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Plant Physiology talk Five
Basic Plant Biochemistry
Carbohydrates
•
Of the macromolecules that we will cover in
this class, those involving carbohydrates are
the most abundant in nature.
•
Via photosynthesis, over 100 billion metric
tons of CO2 and H2O are converted into
cellulose and other plant products.
•
The term carbohydrate is a generic one that
refers primarily to carbon-containing
compounds that contain hydroxyl, keto, or
aldehydic functionalities.
•
Carbohydrates can range in sizes, from
simple monosaccharides (sugars) to
oligosaccharides, to polysaccharides.
Carbohydrates
• Carbohydrates constitute more than 1/2 of organic molecules
• Main role of carbos in nature
 Storage of energy
 Structural support
 Lipid and protein modification:
 membranes asymmetry, recognition by IgG/fertilization/virus
recognition/cell cell communication
Definition: Carbohydrates, Sugars and Saccharides- are all polyhydroxy
 (at least 2 OH) Cn(H20) n = hydrate of carbon
•
Notice that there are two distinct types of monosaccharides, ketoses and
aldoses.
•
The number of carbons is important in general nomenclature (triose = 3, pentose
= 5, hexose =6,
Basic facts
Monosaccharides - Simple sugars
• Single polyhydroxyl
 Can’t be hydrolyzed to simpler form
Trioses - Smallest monosaccharides have three carbon atoms
Tetroses (4C) Pentose (5C) Hexoses (6C) Heptoses (7C) etc…
Disaccharide - two sugars linked together. Can be the same molecule
or two different sugars. Attached together via a glycosidic linkage
Oligosaccharide - 2 to 6 monosaccharides
Polysaccharides - straight or branched long chain monosaccharides.
Bonded together by glycosidic linkages
The functional groups
• Aldehyde: Consists of a carbon atom
bonded to a hydrogen atom and doublebonded to an oxygen atom.
– Polar. Oxygen, more electronegative than carbon, pulls the
electrons in the carbon-oxygen bond towards itself,
creating an electron deficiency at the carbon atom.
• Ketone: Characterized by a carbonyl group (O=C)
linked to two other carbon atoms or a chemical
compound that contains a carbonyl group
– A carbonyl carbon bonded to two carbon atoms
distinguishes ketones from carboxylic acids, aldehydes,
esters, amides, and other oxygen-containing compounds
Classification of monosaccharides
•
Monosaccharides are classified according to
three different characteristics:
– the placement of its carbonyl group,
– the number of carbon atoms it contains
– its chiral handedness.
• If the carbonyl group is an aldehyde, the
monosaccharide is an aldose
• if the carbonyl group is a ketone, the
monosaccharide is a ketose.
• Monosaccharides with three carbon atoms
are called trioses, those with four are
called tetroses, five are called pentoses, six
are hexoses, and so on.
• These two systems of classification are
often combined.
– For example, glucose is an aldohexose (a
six-carbon aldehyde)
carbonyl group
• A functional group composed of
a carbon atom double-bonded to
an oxygen atom: C=O.
• The term carbonyl can also
refer to carbon monoxide as
a ligand in
an inorganic or organometallic
complex.
Classification of monosaccharides
• D-glucose
• is an aldohexose with the formula
(C·H2O)6.
• The red atoms highlight the
aldehyde group
• the blue atoms highlight the
asymmetric center furthest from the
aldehyde; because this -OH is on the
right of the Fischer projection, this
is a D sugar.
Classification of monosaccharides
• The a and b anomers of glucose.
• Note the position of the hydroxyl
group (red or green) on the anomeric
carbon relative to the CH2OH group
bound to carbon 5:
•
Either on the opposite sides (a)
•
Or the same side (b).
Important disaccharides
• Sucrose
• The osmotic effect of a substance
is tied to the number of particles
in solution, so a millilitre of
sucrose solution with the same
osmolarity as glucose will be
have twice the number carbon
atoms and therefore about twice
the energy.
– Thus, for the same osmolarity,
twice the energy can be
transported per ml.
