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Plant Biochemistry
BCH 350
Dr. Wajahat Khan
Office : 67A2
Biochemistry Department
Building Number 5
Phone number: 467-5443
Email: [email protected]
(English Emails only)
Plant Biochemistry 350
•Overview of Plant Biochemistry
•The Plant Cell Wall
Its biochemical composition and formation
•Photosynthesis
•Light phase
cyclic and non-cyclic photophosphorylation
•Dark phase
C3 and C4 pathways
•Respiration and oxidative phophorylation
•Biosynthesis of polysaccharides and chlorophyll
•Nitrogen fixation, transport of nitrogenous
compounds and their stage
Importance of Plants
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ALL of the food we eat comes either directly or
indirectly from plants.
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Oxygen budget: Animal (human!) Respiration
Useful substances in Agriculture, Medicine, & Industry
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Maize, wheat and rice are the main crops that feed the world.
All of these produce starch and all can be stored.
Virtually ALL medicines today have their origin in plant
chemicals
Several 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 flow
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Energy enters as sunlight
Producers convert sunlight to chemical
energy.
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Consumers eat the plants (and each
other).
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Autotrophs
Heterotrophs
Decomposer organisms breakdown the
organic molecules of producers and
consumers
Earth is an open system for energy
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Plants
Small organisms
Sun always provide constant energy!
Earth is a closed system for matter
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All elements go through recycling
Matter & Energy
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The earth is a closed system to matter
Matter cycles through an ecosystem; everything that
was ever here still is
Major Biogeochemical Cycles:
Water/hydrologic
Carbon
Oxygen
Nitrogen
Phosphorous
Sulfur
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THE CARBON CYCLE
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Carbon 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 Cycle
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Plants take in CO2 for photosynthesis and also release
some during respiration.
THE CARBON CYCLE
Other Sources of Atmospheric CO2 Exchanges
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CO2 is also exchanged between the oceans and the atmosphere.
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Fossil Fuel burning (gas, coal, oil)
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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.
Increased pollutant CO2 from fossil fuels creates THE GREEN HOUSE effect
Also 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.
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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 ORGANELLS
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Cell Membrane
Nucleus
Cytoplasm
Mitochondria
Golgi Complex
Ribosomes
Smooth Endoplasmic Reticulum
Rough Endoplasmic Reticulum
Cell Wall
Chloroplast
Central Vacuole
Lysosome
Plant and animal cells
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Both cells have many common components like:
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But Plant Cell has these unique components:
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Nucleus, Mitochondria, ER, Golgi, Ribosome, Plasma membrane,
Cytosol, & Microtubules and microfilaments (cytoskeleton)
Cell wall
Chloroplast
Central Vacuole
By contrast, Animal Cell has
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Centrioles (important for cell division)
Lysosomes (plant cell has peroxisomes and glyoxisomes),
Plant Cell
Cell Membrane
•Every cell is
enclosed by a
cell membrane.
•It controls the
passage of
materials in and
out of the cell.
CELL WALL
(Plant cell only)
•Rigid and
strong wall.
•Protects and
maintains the
shape of the
cell.
NUCLEUS
•The control center of
the cell.
•It contains the DNA
code for the cell coiled
into chromosomes.
CYTOPLASM
•Not a Cell
organelle but
very important
part of the cell
•All organelles
reside (live and
float around in)
the cytoplasm.
MITOCHONDRIA
•This organelle
processes energy
for a cell
•It makes ATP
(ATP = energy)
•Involved in
cellular
respiration
GOLGI COMPLEX
•The protein
packaging and
transport center
of the cell
•Has incoming
and outgoing
vesicles.
RIBOSOMES (Not a Cell organelle
-But important)
•Synthesizes proteins
•Present in cytoplasm
•Present with Rough ER
•No membrane present.
SMOOTH ENDOPLASMIC RETICULUM
•Transports materials
throughout the cell
•Digests lipids
•Produces proteins.
ROUGH ENDOPLASMIC RETICULUM
•Covered with
ribosomes.
•Produces proteins.
•Transports
materials
throughout the cell.
LYSOSOMES
•Breaks down
materials for
digestion
•Contains special
enzymes for
digestion in the
cell
VACUOLE (Plant cell only)
•Most plant cells
have one large one
•Filled with fluid
•Helps maintains
turgor pressure
and shape of cell
CHLOROPLAST (Plant cell only)
•Contains
chlorophyll
•Makes plants green
•Uses light energy
to make ATP &
sugars
•Photosynthesis
occur in this
organelle
CELL WALL
•Rigid and strong
wall
•Protects and
maintains the
shape of the cell
The Cell Wall
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almost all plant cells have a protective
wall of great tensile strength (Primary
±Secondary Cell wall) depending on
growing state of the cell
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10-25 nm in diameter
Consists of long-chain polysaccharides
The composition varies between
different species
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Most common: cellulose in the primary,
lignin in the secondary
The polysaccharide chain folded into
fibers and micro-fibrils
Primary & Secondary wall
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Growing 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:
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hardening of primary cell wall , or
adding secondary cell wall between
plasma membranes and primary wall
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Secondary cell wall may have a
composition similar to that of the primary
wall or be markedly different.
Primary Cell Wall
The cell wall is a network of
1. 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
http://en.wikipedia.org/wiki/File:Plant_cell_wall_diagram.svg
http://en.wikipedia.org/wiki/File:Plant_cell_wall_diagram.svg
• Typically, cellulose, hemicellulose, and
pectin are present in roughly equal amounts.
•Cellulose and cross-linking glycans
provides tensile strength,
• Pectin is the sticky polysaccharide.
Pectin
• The middle lamella is rich in pectin and cements adjacent cells together.
• Proteins Constitutes about 5%.
Features of Cell Wall: Summary
Cell wall is found in all plant cells except sperm and some eggs. It consists
of three zones: (outward Æ inward)
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(1) Middle lamella – mostly pectin, cements adjacent cells together
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(2) Primary cell wall
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Found in all plant cells
Cellulose matrix with hemicellulose, proteins, pectin, lignin, cutin, and wax
Characteristic of undifferentiated cells or ones that still are growing
(3) Secondary cell wall
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Just inside primary cell wall
Characteristic of mature cells
Comprised of hemicellulose and lignin
May have 3 layers
Connections between Cells: Plasmodesmata
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Plasmodesmata (1=plasmodesma) are
microscopic channels through the cell
walls and middle lamella between
adjacent plant cells
Link adjacent plasma membranes and
cytoplasm
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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: Cellulose
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Cellulose, the most abundant polymer on earth, ~ one half of the
organic carbon.
Linear polymer of glucose, with (β1Æ4) 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
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However, many bacteria and fungi have cellulose-hydrolyzing enzymes
(cellulases)
CH2OH
O
C
OH
HO
OH
C
H
OH
C
OH
C
OH
CH2OH
H
C
OH
OH
C
O OH
H
O
H
O
CH2OH
OH
OH
O
H
O
H
CH2OH
OH
CH2OH
OH
OH
CH2OH
Glycans of cell wall: Hemicellulose
Hemicellulose is heterogeneous group of branched
polysaccharides polymers that cross-link cellulose fibrils into
robust network.
