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LECT 6. RESPIRATIONLECT 6. RESPIRATION COMPETENCIESCOMPETENCIESStudents, after mastering the materials of Students, after mastering the materials of Plant Physiology course, should be able to:Plant Physiology course, should be able to:1.1. To explainTo explain the process of respiration (the oxidation of the process of respiration (the oxidation of

substrates particularly carbohydrates or the synthesis substrates particularly carbohydrates or the synthesis of metabolic energy used for plant growth and of metabolic energy used for plant growth and maintenance)maintenance)

2.2. To explain reactions, enzymes and products involved To explain reactions, enzymes and products involved the respirationthe respiration

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The electron transfer system in the inner membrane of mitochondria in plants LECTURE FLOWLECTURE FLOW

QUESTIONSQUESTIONS

SUMMARY OF RESPIRATIONSUMMARY OF RESPIRATION

1.1. DEFINITIONDEFINITION

2.2. THE SITE OF RESPIRATIONTHE SITE OF RESPIRATION

3.3. MAIN STEPS OF RESPIRATIONMAIN STEPS OF RESPIRATION

4.4. COMPARING ENERGY YIELDCOMPARING ENERGY YIELD

5.5. FEEDBACK CONTROL OF RESPIRATIONFEEDBACK CONTROL OF RESPIRATION

6.6. RESPIRATION AND PLANT CARBON BALANCERESPIRATION AND PLANT CARBON BALANCE

7.7. FACTORS AFFECTING RESPIRATIONFACTORS AFFECTING RESPIRATION

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QUESTIONSQUESTIONS1.1. What is the importance of respirationWhat is the importance of respiration2.2. How many steps is the respirationHow many steps is the respiration3.3. What is the first and second step of respirationWhat is the first and second step of respiration4.4. Where does the Where does the glycolysisglycolysis take placetake place5.5. Where does the TCA cycle take placeWhere does the TCA cycle take place6.6. Where does the Electron Transport Chain take placeWhere does the Electron Transport Chain take place7.7. What is the compound exported from What is the compound exported from cytosolcytosol to to

mitochondria in the respirationmitochondria in the respiration8.8. What is the final acceptor of electrons in the respirationWhat is the final acceptor of electrons in the respiration9.9. How many NADP does the How many NADP does the glycolysisglycolysis produceproduce10.10. How many NADP does the TCA cycle produceHow many NADP does the TCA cycle produce11.11. How many ATP does the How many ATP does the glycolysisglycolysis produceproduce12.12. How many ATP does the TCA cycle produceHow many ATP does the TCA cycle produce13.13. What is the meaning of ADP:O ratiosWhat is the meaning of ADP:O ratios

QUESTIONSQUESTIONS

14.14. What is the ADP:O ratio of FADHWhat is the ADP:O ratio of FADH22

15.15. How many ATP does the respiration produces totallyHow many ATP does the respiration produces totally16.16. How efficient is the respiration in the conversion of How efficient is the respiration in the conversion of

energyenergy17.17. What does it mean by feedback control of respiration What does it mean by feedback control of respiration 18.18. How is the effect of tissue stage of development on How is the effect of tissue stage of development on

the respirationthe respiration19.19. How is the effect of OHow is the effect of O22 on the respirationon the respiration20.20. How is the effect of COHow is the effect of CO22 on the respirationon the respiration21.21. How is the effect of plant injury on the respirationHow is the effect of plant injury on the respiration22.22. How is the foliar respiration of trees in term of heightHow is the foliar respiration of trees in term of height23.23. How is the response of foliar respiration to nitrogenHow is the response of foliar respiration to nitrogen24.24. How is the woody respiration in term of wood How is the woody respiration in term of wood

diameterdiameter

Respiration = Dark RespirationRespiration = Dark Respiration

Citric acid cycle (“Krebs cycle”)Citric acid cycle (“Krebs cycle”) Electron transport and ATP synthesisElectron transport and ATP synthesis

Respiration and carbon economy of Respiration and carbon economy of whole plants whole plants

