desired ff t! d i d effect!

Shade on bright days Provide the optimum intensity and duration for the crop

Temperature
Raise temperature by p y
limiting air movement transparent
lets light in, stops heat loss

Water/humidity
Automated watering: g
hydroponics
supply water and nutrients

sprinklers
to water

Extends growing season

supplementary heat blinds to shade and cool

humidifiers
maintain humid atmosphere keep stomata open

The control of abiotic factors is not cheap. Farmers must balance the extra costs of glasshouse cultivation with the extra revenue generated. If it costs more to increase yield than the extra y y yield g generates in income for the farmer, he will soon go out of business. Major costs j The structure
glass/poly tunnel

Major benefit j Extra revenue generated

premium price for crops out of season
Scottish strawberries in April salads through the year

Capital costs for

heating, cooling, ventilation and light units, blinds units blinds, watering/irrigation technology

Exotics grown locally

cuts down transport costs smaller CO2 foot print

Running costs g
heat, light, etc.

Pest control
conditions in glasshouses favour disease

Measurement of photosynthesis ...

It is possible to measure the rate of any reaction by measuring
the rate that a substrate disappears or the rate that a product appears

So, from the following equation th t summarises S f th f ll i ti that i photosynthesis 6CO2 + 6H2O + light energy C6H12O6 + 6O2 Which are the substrates? CO2 and water d t Which are the products? glucose/carbohydrate and O2 l / b h d t d

Traditionally, O2 evolution or CO2 consumption would be used to measure photosynthesis Suggest how each could be measured CO2 use pH change
CO2 is an acid gas remove it and pH i increases

colour change in bicarbonate indicator 14CO 2

O2 evolution bubble count collect gas evolved and measure volume oxygen electrode

Oxygen production can be measured using a photosynthometer O2 collects at the bottom of the flared capillary tube bubble of oxygen is drawn into the capillary tube by the syringe length measured against the scale
flaredendcapillarytube thermometer O2 bubble

syringe scale Ligh htsource shootofpondweed,e.g.Elodea pondwaterordiluteNaHCO pond water or dilute NaHCO3 beakerofwater/waterbath /

The distance moved is proportional to the rate of p photosynthesis, more specifically to the net oxygen y , p y yg produced by photosynthesis! What other information is needed to calculate the volume of oxygen produced and the actual rate of oxygen production? internal diameter/radius of the capillary tube crosscross-sectional area of the capillary tube time What formula would you apply to measure the rate of oxygen production? r2 x distance moved / time Do any factors need controlling so that we are confident in our estimate of photosynthesis? Working in pairs/small groups, describe and explain the effect of each factor and its control.

Factor Temperature

Effect if uncontrolled

Control measure

Light

Carbon dioxide

Other

Factor Temperature

Effect if uncontrolled Temperature influences photosynthesis increases if temperature rises but does not exceed the optimum. p Thermal expansion of gas. Variation in light results in variation in photosynthesis photosynthesis. Note: if light close to photosynthometer, it is likely to heat it. it Variation in CO2 produces variation in photosynthesis. CO2 is usually limiting, but if too high can cause stomatal closure. Amount of plant material Age of material Physiological state well watered or wilted healthy or chlorotic

Control measure Water bath or room temperature, with frequent checks on temperature. Choose optimum.

Light

Exclude extraneous light blinds to windows, room lights windows lights. Standardise wattage and distance from light. Choose saturating light. Immerse in HCO3- solution or allow HCO3- solution to equilibrate with air. Choose saturating CO2.

Carbon dioxide

Other

Standardise! Choose young, good condition material.

Hopefully, in addition to control of temperature, light and CO2 availability, you have decided to control the amount of plant material and its age/condition. A t f l t t i l d it / diti duplicate apparatus, but without the pondweed, could also be included as a control control.

It is possible to investigate the effect of varying temperature or light or CO2 whilst keeping the other factors constant constant. Suggest how you could vary the factor: Temperature
Use a water bath
thermostat control thermometer to check

Light
Vary distance from light
inverse square law I = 1/d2

Room temperature?

Neutral density filters

Light quality () (
Prism to split white light Monochromatic filters
need to check intensity with light meter

CO2
Hydrogen carbonate solution
aquatic p q plant
immerse in solution

terrestrial plant
solution equilibrates with l ti ilib t ith atmosphere

Extension material ...

Examiners sometimes use unfamiliar material to test understanding and application of knowledge. Three examples are given:

Accessory pigments and algal zonation Leaf structure adaptations to photosynthesis and gas exchange Sun and shade adaptations of leaves

Accessory pigments
You already know that: Pigments are classified as either primary pigments (P680 and P700) or accessory pigments Accessory pigments are often present but their colour is i masked b th d k chlorophylls k d by the darker hl h ll
xanthophyll (pale yellow) and carotene ( t (orange) (orange)

Some other accessory pigments are seen more easily in the algae
seaweeds show a zonation of types with depth

Green algae
possess chlorophyll need large amounts of red and blue light green algae near the surface

Brown algae B l
accessory pigment is coloured brown (fucoxanthin) red and blue light absorbed more efficiently brown algae at depths 10-25m g p

Red algae
accessory pigment is coloured red (phycoerythrin) absorb and use the mainly blue light found at depth red algae at greater depths

Adaptation of leaf for photosynthesis

You are familiar with features of gas exchange surfaces and adaptations of specialist cells like palisade, xylem and phloem. I small groups di li d l d hl In ll discuss how the leaf is adapted for photosynthesis. Consider general leaf features as well as specific detail, e.g. palisade mesophyll.

