Plant Biochemistry BCH 350site.iugaza.edu.ps/mawad/wp-content/uploads/All lectures... · 2013. 9. 7. · Stoma (pl. Stomata): Pores in a plant’s cuticle through which water and

Plant BiochemistryBCH 350

Dr. Wajahat KhanOffice : 67A2

Biochemistry DepartmentBuilding Number 5

Phone number: 467-5443Email: [email protected]

(English Emails only)

mailto:[email protected]

•Overview of Plant Biochemistry•The Plant Cell WallIts biochemical composition and formationIts biochemical composition and formation

•Photosynthesis•Light phasecyclic and noncyclic and non--cyclic cyclic photophosphorylationphotophosphorylation

•Dark phaseC3 and C4 pathwaysC3 and C4 pathways

•Respiration and oxidative phophorylation•Biosynthesis of polysaccharides and chlorophyll•Nitrogen fixation, transport of nitrogenous compounds and their stage

Plant Biochemistry 350Plant Biochemistry 350

Importance of PlantsALL of the food we eat comes either directly or indirectly from plants.

Maize, wheat and rice are the main crops that feed the world. All of these produce starch and all can be stored.

Oxygen budget: Animal (human!) RespirationUseful substances in Agriculture, Medicine, & Industry

Virtually ALL medicines today have their origin in plant chemicalsSeveral major industries are based on plants or plant’s products

Plants are a major player in regulation of the Earth’s ecosystem

Plants and Energy flowEnergy enters as sunlightProducers convert sunlight to chemical energy.

PlantsSmall organisms

Consumers eat the plants (and each other).

AutotrophsHeterotrophs

Decomposer organisms breakdown the organic molecules of producers and consumers Earth is an open system for energy

Sun always provide constant energy!Earth is a closed system for matter

All elements go through recycling

Matter & EnergyThe earth is a closed system to matterMatter cycles through an ecosystem; everything that was ever here still is

Major Biogeochemical Cycles:Water/hydrologicCarbonOxygenNitrogen PhosphorousSulfur

THE CARBON CYCLECarbon is the “brick” of all living things.To get carbon, people eat plants.Many cycles for carbon Plants use photosynthesis

THE CARBON CYCLE

The Carbon CyclePlants take in CO2 for photosynthesis and also release some during respiration.

THE CARBON CYCLE

Other Sources of Atmospheric CO2 ExchangesCO2 is also exchanged between the oceans and the atmosphere.

Dissolved CO2 in the oceans is used by marine plants.Cold water takes in CO2 while warm water releases it back to the atmosphere. Currents carry CO2 to the depths of the ocean and back up again.

Fossil Fuel burning (gas, coal, oil)Increased pollutant CO2 from fossil fuels creates THE GREEN HOUSE effectAlso increased deforestation removes plants from the equation.

Fire (natural or otherwise) provides an important source of carbon in the form of carbon monoxide.

The amount of Carbon Dioxide in the atmosphere naturally fluctuates through the year.

In the cold months, respiration and photosynthesis cease and the exchange between plants and atmosphere stops.

CELL ORGANELLS

WHAT IS A CELL ORGANELL?

An organelle is a membrane-bound structure that carries out specific

activities for the cell

CELL ORGANELLSCell MembraneNucleusCytoplasmMitochondriaGolgi ComplexRibosomesSmooth Endoplasmic ReticulumRough Endoplasmic ReticulumCell WallChloroplastCentral VacuoleLysosome

Plant and animal cellsBoth cells have many common components like:

Nucleus, Mitochondria, ER, Golgi, Ribosome, Plasma membrane, Cytosol, & Microtubules and microfilaments (cytoskeleton)

But Plant Cell has these unique components:Cell wallChloroplastCentral Vacuole

By contrast, Animal Cell hasCentrioles (important for cell division) Lysosomes (plant cell has peroxisomes and glyoxisomes),

Plant Cell

Cell Membrane

••Every cell is Every cell is enclosed by a enclosed by a cell membrane.cell membrane.

••It controls the It controls the passage of passage of materials in and materials in and out of the cell.out of the cell.

CELL WALL (Plant cell only)

••Rigid and Rigid and strong wall.strong wall.

••Protects and Protects and maintains the maintains the shape of the shape of the cell.cell.

NUCLEUS

••The control center of The control center of the cell. the cell.

••It contains the DNA It contains the DNA code for the cell coiled code for the cell coiled into chromosomes.into chromosomes.

CYTOPLASM

••Not a Cell Not a Cell organelle but organelle but very important very important part of the cellpart of the cell

••All organelles All organelles reside (live and reside (live and float around in) float around in) the cytoplasm.the cytoplasm.

MITOCHONDRIA

••This organelle This organelle processes energy processes energy for a cell for a cell

••It makes ATP It makes ATP (ATP = energy)(ATP = energy)

••Involved in Involved in cellular cellular respirationrespiration

GOLGI COMPLEX

••The protein The protein packaging and packaging and transport center transport center of the cellof the cell

••Has incoming Has incoming and outgoing and outgoing vesicles.vesicles.

RIBOSOMES (Not a Cell organelle-But important)

••Synthesizes proteinsSynthesizes proteins

••Present in cytoplasmPresent in cytoplasm

••Present with Rough ERPresent with Rough ER

••No membrane present.No membrane present.

SMOOTH ENDOPLASMIC RETICULUM

••Transports materials Transports materials throughout the cellthroughout the cell

••Digests lipidsDigests lipids

••Produces proteins.Produces proteins.

ROUGH ENDOPLASMIC RETICULUM

••Covered with Covered with ribosomesribosomes..

••Produces proteins.Produces proteins.

••Transports Transports materials materials throughout the cell.throughout the cell.

LYSOSOMES

••Breaks down Breaks down materials for materials for digestiondigestion

••Contains special Contains special enzymes for enzymes for digestion in the digestion in the cellcell

VACUOLE (Plant cell only)

••Most plant cells Most plant cells have one large onehave one large one

••Filled with fluidFilled with fluid

••Helps maintains Helps maintains turgorturgor pressure pressure and shape of celland shape of cell

CHLOROPLAST (Plant cell only)

••Contains Contains chlorophyllchlorophyll

••Makes plants greenMakes plants green

••Uses light energy Uses light energy to make ATP & to make ATP & sugarssugars

••Photosynthesis Photosynthesis occur in this occur in this organelleorganelle

CELL WALL

••Rigid and strong Rigid and strong wallwall

••Protects and Protects and maintains the maintains the shape of the cellshape of the cell

The Cell Wallalmost all plant cells have a protective wall of great tensile strength (Primary ±Secondary Cell wall) depending on growing state of the cell

10-25 nm in diameterConsists of long-chain polysaccharides The composition varies between different species

Most common: cellulose in the primary, lignin in the secondary

The polysaccharide chain folded into fibers and micro-fibrils

Primary & Secondary wallGrowing cells have primary cell walls that are usually thin and extensible, although tough.

Mature cells no longer needs to be extensible: a rigid, secondary cell wall is produced by either:

hardening of primary cell wall , or adding secondary cell wall between plasma membranes and primary wall

Secondary cell wall may have a composition similar to that of the primary wall or be markedly different.

Primary Cell WallThe cell wall is a network of1. microfibril threads (chains of

cellulose)2. cross-linking polysaccharides

(hemicellulose and/or others) 3. matrix of mainly acidic

polysaccharides (pectins)4. calcium bridges pectin chains

• Typically, cellulose, hemicellulose, and pectin are present in roughly equal amounts.

•Cellulose and cross-linking glycansprovides tensile strength,• Pectin is the sticky polysaccharide.

• The middle lamella is rich in pectin and cements adjacent cells together.• Proteins Constitutes about 5%.

http://http://en.wikipedia.org/wiki/File:Plant_cell_wall_diagram.svgen.wikipedia.org/wiki/File:Plant_cell_wall_diagram.svg

PectinPectin

Features of Cell Wall: SummaryCell wall is found in all plant cells except sperm and some eggs. It consists

of three zones: (outward inward)

(1) Middle lamella – mostly pectin, cements adjacent cells together

(2) Primary cell wallFound in all plant cellsCellulose matrix with hemicellulose, proteins, pectin, lignin, cutin, and waxCharacteristic of undifferentiated cells or ones that still are growing

(3) Secondary cell wallJust inside primary cell wallCharacteristic of mature cellsComprised of hemicellulose and ligninMay have 3 layers

Connections between Cells: PlasmodesmataPlasmodesmata (1=plasmodesma) are microscopic channels through the cell walls and middle lamella between adjacent plant cells Link adjacent plasma membranes and cytoplasm

Desmotubule: modified endoplasmic reticulum strands lined by plasma membrane

They enable regulated intercellular transport and communication between them (800-900 Da, soluble sugar, AA, N)

Glycans of cell wall: CelluloseCellulose, the most abundant polymer on earth, ~ one half of theorganic carbon. Linear polymer of glucose, with (β1 4) linkages and alternate rotation (180°), to form long straight chains (2-250K residues).About 36 cellulose chains are associated by hydrogen bonds to a crystalline lattice structure known as a microfibril. These structures are impermeable to water, of high tensile strength, very resistant to chemical and biological degradations

However, many bacteria and fungi have cellulose-hydrolyzing enzymes (cellulases)

O

C

HOH

OH

CH2OH

O

C

HOHOH

OH

CH2OH

O

C

HOH

OH

CH2OH

O

C

HOH

OH

CH2OH

O

C

HOH

OH

CH2OH

O

C

HOH

OH

CH2OH

OH

Glycans of cell wall: HemicelluloseHemicellulose is heterogeneous group of branched

polysaccharides polymers that cross-link cellulose fibrils into robust network.

defined as those which can be extracted by alkaline solutions. They all have a long linear backbone composed of one type of sugar (glucose, xylose, or mannose) with several branches.

Glycans of cell wall: PectinPectins are a heterogeneous group of branched polysaccharides that contain

many negatively charged galacturonic. They form negatively charged, hydrophilic network that gives compressive strength to primary walls; cell-cell adhesion.

Pectin is a soluble compound in the absence of Ca2+/Mg2+ , but forms amorphous deformable gel in their presence (effect of free carboxyl groups).

Food industries use of this property when preparing jellies and jams.

Glycans of cell wall: LigninThe most common additional polymer in secondary walls is lignin

Found mostly in the walls of the xylem vessels and fiber cells of woody tissues.