• As a non-reducing sugar, sucrose
is less reactive and more likely to
survive the journey in the phloem.
• Invertase (sucrase) is the only
enzyme that will touch it and
this is unlikely to be present in
the phloem sieve tubes.
Important disaccharides
• Maltose
• Malt sugar or corn sugar consists
of two glucose molecules linked
by an a-1,4-glycosidic bond
• It comes from partial hydrolysis
of starch by the enzyme amylase,
which is in saliva and also in grains
(like barley)
• Maltose is an important
intermediate in the digestion of
starch. Starch is used
by plants as a way to
store glucose. After cellulose,
starch is the most abundant
polysaccharide in plant cells.
Important plant saccharides
•
Raffinose is a trisaccharide composed
of galactose, fructose, and glucose.
•
Raffinose can be hydrolyzed to Dgalactose and sucrose by the enzyme αgalactosidase (a-GAL), an enzyme not
found in the human digestive tract. a-GAL
also hydrolyzes other a-galactosides such
asstachyose, verbascose, and galactinol, if
present. The enzyme does not cleave βlinked galactose, as in lactose.
•
The raffinose family
of oligosaccharides (RFOs) are alphagalactosyl derivatives of sucrose, and the
most common are raffinose, stachyose,
verbascose.
RFOs are almost ubiquitous in
the plant kingdom, being found in a large
variety of seeds from many different
families, and they rank second only to
sucrose in abundance as soluble
carbohydrates.
•
Carbohydrates-make up 16-25% of sap.
• The major organic transport
materials are sucrose, stachyose
(sucrose-gal), raffinose (stachyosegal).
• These are excellent choices for
transport materials for two reasons:
• (a) they are non-reducing sugars (the
hydroxyl group on the anomeric
carbon, the number one carbon, is
tied up) which means that they are
less reactive and more chemically
stable.
• (b) the linkage between sucrose and
fructose is a "high-energy" linkage
similar to that of ATP. Thus, sucrose
is a good transport form that
provides a high energy, yet stable
packet of energy;
Important Polysaccharides:
Starch - energy reservoir
in plants - made of two
polysaccharides
Amylose -long unbranched
glucose a (1,4) with
open reducing end large
tight helical forms.
Test by iodination..
Important Polysaccharides:
Starch - energy reservoir in plants - made of two polysaccharides
– Amylose -long unbranched glucose a (1,4) with open reducing end large tight
helical forms. Test by iodination.
– Amylopectin - polymer of a(1,4) and a (1,6) branches. Not helical.
Plant Starch (Amylose and Amylopectin)
•
Starch contains a mixture of amylose and amylopectin
•
Amylose is an unbranched polymer (forms a-helix) of D-glucose molecules linked by a1,4-glycosidic bonds
•
Amylopectin is like amylose, but has extensive branching, with the branches using a-1,6glycosidic bonds
Cellulose
• Linear glucan chains of
unbranched (1-4)-blinked-D-glucose in which
every other glucose
residue is rotated 180°
with respect to its two
neighbors and contrasts
with other glucan
polymers such as:
• starch (1-4-a-glucan)
• callose (1-3-b-glucan).
Cellulose
• This means that cellobiose, and not glucose, is the basic
repeating unit of the cellulose molecule. Groups of 30 to 40
of these chains laterally hydrogen-bond to form crystalline
or para-crystalline microfibrils.
Proteins
Basic facts
Amino acids
• -20 common amino acids there are others
found naturally but much less frequently
• Common structure for amino acid
• COOH, -NH2, H and R functional groups all
attached to the alpha carbon
Proteins: Three-dimensional structure
• Background on protein composition:
• Two general classes of proteins
 Fibrous - long rod-shaped, insoluble proteins. These
proteins are strong (high tensile strength).