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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: Pectin
Pectins 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.
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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.
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Glycans of cell wall: Lignin
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The most common additional polymer in secondary walls is
lignin
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Found mostly in the walls of the xylem vessels and fiber cells of
woody tissues.
Lignin causes the walls to become thick, stiff, and
incompressible
Lignin is a ploymer of cross-linked coumaryl, coniferyl, and
sinapyl alcohols
Functions of The Cell Wall
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Cell wall is thicker, stronger and more rigid than similar
components around animal cells. It forms barrier against
pathogens and deters herbivores
The wall is responsible for:
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In growing state, the wall has dynamic nature that allows
expansion.
In Mature state, the wall determines cell morphology
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Osmoregulation (see later)
Cell adhesion, protection and support
Intercellular communication through plasmodesmata
Regulated exchange of selected molecules and fluids
Secondary cell wall may contain lignin for greater support
Specialized cells have unique cell wall adaptations
depending on function and environment
Cell Wall, Shape & Classification
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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 & Turgor
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Cell walls is made of neutral and charged polysaccharides Æ
absorbs H2O
Its environment is hypotonic to the cell Æ H2O must flow to the
cell
Increased H2O inside the cell Æ Turgor Pressure
If a plant cell is turgid, It is very firm, a healthy state in most plants
If a plant cell is flaccid, It is in an isotonic or hypertonic
environment
OSMOSIS AND TURGOR PRESSURE
PLANT CELL
„ Hypertonic solution → Plasmolysed cell
„ Isotonic solution → Non-turgid or wilted cell
„ Hypotonic solution → Turgid cell (Usual environment)
ANIMAL CELL
„ Hypertonic solution → Cell shrinks
„ Isotonic solution → Normal (Usual environment)
„ Hypotonic solution → Cell swells and may burst
OSMOSIS AND TURGOR PRESSURE
PHOTOSYNTHESIS
THE BASICS OF PHOTOSYNTHESIS
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Organisms can be classified based on how they obtain
energy into autotrophs & heterotrophs.
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Autotrophs generate their own organic matter through
photosynthesis
Sunlight energy is transformed to energy, stored in the form of
chemical bonds
Almost all plants are photosynthetic autotrophs (also some
bacteria and protists)
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May occur in stems of plants that do not have leaves
plants
Ferns
Horsetails
Euglena
Cyanobacteria
Photosynthesis
Location: Chloroplasts
„ Energy comes from Photons from SUN and
H2O splitting
„ Process: two main sets of reactions
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1) Light Reactions: capture energy to synthesize ATP
and NADH
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It uses Electron transport chain & Photorespiration
2) Calvin cycle to fix CO2
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Several mechanisms of C-fixation
Structure of a leaf
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/leafstru.gif
http://www.emc.maricopa.edu/faculty/farabee/BIOBK/leafstru.gif
Important structures in a leaf
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Two structures important for photosynthesis are
Stoma (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 40200) arranged to receive maximum amount of light.
O2
CO2
Guard cell
Guard cell
Stoma
Mesophyll cell
Plastids
Plastids are a family of organelles
surrounded by double membrane
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All are maternally inherited
Have their own DNA and ribosomes
Have their own unique functions
Divide by binary fission (like bacteria)
There are several types, the most abundant ones are:
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Chloroplasts - photosynthetic; green due to chlorophyll content
Chromoplasts: contain pigments other than chlorophyll (in fruits, leaves,
flowers)
Leucoplasts: involved in lipid biosynthesis
Amyloplasts: store starch (colourless)
Etioplasts: intermediate state in production of chloroplasts, in tissue
exposed to light for the first time
The Chloroplast
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Usually lens-shaped, an organelle needed for photosynthesis
Has internal membrane system arranged into flattened sacs
(=thylakoids) Æ2 compartments: thylakoid space and stroma
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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?
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All organisms use cellular respiration to extract
energy from organic molecules.
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Aerobic respiration: C6H12O6 + 6O2 Æ 6CO2 + 6H2O
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ΔG = -686kcal/mol of glucose
Plants and certain algae/bacteria use
photosynthesis to synthesize organic molecules
(sugar) using light energy, CO2, & H2O.
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Photosynthesis: 6CO2 + 6H2O Æ C6H12O6 + 6O2
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Energy for this reaction comes from the sun
Photosynthesis & Respiration
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Both 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 Sun
Energy from food
Released Eergy
is collected to
make ATP
2 H2O
6 CO2
O2
Glucose
O2
Glucose
2 H2O
6 CO2
OXIDATION AND REDUCTION
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Remember OIL RIG
OIL: Oxidation Is Loss of electron
look for Oxygen addition or dehydrogenation
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RIG: Reduction is Gain of electron
look for Oxygen removal or hydrogenation
Reduction
6 CO2 + 6 H2O
C6H12O6 + 6 O2
Oxidation
Oxidation & Reduction
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Both 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:
Reduction
6 CO2 + 6 H2O
C6H12O6 + 6 O2
Oxidation
Photosynthesis
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A series of chemical reactions that enable plants, algae, and some
bacteria to covert CO2 and H2O into Sugars using SUN light .
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Photosynthesis is two separate sets of reactions
1. Light Reaction
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6CO2 + 6H2O Æ C6H12O6 + 6O2
Photosynthesis 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).
Produces energy from solar power (photons) in the form of ATP and
NADPH.
2. Calvin Cycle or dark Reaction or Carbon Fixation Reaction.
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Uses energy (ATP and NADPH) from light reaction to make sugar
(glucose).
An overview of photosynthesis
Photosynthesis has two processes: each with
multiple steps
„ Light reactions
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convert solar energy to chemical energy
Light
light energy drives transfer of electrons to
NADP+ forming NADPH
ATP is generated by photophosphorylation
occur at the thylakoids
Calvin cycle
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Named after Melvin Calvin who illustrated
many of its steps in the 1940s
Incorporates CO2 from the atmosphere into
an organic molecule (carbon fixation)
Uses energy from the light reaction to
reduce the new carbon to a sugar
Occurs in the stroma of the chloroplast
Chloroplast
NADP+
ADP
+P
Tracking atoms through photosynthesis
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Does the O2 come from the CO2 or the H2O?
Some bacteria can use H2S (hydrogen sulfide) instead of H2O in
photosynthesis
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produce yellow globules of sulfur as waste Æ H2S is split, producing sulfur
Other scientists used oxygen isotope (18O ): either CO2 or H2O
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18O
label appears in O2 only when H218O is used
6 CO2 + 12 H2O
Reactants
C6H12O6 + 6 H2O + 6 O2 Products
Outline of Light-Dependent Reactions
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How Is Light Energy Converted to Chemical Energy?