Leaf structure
The leaf is the main photosynthetic organ Basic leaf structure was covered in Cells, Exchange and Transport. Briefly: It is broad and thin
large surface area short diffusion path

It has a network of veins/vascular tissue

transport nutrients and water to leaf transport photosynthate away from leaf

It has chlorenchyma thats cells with p , p y chloroplasts, i.e. photosynthetic tissue Stomata are present for gas exchange

it must absorb light it must absorb carbon dioxide and remove waste oxygen

contains pigments large surface area thin leaf minimises diffusion distances gas exchange pores or stomata are present vascular tissue present to transport water and organic nutrients

it must have a water supply and be able to export organic nutrients it has made

upper epidermis

vascular bundle collenchyma

palisade mesophyll spongy mesophyll lower epidermis with stomata xylem

parenchyma

phloem

palisade mesophyll
long cylindrical cells, fewer cell walls for light to arranged at right angles to pass through and be upper epidermis attenuated thin cell walls long narrow air spaces between the cells numerous chloroplasts restricted to a thin layer of cytoplasm cyclosis low diffusion resistance large surface area for gas exchange rapid photosynthesis large vacuole pushes chloroplasts to periphery prevents damage due to excessive light

spongy mesophyll

irregular shape and irregularly packed p present, but chloroplasts p fewer in number moist cell walls help with gas exchange

large air spaces and large surface area for gas g exchange photosynthesis but less p y than in palisade create humid internal atmosphere and so increases chance of water loss from the leaf

xylem

cells elongate, dead h ll hollow cylinders j i d li d joined end-toend-to-end walls strengthened with lignin pits present

fine tubes aid water flow rather like pipe sections th lik i ti prevents collapse during transpiration allows movement of water and dissolved minerals out of xylem i l t f l

phloem

translocates organic nutrients cells elongated thin cytoplasm sieve plates connect the cells

this is an active process, requiring energy fine tubes help flow low resistance to flow form long tubes for transport and allow free llow movement mo ement from one sieve tube to another

stomatal structure
guard cells surround th d ll d the stoma (singular) each guard cell is sausagesausageshaped and contains chloroplasts cell wall facing the stoma is thicker than the rest of the cell wall during opening, the guard cell absorbs water thick inner cell wall resists stretching so cell becomes more sausage-shaped sausagesausage-shaped guard cells contain chloroplasts

thin outer cell wall

thicker cell wall facing the stoma

Sun and shade-adapted plants shadeSome plants are adapted to bright light whilst others are adapted to low light. Even on the same plant:
some leaves will be adapted to bright light
e.g. those i th crown and e.g. th in the d

some to shade
e.g e g. those at the base e.g.

What adaptations do you expect sun-adapted and sunshadeshade-adapted leaves to have?

Photosynthesis

Sun leaf note the relatively high compensation point and saturation point

Shade leaf note the relatively low compensation point and saturation point. Also the dip at high light intensities

Light intensity / relative

SunSun-adapted Smaller surface area Leaf blade is thicker Palisade layer may b P li d l be 23 cells thick Fewer chloroplasts Less accessory pigment High LCP High LSP

Shade-adapted ShadeSurface area greater Thinner leaf blade Palisade layer only 1 P li d l l cell thick More chloroplasts More accessory pg pigment Low LCP Low LSP (may even suffer at the highest intensities)

Summary
Photosynthesis is an y important biological process
it produces the carbohydrate and oxygen relied upon by many organisms takes place in chloroplasts

Chloroplasts are organelles

surrounded by an envelope membranous thylakoids contain the photosynthetic pg pigment and electron transport chain thylakoids grouped into grana liquid li id component or stroma t t contains Calvin cycle enzymes

Green plants are autotrophs p p

selfself-feeders make their complex organic nutrients from CO2 and water ti t f d t use light energy to drive the p process

Animals, fungi, protoctists and most bacteria are heterotrophs h t t h

must have organic nutrients supplied in their food pp

Photosynthetic pigments are organic molecules

absorb some wavelengths of b b l th f light but not others include chlorophyll, carotene p y and xanthophyll

Photosynthesis is a two-stage twoprocess

primary pigments are P680 and P700

types of chlorophyll a

accessory pigments absorb light energy

pass it to primary pigment

return to P700 via electron transport chain lost l t energy li k d t ATP linked to production

nonnon-cyclic photophosphorylation
involves both PSI and PSII light absorbed by PS
highhigh-energy electrons emitted

LightLight-dependent stage
takes place in the thylakoids
light energy is harvested used to produce ATP and reducing power (reduced NADP)

PSII electrons pass to PSI

via electron t i l t transport chain t h i lost energy linked to ATP production

cyclic photophosphorylation
involves light-harvesting lightclusters called photosystem I P700 as primary pigment light absorbed passed to P700 high energy electrons emitted

electrons re-energised by PSI repass NADP

PSII has water-splitting waterenzyme: H2O 2H+ + 2e- + O2 electrons from water replace those lost by P680 H+ pass to NADP, reducing it

Chemiosmosis responsible for ATP production

energy from electrons used to pump H+ into the thylakoids from the stroma
generates proton gradient

splits into 2xC3 (GP)

GP then reduced to triose phosphate (TP)

uses ATP and reduced NADP

H+ flows into stroma via ATP synthetase

LightLight-independent stage
takes place in the stroma uses ATP and reducing power from the lightlightdependent stage In the Calvin cycle, CO2 is added to ribulose bisphosphate uses ribulose bisphosphate carboxylase (RUBISCO) b l C6 intermediate produced

most TP used to regenerate RuBP, but some used for carbohydrate, carbohydrate lipid and amino acid synthesis

Photosynthesis affects levels of Calvin cycle intermediates

in steady-state conditions steadyreach an equilibrium

changing conditions, results in change

e.g. go from light to dark RUBP,GP and TP

Paradoxically, Paradoxically conditions that favour photosynthesis can actually reduce it!