 Globular - compact spherical shaped proteins usually
water-soluble. Most hydrophobic amino acids found in
the interior away from the water. Nearly all enzymes
are globular…
 Proteins can be simple - no added groups or modifications, just
amino acids
 Or proteins can be conjugated. Additional groups
covalently bound to the amino acids. The naked
protein is called the apoprotein and the added group is
the prosthetic group. Together the protein and
prosthetic group is called the holoprotein. Ex.
chlorophyll
Four levels of protein structure
• Primary structure: amino acid only. The actual amino acid
sequence is specified by the DNA sequence. The primary
structure is used to determine genetic relationships with
other proteins - AKA homology. Amino acids that are not
changed are considered invariant or conserved.
Primary
sequence is also
used to
determine
important
regions and
functions of
proteins domains.
Four levels of protein structure
• Secondary structure: This level is only concerned with the
local or close in structures on the protein - peptide
backbone. The side chains are not considered here, even
though they have an affect on the secondary structure.
Two common
secondary
structures - alpha
helix and beta
pleated sheet
Non- regular
repeating structure
is called a random
coil.
- no specific
repeatable pattern
Four levels of protein structure
Tertiary structure - the overall three-dimensional shape
that a protein assumes. This includes all of the secondary
structures and the side groups as well as any prosthetic
groups. This level is also where one looks for native vs.
denatured state. The hydrophobic effect, salt bridges
And other
molecular
forces are
responsible
for
maintaining
the tertiary
structure
Four levels of protein structure
• Quaternary structure: The overall interactions of
more than one peptide chain. Called subunits.
Each of the sub units
can be different or
identical subunits,
hetero or homo – x
mers (ex.
Heterodimer is a
protein composed of
two different
subunits).
Lipids
Lipids fats oils…. Greasy molecules, mmmmm donuts.
Several levels of complexity:
• Simple lipids - a lipid that cannot be broken down to smaller
constituents by hydrolysis.
– Fatty acids, waxes and cholesterol
• Complex lipids - a lipid composed of different molecules held
together mostly by ester linkages and susceptible to cleavage
reactions.
– acylglycerols - mono, di and triacyl glycerols ( fatty acids and
glycerol)
– phospholipids (also known as glycerophospholipids) - lipids which
are made of fatty acids, glycerol, a phosphoryl group and an
alcohol. Many also contain nitrogen
– glycolipids (also known as glycosphingolipids): Lipids which have
a spingosine and different backbone than the phospholipids
General Structure
• glycerol (a type of alcohol with a
hydroxyl group on each of its three
carbons)
• Three fatty acids joined by
dehydration synthesis.
• Since there are three fatty acids
attached, these are known as
triglycerides.
General Structure
-
The longer the fatty acids the higher
the melting point.
-
Again the more hydrophobic
interactions effects the more the
energy it takes to break the order.
Decreases in the packing efficiency
decreases the mp
-
The van der Waals forces then come
apart more easily at lower
temperatures.
-
Animal alter the length and unsaturated
level of the fatty acids in lipids
(cholesterol too) to deal with the cold
temps
Saturated or not – the power of H
•
The terms saturated, monounsaturated, and poly-unsaturated
refer to the number of hydrogens
attached to the hydrocarbon tails of
the fatty acids as compared to the
number of double bonds between
carbon atoms in the tail.
•
Oils, mostly from plant sources, have
some double bonds between some of
the carbons in the hydrocarbon tail,
causing bends or “kinks” in the shape of
the molecules.
•
Because some of the carbons share
double bonds, they’re not bonded to as
many hydrogens as they could if they
weren’t double bonded to each other.
Trans and Cis
•
In unsaturated fatty acids, there are two
ways the pieces of the hydrocarbon tail can
be arranged around a C=C double bond.
•
TRANS
– The two pieces of the molecule are on
opposite sides of the double bond, that is,
one “up” and one “down” across from each
other.
•
CIS
– the two pieces of the carbon chain on
either side of the double bond are either
both “up” or both “down,” such that both
are on the same side of the molecule
Trans and Cis
•
Naturally-occurring unsaturated vegetable
oils have almost all cis bonds
– but using oil for frying causes some of the
cis bonds to convert to trans bonds.