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Captured sunlight energy is stored as chemical energy in two
carrier molecules: ATP & NADPH
The pathways are different in plants versus bacteria, but the
processes are similar
In Plants
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Light Is First Captured by Pigments in Chloroplasts
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Pigments are bound to proteins of the Thylakoid Membranes
Photosystem II pass electrons through ETC to generates ATP
Photosystem I pass electron through carriers to generates NADPH
Splitting Water maintains the flow of electrons through the
photosystems
Light & the Electromagnetic Spectrum
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Light is electromagnetic radiation that has a dual nature:
„ wave - explains physical properties of light itself
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Two 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)
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Always, λ ∗ ν = c (speed of light in vacuum = 2.998 x 108 m/s)
Electric
λ=wavelength
Direction
Magnatic
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Particulate (photon) - explains how light interacts with matter
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Photon has a discrete energy: E = hν = hc /λ ; where h is Planck’s constant=6.63 x 10-34 J s
Only discrete energies of light are absorbed by matter, i.e., light is quantized
γ-rays
x-rays
Cancer treatment
Radar
TV
FM
AM
Region
UV Infrared Microwave Radiowave
imaging Illumination Heating Cooking
Electronic
Nuclear
Visible
700 nm
400 nm
Regions of the Electromagnetic Spectrum
Vibrational
Signal Transmission
Uses
Transition
Rotational
Energy
Wavelength
0.1
pm
0.01
nm
10
nm
N
106 K
1.0
μm
1.0
mm
Y
104 K
0.1
m
1.0
m
N
102 K
100
m
Y
1K
Penetrate
Atmosphere?
Temperature of
emitting bodies
Chlorophylls
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The principle photoreceptor in
photosynthesis is Chlorophyll
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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
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Mg2+ is in the center, not Fe2+
Ring V is fused to pyrrole ring III
Hydrocarbon tail
Ring IV is partially reduced
Chlorophylls
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Ratio 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 chlorophyllbinding 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 & Light
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Free 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 chromophore
open)
Chlorophyll & Pigments & Light
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All 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)
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The Color of the pigment comes from the wavelengths
of light reflected NOT absorbed
Accessory Pigments (Light Antenna)
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Observation: photosynthetic organism have more
chlorophyll molecules than is needed by reaction centers
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Chlorophyll also function to gather light
Light 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.htm
Fall Colors
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Leaves 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 Light
When a pigment absorbs light
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It goes from a ground state to an excited state, which is
unstable
e–
Energy of election
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Heat
Photon
(fluorescence)
Photon
Figure 10.11 A
Excited
state
Chlorophyll
molecule
Ground
state
Excited chlorophyll & Pigments
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If a Pigment absorbs light, it must release its energy to return
to its ground state
This can be accomplished via four common mechanisms:
1.
2.
3.
4.
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Dissipated as Heat (the most common route in general)
Transferred to another molecules (required special arrangements)
Emitted as Fluorescence (required special molecules)
Trigger a Chemical Reaction (special molecules)
Efficiency of photosynthesis is nearly 100% due to special
arrangement of proteins in the thylakoids membrane
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Such arrangement prevents dissipation of energy as heat Æonly
the other three mechanisms are important for photosynthesis
How light is harvested
When any antenna molecule absorbs a photon, it initiates a series of
energy transfers eventually reaching a particular chlorophyll a in the
reaction center
„ At the REACTION CENTER , energy from light excites
an electron in chlorophyll, which initiates a series
of reactions leading to generation of ATP and NADPH
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Photosystem I and Photosystem II
Photosystem I (PS I)
It needs light of longer
wave lengths
(lambda > 700 nm)
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Photosystem II (PS II)
It becomes active when
exposed to shorter wave
lengths (lambda < 680 nm)
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Photosystem II
http://www.sirinet.net/~jgjohnso/lightreactionproject.html
Photosynthesis Stages
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2-Stage Process
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Light Reactions
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Require Light to Occur
Involves the Actual Harnessing of Light Energy
Occur in\on the Grana
Dark Reactions
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Do not Need Light to Occur
Involve the Creation of the Carbohydrates
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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 Transfer
„ When 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
http://www.sirinet.net/~jgjohnso/lightreactionproject.html
Light Reactions
Photophosphorylation
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
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http://www.sirinet.net/~jgjohnso/lightreactionproject.html
Adenosine-5'-triphosphate (ATP)
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ATP = Adenosine-(PO4-)-(PO4-)-(PO42-)
3 Phosphate Groups Stuck off the End
of an Adenosine Molecule
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Fairly Simple Compound Containing Nitrogen
The String of 3 Phosphate Groups Is Held Together by
Covalent Bonds
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Plays an important role in cell biology as a coenzyme
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Transports chemical energy within cells
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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 Strong
„ Think of the Bond Like a Rope in a Tug-of-War with 2
People Pulling on the Rope in Opposite Directions
„ If someone Comes along and Cuts the Rope the 2 People
Will Go Flying
„ They Go Flying off because Lots of Energy Was Being
Stored in the Rope and the Energy Was Released as the
People Fell
„ When the Bond that Attaches 1 of the Phosphate Groups
onto ATP Is Broken, It Becomes ADP
„
Energy Molecules
ATP 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 Reaction
ATP and NADPH2 Are Renewable or Recyclable Energy
Sources
Light Reactions
„
„
„
„
„
Photolysis (Hill Reaction)
The 2 Water Molecules Are Split into Hydrogen and Oxygen
The Hydrogen Is Attached to a Molecule Called Nicotinamide
Adenine Dinucleotide Phosphate (NADP)
Produces NADPH2
The Oxygen Is Given
off as Oxygen Gas
2 H20 + NADP + light
Æ NADPH2 + O2
http://www.sirinet.net/~jgjohnso/lightreactionproject.html
LIGHT REACTION
„
Produces NADPH, ATP, and oxygen
H2O
CO2
Light
NADP+
ADP
CALVIN
CYCLE
LIGHT
REACTIONS
ATP
NADP
H
[CH2O] (sugar)
O2
Primary
acceptor
4
Primary
2
acceptor
Pq
+
2 H3
+
O2
1
Light
H2O
e
E le c
tron
tran
s
port
ch
8
Fd
e
ain
Cytochrome
complex
e–
e–
El
Tra ectro
n
n
ch spo7r
ai n t
e–
NADP
reductase
+
PC
5
P680
Light
6
Figure 10.13
Photosystem II
(PS II)
Photosystem-I
(PS I)
NADPH
+ H+
P700
ATP
NADP+
+ 2 H+
Light Reaction
„
A mechanical analogy for the light reactions
Photosynthesis: The Main Players
„
In eukaryotes, photosynthesis is carried out by four protein complexes,
all located in the thylakoid membrane
„
„
„
„
Photosystem II or P680: pass electrons & splits H2O
Cytochrome b6f complex : the electric connector (plastoquinone &
plastocyanin)
Photosystem I or P700 generates NADPH
Proton translocating ATP synthase complex (CF1 & CF0)
Electron Flow During Light Reaction
„
During the light reaction, there are two possible routes for
electron flow
„
„
Noncyclic Electron Flow
„
„
„
„
All reactions Occurs in the Thylakoid membranes
Uses PS II and PS I (P680 & P700)
Uses Electron Transport Chain (ETC)
Generates O2, ATP and NADPH
Cyclic Electron Flow
„
„
„
Uses Photosystem I only: P700 reaction center
Uses Electron Transport Chain (ETC)
Generates ATP only
Noncyclic Electron Flow-1
„
„
„
Accessory 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-2
„
„
„
Electrons ejected from P680
are replaced with electrons
abstracted from H2O
Splitting of H2O is carried out
by an Mn-containing Enzyme
complex (5 complex steps).