RUBISCO is both a carboxylase and oxygenase

competition for active site when CO2 is low and O2 is high it is an oxygenase consumes RuBP and is wasteful of energy

at CO2 saturation, another factor limits photosynthesis

Light intensity
as light intensity increases photosynthesis increases light compensation and saturation points are evident at light saturation point, another factor limits photosynthesis

A variety of factors affect photosynthesis

the one in least supply limits the overall process CO2 concentration, light intensity and temperature are key factors CO2
photosynthesis i h h i increases as CO2 concentration increases it is a substrate CO2 compensation and saturation points are evident

Temperature
at low temperatures photosynthesis is limited, raising t i i temperature t increases rate to an optimum above optimum rate declines due to enzyme denaturation Note: at low light intensities temperature has no effect
photochemical processes are not affected by temperature

Investigating photosynthesis
p possible to measure photosynthesis by
CO2 uptake or O2 production

Accessory pigments
increase the range of g wavelengths of light that can be used
zonation of algae green brown red

must identify
independent variable, dependent variable and control variables

The leaf is adapted to photosynthesis

broad, thin leaf for light absorption and gas exchange stomata p palisade cells vascular tissue

If investigating effect of temperature on O2 production

IV is temperature DV is O2 production and CVs are light intensity, CO2 concentration, amount of plant material ...

Some leaves are adapted for full f ll sun, others f shaded th for h d d conditions
sun leaves have higher LCP and LSP than shade leaves

Respiration
Select topic by clicking on the button General introduction Role of ATP Structure of ATP Stages of respiration Respiratory substrates p y Measurement of respiration Summary

Introduction ...
All living things need energy if they are to stay alive. It is needed for a range of living or biological processes such as: h
Active transport Mechanical work
muscle contraction

Anabolic reactions
synthesis complex molecules from simpler building blocks

Bioluminescence and electrical discharge

firefly and electric eel

Maintenance of b d t M i t f body temperature t

As you are aware, the ultimate source of the energy used by most organisms is the sun. Plants harvest solar energy
convert it into chemical potential energy of photosynthetic products products.

Photosynthetic products are source of energy for

plants themselves and heterotrophs that feed on the plants.

Respiration converts the chemical potential energy of organic matter into chemical potential energy of ATP.

ATP is used by cells for work. It is the universal energy currency of the cell. Energy released by respiration is transferred to the highhigh-energy bond of ATP, which can then be used for work. k We will briefly look at the role of respiration (ATP) in
active t ti transport and t d metabolic reactions

then l k t th t t th look at the structure of ATP and its production f d it d ti by respiration.

Active transport
You are familiar with active transport from your AS and so are the experts! So What is active transport? The movement of materials against their concentration gradient What is needed for it to occur? A carrier and energy in the form of ATP

Many materials are moved against a concentration gradient: nutrients (digestive system) salt (loop of Henl in kidney) sodium and potassium (in nerve and other sensitive cells) to name but a f few.

SodiumSodium-potassium pump
Most cells have sodium-potassium pumps in their sodiumplasma membranes. They pump sodium ions out and potassium ions in. They are essential for creating the resting potential of nerve cells, without which action potentials/nerve ll ith t hi h ti t ti l / impulses could not be generated!

Sodium and potassium ions enter their sites

ATP is split, providing energy to move more ions

ADP + Pi K+ Na+ ATP

NA+ and K+ ions moved to other side of membrane

Na+ and K+ ions released

Role in metabolism
Cells need ATP energy to drive metabolism. Protein synthesis and DNA replication are anabolic reactions
build large complex molecules from simpler building blocks protein is built from amino acid monomers DNA is built from nucleotide monomers
nucleotides are a sugar, phosphate, base combination

Amino acids and nucleotides have to be activated with two extra phosphate groups before they can be built into a new protein or DNA.

Protein synthesis ...

Amino acids are activated by
amino-acylamino-acyl-tRNA synthetases attach a specific amino acid to its tRNA.

A high-energy covalent bond is made between highthe carboxyl end of the amino acid and the 3 end of the tRNA makes an aminoacyl tRNA k i l tRNA.

Bond formation gets the energy from ATP.

AA AA + + P P P Adenosine + P Adenosine + P P

Amino acid tRNA

ATP

Aminoacyl tRNA

AMP

Pi

DNA replication ...

Nucleotides are activated by the addition of two extra phosphates from ATP
forms a nucleoside triphosphate

DNA polymerase removes the two phosphates

forms a phosphodiester bond between the remaining phosphate and the deoxyribose of th growing DNA molecule. th d ib f the i l l

The DNA molecule has unzipped (hydrogen bonds holding complementary bases have been broken by a helicase enzyme) and the organic bases are exposed. A ti t d nucleotides ( d Activated l tid (nucleoside t i h l id triphosphates) b h t ) base pair with th i i ith their complementary bases (AT; GC) and the DNA polymerase removes the two phosphates from the activated nucleotides. This provides the energy to make the phosphodiester bond between the remaining phosphate and the growing DNA molecule.

ATP structure
Energy is not made directly available to cells
if all the energy contained within a glucose molecule were released all at once l d ll t it could not be used and much would be wasted

It is transferred into ATP

abbreviation for adenosine triphosphate rip the immediate source of energy for metabolism

ATP is made from

an adenine nucleotide ribose three phosphate groups

It is a phosphorylated nucleotide ATP is known as the cells energy currency it is used b all li i organisms t carry energy f d by ll living i to from
energyenergy-releasing reactions to energyenergy-consuming reactions

ATP structure: t t
adenine d i adenosine ribose

AMP ADP ATP

ATP is made from ADP and inorganic phosphate

uses energy from energy-releasing reactions energye.g. respiration.

catalysed by an ATP synthase or synthetase

Once made, th ATP moves away from the site of O d the f th it f synthesis to the place where it is needed
ATP is broken down into ADP and inorganic phosphate releases the energy contained within the bond catalysed by an ATPase

The energy released is then used for work. The ADP and inorganic phosphate is recycled recycled.