•
If oil is used only once like when you fry an
egg, only a few of the bonds do this so it’s not
too bad.
•
However, if oil is constantly reused, like in
fast food French fry machines, more and
more of the cis bonds are changed to trans
until significant numbers of fatty acids with
trans bonds build up.
•
The reason this is of concern is that fatty
acids with trans bonds are carcinogenic!
Phospholipids
•
Made from glycerol, two fatty
acids, and (in place of the third
fatty acid) a phosphate group with
some other molecule attached to
its other end.
•
The hydrocarbon tails of the fatty
acids are still hydrophobic, but
the phosphate group end of the
molecule is hydrophilic because of
the oxygens with all of their pairs
of unshared electrons.
•
This means that phospholipids are
soluble in both water and oil.
•
Plant Biotechnology, GMOs,
and the Environment
Why GMOs?
• “For centuries, humankind has made improvements
to crop plants through selective breeding and
hybridization — the controlled pollination of plants.
• Plant biotechnology is an extension of this
traditional plant breeding with one very important
difference —
– plant biotechnology allows for the transfer of a
greater variety of genetic information in a more
precise, controlled manner.”
Indeed
Figure why?
9.1
• The Earth is currently
experiencing the most population
increase in Human history.
• 2.5 billion in 1955 to 6 billion in
1999
• At current rate, will double within
30 years!
• Fastest growing nations have
growth rates at or above 4% - this
will double the countries
population every 17 years
Indeed why?
• Hunger, starvation, and malnutrition are endemic in
many parts of the world today.
• Rapid increases in the world population have
intensified these problems!
• ALL of the food we eat comes either directly or
indirectly from plants.
• Can’t just grow more plants, land for cultivation has
geographic limits
– Also, can destroy ecosystems!
•
Increasing
crop
yields
Figure
11.13
To feed the increasing
population we have to increase
crop yields.
• Fertilizers - are compounds to
promote growth; usually applied
either via the soil, for uptake
by plant roots, or by uptake
through leaves. Can be organic
or inorganic
• Have caused many problems!!
• Algal blooms pollute lakes near
areas of agriculture
Increasing
crop
yields
Figure 11.13
• Algal blooms - a relatively
rapid increase in the population
of (usually) phytoplankton algae
in an aquatic system.
• Causes the death of fish and
disruption to the whole
ecosystem of the lake.
• International regulations has led
to a reduction in the
occurrences of these blooms.
Chemical
pest
control
Figure 11.17
• Each year, 30% of crops are lost to insects and other crop
pests.
• The insects leave larva, which damage the plants further.
• Fungi damage or kill a further 25% of crop plants each year.
• Any substance that kills organisms that we consider
undesirable are known as a pesticide.
• An ideal pesticide would:-
–
–
–
–
Kill only the target species
Have no effect on the non-target species
Avoid the development of resistance
Breakdown to harmless compounds after a short time
Chemical
pest
control
Figure 11.17
• DDT was first developed in the 1930s
• Very expensive, toxic to both harmful and
beneficial species alike.
• Over 400 insect species are now DDT
resistant.
• As with fertilizers, there are run-off
problems.
• Affects the food pyramid.
– Persist in the environment
•
Chemical
pest
control
Figure
11.18
DDT persists in the food chain.
• It concentrates in fish and fisheating birds.
• Interfere with calcium
metabolism, causing a thinning in
the eggs laid by the birds –
break before incubation is
finished – decrease in population.
• Although DDT is now banned, it
is still used in some parts of the
world.
Plant Biotechnology
• The use of living cells to make products
such as pharmaceuticals, foods, and
beverages
• The use of organisms such as bacteria to
protect the environment
• The use of DNA science for the production
of products, diagnostics, and research
Genetically modified crops
• All plant characteristics, such as size, texture, and
sweetness, are determined on the genetic level.
•
•
•
•
•
•
Also:
The hardiness of crop plants.
Their drought resistance.
Rate of growth under different soil conditions.
Dependence on fertilizers.
