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Signal Transduction
Pathways
Signal Transduction Pathways
 link cellular responses to plant hormonal signals
environmental stimuli
 Binding of a hormone to a membrane receptor may
stimulate production of second messengers
 The activation of protein kinases, which in turn activate
other proteins is a common component of signal
transduction in plants
 Hormones may enter the cell to bind with a receptor,
and environmental stimuli can also trigger signaltransduction pathways
Signal Transduction Components
Stimulus
Hormones, physical environment, pathogens
Receptor
On the plasmamembrane, or internal
Secondary messengers
Ca2+, G-proteins, Inositol Phosphate
Effector molecules
Protein kinases or phosphatases
Transcription factors
Response
Stomatal closure
Change in growth direction
Signal transduction
Simplified model
STIMULUS
Ca2+
Plasma
membrane
R
Ca2+
Phos
Kin
Nuclear
membrane
R
TF
DNA
Light in Plants
We see visible light (350-700 nm)
Plants sense Ultra violet (280) to Infrared (800)
Examples
Seed germination - inhibited by light
Stem elongation- inhibited by light
Shade avoidance- mediated by far-red light
There are probably 4 photoreceptors in plants
PHYTOCHROMES
The structure of Phytochrome
A dimer of a 1200 amino acid protein with several domains and 2
molecules of a chromophore.
Chromophore
660 nm
730 nm
Pr
Pfr
Binds to membrane
• The two variations of the phytochrome are photoreversible
•The Pr to Pfr interconversion acts as a switch controlling the various events in
the life of a plant
Ecological Significance of Phytochrome as a
Photoreceptor
Phytochrome tells the plant that light is present by the conversion of Pr, which
is the form the plant synthesizes, to Pfr in the presence of sunlight
Pfr triggers the breaking of seed dormancy
The relative amounts of red and far-red light, is communicated to a plant by
the ratio of the two forms of phytochrome
The widespread response to the photoconversion of the phytochrome involves
signal-transduction pathways
 Phytochrome tells the plant that light is present by the conversion of Pr, which
is the form the plant synthesizes, to Pfr in the presence of sunlight
 Pfr triggers the breaking of seed dormancy
 The relative amounts of red and far-red light, is communicated to a plant by
the ratio of the two forms of phytochrome
 The widespread response to the photoconversion of the phytochrome
involves signal-transduction pathways
Photochromes may entrain the biological
clock
 In darkness, the Phytochrome ratio shifts towards Pr, because Pfr is converted
to Pr in some plants, and also because Pfr is degraded and new pigment is
synthesized as Pr
 Role of Phytochrome may be to synchronize the biological clock by signaling
when the sun sets and rises
Signal Transduction of Phytochrome
Membrane
Pfr
Ga
G protein a subunit
Pr
Guanylate cyclase
cGMP
Ca2+/CaM Calmodulin
CAB, PS II
ATPase
Rubisco
FNR
PS I
Cyt b/f
Chloroplast biogenesis
Cyclic
guanidine
monophosphate
CHS
Anthocyanin synthesis
Light-Regulated Elements (LREs)
The promotor of chalcone synthase-first enzyme in anthocyanin synthesis
Promoter has 4 sequence motifs which participate in light regulation.
If unit 1 is placed upstream of any transgene, it becomes light regulated.
-252
-230
IV
III
-159
II
-131
+1
I
Unit 1
5’-CCTTATTCCACGTGGCCATCCGGTGGTGGCCGTCCCTCCAACCTAACCTCCCTTG-3’
Transcription
Factors
bZIP
Myb
Light-Regulated Elements (LREs)
 There are at least 100 light responsive genes (e.g.
photosynthesis)
 There are many cis-acting, light responsive regulatory elements
 7 or 8 types have been identified of which the two for CHS are
examples
 No light regulated gene has just 1.
 Different elements in different combinations and contexts
control the level of transcription
 Trans-acting elements and post-transcriptional modifications
are also involved.