Energy-producing reactions (respiration) allow energy to be transferred into the bond energy of ATP
31 kJ mol-1 31 kJ mol-1 14 kJ mol-1

ATP

ADP + P

AMP + P

Adenosine + P

31 kJ mol-1

31 kJ mol-1

14 kJ mol-1

Energy-consuming Energy consuming reactions (work e g active transport) use the energy (work, e.g. released from the bond energy of ATP. Realistically, it is only the first part of this sequence that is important for respiration, ATP production and use for work.

respiration drives ATP production

ATP

ATP synthetase energy used

ADP +

adenine ribose phosphate high hi h energy b d bond low energy bond

ATP is made from ADP and inorganic phosphate using energy from energyenergy releasing reactions such as respiration. ATP synthetase catalyses this process.

work uses ATP to power it

ATP

energy released ATPase

ADP +

adenine ribose phosphate high hi h energy b d bond low energy bond

ATP is broken down into ADP and inorganic phosphate, phosphate releasing the energy contained within the bond. ATPase catalyses this process. The energy is then i th used f d for work. k

ATP is the energy carrier .

ATP

energy released l d ATPase ATP synthetase energy used gy

ADP +

adenine ribose phosphate high hi h energy b d bond low energy bond

ATP is made ATP is used i d ADP and Pi are recycled

Stages of respiration
Respiration is the process whereby organic molecules are broken down (oxidised) and the energy released used as f l f synthesis of ATP l d d fuel for th i f ATP. The main fuel is glucose, although other fuels can be used lipid protein Respiration/oxidation of energy rich organics like glucose can happen:
in the presence of oxygen in the absence of oxygen

Aerobic
in the presence of oxygen glucose is completely oxidised a lot of energy is released for the synthesis of a lot of ATP glucose + oxygen carbon dioxide + water + energy (2870 kJ)

Anaerobic A bi
in the absence of oxygen glucose i not f ll oxidised l is t fully idi d only a little energy is released, so much less ATP is produced glucose glucose ethanol + carbon dioxide + energy (150 kJ) lactate + energy (150 kJ)

Overall aerobic respiration can be summarised by an equation: word equation glucose + oxygen carbon dioxide + water + energy chemical equation C6H12O6 + 6O2 6CO2 + 6H2O + 2870 kJ

Glucose breakdown is split into several stages:

Glycolysis Link reaction Krebs cycle Oxidative phosphorylation

Glycolysis takes place in the cytoplasm and does not require O2 Link, Krebs and oxidative phosphorylation take place in the mitochondrion and do require O2

The coenzymes NAD and coenzyme A are important in respiration. NAD

is an abbreviation for nicotinamide adenine dinucleotide transfers hydrogen from the respiratory substrates to oxidative phosphorylation

Coenzyme A
often abbreviated to CoA transfers acetate from the link reaction into Krebs cycle

Click to select topic on aspects of the stages of respiration Glycolysis Link reaction Krebs cycle Oxidative h O id ti phosphorylation h l ti Experimental evidence for chemiosmosis Mitochondrial structure and function Anaerobic respiration Energy release in respiration Efficiency of respiration

Glycolysis ...
Glycolysis is the first stage of respiration It takes place in the cytoplasm It is the first stage of both aerobic and anaerobic respiration and doesnt need O2 therefore it is doesn anaerobic (sometimes called anaerobic glycolysis) bi ( ti ll d bi l l i ) Essentially the stages of glycolysis split glucose (C6) to produce 2 molecules of pyruvate (C3) 2ATP (net) (C3), and 2 reduced NAD (sometimes referred to as NADH+ + H+ or NADH2)

Glycolysis includes a phosphorylation stage and an oxidation stage. In the phosphorylation stage
Glucose is phosphorylated by adding 2 phosphates
uses up 2 molecules of ATP l l f produces hexose bisphosphate

The hexose bisphosphate is then split

produces 2x molecules of triose phosphate (TP)

In the oxidation stage

Each triose phosphate is oxidised producing pyruvate Each triose phosphate loses 2 hydrogens
catalysed by a dehydrogenase hydrogen picked up b th coenzyme NAD h d i k d by the
reduces it

remember what NAD is an abbreviation for? nicotinamide adenine dinucleotide

During the oxidation of triose phosphate enough energy is released to make 4x ATP

 glycolysis
2ATP

Phosphorylation stage

2ADP

NAD

ADP ATP

NAD

ADP ATP ADP + Pi ATP

Oxidation O id ti stage

NADred

ADP + Pi NADred ATP

Under aerobic conditions

reduced NAD is transferred into the mitochondrion and onto the electron transport chain t th l t t t h i pyruvate is actively transported into the matrix of the mitochondrion for completion of
link reaction and Krebs cycle y

The transfer across the mitochondrial membranes is active

reduces the overall efficiency of the respiration (see later)

Link reaction
Link reaction takes place in the mitochondrial matrix It connects glycolysis to Krebs cycle
happens when oxygen is available

A carbon atom is removed

pyruvate is decarboxylated

2H is removed and collected by NAD

pyruvate is oxidised NAD is reduced

The resulting 2 carbon molecule

combines with coenzyme A produces acetyl coenzyme A (acetyl CoA) CoA)

Acetyl CoA feeds into Krebs

also takes place in mitochondrial matrix

Reduced NAD feeds into electron transport chain or oxidative phosphorylation id ti h h l ti

takes place in the inner membrane of mitochondrion

link reaction

NAD

CO2 CoA

NAD red

CoA

Krebs cycle
Krebs cycle takes place in the mitochondrial matrix
it completes the oxidation of respiratory substrate

In K b I Krebs cycle l
the acetyl group is added to oxalo-acetate oxaloproduces citrate

citrate is successively decarboxylated and dehydrogenated hydrogen is picked up by coenzymes

removal of CO2 and hydrogen/reducing power transferred into electron transport chain or oxidative phosphorylation

Overall each acetyl unit entering Krebs produces

2x CO2, 1x ATP, 1 ATP, 3x reduced NAD and 1x reduced FAD FAD.