Energetics:
„
„
„
Production of O2 requires the
oxidation of 2 H2O
Transfer of 1 electron from H2O
to NADP+ requires two
photochemical events
A minimum of 8 photons are
required per 1 O2 produced
„
(2 x 2 x2)
Noncyclic Electron Flow-3
Energetics:
Splitting of H2O release 2
electrons, two protons, and ½
O2 Æ 4 protons are
generated per O2
Protons are pumped into
thylakoid space through
ETCÆ membrane potential
Membrane potential is used
to synthesize ATP via
chemiosmosis
„
Each ejected electron is passed through a chain of electron carriers
(oxidation-reduction steps:
„
Plastoquinone (Pq) Æ cytochrome complex Æplastocyanin (Pc)
Noncyclic Electron Flow-4
„
In continuation of electron flow, plastocyanin (Pc) regenerates
Photosystem I (P700), which ejects electrons from its chlorophyll a
molecule
„
Photosystem I can be excited independently by light
Noncyclic Electron Flow-5
„
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
„
„
Nocyclic flow is
called the Z-scheme
The Overall scheme
for PS is deceptively
simple:
„
„
Energy by photons to
and splitting of H2O
is used generates O2,
ATP, and NADPH
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
Cyclic Electron Flow
„
„
„
Most electrons passing PS-I are used to reduce NADP+
Some electrons may pass via the cytochrome complex back to P700 Æ Cyclic flow
of electrons
This 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 Flow
„
Noncyclic electron flow
„
„
„
„
Cyclic electron flow
„
„
„
Involves PS-II and PS-I
Involves splitting of H2O and production of O2
produces ATP and NADPH in roughly equal amounts
Involves PS-I only
generates 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 thylakoid
membrane
„
„
„
„
PSI is located mainly in the unstacked membrane Æ 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 Chloroplasts
„
„
„
Remember the definition of Photosynthesis &
Respiration.
Mechanisms for ATP generation are similar in
chloroplasts and mitochondria: chemiosmosis
Each time electrons pass through the cytchrome system,
protons are pumped across thylakoid membrane
„
„
„
Move from stroma into the thylakoid space
This generates H+ gradient across the membranes Æ
proton-motive force
Flow of H+ back across thylakoid membrane energizes
ATP synthase: ADP Æ ATP
Chemiosmosis in Chloroplasts
Calvin Cycle
„
„
„
„
„
Also called: the dark reaction, Carbon Fixation, or C3cycle.
It is a set of complex reactions that occurs in the stroma.
Uses ATP and NADPH from light reaction to add 1 CO2
to ribulose bisphosphate (RuBP)
To produce one glucose molecule, the cycle must turn 6
times: 6 CO2 Æ1 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 Cycle
Calvin cycle can be divided into three stages:
„ 1= Carbon fixation stage (CARBOXYLATION)
„
„
6 Ribulose bisphosphate (RuBP) + 6 CO2 Æ yield twelve 3carbon 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 molecules
2 of 12 G3P molecules used to synthesize 1 glucose
First Stage: CO2-Fixation (CARBOXYLATION
„
The enzyme that catalyzes the first step in Calvin cycle is
Ribulose bisphosphate carboxylase
„
„
„
It is the most important enzyme in nature that carries “True” CO2
fixation reaction
The most abundant protein in the biosphere (~15- 50% of leaf
proteins)
Its overall reaction is believed to proceed
as follows:
-
H
O
H C O P OO
O C
H C OH
H C OH
-
H
O
H C O P OH
O
Ribulose-1,5bis-phosphate
+
O
H
P OH
H C O O
O CO
2
HC O PO
O
O
O C
Carboxylase HO C C C OH
O
O C
H C OH
-
O
H C O P OH
O
Enediolate
intermediate
H C OH
-
O
H C O P OH
O
β-Keto
intermediate
O
-
C
H C OH
H2O
O
O
-
O
H C O P OH
O
C
O
-
H C OH
-
O
H C O P OH
O
3-Phosphoglycerate
RuBP Carboxylase
„
RuBP Carboxylase in plants is a very
complex enzyme consisting of:
„
8 large catalytic subunits (477 residues,
each (blue &cyan)
„
„
8 small subunits (123 residues, each
(red)).
„
„
„
Encoded by nuclear genes
The large subunit has the catalytic
activity
„
„
Encoded by Chloroplast gene
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 10^11
tons of CO2 per year
Crude oil consumption:
3*10^9 tons per year
RuBisCO
„
RuBP 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 Æ the oxygenase reaction is
significant only under conditions in which CO2 levels are low and O2 levels are high
„ Oxygenase reaction is responsible for Photorespiration (see later).
-
H
H C
CO2
-
H
O
H C O P OO
O C
H C OH
H C OH
-
O
H C O P OH
O
Ribulose-1,5bis-phosphate
-
H
O
H C O P OO
O C
H+
Ca r
HO C
se
a
l
y
box
C OH
H C OH
Enediolate
intermediate
-
H C OH
O
H C O P OH
O
O
C
O
O
-
H C OH
H2O
O
H C O P OH
O
C
O
-
H C OH
-
O
H C O P OH
O
3-Phosphoglycerate
β-Keto
intermediate
-
O
H C O P OH
O
O C
O
P OOO
O
C
O
O2
Ox
yg
en
a se
-
H
O
H C O P OO
HO C O O
O
-
O
H C O P OH
O
-
H C OH
O C
H C OH
C
O
H2O
-
O
-
C
O
-
O
H C O P OH
O
O
H C O P OH
O
3-Phosphoglycerate
2-Phosphoglycolate
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 Æ different enzymes
Chloroplast 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 consumption
„
We know from the first stage that:
„
„
„
6 Ribulose bisphosphate (RuBP) + 6 CO2 Æ yield twelve 3-carbon phosphoglyceric acid (PGA) molecules
These sets of reactions must run 12 times per glucose molecule synthesized
The sequence of events are proposed to proceed as follows:
Phosphoglycerate
Kinase
O
C
O
-
ATP
H C OH
H C O PO3
H
ADP
O
Dehydrogenase
C
O
3-Phosphoglycerate
H
-
PO3 2
H C OH
-2
Triose Phosphate
Isomerase
H C O PO3
H
NADPH
O
C
H
H C OH
-2
1,3 BisphosphoGlycerate
NADP+
Pi
H C OH
C O
-2
H C O PO3
H
D-Glyceraldehyde
3-Phosphate
H C O PO3
H
-2
Dihydroxyacetone
Phosphate
Third stage of Calvin cycle
„
The second stage produces 12 G3P molecules
„
„
2 molecules are transported across the membrane to be used in the
synthesis of 1 glucose
The remaining 10 molecules are converted into 6 RuBP molecule
6 CO2
12
Inner chloroplast
Membrane
3-Phospho
Glycerate
6 RuBP
Triose
Phosphate
10
2
Triose
Phosphate
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)
Glyceraldehyde P
Carbons
Dihydroxyacetone P
Fructose 1,6-bisphosphate
Dihydroxyacetone P
C3 + C6
C4 + C5
Glyceraldehyde P
C3 + C4
C7
Erythrose 4-P
Xylulose 5-P
C3 + C7
C5 + C5
5 C3
3 C5
P
Ribose 5-P
C6
P
Fructose 6-P
Sedoheptulose 1,7-bisphosphate
Glyceraldehyde P
C3 + C3
Sedoheptulose 7-P
Xylulose 5-P
Regeneration of (RuBP)
„
These reactions are similar
to Pentose Phosphate
Pathway reactions, but
running backward.