Plant Hormones
 Signal was a mobile substance, which was capable of transmitting through
a block of gelatin separating the tip from the rest of the coleoptile (Boysen –
Jensen)
 Chemical produced in the tip was promoting growth and was in higher
concentration on the side away from the light (Went)
 Chemical signals that coordinate the parts of the organism, and are
translocated through the body, where minute concentrations are able to
trigger responses in target cells and tissues → Plant hormone
Plant hormones help coordinate growth, development, and
responses to environmental stimuli
 Depending on the site of action, the developmental stage, and relative
hormone concentration, the effects of the hormone will vary
 effective in small concentrations
 They may act by affecting the expression of genes, the activity of enzymes,
or the properties of membranes
Signal-transduction pathways
link cellular responses
to plant hormonal
signals environmental stimuli
Binding of a hormone to a membrane receptor may stimulate production
of second messengers
The activation of protein kinases, which in turn activate other proteins is a
common component of signal transduction in plants
Hormones may enter the cell to bind with a receptor, and environmental
stimuli can also trigger signal-transduction pathways
Plant growth regulators and their impact on
plant development
Hormone
Response
(not a complete list)
Auxin
Abscission suppression; apical dominance; cell elongation;
fruit ripening; tropism; xylem differentiation
Cytokinin
Bud activation; cell division; fruit and embryo development;
prevents leaf senescence
Gibberellin
Stem elongation; pollen tube growth; dormancy breaking
Abscisic Acid
Initiation of dormancy; response to stress; stomatal closure
Ethylene
Fruit ripening and abscission; initiation of root hairs;
wounding responses
Abscisic Acid (ABA) responsive genes
ABA is involved in two distinct processes
1/ Control of seed development and germination
2/ Stress responses of the mature plant
DROUGHT
IN SALINITY
A suite of stress response genes are turned on
COLD
The signal transduction pathway
is still poorly understood but
certain common regulatory
elements have been found in the
promoters of ABA responsive
genes.
CH3
CH3
CH3
OH
O
CH3
COOH
Promoter studies of ABA responsive elements in Barley
Section of the upstream region of a barley ABA responsive gene
CCGGCTGCCCGCCACGTACACGCCAAGCACCCGGTGCCATTGCCACCGG
-104
-56
(Shen and Ho 1997)
Minimal
promoter
Reporter
gene (GUS)
ABA responsiveness
GUS activity in the presence of ABA
related to no ABA
1x
38x
24x
55x
87x
ABA responsive elements
GCCACGTACANNNNNNNNNNNNNNNNNNNNTGCCACCGG--------
ACGCGTCCTCCCTACGTGGC-----------------------------------
Plant Disease Resistance
Importance of pests and pathogens
Complete v.s. partial resistance
Gene for gene theory
Cloned resistance genes
A model of Xa21, blight resistance gene
The arms race explained
Complete and Partial Resistance
There are two fundamentally different
mechanisms of disease resistance.
Complete resistance
Partial Resistance
vertical resistance
Highly specific (race
specific)
Involves evolutionary
genetic interaction (arms
race)
between host and one
species of pathogen.
QUALITATIVE
horizontal resistance
Not specific- confers
resistance to a range of
pathogens
QUANTITATIVE
Complete and Partial Resistance
Complete resistance
Partial resistance
Frequency %
Frequency %
40
30
25
30
20
20
15
10
10
5
0
0
1
2
3
4
5
6
7
8
Disease severity class
9
10
1
2
3
4
5
6
7
8
Disease severity class
9
10
Gene-for-Gene theory of Complete
Resistance
Pathogen has
virulence (a)
and avirulence
(A) genes
A
a
Plant has resistance gene
RR
rr
If the pathogen has an Avirulence gene and the host a Resistance gene,
then there is no infection
Gene-for-Gene theory of Complete
Resistance
The Avirulence gene codes for an Elicitor molecule or protein controlling
the synthesis of an elicitor.
The Resistance gene codes for a receptor molecule which ‘recognises’
the Elicitor.
A plant with the Resistance gene can detect the pathogen with the
Avirulence gene.
Once the pathogen has been detected, the plant responds to destroy the
pathogen.
Both the Resistance gene and the Avirulence gene are dominant
Gene-for-Gene theory of Complete
Resistance
What is an elicitor?