Lignin causes the walls to become thick, stiff, and incompressibleLignin is a ploymer of cross-linked coumaryl, coniferyl, and sinapyl alcohols

Functions of The Cell WallCell wall is thicker, stronger and more rigid than similar components around animal cells. It forms barrier against pathogens and deters herbivoresThe wall is responsible for:

Osmoregulation (see later)Cell adhesion, protection and supportIntercellular communication through plasmodesmataRegulated exchange of selected molecules and fluids

In growing state, the wall has dynamic nature that allows expansion.In Mature state, the wall determines cell morphology

Secondary cell wall may contain lignin for greater supportSpecialized cells have unique cell wall adaptations depending on function and environment

Cell Wall, Shape & Classification

A) A trichome, or hair, on the upper surface of a leaf is shaped by the local deposition of a tough, cellulose-rich wall. (B) Surface view of tomato leaf (like the pieces of a jigsaw puzzle). The outer cell wall is reinforced with a cuticle and waxes that waterproof the leaf and help defend it against pathogens. (C) Secondary cell wall that creating robust tubes for the transport of water throughout the plant (view into young xylem)

Cell wall & TurgorCell walls is made of neutral and charged polysaccharides absorbs H2OIts environment is hypotonic to the cell H2O must flow to the cellIncreased H2O inside the cell Turgor Pressure If a plant cell is turgid, It is very firm, a healthy state in most plantsIf a plant cell is flaccid, It is in an isotonic or hypertonic environment

OSMOSIS AND TURGOR PRESSUREPLANT CELL

Hypertonic solution → Plasmolysed cellIsotonic solution → Non-turgid or wilted cellHypotonic solution → Turgid cell (Usual environment)

ANIMAL CELLHypertonic solution → Cell shrinksIsotonic solution → Normal (Usual environment) Hypotonic solution → Cell swells and may burst

OSMOSIS AND TURGOR PRESSURE

PHOTOSYNTHESIS

THE BASICS OF PHOTOSYNTHESISOrganisms can be classified based on how they obtain energy into autotrophs & heterotrophs.

Autotrophs generate their own organic matter through photosynthesisSunlight energy is transformed to energy, stored in the form of chemical bonds

Almost all plants are photosynthetic autotrophs (also some bacteria and protists)

May occur in stems of plants that do not have leaves

plants Ferns Euglena CyanobacteriaHorsetails

Photosynthesis

Location: ChloroplastsEnergy comes from Photons from SUN and H2O splittingProcess: two main sets of reactions

1) Light Reactions: capture energy to synthesize ATP and NADH

It uses Electron transport chain & Photorespiration2) Calvin cycle to fix CO2

Several mechanisms of C-fixation

Structure of a leaf

http://http://www.emc.maricopa.edu/faculty/farabee/BIOBK/leafstru.gifwww.emc.maricopa.edu/faculty/farabee/BIOBK/leafstru.gif

Important structures in a leafTwo structures important for photosynthesis areStoma (pl. Stomata): Pores in a plant’s cuticle through which water and gases are exchanged between the plant and the atmosphere.Mesophyll cells: Contain a lot of chloroplasts (between 40-200) arranged to receive maximum amount of light.CO2 O2

Guard cell Guard cell

Mesophyll cellStoma

PlastidsPlastids are a family of organelles

surrounded by double membraneAll are maternally inheritedHave their own DNA and ribosomesHave their own unique functionsDivide by binary fission (like bacteria)

There are several types, the most abundant ones are:Chloroplasts - photosynthetic; green due to chlorophyll contentChromoplasts: contain pigments other than chlorophyll (in fruits, leaves, flowers)Leucoplasts: involved in lipid biosynthesisAmyloplasts: store starch (colourless)Etioplasts: intermediate state in production of chloroplasts, in tissue exposed to light for the first time

The ChloroplastUsually lens-shaped, an organelle needed for photosynthesisHas internal membrane system arranged into flattened sacs (=thylakoids) 2 compartments: thylakoid space and stroma

thylakoids stacked forming grana (1 granum)Contains the green pigment chlorophyll & pigments of other colors (red, blue, yellow/brown) depending on light conditions, chloroplasts can move within the cells e.g. to the surface to catch more light in low light conditions.

How do cells harvest energy?All organisms use cellular respiration to extract energy from organic molecules.

Aerobic respiration: C6H12O6 + 6O2 6CO2 + 6H2OΔG = -686kcal/mol of glucose

Plants and certain algae/bacteria use photosynthesis to synthesize organic molecules (sugar) using light energy, CO2, & H2O.

Photosynthesis: 6CO2 + 6H2O C6H12O6 + 6O2Energy for this reaction comes from the sun

Photosynthesis & RespirationBoth respiration and photosynthesis handle this large energy in small steps rather than all at once.Both processes collect the released energy to synthesize ATP

Energy from food

O2 2 H2OGlucose 6 CO2

Energy from Sun

2 H2O O26 CO2 Glucose

Released Eergy is collected to make ATP

OXIDATION AND REDUCTIONRemember OIL RIGOIL:OIL: Oxidation Is Loss of electron

look for Oxygen addition or dehydrogenation

RIG:RIG: Reduction is Gain of electronlook for Oxygen removal or hydrogenation

6 CO2 + 6 H2O C6H12O6 + 6 O2

Reduction

Oxidation

Oxidation & ReductionBoth respiration and photosynthesis shuttle electrons through a series of electron carriers to a final electron acceptor.

NADH, FADH2, Chlorophylls, Quinones, Cytchromes, etc.

All reactions involved oxidation/reduction steps:

6 CO2 + 6 H2O C6H12O6 + 6 O2

Reduction

Oxidation

PhotosynthesisA series of chemical reactions that enable plants, algae, and some bacteria to covert CO2 and H2O into Sugars using SUN light .

6CO2 + 6H2O C6H12O6 + 6O2Photosynthesis is anabolic (construction of molecules from smaller units) & endergonic (absorbing energy).It takes place in the leaves of all green plant, & reaction centers of algae & bacteria (if any).

Photosynthesis is two separate sets of reactions1. Light Reaction

Produces energy from solar power (photons) in the form of ATP and NADPH.

2. Calvin Cycle or dark Reaction or Carbon Fixation Reaction.Uses energy (ATP and NADPH) from light reaction to make sugar (glucose).

An overview of photosynthesisPhotosynthesis has two processes: each with

multiple stepsLight reactions

convert solar energy to chemical energylight energy drives transfer of electrons to NADP+ forming NADPHATP is generated by photophosphorylationoccur at the thylakoids

Calvin cycle Named after Melvin Calvin who illustrated many of its steps in the 1940sIncorporates CO2 from the atmosphere into an organic molecule (carbon fixation)Uses energy from the light reaction to reduce the new carbon to a sugarOccurs in the stroma of the chloroplast

LightChloroplast

NADP+

ADP+ P

Tracking atoms through photosynthesisDoes the O2 come from the CO2 or the H2O?Some bacteria can use H2S (hydrogen sulfide) instead of H2O in photosynthesis

produce yellow globules of sulfur as waste H2S is split, producing sulfurOther scientists used oxygen isotope (18O ): either CO2 or H2O

18O label appears in O2 only when H218O is used

6 CO2 + 12 H2O

C6H12O6 + 6 H2O + 6 O2

Reactants

Products

Outline of Light-Dependent ReactionsHow Is Light Energy Converted to Chemical Energy?

Captured sunlight energy is stored as chemical energy in two carrier molecules: ATP & NADPHThe pathways are different in plants versus bacteria, but the processes are similar

In PlantsLight Is First Captured by Pigments in Chloroplasts

Pigments are bound to proteins of the Thylakoid MembranesPhotosystem II pass electrons through ETC to generates ATPPhotosystem I pass electron through carriers to generates NADPHSplitting Water maintains the flow of electrons through the photosystems

Light & the Electromagnetic SpectrumLight is electromagnetic radiation that has a dual nature:

wave - explains physical properties of light itselfTwo waves one electrical and one magnetic propagating at 90-degrees to each other.Wavelength (λ) - distance between successive crests of a wave Frequency (ν)- number of wave crests passing a point in 1 second; one hertz= one cycle per second (1/s)

Always, λ ∗ ν = c (speed of light in vacuum = 2.998 x 108 m/s)

Particulate (photon) - explains how light interacts with matterPhoton has a discrete energy: E = hν = hc /λ ; where h is Planck’s constant=6.63 x 10-34 J sOnly discrete energies of light are absorbed by matter, i.e., light is quantized

Direction

λ=wavelengthElectric

Magnatic

Regions of the Electromagnetic Spectrum

Radar TV FM AM

Energy

Wavelength

Cancer treatment imaging Illumination Heating Signal TransmissionCooking Uses

γ-rays x-rays UV Infrared Microwave Radiowave

400

nm

700

nm

Region

0.1 0.01 10 1.0 1.0 0.1 1.0 100pm nm nm μm mm m m m

N Y N Y

106 K 104 K 102 K 1K

PenetrateAtmosphere?

Temperature of emitting bodies

Nuclear Electronic Vibrational Rotational Transition

Visible

ChlorophyllsThe principle photoreceptor in photosynthesis is Chlorophyll

Chlorophyll a & b in plant, bacteriochlorophyll a & b in bacteria

Chlorophyll is similar to the heme group of globins and cytochromes, but with very significant differences

Mg2+ is in the center, not Fe2+Ring V is fused to pyrrole ring IIIHydrocarbon tailRing IV is partially reduced

ChlorophyllsRatio of Chlorophyll a:b in plant (3:1)

Only chl-a is a constituent of the photosynthetic reaction centers, hence central photosynthesis pigment

Chlorophyll molecules are bound to chlorophyll-binding proteins.

In a complex with proteins the absorption spectrum of the bound chlorophyll may differ considerably from the absorption spectrum of the free chlorophyll

The same applies for other light-absorbing substances, (carotenoids, xanthophylls etc)

Chlorophyll & Pigments & LightFree absorbing substances are called chromophore (Greek, carrier of color) and the chromophore-protein complexes are called pigments.

Pigments are often named after the wavelength of their absorption maximum.

Chlorophyll-a 700 means a pigment of chl-a with an absorption maximum of 700 nm.