Hans Krebs, 1953 Nobel Prize

Biochemistry of respirationBiochemistry of respiration GlycolysisGlycolysis (“(“glycoglyco”= sugar; “”= sugar; “lysislysis”= “untie”)”= “untie”)

CH2O + O2 → CO2 + H2O + energy

GlycolysisGlycolysis –– break a 6break a 6--carbon sugar into two 3carbon sugar into two 3--Carbon sugars (Carbon sugars (triosetriose phosphate) phosphate) –– takes some takes some energy energy –– then strip electrons from these 3then strip electrons from these 3--C C sugars sugars –– releases a bit of energy in the form of releases a bit of energy in the form of ATP and NADH. “Leftover” products: 3C sugars ATP and NADH. “Leftover” products: 3C sugars PyruvatePyruvate and and MalateMalate (still embody substantial free (still embody substantial free energy)energy)

Citric acid/Citric acid/krebskrebs cycle cycle complete oxidation of complete oxidation of pyruvatepyruvate//malatemalate to produce COto produce CO22, H, H22O, reducing O, reducing power (NADH, FADHpower (NADH, FADH22) and ATP) and ATP

Electron Transport Chain Electron Transport Chain launder NADH, FADHlaunder NADH, FADH22

to ATP across inner mitochondrial membrane and to ATP across inner mitochondrial membrane and to Membrane)to Membrane)

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1. DEFINITION1. DEFINITION1.1. RespirationRespiration is the process whereby the is the process whereby the

energy stored in carbohydrates, produced energy stored in carbohydrates, produced during photosynthesis, is released in a during photosynthesis, is released in a controlled manner. controlled manner.

2.2. The energy (free energy) released during The energy (free energy) released during respiration is incorporated into a form (ATP) respiration is incorporated into a form (ATP) that can be readily utilized for the that can be readily utilized for the maintenance and development of the plant. maintenance and development of the plant.

What is respiration ?

3.3. Respiration is tightly coupled to other Respiration is tightly coupled to other pathwayspathways

Biosynthesis of Nucleotides Biosynthesis of Nucleotides

Biosynthesis of Proteins Biosynthesis of Proteins

Biosynthesis of Lipids Biosynthesis of Lipids

Biosynthesis of Cell wall components Biosynthesis of Cell wall components

Biosynthesis of Biosynthesis of PhytohormonesPhytohormones

Biosynthesis of Plant pigmentsBiosynthesis of Plant pigments

2. THE SITE OF RESPIRATION2. THE SITE OF RESPIRATION

1.1. Mitochondria are the main Mitochondria are the main site of ATP synthesis in site of ATP synthesis in eukaryote cells and as such eukaryote cells and as such are vital for the health and are vital for the health and survival of the cellsurvival of the cell

Where does the respiration take place?

2. From a chemical standpoint, respiration is most commonly expressed in terms of the oxidation of the six-carbon sugar glucose.

3.3. This equation represents a coupled This equation represents a coupled redoxredoxreaction that oxidizes completely glucose to COreaction that oxidizes completely glucose to CO22

with with oxygenoxygen serving as the ultimate electron serving as the ultimate electron acceptor and reduced to water. acceptor and reduced to water.

4.4. The substrate for respiration most commonly The substrate for respiration most commonly cited, in a functioning plant cell, is cited, in a functioning plant cell, is glucoseglucose that is that is actually derived from such sources as the actually derived from such sources as the glucose polymer starchglucose polymer starch, , the disaccharide the disaccharide sucrosesucrose, , fructosefructose--containing polymers containing polymers ((fructosansfructosans), and ), and other sugarsother sugars, , as well as as well as lipidslipids(primarily (primarily triacylglyceroltriacylglycerol), ), organic acidsorganic acids, , and on and on occasion, occasion, proteinsproteins

5.5. The amount of energy release is roughly The amount of energy release is roughly 2880 kJ 2880 kJ (686 kcal) per mole (180 g) of glucose oxidized (686 kcal) per mole (180 g) of glucose oxidized that is coupled to the synthesis of ATP. that is coupled to the synthesis of ATP.