Resistance to various pests and diseases.
• Used to do this by selective breeding
Why would we want to modify
an organism?
• Better crop yield, especially under harsh
conditions.
• Herbicide or disease resistance
• Nutrition or pharmaceuticals, vaccine delivery
• “In 2010, approximately 89% of soy and 69% of
corn grown in the U.S. were grown from Roundup
Ready® seed.”
http://www.oercommons.org/courses/detecting-genetically-modified-food-by-pcr/
Roundup Ready Gene
• The glyphosate resistance gene protects food
plants against the broad-spectrum herbicide
Glyphosate - N-(phosphonomethyl) glycine
[Roundup®], which efficiently kills invasive weeds in
the field.
• The major advantages of the "Roundup Ready®”
system include better weed control, reduction of
crop injury, higher yield, and lower environmental
impact than traditional weed control systems.
• Notably, fields treated with Roundup® require less
tilling; this preserves soil fertility by lessening soil
run-off and oxidation.”
Glyphosate - N-(phosphonomethyl) glycine
• An aminophosphonic analogue of
the natural amino acid glycine.
• It is absorbed through foliage
and translocated to actively
growing points. (Meristems!!!)
• Mode of action is to inhibit
an enzyme involved in the
synthesis of the aromatic amino
acids:
• tyrosine,
• tryptophan
• phenylalanine
Glyphosate
Glycine
Glyphosate - N-(phosphonomethyl) glycine
• It does this by inhibiting
the enzyme 5-enolpyruvylshikimate3-phosphate synthase (EPSPS),
which catalyzes the reaction
of shikimate-3-phosphate (S3P)
and phosphoenol pyruvate to form
5-enolpyruvyl-shikimate-3phosphate (ESP).
• ESP
subsequently dephosphorylated to
chorismate, an essential precursor
in plants for these aromatic amino
acids.
Glyphosate
Glycine
Roundup Ready Gene
• Glyphosate functions by
occupying the binding site of
the phosphoenol pyruvate,
mimicking an intermediate
state of the enzyme
substrates complex.
• The "Roundup Ready®” system
introduces a stable gene
alteration which prevents
Glyphosate binding and
allowing the formation of the
essential aromatic amino acids
Roundup Ready Gene
• The shikimate pathway is not present in animals, which
instead obtain aromatic amino acids from their diet.
• Glyphosate has also been shown to inhibit other plant
enzymes
•Also has been found to affect animal
enzymes.
•The United States Environmental
Protection Agency considers glyphosate to
be relatively low in toxicity, and without
carcinogenic or teratogenic effects
•However, some farm workers have reported
chemical burns and contact skin burns
Environmental degradation
• When glyphosate comes into contact with the soil, it can
be rapidly bound to soil particles and be inactivated.
•
Unbound glyphosate can be degraded by bacteria.
– However, glyphosate has been shown to increase the infection
rate of wheat by fusarium head blight in fields that have been
treated with glyphosate.
• In soils, half-lives vary from as little as 3 days at a site in
Texas to 141 days at a site in Iowa.
• In addition, the glyphosate metabolite amino methyl
phosphonic acid has been shown to persist up to 2 years
in Swedish forest soils.
• Glyphosate absorption varies depending on the kind of
soil.
Insect Resistance
• B. thuringiensis (commonly
known as 'Bt') is an insecticidal
bacterium, marketed worldwide
for control of many important
plant pests - mainly caterpillars
of the Lepidoptera (butterflies
and moths) but also mosquito
larvae, and simuliid blackflies
that vector river blindness in
Africa.
• Bt products represent about 1%
of the total ‘agrochemical’
market (fungicides, herbicides
and insecticides)
Genetically modified crops
• 1992- The first commercially grown
genetically modified food crop was a tomato
- was made more resistant to rotting, by
adding an anti-sense gene which interfered
with the production of the enzyme
polygalacturonase.
– The enzyme polygalacturonase breaks
down part of the plant cell wall, which is
what happens when fruit begins to rot.