Note: organic chemicals from other sources can feed into Krebs
acetyl groups from the oxidation of fatty acids organic acids following deamination of amino acids.

CoA

Krebs cycle

CoA

NAD red NAD

NAD NAD red

CO2

FAD red FAD ATP ADP

NAD NAD red

CO2

Oxidative phosphorylation
Oxidative phosphorylation also takes place in the mitochondrion It is the final stage of respiration
involves the transfer of hydrogen and then electrons along a transport chain chain. hydrogen is passed into the transport chain from
reduced NAD and reduced FAD.

used to generate ATP g requires free oxygen.

Hydrogen is split into component H+ and electrons (e-).

H+ stays in solution in the mitochondrion whilst the electrons are transferred along a series of cytochromes (electron carriers) to O2.

When the l t Wh th electrons are transferred to O2, H+ i t f dt is removed from the mitochondrial solution to reduce it to water. water Note:
reducing power in reduced NAD (NADH2) and reduced FAD (FADH2) is used to generate ATP free oxygen is the terminal electron acceptor.

 oxidative phosphorylation

Hydrogen transferred NADH2 FAD NAD FADH2 red ox ox cyt y red Electrons transferred between cytochromes red cyt ox ox cyt y red O2 H 2O ATP ATP ATP

The traditional story was that: that: as the protons, and then electrons are passed along the chain of hydrogen and electron carriers there is a loss of energy. the l th loss of energy is coupled t ATP synthesis f i l d to th i reduced NAD has sufficient energy for 3 ATPs reduced FAD has sufficient energy for 2 ATPs. ff f ATPs.

The carriers were positioned/sequenced in the membranes of the cristae b f th i t

allows the orderly transport from one carrier to the next in the sequence sequence.

Chemiosmosis
The traditional view is not exactly what happens. It is true that:
reduced NAD and reduced FAD pass their hydrogen into the transport chain the h d th hydrogen splits i t H+ i lit into ions and electrons and th t d l t d that the electrons travel down the electron transport chain .

However, However it is now generally accepted that energy is used to pump protons from the matrix into the mitochondrial intermembrane space. p The protons then flow back into the matrix
via an ATP synthase y so generating ATP.

The proposed mechanism, is known as chemiosmosis

essentially the same as that for ATP production in chloroplasts
proposed b P d by Peter Mi h ll i 1961 Mitchell in 1961.

Note:
despite th name d it the
chemiosmosis has nothing to do with movement of water or water potential g p gradients

yield of ATP is reduced from the theoretical maximum

energy used to transport materials
ADP, Pi, ATP, etc. across mitochondrial membranes.

realistic yield equivalents

red NAD = 2 5 ATP 2.5 red FAD = 1.5 ATP

CO2 CO2 ATP

Glucose Glycolysis Pyruvate

O2

Link reaction Acetyl CoA

H+ H+

Oxidative phosphorylation

ADP + Pi
H+

Krebs

Reduced Reduced NAD NAD Reduced FAD

Electron transport t t chain

H+

H+ H+

ATP ADP + Pi
H+

ATP

ATP

H2O

Experimental evidence for chemiosmosis

There are a number of pieces of evidence that support Mitchells chemiosmosis theory:
A pH gradient is actually present across the membranes of the chloroplast and mitochondrion When chloroplasts are illuminated, the solution in illuminated which they are suspended becomes alkaline Thylakoids can synthesis ATP if there is a pH g y y p gradient and they are supplied with ADP and Pi

Existence of pH gradient ...

There is a pH gradient across chloroplast and mitochondrial membranes
pH of the chloroplast stroma is higher than the thylakoid space pH of the mitochondrial matrix is higher than the intemembrane space

Consistent with the idea that H+ ions are actively transported across the membrane
active because a gradient exists and is maintained.

Production of gradient in illuminated chloroplast ...

alkaline acid

When illuminated the medium becomes alkaline and thylakoids become acid (pH 4)

same pH

When isolated chloroplasts are illuminated the pH of the th medium i which th di in hi h they are suspended becomes higher and the thylakoids become lower (pH 4). This observation is consistent with the idea that hydrogen ions are pumped into the thylakoids from th stroma. th l k id f the t

When not illuminated the medium and thylakoids remain the same pH

Production of ATP by thylakoids ...

pH 4 buffer H+ H+

H+

H+

pH 8 buffer + ADP + Pi

pH 4 buffer + ADP + Pi

If isolated chloroplasts are suspended in an acid medium in the dark, the chloroplasts and thylakoids become acid. The chloroplast envelope can be disrupted and the thylakoids separated from the rest of the chloroplast debris. The thylakoids are then placed in buffered medium with ADP and Pi.
if the suspending medium is p g pH 8 ATP is synthesised. if the suspending medium is pH 4 ATP is not made made.

ATP is made

ATP not made

ATP is made by the thylakoids if there is a gradient between the thylakoid and the surrounding medium and ADP + Pi are present

Mitochondrial structure and function ...

The mitochondrion is an organelle of eukaryotic cells It is present in plant and animal cells alike It is sometimes called the powerhouse of the cell
for a very good reason it is where most of the usable energy is generated

During the complete oxidation of glucose

30 out of 32 ATPs are made in the mitochondrion 36 out of 38 ATP if using the theoretical maximum

Mitochondria are generally described as

rodrod-shaped 0.50.5-1.0 m diameter

The number and size depends upon cell activity

active cells have more and larger mitochondria
liver cells have 10002000 per cell 1000

Each it h d i is E h mitochondrion i surrounded b an envelope d d by l

outer membrane is smooth but inner membrane is highly folded
cristae provide a large internal surface area

mitochondria are the site of respiration they are surrounded by a double membrane or envelope the inner membrane is highly folded into cristae (singular crista) ( i l crista) i t there is a fluid filled region called the matrix

link reaction and Krebs cycle take place in the matrix which contains the th necessary enzymes electron transport or oxidative phosphorylation takes place in the inner membrane which b hi h contains the carriers and stalked particles or p ATP synthetase

Can you identify the key structures?