Another common designation is P700 (makes the nature of the chromophoreopen)

Chlorophyll & Pigments & LightAll photosynthetic organisms have Chlorophyll a

Chlorophyll a absorbs Light in Red (660 nm) and Blue (450 nm) Wavelengths

Leaves are green because chlorophyll reflect the Green light (which is detected by our eyes)

The Color of the pigment comes from the wavelengths of light reflected NOT absorbed

Accessory Pigments (Light Antenna)Observation: photosynthetic organism have more chlorophyll molecules than is needed by reaction centers

Chlorophyll also function to gather lightLight Harvesting complex are membrane proteins containing pigments to absorb light energy outside the range of chlorophyll. The most common pigments are Chlorophyll b, Carotenoids, Xanthophylls & Pilins (in water-dwelling algae & Bacteria).

Chlorophyll and Accessory Pigments

Chlorophyll and Accessory Pigments

http://www.biologie.unihttp://www.biologie.uni--hamburg.de/bhamburg.de/b--online/e24/3.htmonline/e24/3.htm

Fall ColorsLeaves contain chlorophyll and other pigments, but they appear green because chlorophyll is the major component.During the fall, the green chlorophyll pigments are greatly reduced revealing the other pigments: Carotenoids and/or Xanthophylls.

Excitation of Chlorophyll by LightWhen a pigment absorbs light

It goes from a ground state to an excited state, which is unstable

Excitedstate

Ener

gy o

f el e

ctio

n

Heat

Photon(fluorescence)

Chlorophyllmolecule

GroundstatePhoton

e–

Figure 10.11 A

Excited chlorophyll & PigmentsIf a Pigment absorbs light, it must release its energy to returnto its ground stateThis can be accomplished via four common mechanisms:

1. Dissipated as Heat (the most common route in general)2. Transferred to another molecules (required special arrangements)3. Emitted as Fluorescence (required special molecules)4. Trigger a Chemical Reaction (special molecules)

Efficiency of photosynthesis is nearly 100% due to special arrangement of proteins in the thylakoids membrane

Such arrangement prevents dissipation of energy as heat only the other three mechanisms are important for photosynthesis

How light is harvestedWhen any antenna molecule absorbs a photon, it initiates a series of energy transfers eventually reaching a particular chlorophyll a in the reaction centerAt the REACTION CENTER , energy from light excites

an electron in chlorophyll, which initiates a series of reactions leading to generation of ATP and NADPH

Photosystem I and Photosystem II

Photosystem I (PS I)It needs light of longer wave lengths (lambda > 700 nm)

Photosystem II (PS II)It becomes active when exposed to shorter wave lengths (lambda < 680 nm)

Photosystem II

http://www.sirinet.net/~jgjohnso/lightreactionproject.html

Photosynthesis Stages

2-Stage ProcessLight Reactions

Require Light to OccurInvolves the Actual Harnessing of Light EnergyOccur in\on the Grana

Dark ReactionsDo not Need Light to OccurInvolve the Creation of the Carbohydrates

Products of the Light Reaction Are Used to Form C-C Covalent Bonds of Carbohydrates Occur in the Stroma

http://www.daviddarling.info/images/chloroplast.jpg

Light Reactions

Electron TransferWhen Light Strikes Magnesium (Mg)

Atom in Center of Chlorophyll Molecule, the Light Energy Excites a Mg Electron and It Leaves Orbit from the Mg Atom

The Electron Can Be Converted to Useful Chemical Energy


Light ReactionsPhotophosphorylation

The Excited Electron (plus Additional Light Energy) eventually Provides Energy so a Phosphate Group Can Be Added to a Compound Called Adenosine Diphosphate (ADP), Yielding Adenosine Triphosphate (ATP)ATP Is an Important

Stored Energy Molecule


Adenosine-5'-triphosphate (ATP)ATP = Adenosine-(PO4-)-(PO4-)-(PO42-)3 Phosphate Groups Stuck off the End

of an Adenosine MoleculeFairly Simple Compound Containing NitrogenThe String of 3 Phosphate Groups Is Held Together by Covalent BondsPlays an important role in cell biology as a coenzymeTransports chemical energy within cells ATP is made from adenosine diphosphate (ADP) or adenosine monophosphate (AMP)Continuously recycled in organisms

Adenosine-5'-triphosphate (ATP)For some Reason, Phosphate

Groups in a String Need a Really, Really Strong Bond to Hold Them Together

So the Ones within the String Are Extremely StrongThink of the Bond Like a Rope in a Tug-of-War with 2 People Pulling on the Rope in Opposite DirectionsIf someone Comes along and Cuts the Rope the 2 People Will Go FlyingThey Go Flying off because Lots of Energy Was Being Stored in the Rope and the Energy Was Released as the People FellWhen the Bond that Attaches 1 of the Phosphate Groups onto ATP Is Broken, It Becomes ADP

Energy MoleculesATP and NADPH2

ATP and NADPH2 Are Common Energy-Carrying Molecules in all Plant and Animal Cells ATP Gives up the Phosphate Group when It Is Involved in a Chemical Reaction

This Gives off a Lot of Energy which Helps the Needed Reaction Occur

Same Thing Happens when NADPH2 Gives off the Hydrogen Atoms as Part of a Reaction

It Provides Energy to Drive that ReactionATP and NADPH2 Are Renewable or Recyclable Energy Sources

Light Reactions

Photolysis (Hill Reaction)The 2 Water Molecules Are Split into Hydrogen and OxygenThe Hydrogen Is Attached to a Molecule Called Nicotinamide Adenine Dinucleotide Phosphate (NADP)Produces NADPH2The Oxygen Is Given off as Oxygen Gas

2 H20 + NADP + lightNADPH2 + O2


http://upload.wikimedia.org/wikipedia/commons/1/18/Thylakoid_membrane.png

LIGHT REACTIONProduces NADPH, ATP, and oxygen

Figure 10.13 Photosystem II(PS II)Photosystem-I

(PS I)

ATPNADP

H

NADP+

ADPCALVINCYCLE

CO2H2O

O2 [CH2O] (sugar)

LIGHTREACTIONS

Light

Primaryacceptor

Pq

Cytochromecomplex

PC

e

P680

e–

e–

O2+

H2O2 H+

Light

ATP

Primaryacceptor

Fde e–

NADP+reductase

ElectronTransportchain

Electron transport chain

P700

Light

NADPH

NADP++ 2 H+

+ H+

1

5

7

2

3

4

6

8

Light ReactionA mechanical analogy for the light reactions

Photosynthesis: The Main PlayersIn eukaryotes, photosynthesis is carried out by four protein complexes, all located in the thylakoid membrane

Photosystem II or P680: pass electrons & splits H2OCytochrome b6f complex : the electric connector (plastoquinone & plastocyanin) Photosystem I or P700 generates NADPHProton translocating ATP synthase complex (CF1 & CF0)

Electron Flow During Light Reaction During the light reaction, there are two possible routes for electron flow

All reactions Occurs in the Thylakoid membranes

Noncyclic Electron FlowUses PS II and PS I (P680 & P700)Uses Electron Transport Chain (ETC)Generates O2, ATP and NADPH

Cyclic Electron FlowUses Photosystem I only: P700 reaction center Uses Electron Transport Chain (ETC)Generates ATP only

Noncyclic Electron Flow-1Accessory pigments in Photosystems absorb light and pass energy to reaction centers containing chlorophyll a

Electrons are ejected from P680 upon absorption of photons (Mg2+ of chlorophyll)

This generates a strong oxidant capable of oxidizing H2O

Noncyclic Electron Flow-2Electrons ejected from P680 are replaced with electrons abstracted from H2OSplitting of H2O is carried out by an Mn-containing Enzyme complex (5 complex steps).Energetics:

Production of O2 requires the oxidation of 2 H2OTransfer of 1 electron from H2O to NADP+ requires two photochemical eventsA minimum of 8 photons are required per 1 O2 produced

(2 x 2 x2)

Noncyclic Electron Flow-3

Each ejected electron is passed through a chain of electron carriers (oxidation-reduction steps:

Plastoquinone (Pq) cytochrome complex plastocyanin (Pc)

Energetics:Splitting of H2O release 2 electrons, two protons, and ½O2 4 protons are generated per O2

Protons are pumped into thylakoid space through ETC membrane potential

Membrane potential is used to synthesize ATP via chemiosmosis


In continuation of electron flow, plastocyanin (Pc) regenerates Photosystem I (P700), which ejects electrons from its chlorophyll amolecule

Photosystem I can be excited independently by light


Ejected electrons from PS-I are passed through another series of electron carriers that ends with the reduction of NADP+ to NADPH

Noncyclic Electron Flow-Summary

In Fact, a complex set of chemical reactions must occur in a coordinated manner for the synthesis of carbohydrates

To produce a sugar molecule, plants use nearly 30 distinct proteins to work within a complicated membrane structure

Nocyclic flow is called the Z-schemeThe Overall scheme for PS is deceptively simple:

Energy by photons to and splitting of H2O is used generates O2, ATP, and NADPH

Cyclic Electron FlowMost electrons passing PS-I are used to reduce NADP+Some electrons may pass via the cytochrome complex back to P700 Cyclic flow of electronsThis process generates ATP only (No NADPH or O2)

Cyclic flow increases [ATP] production relative to that of [NADPH].

Photons from the SUN

Noncyclic and Cyclic Electron FlowNoncyclic electron flow

Involves PS-II and PS-IInvolves splitting of H2O and production of O2produces ATP and NADPH in roughly equal amounts

Cyclic electron flow Involves PS-I onlygenerates only ATP

Why are there two types of electron flow?Calvin cycle consumes more ATP than NADPH

Distribution of PSII & PSI PSII & PSI have characteristic distribution within the thylakoidmembrane

PSI is located mainly in the unstacked membrane access to NADP+PSII is located in mainly the stacked membrane Cytochrome b6f is uniformly distributed

This arrangement permits chloroplast to respond to changes in illumination & prevent direct electron transfer between the systems

ATP Generation in ChloroplastsRemember the definition of Photosynthesis & Respiration.Mechanisms for ATP generation are similar in chloroplasts and mitochondria: chemiosmosisEach time electrons pass through the cytchrome system, protons are pumped across thylakoid membrane

Move from stroma into the thylakoid spaceThis generates H+ gradient across the membranes proton-motive forceFlow of H+ back across thylakoid membrane energizes ATP synthase: ADP ATP

Chemiosmosis in Chloroplasts

Calvin CycleAlso called: the dark reaction, Carbon Fixation, or C3-cycle.It is a set of complex reactions that occurs in the stroma.Uses ATP and NADPH from light reaction to add 1 CO2to ribulose bisphosphate (RuBP)To produce one glucose molecule, the cycle must turn 6 times: 6 CO2 1 glucose (C6H12O6) This cost 18 ATP and 12 NADPH, all of which come from light-dependent reaction.