Genetically modified crops
• Need to build in a:
• Promoter
• Stop signal
ON/OFF Switch
Makes Protein
PROMOTER INTRON CODING SEQUENCE
stop sign
poly A signal
Genetically modified crops
• So to modify a plant:
• Need to know the DNA
sequence of the gene of
interest
• Need to put an easily
identifiable maker gene
near or next to the gene
of interest
• Have to insert both of
these into the plant
nuclear genome
• Good screen process to
find successful insertion
Building the Transgenes
ON/OFF Switch
Makes Protein
PROMOTER INTRON CODING SEQUENCE
Plant Transgene
Plant Selectable
Marker Gene
bacterial genes
•antibiotic marker
•replication origin
Plasmid DNA
Construct
stop sign
poly A signal
Cloning into a Plasmid
• The plasmid carrying genes
for antibiotic resistance,
and a DNA strand, which
contains the gene of
interest, are both cut with
the same restriction
endonuclease.
• The plasmid is opened up
and the gene is freed from
its parent DNA strand.
They have complementary
"sticky ends." The opened
plasmid and the freed gene
are mixed with DNA ligase,
which reforms the two
pieces as recombinant
DNA.
Cloning into a Plasmid
• Plasmids + copies of the
DNA fragment
produce quantities of
recombinant DNA.
• This recombinant DNA
stew is allowed to
transform a bacterial
culture, which is then
exposed to antibiotics.
• All the cells except those
which have been encoded
by the plasmid DNA
recombinant are killed,
leaving a cell culture
containing the desired
recombinant DNA.
So, how do you get the
DNA into the Plant?
Meristems Injections
• The tissue in most plants consisting of
undifferentiated cells (meristematic
cells), found in zones of the plant
where growth can take place.
• Meristematic cells are analogous in
function to stem cells in animals, are
incompletely or not differentiated, and
are capable of continued cellular
division.
• First method of DNA transfer to a
plant.
• Inject DNA into the tip containing
the most undifferentiated cells – more
chance of DNA being incorporated in
plant Genome
• Worked about 1 in 10,000 times!
Tunica-Corpus model of the apical
meristem (growing tip). The
epidermal(L1) and subepidermal (L2)
layers form the outer layers called
the tunica.
The inner L3 layer is called the
corpus.
Cells in the L1 and L2 layers divide
in a sideways fashion which keeps
these layers distinct, while the L3
layer divides in a more random
fashion.
Particle Bombardment
Particle Bombardment
Particle-Gun Bombardment
• DNA- or RNA-coated
gold/tungsten particles are
loaded into the gun and you
pull the trigger.
• Selected DNA sticks to
surface of metal pellets in a
salt solution (CaCl2).
Particle Bombardment
•
A low pressure helium pulse
delivers the coated gold/tungsten
particles into virtually any target
cell or tissue.
•
The particles carry the DNA 
cells do not have to be removed
from tissue in order to transform
the cells
•
As the cells repair their injuries,
they integrate their DNA into
their genome, thus allowing for the
host cell to transcribe and
translate the transgene.
Particle Bombardment
The DNA sometimes was
incorporated into the nuclear
genome of the plant
Gene has to be incorporated
into cell’s DNA where it will
be transcribed
Also inserted gene must not
break up some other
necessary gene sequence
Overview of the Infection Process
Agrobacterium chromosomal DNA
pscA
chvA chvB
T-DNA-inserts into plant genome
for
transfer
to the
plant
vir genes
pTi
tra
bacterial
conjugation
opine catabolism
oriV
Agrobacterium tumefaciens
• Agrobacterium tumefaciens
chromosomal genes: chvA,
chvB, pscA required for initial
binding of the bacterium to
the plant cell and code for
polysaccharide on bacterial
cell surface.
• Virulence region (vir) carried
on pTi, but not in the
transferred region (T-DNA).
Genes code for proteins that
prepare the T-DNA and the
bacterium for transfer.
Agrobacterium tumefaciens
• T-DNA encodes genes for
opine synthesis and for tumor
production.