glyceraldehyde-3-P
TI
dihydroxyacetone-P
AL, FB
fructose-6-P
TK
xyulose-5-P + erythrose-4-P
„
AL, SB
Overall: 5 C3 Æ 3 C5
„
sedoheptulose-7-P
Enzymes:
TI, Triosephosphate Isomerase
„AL, Aldolase
„FB, Fructose-1,6-bisphosphatase
„SB, Sedoheptulose-Bisphosphatase
„TK, Transketolase
„EP, Epimerase
„IS, Isomerase
„PK, Phospho-ribulokinase
TK
„
xylulose-5-P + ribose-5-P
EP
IS
(3) ribulose-5-P
PK
(3) ribulose-1,5-bis-P
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 Reactions
Cease Soon if Light
Energy Is not Available
to Make Light Reaction
Products
„
Exception: some
Xerophytes
http://www.ualr.edu/~botany/thylakoidmembrane.gif
Dark Reactions
2 Main Steps
„
„
„
Carbon Dioxide Fixation
Sugar Formation
Via three stages
1)
Carboxylation
2)
Reduction
3)
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 4Carbon Chain
„
„
C3 Plants
C4 Plants
http://www.science.siu.edu/plant-biology/PLB117/JPEGs%20CD/0127.JPG
Alternative mechanisms of C-fixation
Photorespiration may have drastic effect on the viability of plants:
In hot, dry weather: Stomata closed (preserve their H2O), O2 ↑ ,
CO2↓, Photorespiration ↑Æ 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 climates
„
„
„
Many succulents plants (e.g., Cacti, Pineapple, agaves, etc.)
Stomata open during night, and close during the day
Use Crassulacean Acid Metabolism (CAM)
Carbon Dioxide Fixation
„
C3 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 Reactions
„ Rubisco Is the most Abundant Enzyme
on Earth!
This Occurs in the Mesophyll Cells
„ Palisade or Spongy
Creates a 3-Carbon Product Ready for
Sugar Formation
Called C3 Plants because the 1st Stable
Carbon Chain Made from CO2 Has 3
Carbons
C3 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
C4 Plants
„
„
„
„
‘Hatch-Slack Pathway’
Process of CO2 Fixation for
many Plants of Dry or
Tropical Origins
Plants 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 Rubisco
„ At 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
Carbon Dioxide Fixation
http://www.ualr.edu/~botany/c4pathway.gif
Carbon Dioxide Fixation
„
C4 Plants
„
„
This 4-Carbon Chain Is then
Transported into Bundle
Sheath Cells where the CO2
Is Released and then
Immediately Fixed by
Rubisco as Part of the C3
Cycle
„ 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. Rubisco
„
„
„
„
PEP Carboxylase Works Well at Warm Temperatures
but not Optimally at Cool Temps
This Is the Reason why C4 Grasses Are Referred to as
Warm Season Grasses, but Do not Compete Well with C3
Grasses at Cooler Temps
C4 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 Leaf
In Contrast, C3 Grasses that Keep Stomata Closed in
Dry Sunny Sites Undergo High Amounts of Respiration
C4-Plants
„
At 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 fixation
it 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
C4 Logistics
C4 plants: CO2 Fixation occurs
twice: First in mesophyll, then in
bundle-sheath cells
Mesophyll cells fix CO2 as C4
„
„
-
CO2
O
C
C
CH2
O PO3
O
PEP
„
„
-
O
PEP Carboxylase
O
C
Pi
C
O
O
CH2 C
O
Oxaloacetate
PEP Carboxylase has a very high affinity
for CO2 Æ can fix CO2 when RuBP cannot
Mesophyll cells pump malate into
bundle sheath cells. There:
„
„
Malate Æ Pyruvate + CO2
CO2 is fixed by RuBISCO
C3 & C4 plants
„
„
C4 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 Æ 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 Functions
C4 Photosynthetic Rates Drop Dramatically or Stop
Photorespiration
„
Plants that use the Calvin Cycles are called C3 Plants, because the 1st
Stable Carbon Chain Made from CO2 Has 3 Carbons
„
„
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)
„
„
„
Nearly 80% of plants, e.g. Wheat, Soybeans, Cotton, Tobacco, Grains, Legumes,
Tomatoes, Potatoes, and Peppers
Produces 2-C molecules instead of 3-C sugar molecules.
Produces no sugar molecules or no ATP.
Photorespiration occurs in light only
„
„
Occurs 1 out of 4 reactions under today’s atmospheric CO2
Rate increases with temperature
Photorespiration
„
In 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 C4
photosynthesis
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
-
H
H C
CO2
-
H
O
H C O P OO
O C
H C OH
H C OH
-
O
H C O P OH
O
Ribulose-1,5bis-phosphate
-
H
O
H C O P OO
O C
H+
Ca r
HO C
se
a
l
y
bo x
C OH
H C OH
Enediolate
intermediate
-
H C OH
O
H C O P OH
O
O
C
O
O
-
H C OH
H2O
O
H C O P OH
O
C
O
-
H C OH
-
O
H C O P OH
O
3-Phosphoglycerate
β-Keto
intermediate
-
O
H C O P OH
O
O C
O
P OOO
O
C
O
O2
Ox
yg
en
ase
-
H
O
H C O P OO
HO C O O
O
-
O
H C O P OH
O
-
H C OH
O C
H C OH
C
O
H2O
O
-
C
O
-
O
H C O P OH
O
O
H C O P OH
O
3-Phosphoglycerate
2-Phosphoglycolate
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 Plant
Less 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
http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif
Factors Influencing
Photorespiration
„
Light Intensity
„
„
Increasing 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
http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif
Factors Influencing Photorespiration
„
Temperature
„
„
Photorespiration
Increases with
Temperature
Plants Have
Optimum, Minimum
and Maximum Temp
Ranges
http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif
Factors Influencing
Photorespiration
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 C3
Plants Called Non-Efficient Plants
A Few C3 Plants Have Low Respiration and Similar
Assimilation Rates as C4 Plants
„
„
Sunflower
Peanut
http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif
Factors Influencing
Photorespiration
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 4Carbon Structure no Longer Functions
C4 PS Rates Drop Dramatically or Stop
http://www.botany.hawaii.edu/faculty/webb/BOT311/BOT311-00/PSyn/Image81.gif
Carbon Dioxide Fixation (C3 and C4
Pathways)
„
„
Both Types of Plants Use Energy from ATP and
NADPH2 to Carry out the Reactions
The Energy from ATP Is Given by ATP Giving up Its
3rd Phosphorus
„
„
ATP → ADP + P
The Energy from NADPH2
Giving up Its Hydrogens
„
NADPH2 → NADP + H2
CAM Photosynthesis
„
„
„
Crassulacean Acid
Metabolism
Another Type of PS Carried
out only by Xerophytes
At Night
„
„
„
Stomata Are Open
Plants Fix CO2 into a 4-Carbon
Product
4-Carbon Product Stored
overnight in Vacuole
http://www.ualr.edu/~botany/c4andcam.jpg
CAM Photosynthesis
„
During the Day
„
„
„
„
„
Stomata Are Closed
CO2 Is Released from the 4Carbon Produce
Normal Light and Dark
Reactions occur without
Stomata Opening
Allows the Plants to Conserve
Water during the Day
When Water Is Adequate,
these Plants usually Carry out
C3 PS
http://www.ualr.edu/~botany/c4andcam.jpg
CAM Photosynthesis
„
CAM Plants
„ Cacti, Succulents
„ Crops include Pineapple,
Tequila Agave
http://www.ualr.edu/~botany/c4andcam.jpg
CAM Photosynthesis
„
Alternative mechanisms of C-fixation is found in
Succulent plants of hot, arid environments: cacti,
pineapples, etc.