CLAVIN CYCLE

Calvin Cycle

Summary of Calvin Cycle

Three stages of Calvin CycleCalvin cycle can be divided into three stages:

1= Carbon fixation stage (CARBOXYLATION)6 Ribulose bisphosphate (RuBP) + 6 CO2 yield twelve 3-carbon phosphoglyceric acid (PGA) molecules

2- Synthesis of Glyceraldehyde 3-Phosphate (G3P)(REDUCTION)Phosphoglyceric acid (PGA) molecules are converted into glyceraldehyde 3-Phophate (G3P) molecules

Energy is donated by ATP and NADPH

Three stages of Calvin Cycle

3- Regeneration of Ribulose bis-phosphate (RuBP)(REGENERATION)10 of 12 G3P molecules converted into 6 RuBP molecules2 of 12 G3P molecules used to synthesize 1 glucose

First Stage: CO2-Fixation (CARBOXYLATIONThe enzyme that catalyzes the first step in Calvin cycle is Ribulose bisphosphate carboxylase

It is the most important enzyme in nature that carries “True” CO2fixation reactionThe most abundant protein in the biosphere (~15- 50% of leaf proteins)

Its overall reaction is believed to proceed as follows:

CCCCC

OO

OHOHO

HH

H

HH

H

P

P

O

O-

O-

O

O-

O-

CC

OHO

H

HH P

O

O-

O-

CO O

-

3-Phosphoglycerate

Carboxylase

C

CCCC

O

OHO

HH

H

HH

P

P

O

O-

O-

O

O-

O-

OOH C

O

O-

CO2CCCCC

O

OHOHO

HH

H

HH

P

P

O

O-

O-

O

O-

O-

O-

Ribulose-1,5-bis-phosphate

Enediolateintermediate

β-Ketointermediate

H+

H2O

CC

OHO

H

HH P

O

O-

O-

CO O

-

RuBP CarboxylaseRuBP Carboxylase in plants is a very complex enzyme consisting of:

8 large catalytic subunits (477 residues, each (blue &cyan)

Encoded by Chloroplast gene8 small subunits (123 residues, each (red)).

Encoded by nuclear genesThe large subunit has the catalytic activity

The small subunit probably modulate the activity by increasing Kcat.

Some bacteria contain only the large subunit, with the smallest functional unit being a homodimer, L2.The enzyme has low catalytic activity (Kcat=~ 3 S-1)

RuBisCO PDB 1RCX

The enzyme fixes 10^11tons of CO2 per yearCrude oil consumption: 3*10^9 tons per year

RuBisCORuBP Carboxylase can under certain condition work also as an oxygenase, thereby fixing O2 instead of CO2, hence the name RuBisCO

The enzyme has much higher affinity for CO2 than O2 the oxygenase reaction is significant only under conditions in which CO2 levels are low and O2 levels are highOxygenase reaction is responsible for Photorespiration (see later).

CCCCC

OO

OHOHO

HH

H

HH

H

P

P

O

O-

O-

O

O-

O-

CC

OHO

H

HH P

O

O-

O-

CO O

-

CC OH

H PO

O-

O-

O O-

2-Phosphoglycolate

3-PhosphoglycerateCarboxyl

ase

Oxygenase

C

CCCC

O

OHO

HH

H

HH

P

P

O

O-

O-

O

O-

O-

OOH C

O

O-

C

CCCC

O

OHO

HH

H

HH

P

P

O

O-

O-

O

O-

O-

OOH O O

- CC

OHO

H

HH P

O

O-

O-

CO O

-

CO2

O2

CC

OHO

H

HH P

O

O-

O-

CO O

-

3-Phosphoglycerate

CCCCC

O

OHOHO

HH

H

HH

P

P

O

O-

O-

O

O-

O-

O-



β-KetointermediateH

+

H2O

H2O

Second Stage: Synthesis of G3P (GAP)The second stage of Calvin Cycle is like the Glycolysis running backward, except for

These reactions occur in the stroma of chloroplast different enzymesChloroplast Dehydrogenase uses NADPH as e- donor, while the cytosolic Glycolysis enzyme uses NAD+ as e- acceptor.

This is the most expensive stage in term of ATP and NADPH consumptionWe know from the first stage that:

6 Ribulose bisphosphate (RuBP) + 6 CO2 yield twelve 3-carbon phosphoglyceric acid (PGA) moleculesThese sets of reactions must run 12 times per glucose molecule synthesized

The sequence of events are proposed to proceed as follows:

3-Phosphoglycerate

CC

OHO

H

HH PO3

-2

CO O PO3

-2

CC

OHO

H

HH PO3

-2

CO O

-

CC

OHO

H

HH PO3

-2

CO H

CC

OO

HH PO3

-2

C OH

H

H

PhosphoglycerateKinase

Dehydrogenase Triose PhosphateIsomerase

NADPHATP ADP

NADP+

Pi

1,3 Bisphospho-Glycerate

D-Glyceraldehyde3-Phosphate

DihydroxyacetonePhosphate

Third stage of Calvin cycleThe second stage produces 12 G3P molecules

2 molecules are transported across the membrane to be used in the synthesis of 1 glucoseThe remaining 10 molecules are converted into 6 RuBP molecule

3-PhosphoGlycerate

TriosePhosphate

RuBP

CO2 Inner chloroplastMembrane

TriosePhosphate

6

12

10

2

6

Third stage: Regeneration of (RuBP)Complex series of reactions where five 3C-G3P molecules are rearranged to make three 5C-RuBP molecules

An additional ATP molecule is consumed for generating RuBP (reaction is not shown)

Fructose 1,6-bisphosphate

Fructose 6-P

Dihydroxyacetone PGlyceraldehyde P

Erythrose 4-P Xylulose 5-P

Sedoheptulose 1,7-bisphosphate

Sedoheptulose 7-P

Glyceraldehyde PDihydroxyacetone P

Xylulose 5-P

Glyceraldehyde P

Ribose 5-P

P

P

C3 + C3 C6

C3 + C6 C4 + C5

C3 + C4 C7

C3 + C7 C5 + C5

5 C3 3 C5

Carbons

Regeneration of (RuBP)These reactions are similar to Pentose Phosphate Pathway reactions, but running backward.

Overall: 5 C3 3 C5Enzymes:

TI, Triosephosphate IsomeraseAL, AldolaseFB, Fructose-1,6-bisphosphataseSB, Sedoheptulose-BisphosphataseTK, TransketolaseEP, EpimeraseIS, IsomerasePK, Phospho-ribulokinase

TK

EP

PK

glyceraldehyde-3-P dihydroxyacetone-P

fructose-6-P

xyulose-5-P + erythrose-4-P

sedoheptulose-7-P

xylulose-5-P + ribose-5-P

(3) ribulose-5-P

(3) ribulose-1,5-bis-P

TI

TK

AL, FB

IS

AL, SB

Dark Reactions‘Calvin Cycle’‘Carbon Reactions Pathway’Do not Require Light Energy to Occur

Do Require Energy Captured by Light Reactions

http://www.ualr.edu/~botany/calvincycle.gif

Dark Reactions

Occur at same Time as Light ReactionsCease Soon if Light Energy Is not Available to Make Light Reaction Products

Exception: some Xerophytes

http://www.ualr.edu/~botany/thylakoidmembrane.gif

Dark Reactions2 Main Steps

Carbon Dioxide FixationSugar Formation

Via three stages1) Carboxylation2) Reduction3) Regeneration

Occur in the Stroma of the Chloroplasts

http://courses.cm.utexas.edu/jrobertus/ch339k/overheads-3/ch19_Dark-reactions.jpg

Carbon Dioxide Fixation‘Carbon Dioxide Assimilation’CO2 Is Incorporated into a 3-Carbon or 4-Carbon Chain

C3 PlantsC4 Plants

http://www.science.siu.edu/plant-biology/PLB117/JPEGs%20CD/0127.JPG

Alternative mechanisms of C-fixationPhotorespiration may have drastic effect on the viability of plants:In hot, dry weather: Stomata closed (preserve their H2O), O2 ↑ ,

CO2↓, Photorespiration ↑ photosynthesis ↓ (no glucose synthesis)

Plants have special adaptations to limit the effect of photorespiration

C4-plants in Hot, moist environments.15% of plants (e.g., corn, sugarcane , sorghum, millet, etc.)They store CO2 temporarily as C4 molecule, giving them advantage under high light, high temperature, low CO2 conditions.

CAM plants in hot arid climatesMany succulents plants (e.g., Cacti, Pineapple, agaves, etc.)Stomata open during night, and close during the dayUse Crassulacean Acid Metabolism (CAM)

Carbon Dioxide FixationC3 Plants

Most Plants Use an Enzyme Called RuBP Carboxylase (RuBisCo) to Carry out the CO2 Fixation

Enzymes Are Natural Proteins that Help Catalyze/Carry out ReactionsRubisco Is the most Abundant Enzyme on Earth!