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• Two membranes

• Inner membrane invaginated

• Numbers of mitochondria per cell

vary but usually 100s/cell

• Matrix contains the TCA cycle (and other) soluble enzymes

• Inner membrane contains metabolite transporters and the electron transport chain

Mitochondria have their own DNA and Ribosomes

Mitochondrial DNA• Mitochondria have

some of their own DNA, ribosomes and tRNA; 22 tRNAs & rRNAs (16S and 12S), so mitochondria can make many of their own proteins.

• The DNA is circular and lies in the matrix in structures called "nucleoids". Each nucleoid may contain 4-5 copies of the mitochondrial DNA (mtDNA).

3. MAIN STEPS OF RESPIRATION3. MAIN STEPS OF RESPIRATIONWhat are the stages of glucose oxidation ?

The reactions of glucose oxidation can be subdivided into three stages:

1.Glycolysis

2.The tricarboxylic acid (TCA) cycle, and

3.The electron transport chain (terminal oxidation)

Anaerobic RespirationAnaerobic Respiration

C6H12O6 + O2→2 CH2O5 + 2 H2O + 2 ATPor

Glucose + Oxygen →2 Ethanol + 2 Water + 2 ATP

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3 Main Respiration Steps3 Main Respiration Steps

1.1. GlycolysisGlycolysis

2.2. Glucose (CGlucose (C66HH1212OO66) ) PyruvatePyruvate (C(C33HH44OO33): Breakdown of ): Breakdown of Glucose to a 3Glucose to a 3--Carbon Compound Called Carbon Compound Called calledcalledPyruvatePyruvate

• This occurs in Cytosol• Some ATP and NADH

Are also Formed– Storage Energy Molecules

• NADH is Formed from NAD

• Similar Type of Energy-Storing Rx asNADP + H2 →NADPH2 – NAD + H →NADH

2. 2. Krebs Cycle/Citric Acid CycleKrebs Cycle/Citric Acid Cycle ‘‘TricarboxylicTricarboxylic acid Cycle (TCA Cycle)’ occurs in acid Cycle (TCA Cycle)’ occurs in

Mitochondrial MatrixMitochondrial Matrix A Cyclic Series of A Cyclic Series of RxsRxs that Completely Break that Completely Break

down down PyruvatePyruvate to COto CO22 and Various Carbon and Various Carbon SkeletonsSkeletons

This is the step where CO2 is given off by the Plant

SkeletonsSkeletons Are Used in other Are Used in other Metabolic Pathways to Make Metabolic Pathways to Make various Compoundsvarious Compounds ProteinsProteins LipidsLipids Cell Wall CarbohydratesCell Wall Carbohydrates DNADNA Plant HormonesPlant Hormones Plant PigmentsPlant Pigments Many other Biochemical CompoundsMany other Biochemical Compounds

3. 3. Electron Transport ChainElectron Transport Chain ‘Oxidative ‘Oxidative PhosphorylationPhosphorylation’’ Series of proteins in the mitochondria helps Series of proteins in the mitochondria helps

transfer electrons (etransfer electrons (e--) from NADH to oxygen) from NADH to oxygen Releases a lot of energyReleases a lot of energy

This occurs on Mitochondrial Inner Membrane (Proteins Bound to Membrane)

Released energy is used to drive the reaction ADP + P → ATPMany ATP are made

Oxygen is required for this step

Water is produced

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Overview of aerobic respiration GLYCOLISIS: GLYCOLISIS:

SUCROSE or STARCHSUCROSE or STARCHPYRUVATEPYRUVATE1.1. Sucrose Sucrose Glucose + FructoseGlucose + Fructose2.2. Starch Starch GluGlu 11--PP3.3. GluGlu 11--P P GluGlu 66--PP4.4. Glucose Glucose GluGlu 66--PP5.5. Fructose Fructose FrucFruc 66--PP6.6. GluGlu 66--P P FrucFruc 66--P P 7.7. FruFru 66--P P FrucFruc 1,61,6--P P 8.8. FruFru 1,61,6--P P DihydroxyacetoneDihydroxyacetone

P + P + GlyceraldehydeGlyceraldehyde 33--PP

9.9. Glyceraldehyde 3Glyceraldehyde 3--P P 1,3 1,3 BisphosphoglycerateBisphosphoglycerate

10.10. 1,3 Bisphosphoglycerate 1,3 Bisphosphoglycerate 3 3 phosphoglycerate phosphoglycerate