It is a molecule which induces any plant defence response.
It can be a polypeptide coded for by the pathogen a-virulence gene, a cell wall
breakdown product or low-molecular weight metabolites.
Not all elicitors are associated with gene-for-gene interactions.
What do the Avirulence genes (avr genes) code for?
They are very diverse!
In bacteria, they seem to code for cytoplasmic enzymes involved in the synthesis of
secreted elicitor. In fungi, some code for secreted proteins, some for fungal toxins.
ELICITORS
proteins made by the pathogen a-virulence genes, or
the products of those proteins
Elicitors of Viruses
Coat proteins, replicases, transport proteins
Elicitors of Bacteria
40 cloned, 18-100 kDa in size
Elicitors of Fungi
Several now cloned- diverse and many unknown function
Elicitors of Nematodes
Unknown number and function
Gene-for-Gene theory of Complete
Resistance
What does a resistance gene code for?
The receptor for the specific elicitor associated with the interacting avr gene
Protein structure of
cloned resistance genes N
C
Pto
tomato; bacterial resistance
N
C
Xa21
rice; bacterial resistance
N
C
Hs1 sugar beet; nematode resistance.
Cf9, Cf2 tomato; fungal resistance
N
C
L6 flax; fungal resistance
C
RPS2, RMP1 Arabidopsis; bacterial res.
N tomato; viral resistance
Prf tomato; bacterial resistance
N
Membrane anchor site
Trans-membrane domain
Serine/threonine protein
kinase domain
Conserved motif
Signal peptide
Leucine zipper domain
Leucine-rich repeat
DNA binding site
Model for the action of Xa21
(rice blight resistance gene)
Leucine-rich receptor
Transmembrane domain
Elicitor
Cell Wall
Membrane
Kinase
Signal transduction
([Ca2+], gene expression)
Plant Cell
The arms race explained
An avirulence genes
mutates so that it’s
product is no longer
recognised by the host
resistance gene.
The host resistance gene
mutates to a version
which can detect the
elicitor produced by the
new virulence gene.
It therefore
becomes a
virulence gene
relative to the host,
and the pathogen
can infect.
Hypersensitive Reaction/ Programmed Cell Death
In response to signals, evidence suggests that infected cells
produce large quantities of extra-cellular superoxide and hydrogen
peroxide which may
1. damage the pathogen
2. strengthen the cell walls
Oxidative
3. trigger/cause host cell death
Burst
Evidence is accumulating that host cell also undergo changes in
gene expression which lead to cell death
Programmed Cell Death
Systemic Acquired Resistance
Inducer inoculation
3 days to months,
then inoculate
SAR- long-term resistance to a range of
pathogens throughout plant caused by
inoculation with inducer inoculum
Local
acquired
resistance
Systemic
acquired
resistance
Transgenic plants as a research tool for non-genetic studies
e.g. aequorin transformed plants to study calcium’s role as secondary messenger
The aequorin gene from a luminescent jellyfish produces a protein aequorin.
When combined with a small chromophore, coelentrazine, the complex gives
off blue light at a rate dependent on [Ca2+].
When transformed in to tobacco, this
gene can be used to study the role of
[Ca2+] in signal transduction
Tobacco
Transient increase in
luminescence of
tobacco plant
challenged with
fungal elicitor.
Ca2+ involved in
pathogen recognition
Luminescence
Aequorin
Time
Knight et al. 1991
Transgenic plants to identifying gene function through novel
expression eg -3fatty acid desaturase from Arabidopsis in tobacco
•-3fatty acid desaturase converts 16:2 and 18:2 dienoic fatty acids to 16:3 and
18:3 trienoic acids.
•A greater degree of fatty acid unsaturation (especially in the chloroplast) was
thought to confer greater resistance to cold in plants.
Growth after cold
shock relative to
control
•Transformation of tobacco (which lacks the enzyme) with the enzyme from
Arabidopsis, increases fatty acid unsaturation.
Untransformed
Transformed
-3fatty acid desaturase
transformation confers cold
tolerance, confirming that
unsaturation is important.