This Occurs in the Mesophyll CellsPalisade or Spongy

Creates a 3-Carbon Product Ready for Sugar FormationCalled C3 Plants because the 1st Stable Carbon Chain Made from CO2 Has 3 CarbonsC3 Crops

Wheat, Soybeans, Cotton, Tobacco, Small Grains, Legumes, Tomatoes, Potatoes, Peppers, Cucurbits

http://www.uic.edu/classes/bios/bios100/lecturesf04am/rubisco01.jpg, http://www.palaeos.com/Eukarya/Lists/EuGlossary/Images/Rubisco.gif

Carbon Dioxide FixationC4 Plants‘Hatch-Slack Pathway’Process of CO2 Fixation for many Plants of Dry or Tropical OriginsPlants Use a Different Enzyme Called PEP (Phosphoenolpyruvate) carbooxylase in the Mesophyll Cells for CO2 Fixation

PEP Carboxylase Has a much Higher Affinity for CO2 than Does RubiscoAt Low CO2 Pressures, Rubisco Doesn’t Distinguish Well between O2 and CO2 so Stomata usually Have to Be Wide Open for PS to Occur

Creates a 4-Carbon Product http://www.ualr.edu/~botany/c4pathway.gif

Carbon Dioxide FixationC4 Plants

This 4-Carbon Chain Is then Transported into Bundle Sheath Cells where the CO2Is Released and then Immediately Fixed by Rubisco as Part of the C3Cycle

Bundle Sheath Cells Are Specialized Cells that Surround the Vascular Bundles in the Leaves

Same Fixation with Rubisco as in C3 Plants but Occurs in the Bundle Sheath Cells, not Mesophyll Cells

http://gemini.oscs.montana.edu/~mlavin/b434/graphic/Leafc4a.jpg, http://www.ualr.edu/~botany/c4pathway.gif

PEP Carboxylase vs. RubiscoPEP Carboxylase Works Well at Warm Temperatures but not Optimally at Cool TempsThis Is the Reason why C4 Grasses Are Referred to as Warm Season Grasses, but Do not Compete Well with C3Grasses at Cooler TempsC4 Grasses Have an Edge in Dry Warm Sites or Open Sunny Sites as They Can Keep Leaf Stomata Closed during Mid-Day and Extract every Last CO2 Molecule in the LeafIn Contrast, C3 Grasses that Keep Stomata Closed in Dry Sunny Sites Undergo High Amounts of Respiration

C4-PlantsAt least 19 plant families are C4 plants, e.g. Sugarcane, corn, and millet.

The C-4 pathway is not an alternative to the Calvin or even a net CO2 fixationit is a mechanism for CO2 delivery system under condition of O2 ↑ , CO2↓ to limit photorespiration

The C-4 plants has unique leaf anatomy to capture CO2

http://www.answers.com/topic/grain-millet-early-grain-fill-tifton-7-3-02-jpg-1http://commons.wikimedia.org/wiki/Image:Agave victoriae-reginae lv 2.jpghttp://www.answers.com/main/Record2?a=NR&url=http%3A%2F%2Fcommons.wikimedia.org%2Fwiki%2FImage%3AAgave%2520deserti%2520form.jpg

C4 LogisticsC4 plants: CO2 Fixation occurs twice: First in mesophyll, then in bundle-sheath cellsMesophyll cells fix CO2 as C4

PEP Carboxylase has a very high affinity for CO2 can fix CO2 when RuBP cannot

Mesophyll cells pump malate into bundle sheath cells. There:

Malate Pyruvate + CO2CO2 is fixed by RuBISCO

C C CH2O PO3

O-

O

C C CH2O

O-

OC

O

O-

PEP Oxaloacetate

PEP CarboxylaseCO2

Pi

C3 & C4 plantsC4 Plants Can Produce 3 Times as much Dry Matter per Unit of Water as C3 Plants

In hot environment:C4 Plants have higher CO2 assimilation Rates (2-3 Times) than that of C3 Plants Efficient Plants A Few C3 plants are as efficient as C4 Plants

In cooler Temperatures, C3 Plants have the advantage:

PEP Carboxylase Needed to Incorporate CO2 into the 4-Carbon Structure no Longer FunctionsC4 Photosynthetic Rates Drop Dramatically or Stop

PhotorespirationPlants that use the Calvin Cycles are called C3 Plants, because the 1st Stable Carbon Chain Made from CO2 Has 3 Carbons

Nearly 80% of plants, e.g. Wheat, Soybeans, Cotton, Tobacco, Grains, Legumes, Tomatoes, Potatoes, and Peppers

However, some plants close their stomata on hot, dry, & bright days to save their H2O

As a result, CO2 in leaf is reduced

Rubisco can add O2 instead of CO2 to RuBP (Oxygenase Reaction)Produces 2-C molecules instead of 3-C sugar molecules.Produces no sugar molecules or no ATP.

Photorespiration occurs in light onlyOccurs 1 out of 4 reactions under today’s atmospheric CO2Rate increases with temperature

PhotorespirationIn the "normal" reaction, CO2 is joined with RUBP to form 2 molecules of 3PGA

In the process called photorespiration, O2 replaces CO2 in a non-productive, wasteful reaction

In C4-type plants photorespiration is suppressed

It has long been the dream of biologists to increase the production of certain crop plants, such as wheat, that carry on C3 photosynthesis by genetically re-engineer them to perform C4photosynthesis

It seems unlikely that this goal will be accomplished in the near future due to the complex anatomical and metabolic differences that exist between C3- and C4-type plants http://www.marietta.edu/~spilatrs/biol103/photolab/photresp.html

CCCCC

OO

OHOHO

HH

H

HH

H

P

P

O

O-

O-

O

O-

O-

CC

OHO

H

HH P

O

O-

O-

CO O

-

CC OH

H PO

O-

O-

O O-

2-Phosphoglycolate

3-PhosphoglycerateCarboxyl

ase

Oxygenase

C

CCCC

O

OHO

HH

H

HH

P

P

O

O-

O-

O

O-

O-

OOH C

O

O-

C

CCCC

O

OHO

HH

H

HH

P

P

O

O-

O-

O

O-

O-

OOH O O

- CC

OHO

H

HH P

O

O-

O-

CO O

-

CO2

O2

CC

OHO

H

HH P

O

O-

O-

CO O

-

3-Phosphoglycerate

CCCCC

O

OHOHO

HH

H

HH

P

P

O

O-

O-

O

O-

O-

O-



β-KetointermediateH

+

H2O

H2O

Photorespiration

Respiration Driven by Light Energy

Occurs in Chloroplasts and other Structures in a Photosynthetic Cell

Rubisco can React with Oxygen to Start a slightly Different Series of Reactions

Result in a Loss or no Net Gain of Dry Matter for the PlantLess ATP Is Produced from the Photorespiration

http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif

Factors Influencing Photorespiration

O2 : CO2 Ratio

If Cells Have Low O2 but Higher CO2, Normal photosynthesis i.e. Calvin Cycle Dominates

C4 Plants Have Little Photorespiration because They Carry the CO2 to the bundle Sheath Cells and can Build up High [CO2]

Calvin Cycle Reactions always Favored over Photorespiration

If Cells Have Higher O2 and Lower CO2, Photorespiration Dominates



Light IntensityIncreasing Light Intensity will Increase Energy for the Photorespiration Process and for photosynthesis

C3 Plants Light-Saturate at Lower Light Intensities than C4 Plants

Reach Their ‘Break-Even Point’ at much Lower Light Levels due to Increasing Photorespiration



Temperature

Photorespiration Increases with Temperature

Plants Have Optimum, Minimum and Maximum Temp Ranges



Net Photosynthesis or Net Assimilation Rate

C4 Plants generally Have Net Assimilation Rates about 2 to 3 Times that of C3 Plants

C4 Plants Are often Called Efficient Plants and C3Plants Called Non-Efficient Plants

A Few C3 Plants Have Low Respiration and Similar Assimilation Rates as C4 Plants

SunflowerPeanut



Net Photosynthesis or Net Assimilation Rate

Cooler Temps Are the only Time when C3 Plants Have Higher Net Assimilation Rates than C4 Plants

PEP Carboxylase Needed to Incorporate CO2 into the 4-Carbon Structure no Longer FunctionsC4 PS Rates Drop Dramatically or Stop


Carbon Dioxide Fixation (C3 and C4 Pathways)

Both Types of Plants Use Energy from ATP and NADPH2 to Carry out the ReactionsThe Energy from ATP Is Given by ATP Giving up Its 3rd Phosphorus

ATP → ADP + PThe Energy from NADPH2Giving up Its Hydrogens

NADPH2 → NADP + H2

CAM Photosynthesis

Crassulacean Acid MetabolismAnother Type of PS Carried out only by XerophytesAt Night

Stomata Are OpenPlants Fix CO2 into a 4-Carbon Product4-Carbon Product Stored overnight in Vacuole

http://www.ualr.edu/~botany/c4andcam.jpg

CAM Photosynthesis

During the DayStomata Are ClosedCO2 Is Released from the 4-Carbon ProduceNormal Light and Dark Reactions occur without Stomata OpeningAllows the Plants to Conserve Water during the Day

When Water Is Adequate, these Plants usually Carry out C3 PS


CAM Photosynthesis

CAM PlantsCacti, SucculentsCrops include Pineapple, Tequila Agave


CAM PhotosynthesisAlternative mechanisms of C-fixation is found in Succulent plants of hot, arid environments: cacti, pineapples, etc.

Plant family Crassulaceae crassulacean acid metabolism or CAM plants

These plants open their stomates during the night and close them during the day

CAM plants partition carbon fixation by time

During the night: lower temps and higher humidity CO2 is fixed into C4 molecules, and stored in large vacuoles

During daylight: Higher Temps and lower humidityStomata closed for water conservationNADPH and ATP are availableC4 molecules release CO2 to Calvin cycle

http://www.answers.com/topic/cactus-arizona-jpg-1

Overview of CAM

Comparison between C4 & CAM plants

Regulation of Carbon Dioxide FixationPlant cells have chloroplasts that carry outphotosynthesis: CO2 glucosePlant cells also have mitochondria and carry

out glycolysis, TCA, and oxidative phosphorylation: Glucose CO2

To prevent futile cycling of carbohydrate, cells must regulate the activities of key Calvin cycle enzymes

These enzymes respond indirectly to light activation. light energy is available the Calvin cycle proceeds. If no light available, no fixation of CO2 occur

Among the key changes that regulate Calvin cycle versus respiration are:Environment Factors: Light intensity, temperature, & availability of H2O, CO2, O2Cellular factors: cell state of key metabolites (NADPH, ATP, inhibitors, reducing power etc.)