TEM of a mammalian lung showing a mitochondrion. The double membrane or envelope, folded inner membrane making the cristae and matrix are visible. Public domain image: Louisa Howard.

Intermembrane space
Protons are pumped into the intermembrane space using the energy released by electron transport. The proton flow back into the matrix via an ATP synthetase, generating ATP.

Matrix
Contains the enzymes of link reaction and Krebs cycle. Hydrogen passes to ETC, CO2 excreted.

Crista
Foldings provide a large internal surface area. The membrane contains the electron transport chain and the stalked particles (ATP synthetase).

Envelope
TEM of a mammalian lung showing a mitochondrion. The double membrane or envelope, folded inner membrane making the cristae and matrix are visible. Public domain image: Louisa Howard.

Double membrane that surrounds the mitochondrion. Outer mitochondrion membrane is smooth, inner one is highly folded.

Additional points ...

Mitochondria contain small ribosomes (70S or 18nm diameter) loops of mitochondrial DNA Why do mitochondria have them? Small ribosomes and circular DNA are features of prokaryotes
presence explained by the endosymbiotic theory of cell evolution

Mitochondria Mit h d i make some of th i own protein and k f their t i d

need the DNA and ribosomes for this

Outer membrane: Smooth Relatively permeable to small molecules

Inner membrane: Highly folded Less permeable Studded with 9nm diameter spheres the so-called sostalked particles Spheres are the ATP p synthetase

The inner membrane has the electron transport chain ETC pumps H+ ions into the intermembrane space, so creating a gradient between the intermembrane space and the matrix H+ ions flow back into the matrix through the stalked particles which synthesise ATP

Anaerobic respiration
What happens if oxygen is not available? The last stages cannot take place because the electrons cannot be passed onto oxygen. So
the electron acceptors cannot accept any more from reduced NAD and reduced FAD NAD and FAD cannot be regenerated meaning there is no NAD or FAD to accept hydrogen from the link reaction and Krebs cycle respiration in the mitochondrion stops.

However in anaerobic conditions, glycolysis can continue

if some NAD is made available and pyruvate is removed.

This i Thi is possible via th ibl i the

lactate pathway in animals and ethanol pathway i yeast. th l th in t

Lactate pathway ...

In mammalian muscles short of O2
pyruvate can act as a hydrogen acceptor converted to lactate.

This regenerates NAD and

allows glycolysis to continue with limited production of ATP
2 ATP per glucose, instead of 32 (38) l i t d f

Unfortunately lactate is toxic and

its production cannot continue indefinitely. indefinitely.

 anaerobic respiration

ATP

reduced NAD

NAD

Pyruvate

Lactate

Lactate production can be reversed in humans. When oxygen is available again

lactate is transported to the liver in the blood and converted back into pyruvate or into glycogen via the cori cycle.

The oxygen needed by the hepatocytes to remove the l ti th lactic acid i k id is known as th oxygen d bt. the debt. debt

Ethanol pathway ...

In yeast there is a similar response to anaerobic conditions The products of alcoholic fermentation are different:
ethanol and carbon dioxide

Ethanol is toxic
eventually alcoholic f t ll l h li fermentation stops t ti t

The oxidation of glucose is incomplete and so much energy remains in the ethanol
can be a significant intake of energy for drinkers

 anaerobic respiration

ATP

reduced NAD

NAD

CO2

Pyruvate

Ethanal

Ethanol

Compare the lactate and ethanol pathways by completing the table:

Feature Substrate Anaerobic pathway Lactate Ethanol

Products

Significance/other

Compare the lactate and ethanol pathways by completing the table:

Feature Substrate Glucose Anaerobic pathway Lactate Glucose Ethanol

Realistically, any hexose could be the substrate as they can be converted from one to another. Products Significance/other Lactic acid Pyruvate accepts the hydrogen, regenerating NAD so that glycolysis can continue. Build up is toxic. Causes oxygen debt debt. Ethanol Carbon dioxide Pyruvate converted to ethanal which then accepts the hydrogen regenerating hydrogen, NAD so that glycolysis can continue. Build up is toxic. toxic Ethanol contains a lot of energy calorific intake!

Energy release in respiration

It is possible to compare the energy released in aerobic and anaerobic respiration by producing an ATP balance sheet sheet. All you need to know is how many
ATPs are produced directly in each stage p y g ATPs each reduced NAD and reduced FAD is worth in oxidative phosphorylation. each reduced NAD is worth 2.5 (3) ATPs each reduced FAD worth 1 5 (2) ATPs 1.5 ATPs.
(figures in brackets are the theoretical maximum, which in reality is reduced because energy is required to transport material across the mitochondrial membranes)

Complete the energy b l C l h balance sheet f aerobic and h for bi d anaerobic respiration

Stage
Glycolysis

Aerobic

Anaerobic

subtotal Link/Krebs

subtotal

subtotal Oxidative phosphorylation subtotal b l Total

subtotal

subtotal b l Total

Stage
Glycolysis

Aerobic
2 ATP (net) 2 NADH2

Anaerobic
2 ATP (net)

subtotal Link/Krebs

subtotal

2 x 1 ATP (1 per pyruvate) 2 x 4 NADH2 2 x 1 FADH2

subtotal Oxidative p phosphorylation p y

10 x 2.5 ATP (10 x 3) 2 x 1.5 ATP (2 x 2)

subtotal

subtotal Total

28 (34) 32 (38)

subtotal total

0 2

Efficiency of respiration
It is possible to calculate the efficiency of aerobic and anaerobic respiration Assume: 1 mole of sugar contains 2870kJ of energy. Each high energ phosphate bond of ATP prod ced energy produced contains 31kJ per mole. All 2870kJ is released in aerobic respiration whilst respiration, only 150kJ is released under anaerobic conditions.