„
„
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
„
„
Plant family Crassulaceae Æ crassulacean acid
metabolism or CAM plants
CO2 is fixed into C4 molecules, and stored in large
vacuoles
During daylight: Higher Temps and lower humidity
„
„
„
Stomata closed for water conservation
NADPH and ATP are available
C4 molecules release CO2 to Calvin cycle
Overview of CAM
Comparison between C4 & CAM plants
Regulation of Carbon Dioxide Fixation
Plant cells have chloroplasts that carry out
„ photosynthesis: CO2 Æ glucose
„ Plant cells also have mitochondria and carry
out glycolysis, TCA, and oxidative phosphorylation:
Glucose Æ 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 Æ 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, O2
Cellular factors: cell state of key metabolites (NADPH, ATP, inhibitors,
reducing power etc.)
Summary of Carbon fixation
„
„
Each 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
„
„
C4 plants most adapted to:
„
„
~ 80% of plants
high light intensities, high temperatures, Limited rainfall
CAM plants better adapted to extreme aridity (desert
conditions, low water)
Factors Affecting Photosynthesis
„
6CO2 + 12H2O + Light → C6H12O6 + 6O2 + 6H2O
„
Availability of CO2
„
CO2 Supply Diminishes if Stomata are Closed
Normal [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 Water
„
Water (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
Factors Affecting Photosynthesis
„
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
Factors Affecting Photosynthesis
„
Light Duration (Photoperiod)
„
„
Plants Need Sufficient Length of Light Period to fix
enough carbons for Normal Growth
Longest Days in Northern Hemisphere Occur in June
„ December in Southern Hemisphere
Factors Affecting Photosynthesis
„
Leaf Chlorophyll Content
„
„
Pigment that Captures Light Energy and Begins the
Transformation of that Energy to Chemical Energy
Located in Chloroplasts
„
About 20 to 100 Chloroplasts/Mesophyll Cell in Leaves
http://content.answers.com/main/content/wp/en/thumb/3/34/250px-Leaf.jpg
Factors Affecting Photosynthesis
„
Leaf Chlorophyll Content
„
Chlorosis is Yellowing of Leaf
from Lack of Chlorophyll
„
„
If Chlorophyll Is Reduced, PS Will Be
Reduced
Causes of Chlorosis
„ Nutrient Deficiencies
„ N and Mg Are Parts of the
Chlorophyll Molecule
„ K Needed for Enzyme
Activation in Production of
Chlorophyll
„ Any other Nutrient Deficiencies
that Cause Chlorosis also
Reduce PS
http://toptropicals.com/pics/toptropicals/articles/cultivation/chlorosis/4061.jpg
„ Diseases
Factors Affecting Photosynthesis
„
Temperature
„
„
Increasing Temp will Increase
Rate of PS, within Normal Ranges
Below Normal Ranges, PS Slows
or Stops
„
Cytoplasm (Liquid inside Cells) Slows
Moving
„
„
Cells may Freeze
Chilling can Change Protein and
Membrane Structure
„ Causes Cell Content Leakage
and Death
http://www.semena.org/agro/diseases2/environmental-stresses-e.htm
Factors Affecting Photosynthesis
„
Temperature
„
Above Normal Ranges
„
„
Proteins may Change Shape
Membranes 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
Factors Affecting Photosynthesis
„
Temperature
„
„
„
Above Normal Ranges
„ C4 Plants Optimum PS 75-95°F
„ Can Carry out PS from 55-105°F
„ PEP Enzyme Deactivated below 55°F
C3 Plants Are Called Cool-Season Plants
C4 Plants Are Called Warm-Season Plants
Factors Affecting Photosynthesis
„
Leaf Age
„
„
„
„
„
Young, Mature Leaves Have Greatest Rate and Output of PS
Young, Immature Leaves Have High Rate of PS but Use
more of what They Produce for Their Own Growth
Mature Leaves have Slower PS Rates
Defoliation of Young or Young + Mature Leaves of a Plant
Drains the Plant
Must Pull from Stored Carbs in Stems and Roots to
Regenerate enough Leaves to Provide needed Carbs
RESPIRATION AND
OXIDATVIE
PHOSPHORYLATION
Respiration
„
Free 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
Respiration
„
„
Low-Temperature Oxidation of Carbohydrates
Carried out by Enzymes and Living Systems
Net Reaction Appears as the Reverse of PS
„
„
The Individual Reactions that Occur to Achieve the Net
Effect Are Entirely Different
Reactions Occur in Different Parts of Cells
Net Chemical Reaction
C6H12O6 + 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 Photosynthesis
All Living Plant and Animal Cells Carry out
Respiration
Respiration Occurs
„
„
„
„
At same Time as Photosynthesis
During the Night
In Developing and Ripening Fruit
In Dormant Seeds
Mitochondria
„
„
Occurs in Mitochondria of
Cells
Mitochondria are membraneenclosed 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 Oxygen
Main Type of Respiration that Occurs in most
Situations in Plants and Animals
Involves Complete Breakdown of Glucose back to CO2
and Water
Not all of the Energy in Glucose Is Converted to ATP
Formation
„
„
Only about 40% Efficient
Extra Energy Is Given off as Heat
„
„
In Plants, Heat Quickly Dissipates
For 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
glycolysis
glucose
glycolysis
ATP
NADH
pyruvate
Acetyl CoA
CoA
Complete oxidation of
acetyl CoA to H2O
and CO2 accompanied
by production of large
amounts of NADH
and ATP in
mitochondrion
Adapted from MBOC4,
fig. 2-70 & pp. 383
Citric
acid
cycle
TCA cycle
2 CO2
8 e- (Reducing power as NADH)
oxidative
phosphorylation
ATP
O2
H2O
electron transport &
ox. phosphorylation
3 Main Respiration Steps
1.
Glycolysis
„
„
„
„
„
„
Breakdown of Glucose to a 3-Carbon
Compound Called Pyruvate
(Glucose, C6H12O6, into Pyruvate, C3H4O3)
Occurs in Cytosol
Some ATP and NADH Are also Formed
„ Storage Energy Molecules
NADH Is Formed from NAD
Similar Type of Energy-Storing Rx as NADP +
H2 → NADPH2
„ NAD + H → NADH
http://www.med.unibs.it/~marchesi/glycpth2.gif
Respiration Steps
2.