Summary of Carbon fixationEach method of photosynthesis has advantages and disadvantages Depending on the climate (light, heat, water, CO2, and O2)

C3 plants better adapted to:Cold Temp (below 25C), moderate light, balanced CO2 & O2, and High moisture

~ 80% of plants

C4 plants most adapted to:high light intensities, high temperatures, Limited rainfall

CAM plants better adapted to extreme aridity (desert conditions, low water)

Factors Affecting Photosynthesis6CO2 + 12H2O + Light → C6H12O6 + 6O2 + 6H2O

Availability of CO2CO2 Supply Diminishes if Stomata are ClosedNormal [CO2] Is 400 ppm (0.04%)

Increasing [CO2] can Increase Plant Photosynthetic Rates

Artificial Enhancement usually not Practical in Field Production

Has Been Used Effectively in some Greenhouse Production

Factors Affecting Photosynthesis

Availability of WaterWater (almost always) Is not a Limiting Factor for PS

So Little Is actually Used (Less than 1% of Water Absorbed) and Plants Are Made up of so much Water

Water Stress that Causes Stomata to Close can Slow or Stop PS due to Lack of CO2

http://www.dentalindia.com/CO2b.jpg


Light Quality (Color)Chlorophyll Absorbs Light in Red (660 nm) and Blue (450 nm) Wavelengths

These Are the Photosynthetic Wavelengths of Light

Called Photosynthetically Active Radiation (PAR)

http://www.firstrays.com/plants_and_light.htm


Light Duration (Photoperiod)Plants Need Sufficient Length of Light Period to fix enough carbons for Normal Growth

Longest Days in Northern Hemisphere Occur in JuneDecember in Southern Hemisphere


Leaf Chlorophyll ContentPigment that Captures Light Energy and Begins the Transformation of that Energy to Chemical Energy

Located in ChloroplastsAbout 20 to 100 Chloroplasts/Mesophyll Cell in Leaves

http://content.answers.com/main/content/wp/en/thumb/3/34/250px-Leaf.jpg

Factors Affecting PhotosynthesisLeaf Chlorophyll Content

Chlorosis is Yellowing of Leaf from Lack of Chlorophyll

If Chlorophyll Is Reduced, PS Will Be ReducedCauses of Chlorosis

Nutrient DeficienciesN and Mg Are Parts of the Chlorophyll MoleculeK Needed for Enzyme Activation in Production of ChlorophyllAny other Nutrient Deficiencies that Cause Chlorosis also Reduce PS

Diseases http://toptropicals.com/pics/toptropicals/articles/cultivation/chlorosis/4061.jpg


TemperatureIncreasing Temp will Increase Rate of PS, within Normal RangesBelow Normal Ranges, PS Slows or Stops

Cytoplasm (Liquid inside Cells) Slows Moving

Cells may FreezeChilling can Change Protein and Membrane Structure

Causes Cell Content Leakage and Death

http://www.semena.org/agro/diseases2/environmental-stresses-e.htm


TemperatureAbove Normal Ranges

Proteins may Change ShapeMembranes may Become too Leaky

Leads to PS Stoppage and Possible Cell Death

C3 Plants Have Optimum PS from about 55-75°F

Can Carry out PS from 32-95°F

http://www.bbc.co.uk/science/hottopics/obesity/fat.shtml


TemperatureAbove Normal Ranges

C4 Plants Optimum PS 75-95°FCan Carry out PS from 55-105°FPEP Enzyme Deactivated below 55°F

C3 Plants Are Called Cool-Season PlantsC4 Plants Are Called Warm-Season Plants


Leaf AgeYoung, Mature Leaves Have Greatest Rate and Output of PSYoung, Immature Leaves Have High Rate of PS but Use more of what They Produce for Their Own GrowthMature Leaves have Slower PS RatesDefoliation of Young or Young + Mature Leaves of a Plant Drains the PlantMust Pull from Stored Carbs in Stems and Roots to Regenerate enough Leaves to Provide needed Carbs

RESPIRATION AND OXIDATVIE

PHOSPHORYLATION

RespirationFree Energy Is Released and Incorporated into a Form (ATP) that can Be Readily Used for the Maintenance and Development of the Plant

http://www.biol.lu.se/cellorgbiol/dehydrogenase/pop_sv.html

RespirationLow-Temperature Oxidation of Carbohydrates Carried out by Enzymes and Living Systems

Net Reaction Appears as the Reverse of PSThe Individual Reactions that Occur to Achieve the Net Effect Are Entirely DifferentReactions Occur in Different Parts of Cells

Net Chemical ReactionC6H12O6 + 6O2 + 40 ADP + 40 Phosphates →

6 CO2 + 6 H2O + 40 ATP

Respiration

Respiration Means to Turn Carbohydrates into Usable Chemical energy (ATP) for many other Plant Reactions including PhotosynthesisAll Living Plant and Animal Cells Carry out RespirationRespiration Occurs

At same Time as PhotosynthesisDuring the NightIn Developing and Ripening FruitIn Dormant Seeds

MitochondriaOccurs in Mitochondria of CellsMitochondria are membrane-enclosed organelles distributed through the cytosol of most eukaryotic cells. Their main function is the conversion of the potential energy of food molecules into ATP

http://www.science.siu.edu/plant-biology/PLB117/JPEGs%20CD/0077.JPG

Aerobic Respiration

Requires OxygenMain Type of Respiration that Occurs in most Situations in Plants and AnimalsInvolves Complete Breakdown of Glucose back to CO2and WaterNot all of the Energy in Glucose Is Converted to ATP Formation

Only about 40% EfficientExtra Energy Is Given off as Heat

In Plants, Heat Quickly DissipatesFor Animals, Heat Is Retained to Hold Body Temperature

http://www.kathleensworld.com/mitochondria.jpg

Main Steps of Respiration

Breakdown of simple subunits to acetyl

CoA accompanied by production of limited amounts of ATP and

NADH

Complete oxidation of acetyl CoA to H2O

and CO2 accompanied by production of large

amounts of NADH and ATP in

mitochondrion

Acetyl CoA

glucose

Citric acid cycle

CoA

2 CO2

8 e- (Reducing power as NADH)

oxidative phosphorylation

O2

H2O

ATPATP

glyc

olys

is

pyruvate

ATPATP

NADH

Adapted from MBOC4, fig. 2-70 & pp. 383

glycolysis

TCA cycle

electron transport &ox. phosphorylation

3 Main Respiration Steps

1. GlycolysisBreakdown of Glucose to a 3-Carbon Compound Called Pyruvate(Glucose, C6H12O6, into Pyruvate, C3H4O3)Occurs in CytosolSome ATP and NADH Are also Formed

Storage Energy MoleculesNADH Is Formed from NADSimilar Type of Energy-Storing Rx as NADP + H2 → NADPH2

NAD + H → NADHhttp://www.med.unibs.it/~marchesi/glycpth2.gif

Respiration Steps2. Krebs Cycle/Citric Acid Cycle

‘Tricarboxylic acid Cycle (TCA Cycle)’‘Citric Acid Cycle’ Occurs in Mitochondrial Matrix

A Cyclic Series of Rxs that Completely Break down Pyruvate to CO2 and Various Carbon SkeletonsSkeletons Are Used in other Metabolic Pathways to Make various Compounds

ProteinsLipidsCell Wall CarbohydratesDNAPlant HormonesPlant PigmentsMany other Biochemical Compounds

The Step where CO2 Is Given off by the Plant10 NADH Are Generated

http://www.sp.uconn.edu/~bi107vc/images/mol/krebs_cycle.gif

Respiration Steps3. Electron Transport

Chain‘Oxidative Phosphorylation’Series of Proteins in the Mitochondria Helps Transfer Electrons (e-) from NADH to Oxygen

Releases a Lot of Energy

Occurs on Mitochondrial Inner Membrane (Proteins Bound to Membrane) http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%205/ch05.htm

Respiration Steps

Released Energy Is Used to Drive the Reaction ADP + P →ATP

Many ATP Are Made

Oxygen Is Required for this StepWater Is Produced

http://www.uccs.edu/~rmelamed/MicroFall2002/Chapter%205/ch05.htm

ATP Production during Aerobic Respiration by Oxidative Phosphorylation involving Electron

Transport System and Chemiosmosis

Anaerobic Respiration

Anaerobic Respiration‘Fermentation’Occurs in Low-Oxygen Environments

Wet or Compacted Soils for PlantsAfter Strong Exertion for Animals

ATP Is still Produced from Glucose but not as Efficiently as with Aerobic Respiration

http://www.jracademy.com/~vinjama/2003pics/fermentation%5B1%5D.jpg


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

Glucose + Oxygen → 2 Ethanol + 2 Water + 2 ATPSame Rx Used to Produce Alcohol from Corn or to Make Wine or other Consumed Alcohol

Aerobic:C6H12O6 + 6O2 + 40 ADP + 40 Phosphates → 6 CO2 + 6 H2O + 40 ATP


Only 2 ATP Are Formed instead of 40 from Aerobic Respiration

Plant Soon Runs out of EnergyCan Begin to Suffer from Toxic Levels of Ethanol and Related Compounds

Extended Periods of Anaerobic Respiration will Seriously Reduced Plant Growth and Yields

Anaerobic:C6H12O6 + O2 → 2 CH2O5 + 2 H2O + 2 ATPAerobic:C6H12O6 + 6O2 + 40 ADP + 40 Phosphates → 6 CO2 + 6 H2O + 40 ATP

Factors Affecting Respiration

Kind of Cell or TissueYoung and Developing Cells (Meristematic Areas) usually Have Higher Respiration RatesDeveloping and Ripening Fruit and Seeds, tooOlder Cells and Structural Cells Respire at Lower Rates


TemperatureRespiration generally Has Higher Optimum and Maximum Temps than PS RxsCan Have Net Dry Matter Loss at High Temps where Respiration Exceeds PSTemp Refers to Temp Inside Plant or Animal Cell, not Air Temp

Using Irrigation to Help Cool the Plant Can Keep the Plant in Net Gain Range


OxygenLow O2 Can Reduce Aerobic Respiration and Increase Anaerobic RespirationLow O2 Can Reduce Photorespiration


LightCan Enhance Rate of PhotorespirationDoes not Directly Affect other Forms of Respiration


[Glucose]Adequate Glucose Needed to Carry out RespirationReductions can Occur

Reduced PSReduced Flow of Carbohydrates in Plant

Insect FeedingPhloem Blockages


[CO2]Higher CO2 Levels Reduce Rate of Respiration

Feedback Inhibition

Seldom Occurs except when O2 Levels Are LimitedFlooded, Compacted Soils


[ATP]Higher [ATP] Reduces Rate of Respiration

Feedback Inhibition

Usually Occurs when other Metabolic Processes Have Slowed or Stopped


Plant InjuryInjury will Increase RespirationPlant’s Growth Rate Increases in Attempt to Recover

Mechanical DamageHailMowing, Grazing, Cultivation, Wind

Plant Synthesizes Compounds to Fight PestsInsect FeedingDiseases

Some Herbicides Kill Plants by Disrupting or Affecting Respiration

Generally an Indirect EffectHerbicide Disrupts Enzyme Activity or some other Metabolic Process that will Affect Respiration

Nitrogen Cycles

N2 and lifeAll life requires nitrogen compounds to form proteins and nucleic acids.Air is major reservoir of nitrogen (~ 78%).