• occ (opine catabolism) genes
carried on the pTi and allows
the bacterium to utilize
opines as nutrient
Agrobacterium
can be used to
transfer DNA
into plants
Overall process
– Uses the natural infection mechanism of
a plant pathogen
– Agrobacterium tumefaciens naturally
infects the wound sites in
dicotyledonous plant causing the
formation of the crown gall tumors.
– Capable to transfer a particular DNA
segment (T-DNA) of the tumor-inducing
(Ti) plasmid into the nucleus of infected
cells where it is integrated fully into
the host genome and transcribed,
causing the crown gall disease.
• So the pathogen inserts the new DNA with
great success!!!
Agrobacterium tumafaciens senses
Acetosyringone via a 3-component-like
system
3 components:
ChvE,
VirA,
VirG
Genetically modified crops
• The vir region on the plasmid inserts DNA between
the T-region into plant nuclear genome
• Insert gene of interest and marker in the T-region
by restriction enzymes – the pathogen will then
“infect” the plant material
• Works fantastically well with all dicot plant species
– tomatoes, potatoes, cucumbers, etc
– Does not work as well with monocot plant species - corn
• As Agrobacterium tumefaciens do not naturally
infect monocots
MiniTi T-DNA based vector for plants
a binary vector system
kanr
polylinker
LB
11
RB
ori
T-DNA deleted
bom
modified Ti plasmid
vir
miniTi
bom = basis of mobilization
oriV
MiniTi T-DNA based vector for plants
Disarmed vectors: do not produce tumors; can
be used to regenerate normal plants containing
the foreign gene.
• Binary vector: the vir genes required
for mobilization and transfer to the
plant reside on a modified pTi.
• consists of the right and left border
sequences, a selectable marker
(kanomycin resistance) and a
polylinker for insertion of a foreign
gene.
miniTi
1. ChvE
periplasmic protein binds to sugars, arabinose,
glucose
binds to VirA periplasmic domain
 amplifies the signal
VirA
Periplasmic domain
acetosyringone
Transmitter
Inhibitory domain
sugars
ChvE
receiver
VirG
DNAbinding
2. VirA : Receptor kinase
1. Membrane protein five functional domains:
a) Periplasmic binds ChvE-sugar complex does NOT bind
acetosyringone
b) Transmembrane domain
c) Linker region BINDS acetosyringone NOTE this is on the
cytoplasmic side!
VirA
Periplasmic domain
acetosyringone
Transmitter
Inhibitory domain
sugars
ChvE
receiver
VirG
DNAbinding
2. VirA : Receptor kinase
d) Transmitter domain (His) auto- phosphorylates and then
transfers to the response regulator protein VirG
e) Inhibitory domain  will bleed off the phosphate from the
His in the transmitter domain (to an Asp)
VirA
Periplasmic domain
acetosyringone
Transmitter
Inhibitory domain
sugars
ChvE
receiver
VirG
DNAbinding
3. VirG : Response Regulator
• Receiver domain that is phosphorylated on an Asp residue by
the His on the transmitter domain of VirA
• Activates the DNA binding domain to promote transcription
from Vir-box containing promoter sequences (on the Ti
plasmid)
VirA
Periplasmic domain
acetosyringone
Transmitter
Inhibitory domain
sugars
ChvE
receiver
VirG
DNAbinding
sugars
VirA
Periplasmic domain
ChvE
receiver
acetosyringone
Transmitter
Inhibitory domain
VirG
DNAbinding
Summary
Agrobacteria are biological vectors for
introduction of genes into plants.
•Agrobacteria attach to plant cell
surfaces at wound sites.
•The plant releases wound signal
compounds, such as acetosyringone.
•The signal binds to virA on the
Agrobacterium membrane.
•VirA with signal bound activates virG.
Summary
•Activated virG turns on other vir genes,
including vir D and E.