Transgenic plants to identify gene function through over expression
e.g. over-expression of antioxidant proteins
The Halliwell-Asada pathway
O2.-
Superoxide Dismutase
H2O2
Ascorbate peroxidase
H2O
MDHA
Ascorbate
DHA
Dehydroascorbate
reductase
GSSG
GSH
NADP+
Glutathione reductase
NADPH
The Halliwell-Asada
pathway is important in
detoxifying reactive oxygen
intermediates. These are
produced naturally by the
electron-transport chains of
mitochondria and especially
chloroplasts. Most stresses
cause increases in
superoxide or hydrogen
peroxide production.
Transgenic experiments
have investigated the
importance of these
enzymes in stress
resistance.
Transgenic plants to identify gene function through over
expression
e.g. over-expression of antioxidant proteins
Gene Construct
Host
Superoxide Dismutase
Chloroplastic
Tobacco
Mitochondrial
Cytosolic
Tomato
Potato
Alfalfa
Tobacco
Alfalfa
Potato
Plant Phenotype
No protection from MV or O3
Reduced MV damage and photoinhibition
Reduced MV damage by no protection of photoinhibition
No protection from photoinhibition
Reduced MV damage
Reduced aciflurofen, freezing and drought damage
Reduced MV damage in the dark
Reduced freezing and drought damage
Reduced MV damage
Ascorbate Peroxidase
Cytosoloc
Tobacco
Chloroplastic
Tobacco
Reduced MV damage and photoinhibition
Reduced MV damage and photoinhibition
Glutathione Reductase
E. coli in c.plast Tobacco
Poplar
E. coli in cytosol Tobacco
Reduced MV and SO2 damage, not O3
Reduced photoinhibition
Reduced MV damage
Pea
Tobacco
Reduced O3 damage, variable with MV
MV = methyl viologen = paraquat
Allen et al. 1997
Transgenic Plants to identifying gene function through gene
repression
e.g. polygalacturinase and fruit ripening in tomato
•Polygalacturinase breaks down cell walls.
•It’s expression is considerably enhanced in ripening fruit (it makes the fruit soft).
•Transformation of tomatoes with the anti-sense version (the gene in the opposite
direction), reduces the expression of polygalacturinase.
Sense and anti-sense
mRNAs hybridise in
the cytoplasm and
cause large
Anti-sense mRNA
reductions in
expression
Sense mRNA
Polygalacturinase
activity
Result- tomatoes don’t soften so quickly- FLAVR SAVR TOMATO
Untransformed
Transformed
Time
Transgenic plants to study of promoter function through reporter
gene studies
e.g. ABA responsive promoter from barley
Section of the upstream region of a barley ABA responsive gene
CCGGCTGCCCGCCACGTACACGCCAAGCACCCGGTGCCATTGCCACCGG
-104
-56
(Shen and Ho 1997)
Minimal
promoter
Reporter
gene (GUS)
ABA responsiveness
GUS activity in the presence of ABA related to no ABA
1x
38x
24x
55x
87x
Mutants and Plant Genetics
DNA damage- X and Gamma rays, sodium azide (NaN3)
Transposons and T-DNA tagging
The Ac transposable element of maize
11-bp inverted
repeats
Cis-determinants
for excision
Exons of
transposase gene
Introns
A transposon can move at random throughout a plant genome. It is
cut out of its site and reinserted into another site by the action of
an endonuclease and the transposase.
Insertion into a functional gene causes mutation.
Transposons and T-DNA tagging
Transposons have only been found in a few plants (e.g. Maize,
Antirrhium). But, they can be introduced by transformation. The Ac
transposon has been introduced to tobacco, Arabidopsis, potato,
tomato, bean and rice.
Mutations using transposons or T-DNA (both of which insert
randomly into nuclear DNA) are produced by transformation
methods described earlier. Large numbers of plants are screened for
an observable phenotype (e.g. lack of response to light).
Screen
Identify mutated
gene
Transposons and T-DNA tagging
The gene into which the insert has occurred can be recovered by PCR
Mutated ORF
Insertion (Transpososn or T-DNA)
Restrict
Ligate
PCR amplify using primers
homologous to and facing out of
insert