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
Skeletons
„
Skeletons Are Used in other Metabolic Pathways
to Make various Compounds
„
Proteins
„
Lipids
„
Cell Wall Carbohydrates
„
DNA
„
Plant Hormones
„
Plant Pigments
„
Many other Biochemical Compounds
„
The Step where CO2 Is Given off by the Plant
„
10 NADH Are Generated
http://www.sp.uconn.edu/~bi107vc/images/mol/krebs_cycle.gif
„
Respiration Steps
Electron Transport
Chain
3.
„
„
„
‘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 Step
Water 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 Plants
After 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
Anaerobic Respiration
„
C6H12O6 + O2 → 2 CH2O5 + 2 H2O + 2 ATP
or
„
„
Glucose + Oxygen → 2 Ethanol + 2 Water + 2 ATP
Same 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
Anaerobic Respiration
„
Only 2 ATP Are Formed instead of 40 from
Aerobic Respiration
„
„
„
Plant Soon Runs out of Energy
Can 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 ATP
Aerobic:
C6H12O6 + 6O2 + 40 ADP + 40 Phosphates → 6 CO2 + 6 H2O + 40 ATP
Factors Affecting Respiration
„
Kind of Cell or Tissue
„
„
„
Young and Developing Cells (Meristematic Areas)
usually Have Higher Respiration Rates
Developing and Ripening Fruit and Seeds, too
Older Cells and Structural Cells Respire at Lower Rates
Factors Affecting Respiration
„
Temperature
„
„
„
Respiration generally Has Higher Optimum and
Maximum Temps than PS Rxs
Can Have Net Dry Matter Loss at High Temps where
Respiration Exceeds PS
Temp 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
Factors Affecting Respiration
„
Oxygen
„
„
Low O2 Can Reduce Aerobic Respiration and Increase
Anaerobic Respiration
Low O2 Can Reduce Photorespiration
Factors Affecting Respiration
„
Light
„
„
Can Enhance Rate of Photorespiration
Does not Directly Affect other Forms of Respiration
Factors Affecting Respiration
„
[Glucose]
„
„
Adequate Glucose Needed to Carry out Respiration
Reductions can Occur
„
„
Reduced PS
Reduced Flow of Carbohydrates in Plant
„
„
Insect Feeding
Phloem Blockages
Factors Affecting Respiration
„
[CO2]
„
Higher CO2 Levels Reduce Rate of Respiration
„
„
Feedback Inhibition
Seldom Occurs except when O2 Levels Are Limited
„
Flooded, Compacted Soils
Factors Affecting Respiration
„
[ATP]
„
Higher [ATP] Reduces Rate of Respiration
„
„
Feedback Inhibition
Usually Occurs when other Metabolic Processes Have
Slowed or Stopped
Factors Affecting Respiration
„
Plant Injury
„
„
Injury will Increase Respiration
Plant’s Growth Rate Increases in Attempt to Recover
„
„
„
„
Plant Synthesizes Compounds to Fight Pests
„
„
„
Mechanical Damage
Hail
Mowing, Grazing, Cultivation, Wind
Insect Feeding
Diseases
Some Herbicides Kill Plants by Disrupting or Affecting
Respiration
„
„
Generally an Indirect Effect
Herbicide Disrupts Enzyme Activity or some other Metabolic Process that
will Affect Respiration
Nitrogen Cycles
N2 and life
„
„
All 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 N2
fixation that convert N2 into NH3
or NO2, or NO3
Forms of Nitrogen
„
Gas (N2): Very Abundant, but mostly “unavailable”
„
Inorganic nitrogen:
„
„
„
„
„
„
NH3 =Ammonia
NH4+ =Ammonium
NO3- =Nitrate
NO2- =Nitrite
all nutrients, but toxic at high levels
Organic nitrogen
„
Livings things and their proteins, nucleic acids, urea, and
other nitrogenous molecules.
Nitrogen: Oxidation & reduction
„
Nitrogen is present in several oxidation states (N, atomic
number 7, Atomic Mass, 14)
„
N: 5 electrons in the outer shell Æ from (+5 oxidation) to (-3
oxidation)
Oxidation
Ion/
molecule
NH3
NH4+
N2
N2 O
NO
NO2NO3-
Name
ammonia
ammonium
diatomic N
nitrous oxide
nitric oxide
Nitrite
nitrate
Oxidation
State
-3
-3
0
+1
+2
+3
+5
Nitrogen Fixation-1
„
The 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 exergonic
reaction:
„
„
N2 + 3H2 Æ 2NH3 ; ΔG= -33.5 kJ/mol
The 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.
Oxygen
Hydrogen
N
N
Hydrogen
N
N
N
Oxygen
N
Nitrogen gets “fixed” when it is combined with oxygen or
hydrogen.
Nitrogen Fixation-2
„
By 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 fixation
Industrial fixation
Biological 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.
N
N
Atmospheric
Nitrogen (N2)
N
N
Ammonia (NH3)
Nitrates (NO3)
Nitrogen combines
with Hydrogen to make
Ammonia
Nitrogen combines
with Oxygen to make
Nitrates
Atmospheric
Fixation
Lightning “fixes” Nitrogen!
(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.
N
N
O
Nitrogen
combines
with Oxygen
Nitrogen oxides forms
(N2O)
(NO3)
Nitrogen
oxides dissolve
in rain and
change to
nitrates
Plants use
nitrates to
grow!
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.
NN
N
H
H3
Industrial Plant combines
nitrogen and hydrogen
(NH3)
Ammonia is formed
Ammonia is used a fertilizer in soil
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 Fixation
„
„
Biological 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 fixation
„
Biological N2 fixation= Conversion of N2 gas to
ammonium via the following reaction:
N2+10 H++ 8 e- + 16 ATP Æ 2 +NH4+ H2+16 ADP +16 Pi
„
„
A very energetically expensive reaction
The reaction require:
Nitrogenase enzyme (of any suitable organism)
„ Large supply of energy
„ Anoxic site
„
„
oxygen binds to and inactivates the nitrogenase enzyme
Free Living Bacteria
Highly specialized bacteria live in the soil and have the
ability to combine atmospheric nitrogen with hydrogen to
make ammonia (NH3).
N
N
H
NH3
Free-living bacteria live
in soil and combine
atmospheric nitrogen with
hydrogen
(NH3)
Nitrogen changes into
ammonia
Bacteria
Legume plants
Symbiotic
Relationship Bacteria
Bacteria 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.
N
NH3
N
Roots with nodules
where bacteria live
Nitrogen changes into
ammonia.
The Nitrogenase Complex
All nitrogen fixing species (symbionts & non- symbionts) contain
the nitrogenase complex
„ Crucial components of the complex are two proteins:
„
„
„
1) nitrogenase reductase (Fe-4S protein); homodimer
2) nitrogenase (Fe-Mo protein); tetramer (A2B2)
The nitrogenase complex is anaerobic!