•vir D cuts at a specific site in the Ti plasmid
(tumor-inducing), the left border. The left
border and a similar sequence, the right
border, delineate the T-DNA, the DNA that
will be transferred from the bacterium to
the plant cell
•Single stranded T-DNA is bound by vir E
product as the DNA unwinds from the vir D
cut site. Binding and unwinding stop at the
right border.
Summary
•The T-DNA is transferred to the
plant cell, where it integrates in
nuclear DNA.
•T-DNA codes for proteins that
produce hormones and opines.
Hormones encourage growth of the
transformed plant tissue. Opines feed
bacteria a carbon and nitrogen source.
And then?.......
• What is the last step?..........................
Tissue culture
The basics!
What is Plant Tissue Culture?
Of all the terms which have been applied to the process,
"micropropagation" is the term which best conveys the
message of the tissue culture technique most widely in use
today.
The prefix "micro" generally refers to the small size of the
tissue taken for propagation, but could equally refer to the
size of the plants which are produced as a result.
Relies on two plant hormones
Auxin
Cytokinin
Biosynthesis of cellulose
• synthesized at the plasma
membrane by rosette
terminal complexes (RTCs).
• RTC - hexameric protein
structures, approximately
25 nm in diameter
• Contain the Cellulose
synthase enzymes that
synthesise the individual
cellulose chains
Biosynthesis of cellulose
• Each RTC floats in the cell's
plasma membrane and "spins"
a microfibril into the cell
wall.
• RTCs contain at least three
different cellulose synthases,
encoded by CesA genes
Biosynthesis of cellulose
• Requires chain initiation and
elongation, and the two processes
are separate.
• CesA glucosyl transferase initiates
cellulose polymerization using
a steroid primer, sitosterol-betaglucoside, and UDP-glucose.
• Cellulose synthase utilizes UDP-Dglucose precursors to elongate the
growing cellulose chain.
• A cellulase functions to cleave
the primer from the mature
chain.
Protoplast to Plant
• Callus: Induced by
• 2, 4 dichlorphenoxyacetic
acid (2,4D)
• Unorganized, growing mass
of cells
• Dedifferentiation of explant
– Loosely arranged thinned
walled, outgrowths
– No predictable site of
organization or
differentiation
Protoplast to Plant
• 2, 4 dichlorphenoxyacetic acid
(2,4D)
• Stops synthesis of cellulose
• Knocks out every other rosette
• Makes b 1,3 linked glucose
– Callose
• Temporarily alters the cell wall
Auxin (indoleacetic acid)
Produced in apical and root meristems, young
leaves, seeds in developing fruits
• cell elongation and expansion
• suppression of lateral bud growth
• initiation of adventitious roots
• stimulation of abscission (young fruits) or delay
of abscission
• hormone implicated in tropisms (photo-, gravi-,
thigmo-)
Cytokinin (zeatin, ZR,
IPA)
Produced in root meristems, young leaves, fruits,
seeds
• cell division factor
• stimulates adventitious bud
formation
• delays senescence
• promotes some stages of root
development
Organogenesis
The formation of organs
from a callus
• Rule of thumb:
Auxin/cytokinin 10:1100:1 induces roots.
• 1:10-1:100 induces
shoots
• Intermediate ratios
around 1:1 favor
callus growth.
Edible Vaccines
Transgenic Plants Serving Human Health Needs
• Works like any vaccine
• A transgenic plant with a pathogen protein gene is
developed
• Potato, banana, and tomato are targets
• Humans eat the plant
• The body produces antibodies against pathogen protein
• Humans are “immunized” against the pathogen
• Examples:
Diarrhea
Hepatitis B
Measles
Genetically modified crops
• Issues:
• Destroying ecosystems – tomatoes are now
growing in the artic tundra with fish antifreeze
in them!
• Destroying ecosystems – will the toxin now
being produced by pest-resistance stains kill
“friendly” insects such as butterflies.
• Altering nature – should we be swapping genes
between species?
Genetically modified crops
• Issues:
• Vegetarians – what about those
tomatoes?
• Religious dietary laws – anything from a
pig?
• Cross-pollination – producing a superweed
The End!
Any Questions?