Efficiency can be calculated as: usable energy produced x 100/ energy released Where:
usable energy is that transferred to ATP energy released is the total energy released Calculate the efficiency of aerobic and anaerobic respiration

Aerobic respiration:
usable energy produced x 100 / energy released Best: es (38 x 31) x 100 / 2870 = 41.0% Realistic: (32 x 31) x 100 / 2870 = 34.6%

Anaerobic respiration:
usable energy produced x 100 / energy released Best/realistic: es / ea s c (2 x 31) x 100 / 150 = 41.3%

When you have performed the calculations you might be surprised to find that both aerobic and anaerobic respiration h i ti have about 40% efficiency, i 40% of th b t ffi i i.e. f the energy released is trapped in ATP** That said, only about 5% of the energy contained in said glucose is actually released under anaerobic co d t o s conditions what happens to the rest? The 60% or so is lost as heat energy! lost Energy still present in the lactate or ethanol!
**depends upon your calculated value for ATP produced!

Respiratory substrates
A respiratory substrate is an organic molecule that can be respired (oxidised) to produce usable energy in th form of ATP. i the f f ATP
carbohydrates (glucose) are important respiratory substrates
only one for some tissue e.g. brain cells and red blood cells.

other substrates can be respired under aerobic conditions

lipids protein.

Lipid as respiratory substrate ...

Lipids can be hydrolysed to produce
fatty acids and glycerol fatty acids
oxidised to produce acetate units fed into K b f d i t Krebs cycle via acetyl CoA l i t lC A

glycerol
converted to pyruvate fed into Krebs cycle via the link reaction.

Amino acids can be deaminated in the liver

carboxylic acid fed into Krebs cycle directly or via pyruvate and the link reaction.

The relationship between the respiratory substrates and respiration is summarised in the following diagram. di Annotate your copy of it to indicate the reactions that occur. occur

glucose

triose phosphate reducing power ATP pyruvate CO2 glycerol lipid fatty acid

amino acid organic acid

reducing power

Acetyl CoA

C4 acid

C6 acid CO2 C5 acid CO2 C4 acid

reducing power

ATP

The various respiratory substrates contain different amounts of energy:

carbohydrate and protein contain about 17 kJ g-1 lipid contains > 2x as much, at around 38 kJ g-1.

Lipid is Li id i much more energy-d h energy-dense b because

it is much more highly reduced reducing power (h d d i (hydrogen content) i th source of t t) is the f most of the ATP energy during oxidative p phosphorylation. p y
more hydrogen = more energy = more ATP

Measurement of respiration rate ...

It is possible to measure the rate of any reaction by measuring
the rate that a substrate disappears or the rate that a product appears.

So, from the following equation th t summarises S f th f ll i ti that i respiration C6H12O6 + 6O2 6CO2 + 6H2O + energy Which are the substrates? glucose/carbohydrate and O2 l / b h d t d Which are the products? CO2 and water d t

Traditionally, either O2 consumption or CO2 production would be used to measure respiration Suggest how each could be measured O2 consumption Oxygen electrode Use respirometer and CO2 scrubber to measure O2 used d CO2 production pH change
CO2 is an acid gas, add it and pH drops

Use bicarbonate indicator time how long to change

It would also be possible to measure p

glucose/carbohydrate consumption, water and heat production

Glucose could be monitored with

a digital glucose monitor or quantitative Benedict's could be problematic

Water can be detected by simple chemical tests

anhydrous copper sulphate
white t bl hit to blue

anhydrous cobalt chloride

blue to pink

could be problematic water evaporates from cells...

Heat production could be monitored

insulation required...

A traditional way of measuring respiration is to measure the amount of oxygen used the more oxygen consumed in unit time, the faster the yg yg rate of respiration. There are several types of simple respirometer available, one type is illustrated in the diagram:
Manometer M t fluid/ink

Living organism (small invertebrate) in mesh wick CO2 scrubber e.g. NaOH

Respiration removes O2 and produces CO2 d d CO2 is removed by the scrubber The volume of gas in the chamber drops Manometer fluid moves M t fl id towards the chamber Movement proportional p p to O2 uptake

The distance moved is proportional to the rate of respiration, more specifically to the oxygen consumption What other information is needed to calculate the volume of oxygen consumed and the actual rate of oxygen consumption? internal diameter/radius of the capillary tube crosscross-sectional area of the capillary tube time What formula would you apply to measure the rate of oxygen consumption? p r2 x distance moved / time Do any factors need controlling so that we are confident in our estimate of respiration? Working in p p g pairs/small g p groups, describe and explain the effect of each factor and its control.

Factor Temperature

Effect if uncontrolled

Control measure

Atmospheric pressure

Volume of reagents and glassware

Other

Factor Temperature

Effect if uncontrolled Temperature influences respiration increases if temperature rises but does not exceed the optimum. p Thermal expansion of gas. Influences volume of gas and movement of the manometer fluid or ink high pressure moves ink towards the chamber, chamber low pressure away away. More/less CO2 scrubber in large/small respirometer affects efficiency of CO2 removal. More efficient with more scrubber and less volume Amount of respiring material Age of material Physiological t t Ph i l i l state

Control measure Water bath or room temperature, with frequent checks on temperature. Choose optimum. p Using a (de)compression chamber is not realistic! Use a control minus organism movement in control used to compensate... compensate Standardise!