Even though air is a large source of N2 , most living things cannot use this form, and it must be converted to other forms

There are several modes of N2fixation that convert N2 into NH3or NO2, or NO3

Forms of NitrogenGas (N2): Very Abundant, but mostly “unavailable”

Inorganic nitrogen:NH3 =Ammonia NH4+ =Ammonium NO3- =Nitrate NO2- =Nitrite

all nutrients, but toxic at high levels

Organic nitrogenLivings things and their proteins, nucleic acids, urea, and other nitrogenous molecules.

Nitrogen: Oxidation & reductionNitrogen is present in several oxidation states (N, atomic number 7, Atomic Mass, 14)

N: 5 electrons in the outer shell from (+5 oxidation) to (-3 oxidation)

Ion/molecule

Name Oxidation State

NH3 ammonia -3NH4+ ammonium -3N2 diatomic N 0N2O nitrous oxide +1NO nitric oxide +2NO2- Nitrite +3NO3- nitrate +5

Oxidation

Nitrogen Fixation-1The problem: N2 is inert gas, chemically unreactive= can’t bond easily with other things.

Lavoisier named it “Azote” ; meaning “without life”.

The reduction of nitrogen to ammonia is an exergonicreaction:

N2 + 3H2 2NH3 ; ΔG= -33.5 kJ/molThe N≡N triple bond, however, is very stable, with a bond energy of 930 kJ/mol.

Atmospheric nitrogen is almost chemically inert under normal conditions

Nitrogen Fixation“Nitrogen Fixation” is the process that causes the strong two-atom nitrogen molecules found in the atmosphere to break apart so they can combine with other atoms.

Nitrogen gets “fixed” when it is combined with oxygen or hydrogen.

N

N

NN

NOxygen Hydrogen

Oxygen

Hydrogen

N

Nitrogen Fixation-2By definition: “Nitrogen Fixation” is the process that break up N2 molecules found in the atmosphere so that N can combine with other atoms.

Nitrogen gets “fixed” when it is combined with oxygen or hydrogen. Nitrogen fixation is the process that converts N2 into either: NH3, NO3-, or NO2- (usable forms).

Nitrogen fixation is a ubiquitous process, even though requires a lot of energy.

Atmospheric fixationIndustrial fixationBiological fixation

There are three ways that nitrogen gets “fixed”!

(a) Atmospheric Fixation

(b) Industrial Fixation

(c) Biological Fixation

Bacteria

Atmospheric nitrogen is converted to ammonia or nitrates.

Ammonia (NH3)Nitrogen combines

with Hydrogen to make Ammonia

Nitrates (NO3)Nitrogen combines

with Oxygen to make Nitrates

Atmospheric Nitrogen (N2)

N

N

N

N

Atmospheric Fixation

(Only 5 to 8% of the Fixation Process)

The enormous energy of lightning breaks nitrogen

molecules apart and enables the nitrogen atoms to combine with oxygen forming nitrogen oxides (N2O). Nitrogen oxides dissolve

in rain, forming nitrates. Nitrates (NO3) are carried to

the ground with the rain.

Lightning “fixes” Nitrogen!

Nitrogen combines with Oxygen

Nitrogen oxides forms

Nitrogen oxides dissolve in rain and change to nitrates

Plants use nitrates to grow!

(NO3)

N N O

(N2O)

http://www.specialedprep.net/MSAT SCIENCE/Images/Nitriteion.jpg

Industrial Fixation

Under great pressure, at a temperature of 600 degrees Celcius, and with the use of a catalyst, atmospheric

nitrogen (N2) and hydrogen are combined

to form ammonia (NH3). Ammonia can be used as a fertilizer.

Industrial Plant combines nitrogen and hydrogen

Ammonia is formed

Ammonia is used a fertilizer in soil

(NH3)

NN H

N H3

Biological Fixation(where MOST nitrogen fixing is completed)

There are two types of “Nitrogen Fixing Bacteria”

Free Living Bacteria(“fixes” 30% of N2) Symbiotic Relationship Bacteria

(“fixes” 70% of N2)

Biological FixationBiological fixation accounts for the most fixed nitrogen in the biosphere (~10^6 metric tons/year)

Only in certain organisms can fix atmospheric nitrogen. These are divided into:

Non-symbiotic N-fixation carried out by Free-living Organisms (bacteria, Cyanobacteria, & Blue-green algae)

Highly specialized bacteria live in the soil and have the ability to combine atmospheric nitrogen with hydrogen to make ammonia (NH3).

Aerobic (Azotobacter) & Anaerobic (some Clostridium species)

Symbiotic N-fixation carried out by organism living in symbiosis with certain plants (~70% of biological fixation)

symbiosis in legumes (soybeans, alfalfa) = (Rhizobium) with other plants =(Frankia, Azospirillium)

Biological N2 fixationBiological N2 fixation= Conversion of N2 gas to ammonium via the following reaction:

N2+10 H++ 8 e- + 16 ATP 2 +NH4+ H2+16 ADP +16 PiA very energetically expensive reaction

The reaction require:Nitrogenase enzyme (of any suitable organism)Large supply of energyAnoxic site

oxygen binds to and inactivates the nitrogenase enzyme

Free Living BacteriaHighly specialized bacteria live in the soil and have the

ability to combine atmospheric nitrogen with hydrogen to make ammonia (NH3).

Free-living bacteria live in soil and combine

atmospheric nitrogen with hydrogen

Nitrogen changes into ammonia

N N H

NH3

(NH3)

Bacteria

Symbiotic Relationship BacteriaBacteria live in the roots of legume family plants and provide the plants with

ammonia (NH3) in exchange for the plant’s carbon and a

protected home.

Legume plants

Roots with nodules where bacteria live

Nitrogen changes into ammonia.

NH3

N

N

The Nitrogenase ComplexAll nitrogen fixing species (symbionts & non- symbionts) contain

the nitrogenase complex Crucial components of the complex are two proteins:

1) nitrogenase reductase (Fe-4S protein); homodimer2) nitrogenase (Fe-Mo protein); tetramer (A2B2)

The nitrogenase complex is anaerobic!

N2

NH3ATP ADP

Electrons from reduced source Nitrogenase

(Mo-Fe protein)

Nitrogenase Reductase

(Fe-S protein)

Nitrogenase Complex

The Nitrogenase ReductaseDinitrogenase reductase (Mr 60,000) is a dimer of two identical subunits.

provides electrons with high reducing power Electron transfer from the reductase to the nitrogenase is coupled with ATP hydrolysis

Ribbon diagram of Nitrogenase complexgray and pink are the dinitrogenase subunitsblue and green are the dinitrogenase reductasesubunits. (bound ADP red, Fe atoms orange, S atoms yellow)

The NitrogenaseDinitrogenase (Mr 240,000) is a tetramer with two copies of two different subunits.

has two binding sites for the reductase.uses e- to reduce N2 to NH3 highly sensitive to oxygen

Ribbon diagram of Nitrogenase complexgray and pink are the dinitrogenase subunitsblue and green are the dinitrogenase reductasesubunits. (bound ADP red, Fe atoms orange, S atoms yellow)

N2-fixation by the Nitrogenase ComplexThe process requires eight electrons:

Six for the reduction of N2 and two to produce one molecule of H2 as an obligate stepAll electrons are transferred one at a time

The process:(repeated 8 times to transfer eight electrons)

First, Reducatse is reduced by ferrodoxin or flavodoxin (electron source)Then, reduced reductase binds 2 ATPs and change its conformationReducatse (+2ATP) binds to the dinitrogenaseand transfers a single electron to it release ADP and becomes Oxidized

Highly reduced Dinitrogenase then carries out nitrogen fixation and generates NH3 & H2

Other Substrates of the NitrogenaseNitrogenase is able to reduce other substrates beside N2

At least one H2 is produced during N2 fixation: (obligatory step):This reaction is used to measure the activity of nitrogenase.

In the presence of sufficient concentrations, acetylene is reduced to ethylene by the nirogenase:

HC≡CH + 2e- + 2H+ H2C=CH2Other reactions catalyzed by the nitrogenase

N3- N2 + NH3 (Azide reduction)N2O N2 + H2O (Nitrous oxide reductionATP ADP + Pi (ATP hydrolysis)

Nitrogenase is extremely sensitive to oxygen. Therefore N2 fixation can proceed only at very low oxygen concentrations

Nitrogenase & OxygenThe nitrogenase complex is extremely sensitive to the presence of oxygen.

The reductase is inactivated in air, with a half-life of 30 secondsdinitrogenase has a half-life of 10 minutes in air.

Free-living bacteria that fix nitrogen cope with this problem in a variety of ways.

Some live only anaerobically or repress nitrogenasesynthesis when oxygen is present.

Other species solve this problem via the symbiotic relationship, especially between leguminous plants and the nitrogen-fixing bacteria.

Nitrogen-fixing nodulesThe bacteria in root nodules receive carbohydrate and citric acid cycle intermediates from cellBacteria fix 100X more nitrogen than their free-living cousins in soils.

To solve the oxygen-toxicity problem, plants produce a protein called: leghemoglobin

It is a heme-protein that has high affinity for O2Leghemoglobin binds all available oxygen and efficiently delivers it to the bacterial electron-transfer system.

The efficiency of the symbiosis between plants and bacteria is evident in the enrichment of soil nitrogen (the basis of crop rotation)

From: Plant Biochemistry 3rd ed –H. Heldt (Elsevier, 2005)

The Nitrogen CycleAnimals can not fix N2. They get their nitrogen by eating plants or by eating something that eats plants.

Nitrogen Fixation is very expensive process

In the biosphere, the nitrogen cycle is a vast collection of metabolic processes of different species function interdependently to salvage and reuse biologically available nitrogen.