11.11. 3 phosphoglycerate 3 phosphoglycerate 2 2 phosphoglycerate phosphoglycerate

12.12. 2 phosphoglycerate 2 phosphoglycerate Phosphoenol pyruvate Phosphoenol pyruvate (PEP)(PEP)

13.13. PEP PEP PyruvatePyruvate

Anaerobic Respiration (Fermentation)Pyruvate Lactate or Pyruvate Ethanol

Citric Acic Cycle (Krebs Cycle)1. Pyurvate Acetyl-CoA2. Acetyl-CoACitrate3. Citrate Isocitrate4. Isocitratea-ketoglutarate5. a-ketoglutarate Succinyl-

CoA6. Succinyl-CoA Succinate7. Succinate Fumarate8. Fumarate Malate9. Malate Oxaloacetate

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The electron transport chain (terminal oxidation)

Terminal Oxidation can be divided into four large, multi-subunit protein complexes

-complex I is a NADH-ubiquinone reductase

-complex II is succinatedehydrogenase (part of the TCA cycle)

-complex III is the ubiquinone -cytochrome c reductase

-complex IV is cytochromeoxidase

-complex I is a NADH-ubiquinone reductase

-complex II is succinate dehydrogenase (part of the TCA cycle)

-complex III is the ubiquinone -cytochrome c reductase

-complex IV is cytochrome oxidase

http://www.bmb.leeds.ac.uk/illingworth/oxphos/

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Organization of mitochondrial electron Organization of mitochondrial electron transport chaintransport chain

NADH NADH DehydrogenaseDehydrogenase (complex I)(complex I) oxidizes NADHoxidizes NADH transfers transfers ee-- to to UbiquinoneUbiquinone (UQ)(UQ) pumps 1H+ per epumps 1H+ per e--

SuccinateSuccinate DehydroganaseDehydroganase (complex II)(complex II) oxidation of oxidation of succinatesuccinate (from citric acid cycle) (from citric acid cycle) ee-- are transferred via FADHare transferred via FADH22

does not pump protonsdoes not pump protons CytochromeCytochrome bc1 complex bc1 complex (complex III)(complex III)

oxidizes reduced UQ (= oxidizes reduced UQ (= ubiquinolubiquinol) ) pumps 1H+ per pumps 1H+ per ee--

CytochromeCytochrome c c oxidaseoxidase (complex IV)(complex IV) reduces Oreduces O22 to Hto H22OO pumps 1H+ per pumps 1H+ per ee--

ATP ATP synthasesynthase (complex V)(complex V) uses electrochemical proton gradient to synthesize ATPuses electrochemical proton gradient to synthesize ATP

Substrate ADP/O

NADH (Malate) 2.4-2.7

NADH (Succinate) 1.6-1.8

NADH (External) 1.6-1.8

NADH (Ascorbate)* 0.8-0.9

Table. ADP/O ratios in isolated plant mitochondria

*Artificial electron donorADP/O = number of ATPs synthesized per two electrons transferred to oxygen

4. COMPARING ENERGY YIELD4. COMPARING ENERGY YIELD GlycolysisGlycolysis (per glucose):(per glucose): Net: 2ATP, 2NADHNet: 2ATP, 2NADH

Krebs (per glucose):Krebs (per glucose): 2ATP, 8NADH, 2FADH22ATP, 8NADH, 2FADH2

Total: 4ATP, 10NADH equivalentsTotal: 4ATP, 10NADH equivalents ATP:NADH ratio ~3 in MitochondriaATP:NADH ratio ~3 in Mitochondria Thus 4ATP + 10NADHx3 = Thus 4ATP + 10NADHx3 = 34 ATPs per glucose 34 ATPs per glucose

(more or less)(more or less) (34 x 50.2 kJ/mol)/(2880 kJ/mol )= 59% conversion (34 x 50.2 kJ/mol)/(2880 kJ/mol )= 59% conversion

efficiency! (versus around 4% for efficiency! (versus around 4% for glycolysisglycolysis alone)alone)