Atmospheric pressure

Volume of reagents and glassware

Other

Standardise! Choose young, good condition material. t i l

So, weve probably arrived at a situation where we say we must

control temperature
use a water bath.

take account of atmospheric pressure

cant realistically control it, need a duplicate apparatus but with the invertebrate replaced by
the same volume/mass of inert material glass bead? or, f hl kill d i freshly killed invertebrate? t b t ?

We will also want replicates

Glass beads

Living organism

NaOH

NaOH

Water bath

A water bath can be used to control temperature

much better than simply relying upon room temperature t t
can and does fluctuate.

Fluctuations in temperature
cause fluctuation in respiration rate affect the volume of gas in the respirometer
and so the accuracy of measurement an increase in temperature causes thermal expansion p p cooling causes thermal contraction.

Atmospheric pressure needs to be taken account of

cannot realistically be controlled.

So a duplicate apparatus is used

the invertebrate replaced by the same volume/mass of inert i t material t i l
e.g. glass beads.

If pressure (or indeed temperature) changes

causes movement of the ink bubble of the control apparatus a similar effect should occur in the treatment apparatus so can be taken account of.

Volume of CO2 scrubber and surface area

affects the efficiency of CO2 absorption should be kept the same in both the control and treatment respirometers. respirometers.

Volume of th glassware could i fl V l f the l ld influence th the accuracy of readings

small change in a large respirometer is more difficult to measure than the same change in a smaller respirometer.

A greater mass of invertebrate will result in a greater uptake of O2

record results per unit mass?

Uptake could also be affected by

activity level metabolic rate
often influenced by age age.

Should be considered when

replicate readings are taken or when investigating the effect of changing a factor (e.g. temperature) on respiration. p ) p

Summary
Plants, animals and microorganisms must respire
need ATP for
active transport ti t t
sodium potassium pump glucose

immediate source of energy for cellular reactions

During respiration
substrates like glucose are b t t lik l oxidised
energy released used to produce ATP

metabolic reactions
DNA synthesis protein synthesis

ATP is a phosphorylated nucleotide

made f d from
adenine, ribose and three phosphates

Under aerobic conditions oxidation is complete Respiration divided into four stages:
glycolysis link reaction Krebs cycle and y oxidative phosphorylation

transfers energy
from energy producing reactions to energy consuming reactions

Respiration is
controlled b enzymes t ll d by involves coenzymes like NAD and acetyl coenzyme A

universal energy currency

Glycolysis
takes place in the cytoplasm p y p starts with phosphorylation of glucose to hexose bisphosphate hexose bisphosphate splits to give 2x triose phosphate further oxidised to pyruvate yields
2x ATP (net) and 2x reduced NAD

decarboyxlated to produce acetate and reduced NAD acetate combined with coenzyme A to be carried to next stage

Krebs cycle
takes place in acetate (2C) added to oxaloacetate (4C) and produces citrate (6C) citrate is then reconverted to oxaloacetate
involves a series of
decarboxylations dehydrogenations and substrate level phosphorylation

Under aerobic conditions pyruvate is actively transported into the mitochondrial matrix Link reaction
pyruvate enters the link reaction

produces (per acetate)

3x reduced NAD, 1x reduced FAD, 1x ATP, 2x CO2

Oxidative phosphorylation
where most of the ATP made takes place in the inner membrane (cristae) (cristae) reducing power (h d d i (hydrogen) ) fed into oxidative phosphorylation from
reduced NAD and reduced FAD

Mitchells chemiosmosis theory supported by experimental evidence:

pH gradient exists across membranes of the mitochondrion and chloroplast illumination of chloroplast produces a pH gradient isolated thylakoids (pH 4) will make ATP if put into a medium of pH 8 and supplied with ADP and Pi

hydrogen, hydrogen then electrons pass along carriers energy in the hydrogen and e- is used to produce ATP protons pumped into the intermembrane space flow back into the matrix via an ATP synthetase (stalked particle) chemiosomosis

Mitochondrion has an envelope

inner membrane highly folded into cristae cristae have the carriers and ATP synthetase

matrix has
link and Krebs cycle enzymes small ribosomes and circular DNA

liver can convert lactate to carbohydrate and lipid

ethanol pathway
pyruvate converted to ethanal ethanal reduced to ethanol ethanol still contains much energy

Aerobic respiration
Under anaerobic conditions link, Krebs li k K b cycle and l d oxidative phosphorylation cannot continue Limited ATP production is possible if glycolysis continues NAD must be regenerated
lactate and ethanol pathway regenerate NAD t

Respiration is an inefficient process

aerobic respiration releases 2870 kJ per mole glucose
only 35% of this gets into ATP bond energy
assumes 32 ATP produced rather than the theoretical a e a e eo e ca maximum of 38 ATP

lactate pathway
py pyruvate reduced to lactate note energy still present in lactate

anaerobic respiration only releases 150 kJ per mole and

produces 2 ATP so efficiency is about 40%

Glucose is not the only respiratory substrate

Under aerobic conditions lipids and amino acids can be respired
Lipids are digested into glycerol and fatty acids glycerol i converted t l l is t d to pyruvate and
fed into Krebs via link reaction

Lipids are more highly reduced than amino acids, so produce more energy

Respiration is investigated using a respirometer

A simple respirometer consists of a chamber, CO2 scrubber and capillary tube with ink/manometer fluid S ou d de t y Should identify
independent variable dependent variable and control variables t l i bl

fatty acids are split into acetate units and

fed into K b i f d i t Krebs via acetyl t l CoA

Amino acids are deaminated the organic acid produced

either fed in via pyruvate and the link reaction or into Krebs cycle directly

If investigating effect of temperature on respiration

IV is temperature DV is O2 consumption and CVs are pressure O2 pressure, concentration, amount of material ...