The NITROGEN CYCLE

NitrateNO3-

NitriteNO2-

AmmoniaNH4+

Reduction by most plants & someanaerobic bacteria

N2

Nitrification(e.g. Nitrosomonas)

Nitrification(e.g. Nitrobacter)

Denitrification Nitrogen fixation(some bacteria)

Amino acids& reducednitrogen

compounds

Synthesis: (microorganisms, plants & animals

Degradation:Animals & microbes

more oxidized more reduced

Key terms of The Nitrogen CycleNitrogen Fixation: Conversion of N2 to ammonia (NH3)

By any bacteria in soil/water having the nitrogenase complex, e.g. Rhizobium in root nodules of legumes.

Nitrification: Conversion of ammonia to nitrite (NO2-) and then nitrate (NO3-).

Both reactions carried out by bacteria Assimilation: Conversion of NH3, NO2-,, NO3- (inorganic) into organic compounds (proteins, DNA, & other forms)

All living cells (plants, animals, & bacteria). Ammonification: Conversion of the amine groups of organic compounds into simpler compounds (often, ammonia NH3).

Mostly via decay processes carried out by decomposer bacteriaDenitrification: Conversion of NH3, NO2-,, NO3- to N2

Mostly by anaerobic bacteria in waterlogged soil, bottom sediments of lakes, swamps, bogs and oceans.

Overview of the N-cycleThe first product of biological fixation is ammonia (NH3 or +NH4 ).

In principle: this ammonia can be used by most living organisms.However, soil bacteria and plant are in fierce competition for NH3

Bacteria are more abundant and active, but plants have their ways. In either case, Nitrification proceeds: NH3 -NO2 -NO3

Plants and many bacteria can also reduce nitrate and nitrite ammonia (reductases). -NO3 NO2 NH3The new ammonia is incorporated into organic molecules by plants& bacteria. (Assimilation).When organisms die, microbial degradation of their proteins returns ammonia to restart the cycle. Some bacteria can convert nitrate to N2 under anaerobic conditions (denitrification)

Nitrification and DenitrificationNitrification: Conversion of ammonia into nitrite by the Nitrosomonas bacteria. Nitrite is then converted to nitrate by Nitrobacter. Denitrification: Occurs wherein nitrate is converted to nitrite then to ammonia then Bacillus, Psuedomonas and Clostridiumconvert it to nitrogen gas, nitrous oxide and nitric oxide, all nontoxic and are released in the process.

NitrificationNitrification is the biological oxidation of ammonia with oxygen into nitrite followed by the oxidation of these nitrites into nitrates. Degradation of ammonia to nitrite is usually the rate limiting step of nitrification. Nitrification is an important step in the nitrogen cycle in soil. Nitrogen is the most important mineral nutrient in the soil

Nitrogen is frequently limiting in in terrestrial systems.Competition for NH3 might be the driving force for nitrification

In plants: high levels of ammonium are toxic NH3 affects the transmembrane proton gradients required for photosynthesis, respiratory chain, and transport metabolites to vacuoles.

NH3 is membrane permeable and diffuses freely across a membranePlants can store high levels of nitrate without any effect.

Nitrifying bacteria are very abundant, and drive their energy by converting NH3 into NO3

Two bacterial species involved (both use O2)Plants can absorb and use this nitrate.

Nitrifying bacteriaNitrifiers (heterotrophs & autotroph) are delicate organisms and extremely susceptible to a variety of inhibitors.

They are extremely slow growingNitrifying bacteria need a relatively clean

environment with a continuous supply of ammonia and oxygen.

Two bacterial species are required for nitrification: 1) Ammonia-Oxidizing Bacteria: Nitrosomonas

Present in large number They are chemoautotrophs

(require ammonia and CO2) , and found in a great variety of soils, oceans, rivers, lakes, and sewage disposal systems.

2) Nitrite Oxidation Microorganism: NitrobacterAerobic, but occasionally also anaerobicThey are widely distributed in soils, fresh water, seawater, mud layers, sewage disposal systems, and inside stones of buildings, rocks, and inside concrete surfaces.

Nitrate AssimilationNO3 NO2 NH4+ amino acidsnitrate nitrite ammonium

Requires large input of energy Forms toxic intermediates

Mediated by enzymes (Reductases) that are closely regulatedNitrate levels, light intensity, and concentration of carbohydrates all influence the activity of nitrate reductases at the transcription and translation levels

These factors stimulate a protein, phosphatase, that dephosphorylates several serine residues on the nitrate reductaseprotein thereby activating the enzyme

Roots and LeavesNitrate is assimilated in the leaves and also in the roots. In many plants, when the roots receive small amounts of nitrate, this nitrate is reduced primarily in the roots

The transport of nitrate into the root cells proceeds as symport : (secondary active transport)Root cells contain several nitrate transporters in their plasma membrane (different affinities for different conditions).nitrate assimilation in the roots often plays a major role at an early growth state of these plants.

As nitrate supply increases, nitrate is transported to the leaves by the xylem vessels for storage & assimilation.

Large quantities of nitrate can be stored in a leaf vacuole. Sometimes this vacuolar store is emptied during the day and replenished during the night.

Nitrate ReductaseNitrate reductase is found in the cytosol (not chloroplast).

Several Forms depending on the species.

The most common form of this enzyme uses only NADH as an electron donor; Other forms use NADPH or both (NADPH or NADH)

The process involves an electron transport chain from NADH to one flavin adenine dinucleotide molecule (FAD) one cytochrome-b557) one cofactor containing molybdenum

Nitrate Reductase

FAD Cyt-b557 MoCONADH + H+

NAD+

-NO3

-NO2 + H2O

Nitrate Reductase: StructureNitrate reductase (1)- is a large and complex enzyme with multiple subunits and a mass of ~800 kDa.

In higher plants, it is composed of two identical subunits, the MW of each subunit varies (99 -104 kDa) depending on the species

The nitrate reductases of three prosthetic groups: FAD, Heme, Cofactor containing Molybdenum, called pterin

The protein can be cleaved by limited proteolysis into three domains, each of which contains only one of the redox carriers.

Nitrate reductase enzymes are a group of enzymes that reduce nitrate to nitrite.

FAD Heme MoCODomain Domain Domain

HOOC- -NH2 N NH

N

N

O

NH2

C CSS

Mo4+

CHOH

CH2O PO-

O-O

Pterine

Nitrite ReductaseThe reduction of nitrite to ammonia proceeds in the plastids

Nitrite (NO2-) is highly reactive Plant cells immediately transport it into chloroplasts of leaves and plastids in roots

In these organelles, nitrite reductase reduces nitrite to ammonium

The reduction requires six electrons.Reduced ferredoxin is the electron donor for this enzyme.Ferredoxin is regenerated by electrons supplied by photosystem I.

Nitrite Reductase6 FerredoxinReduced

6 FerredoxinOxidized

-NO2 + 8 H+

+NH4 + 2H2OPhotosystem-I 4 Fe-4S FAD Siroheme

6 e-

Light

Nitrite ReductaseChloroplast and root plastids contain different forms of the enzyme, but both forms consist of a single polypeptide containing: a covalently bound 4Fe-4S, one molecule of FAD, and one special heme called siroheme.

Siroheme is a cyclic tetrapyrrole with one Fe-atom in the center. (Heme with different groups)

The heme does redox reactions and electron flow, just like the other hemes

One electron transfer mechanism (repeated six times)

Nitrite Reductase6 FerredoxinReduced

6 FerredoxinOxidized

-NO2 + 8 H+

+NH4 + 2H2OPhotosystem-I 4 Fe-4S FAD Siroheme

6 e-

Light

Ammonium AssimilationAmmonium is highly toxic, yet essential to both animals and plants.

Animal & Plant cells rapidly assimilate into amino acids.In plants: this requires the action of two enzymes:

Ammonium Assimilation: TransaminationOnce assimilated into glutamine and glutamate, nitrogen is incorporated into other amino acids via transaminationreactions

Best known is aspartate aminotransferaseTrnsfers amino group of glutamate to the carboxyl atom of oxaloacetate aspartate + α-ketoglutarateAspartate is involved in the transport of carbon from mesophyllto bundle sheath of C4 carbon fixation All aminotransferases require vitamin B6 to act as a cofactor

C CR1NH3

+

H O

O-

C CR2O

O

O-

C CR2NH3

+

H O

O-

C CR1O

O

O-

+ +Transaminase

Ammonium & Nitrate Assimilation

From: Plant Biochemistry 3rd ed – H. Heldt (Elsevier, 2005)

Ammonia Integration in Animals

DenitrificationDenitrification converts nitrates (NO3) in the soil to atmospheric nitrogen (N2) replenishing the atmosphere.Denitrifying bacteria live deep in soil and in aquatic sediments where conditions make it difficult for them to get oxygen. The denitrifying bacteria use nitrates as an alternative to oxygen, leaving free nitrogen gas as a byproduct. They close the nitrogen cycle!

EutrophicationAgriculture is responsible for increased nitrogen fixation on earth.

Fertilizers & Growing of legumes (soybeans and alfalfa)When denitrifying

bacteria can’t keep up with all the nitrates from fertilizers and legumes nitrogen enrichment in ecosystems.

Nitrates and ammonia are very soluble in water, and can easily washed (leached) from free draining soils

There, algae benefit from the extra nitrogen leading to Algal BloomsAlgae absorb all the oxygen from lakes and ponds killing the organisms in the water.

Too much nitrates in water is called eutrophication.

Plant Biochemistry�BCH 350Dr. Wajahat Khan�Office : 67A2�Biochemistry Department�Building Number 5��Phone number: 467-5443�Email: [email protected]�(EngImportance of PlantsPlants and Energy flowMatter & EnergyTHE CARBON CYCLETHE CARBON CYCLEThe Carbon CycleTHE CARBON CYCLEOther Sources of Atmospheric CO2 ExchangesWHAT IS A CELL ORGANELL?CELL ORGANELLSPlant and animal cellsPlant CellCell MembraneCELL WALL �(Plant cell only)NUCLEUSCYTOPLASMMITOCHONDRIAGOLGI COMPLEXRIBOSOMES (Not a Cell organelle� -But important)SMOOTH ENDOPLASMIC RETICULUMROUGH ENDOPLASMIC RETICULUMLYSOSOMESVACUOLE (Plant cell only)CHLOROPLAST (Plant cell only)CELL WALLThe Cell WallPrimary & Secondary wallPrimary Cell WallFeatures of Cell Wall: SummaryConnections between Cells: PlasmodesmataGlycans o

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