Nitrogenase Complex
Electrons from
reduced source
Nitrogenase
Reductase
(Fe-S protein)
ATP
ADP
Nitrogenase
(Mo-Fe protein)
N2
NH3
The Nitrogenase Reductase
„
„
Dinitrogenase 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 complex
gray and pink are the dinitrogenase subunits
blue and green are the dinitrogenase reductase
subunits.
(bound ADP red, Fe atoms orange, S atoms
yellow)
The Nitrogenase
„
Dinitrogenase (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 complex
gray and pink are the dinitrogenase subunits
blue and green are the dinitrogenase reductase
subunits.
(bound ADP red, Fe atoms orange, S atoms
yellow)
N2-fixation by the Nitrogenase Complex
„
The process requires eight electrons:
„
„
„
The process:(repeated 8 times to transfer
eight electrons)
„
„
„
„
Six for the reduction of N2 and two to produce
one molecule of H2 as an obligate step
All electrons are transferred one at a time
First, Reducatse is reduced by ferrodoxin or
flavodoxin (electron source)
Then, reduced reductase binds 2 ATPs and
change its conformation
Reducatse (+2ATP) binds to the dinitrogenase
and transfers a single electron to it Æ release
ADP and becomes Oxidized
Highly reduced Dinitrogenase then carries
out nitrogen fixation and generates NH3 &
H2
Other Substrates of the Nitrogenase
Nitrogenase 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+ Æ H2C=CH2
„
Other reactions catalyzed by the nitrogenase
„
„
„
„
N3- Æ N2 + NH3 (Azide reduction)
N2O Æ N2 + H2O (Nitrous oxide reduction
ATP Æ ADP + Pi (ATP hydrolysis)
Nitrogenase is extremely sensitive to oxygen. Therefore N2 fixation
can proceed only at very low oxygen concentrations
Nitrogenase & Oxygen
„
The nitrogenase complex is extremely sensitive to
the presence of oxygen.
„
„
„
The reductase is inactivated in air, with a half-life of 30
seconds
dinitrogenase 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 nitrogenase
synthesis when oxygen is present.
Other species solve this problem via the symbiotic
relationship, especially between leguminous plants and
the nitrogen-fixing bacteria.
Nitrogen-fixing nodules
„
„
„
The bacteria in root nodules receive
carbohydrate and citric acid cycle
intermediates from cell
Bacteria 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 O2
Leghemoglobin 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 Cycle
„
„
„
Animals 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
more oxidized
more reduced
Reduction by most
plants & some
anaerobic bacteria
Nitrate
NO3-
Denitrification
Nitrification
(e.g. Nitrobacter)
Synthesis:
(microorganisms,
plants & animals
N2
Nitrogen fixation
(some bacteria)
Ammonia
NH4+
Amino acids
& reduced
nitrogen
compounds
Degradation:
Animals & microbes
Nitrite
NO2-
Nitrification
(e.g. Nitrosomonas)
Key terms of The Nitrogen Cycle
„
Nitrogen Fixation: Conversion of N2 to ammonia (NH3)
„
„
Nitrification: Conversion of ammonia to nitrite (NO2-) and then
nitrate (NO3-).
„
„
All living cells (plants, animals, & bacteria).
Ammonification: Conversion of the amine groups of organic
compounds into simpler compounds (often, ammonia NH3).
„
„
Both reactions carried out by bacteria
Assimilation: Conversion of NH3, NO2-,, NO3- (inorganic) into
organic compounds (proteins, DNA, & other forms)
„
„
By any bacteria in soil/water having the nitrogenase complex, e.g.
Rhizobium in root nodules of legumes.
Mostly via decay processes carried out by decomposer bacteria
Denitrification: 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-cycle
„
The 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 Æ -NO2 Æ -NO3
Plants and many bacteria can also reduce nitrate and nitrite
ammonia (reductases). -NO3 Æ NO2 Æ NH3
The 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 Denitrification
Nitrification: 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 Clostridium
convert it to nitrogen gas, nitrous
oxide and nitric oxide, all nontoxic
and are released in the process.
Nitrification
„
„
„
„
„
„
Nitrification 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.
„ NH is membrane permeable and diffuses freely across a membrane
3
„ Plants 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 bacteria
„
„
Nitrifiers (heterotrophs & autotroph) are delicate organisms and extremely
susceptible to a variety of inhibitors.
„ They are extremely slow growing
„ Nitrifying 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: Nitrobacter
„ Aerobic, but occasionally also anaerobic
„ They are widely distributed in soils, fresh water, seawater, mud layers,
sewage disposal systems, and inside stones of buildings, rocks, and inside
concrete surfaces.
Nitrate Assimilation
NO3
nitrate
NO2
nitrite
NH4+
amino acids
ammonium
„
Requires large input of energy
Forms toxic intermediates
„
Mediated by enzymes (Reductases) that are closely regulated
„
„
„
Nitrate 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 reductase
protein thereby activating the enzyme
Roots and Leaves
„
„
Nitrate 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 Reductase
Nitrate 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) Æ one cytochrome-b557) Æ
one cofactor containing molybdenum
+
NADH + H
+
NAD
Nitrate Reductase
FAD
Cyt-b557
MoCO
-
NO3
-
NO2 + H2O
Nitrate Reductase: Structure
„
„
„
„
Nitrate 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
4+
nitrite.
Mo
HOOC-
FAD Heme
Domain Domain
MoCO
Domain
-NH2
O
S
N C
N
H
N
N
H2N
Pterine
S
O
C
CH CH2 O P - O
O
OH
Nitrite Reductase
„
The reduction of nitrite to ammonia proceeds in the plastids
„
„
Nitrite (NO2-) is highly reactive Æ 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 Reductase
Light
Photosystem-I
6 Ferredoxin
Reduced
6 Ferredoxin
Oxidized
4 Fe-4S
FAD
6 e-
-
Siroheme
NO2 + 8 H+
+
NH4 + 2H2O
Nitrite Reductase
„
„
Chloroplast 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 Reductase
Light
Photosystem-I
6 Ferredoxin
Reduced
6 Ferredoxin
Oxidized
4 Fe-4S
FAD
6 e-
-
Siroheme
NO2 + 8 H+
+
NH4 + 2H2O
Ammonium Assimilation
„
„
Ammonium 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: Transamination
„
Once assimilated into glutamine and glutamate, nitrogen is
incorporated into other amino acids via transamination
reactions
H
R1 C
C +
+
NH3
„
O
O
O
-
R2 C
O
C
O
-
H
O
Transaminase
R1 C
O
+
C
O
-
R2 C
O
C
+
NH3
O
-
Best known is aspartate aminotransferase
„
„
„
Trnsfers amino group of glutamate to the carboxyl atom of
oxaloacetate Æ aspartate + α-ketoglutarate
Aspartate is involved in the transport of carbon from mesophyll
to bundle sheath of C4 carbon fixation
All aminotransferases require vitamin B6 to act as a cofactor
Ammonium & Nitrate Assimilation
From: Plant Biochemistry 3rd ed – H. Heldt (Elsevier, 2005)
Ammonia Integration in Animals
Denitrification
„
„
„
Denitrification 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!
Eutrophication
Agriculture 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 Æ 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 Blooms
„ Algae absorb all the oxygen from lakes and ponds killing the organisms
in the water.
„ Too much nitrates in water is called eutrophication.
„