1 mol glucose givesΔGo = -2880 kJ/mol

1 mol ATP takesΔGo = 50.2 kJ/mol

ENERGY PRODUCTIONENERGY PRODUCTIONSite/Process Quantity ADP/O ATP

•Glycolysis 2 ATP 2•TCA Cycle 2 ATP 2•Cytosol 2 NADH 2.5 5•Mitochondrial Matrix 8 NADH 2.5 20•Mitochondrial Matrix 2 FADH2 1.5 3

TOTAL 32Conversion Efficiency(32 x 50.2 kJ/mol)/(2880 kJ/mol )= 55.8%

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Complex IV, the cytochrome c oxidase, is specifically inhibited by cyanide (CN-

), azide (N3-), and carbon monoxide (CO). Cyanide and azide bind tightly to the

ferric form of cytochrome a3, whereas carbon monoxide binds only to the ferrous form. The inhibitory actions of cyanide and azide at this site are very potent, whereas the principal toxicity of carbon monoxide arises from its affinity for the iron of hemoglobin

5. FEEDBACK CONTROL OF RESPIRATION5. FEEDBACK CONTROL OF RESPIRATION

Demand regulation Demand regulation Low amounts of ADP dramatically reduce the rate Low amounts of ADP dramatically reduce the rate

of mitochondrial respirationof mitochondrial respiration The rate of respiration increases when energy The rate of respiration increases when energy

demand for growth, maintenance and transport demand for growth, maintenance and transport processes is high that consume rapidly ATP processes is high that consume rapidly ATP leading to the production of ADPleading to the production of ADP

RESPIRATION AND PLANT CARBON RESPIRATION AND PLANT CARBON BALANCEBALANCE

• On a whole-plant basis, respiration consumes from 30% to 70% of total fixed carbon

• Leaves account for about half of the total

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FACTORS AFFECTING RESPIRATIONFACTORS AFFECTING RESPIRATION1.1. Kind of Cell or TissueKind of Cell or Tissue.. Young and developing cells (Young and developing cells (meristematicmeristematic areas) usually have higher areas) usually have higher

respiration rates. respiration rates. Developing and ripening fruit and seeds, too. Older cells and structural Developing and ripening fruit and seeds, too. Older cells and structural

cells respire at lower ratescells respire at lower rates2.2. TemperatureTemperature Respiration generally has higher optimum and maximum temps than PS Respiration generally has higher optimum and maximum temps than PS RxsRxs

3.3. OxygenOxygen Low OLow O22 can reduce aerobic respiration and increase anaerobic respirationcan reduce aerobic respiration and increase anaerobic respiration Low OLow O22 can reduce photorespirationcan reduce photorespiration

4.4. COCO2 2 Higher COHigher CO22 levels reduce rate of respiration (feedback inhibition). Seldom levels reduce rate of respiration (feedback inhibition). Seldom

occurs except when O2 levels are limited (flooded, compacted soils)occurs except when O2 levels are limited (flooded, compacted soils)5.5. Plant InjuryPlant Injury Injury will Increase RespirationInjury will Increase Respiration

Kind of Cell or Tissue Kind of Cell or Tissue

Temperature EffectTemperature Effect

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QUESTIONSQUESTIONS1.1. What is respiration ?What is respiration ?2.2. Where does the respiration take placeWhere does the respiration take place3.3. What are the stages of glucose oxidation ? What are the stages of glucose oxidation ? 4.4. Where does the TCA cycle take placeWhere does the TCA cycle take place5.5. What does it happen in respiration when OWhat does it happen in respiration when O22 is absent ? is absent ? 6.6. What event does it take place in What event does it take place in cytosolcytosol in respiration ? in respiration ? 7.7. What does the reaction produce FADH ? What does the reaction produce FADH ? 8.8. What is the molecule transported from What is the molecule transported from cytosolcytosol to mitochondrion to mitochondrion

matrix ?matrix ?9.9. How many are ATP produced in respiration ?How many are ATP produced in respiration ?10.10. What does it mean by ADP:O ratio in respiration ? What does it mean by ADP:O ratio in respiration ?