signal transduction pathways. link cellular responses to plant hormonal signals environmental...
Post on 21-Dec-2015
221 views
TRANSCRIPT
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 signal-transduction pathways
Stimulus Hormones, physical environment, pathogensReceptor On the plasmamembrane, or internalSecondary messengers Ca2+, G-proteins, Inositol PhosphateEffector molecules Protein kinases or phosphatases Transcription factorsResponse Stomatal closure Change in growth direction
Signal Transduction Components
STIMULUS
R
R
Ca2+
Ca2+
G-prot
Kin
Phos
TF
Plasmamembrane
Nuclear membrane
DNA
Signal transductionSimplified model
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
We will deal with the best understood; PHYTOCHROMES
Light in Plants
Pr Pfr
The structure of Phytochrome
660 nm
730 nm
Binds to membrane
A dimer of a 1200 amino acid protein with several domains and 2
molecules of a chromophore. Chromophore
Signal Transduction of Phytochrome
PrPfr G
Ca2+/CaM cGMP
CAB, PS IIATPaseRubisco
FNRPS I
Cyt b/f
CHS
Chloroplast biogenesis Anthocyanin synthesis
Membrane
G protein subunit
Calmodulin
Guanylate cyclase Cyclic guanidine monophosphate
IV III II I
-252 -230 -159 -131 +1
Promoter has 4 sequence motifs which participate in light regulation.If unit 1 is placed upstream of any transgene, it becomes light regulated.
5’-CCTTATTCCACGTGGCCATCCGGTGGTGGCCGTCCCTCCAACCTAACCTCCCTTG-3’
bZIP Myb TranscriptionFactors
Unit 1
Light-Regulated Elements (LREs)
e.g. the promotor of chalcone synthase-first enzyme in anthocyanin synthesis
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.
Light-Regulated Elements (LREs)
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 processes1/ Control of seed development and germination2/ Stress responses of the mature plant
DROUGHT IN SALINITY A suite of stress response genes are turned on
COLD
CH3 CH3CH3
CH3
COOHOH
O
The signal transduction pathway is still poorly understood but certain common regulatory elements have been found in the promoters of ABA responsive genes.
Section of the upstream region of a barley ABA responsive geneCCGGCTGCCCGCCACGTACACGCCAAGCACCCGGTGCCATTGCCACCGG-104 -56
Minimal promoter
Reporter gene (GUS)
Promoter studies of ABA responsive elements in Barley
(Shen and Ho 1997)
ABA responsivenessGUS activity in the presence of ABA
related to no ABA
1x38x24x55x87x
ABA responsive elements
GCCACGTACANNNNNNNNNNNNNNNNNNNNTGCCACCGG--------
ACGCGTCCTCCCTACGTGGC-----------------------------------
Importance of pests and pathogensComplete v.s. partial resistance
Gene for gene theoryCloned resistance genes
A model of Xa21, blight resistance geneThe arms race explained
Plant Disease Resistance
Complete and Partial Resistance
There are two fundamentally different mechanisms of disease resistance.
Complete resistance
vertical resistanceHighly specific (race
specific)Involves evolutionary genetic interaction
(arms race)between host and one species of pathogen.
QUALITATIVE
Partial Resistance
horizontal resistanceNot specific- confers
resistance to a range of pathogens
QUANTITATIVE
Complete and Partial Resistance
There are two fundamentally different mechanisms of disease resistance.
Complete resistance
0
10
20
30
40
1 2 3 4 5 6 7 8 9 10
Disease severity class
Frequency %
0
5
10
15
20
25
30
1 2 3 4 5 6 7 8 9 10
Partial resistance
Disease severity class
Frequency %
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 avirulence 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.
ELICITORSElicitors are proteins made by the pathogen
avirulence genes, or the products of those proteins
Elicitors of VirusesCoat proteins, replicases, transport proteins
Elicitors of Bacteria40 cloned, 18-100 kDa in size
Elicitors of FungiSeveral now cloned- diverse and many unknown function
Elicitors of NematodesUnknown 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
Membrane anchor site
Serine/threonine proteinkinase domain
Signal peptide
Leucine-rich repeat
Transmembrane domain
Conserved motif
Leucine zipper domain
DNA binding site
C
C
C
C
C
N
N
N
N
N
Ptotomato; bacterial resistance
Xa21 rice; bacterial resistance
Hs1 sugar beet; nematode res.Cf9, Cf2 tomato; fungal resistance
L6 flax; fungal resistance
RPS2, RMP1 Arabidopsis; bac. res.N tomato; viral resistancePrf tomato; bacterial resitance
Protein structure ofcloned resistance genes
Kinase
Signal transduction([Ca2+], gene expression)M
embr
ane
Leucine-rich receptor
Elicitor
Transmembrane domain
Plant CellCell Wall
Model for the action of Xa21 (rice blight resistance gene)
The arms race explained
An avirulence genes mutates so that it’s product is no longer recognised by the host resistance gene.
It therefore becomes a virulence gene relative to the host, and the pathogen can infect.
The host resistance gene mutates to a version which can detect the elicitor produced by the new virulence gene.
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 may1. damage the pathogen2. strengthen the cell walls Oxidative3. 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
Local acquired resistance
Systemic acquired resistance
SAR- long-term resistance to a range of pathogens throughout plant caused by inoculation with inducer inoculum
Targets for crop improvementsGenetics of improvement
Molecular mappingMapping a qualitative trait
Marker assisted selection for aroma in riceMarker assisted selection for multiple resistant
genesMapping quantitative traits
QTLs and marker assisted selection
Marker Assisted Selection
Targets for ImprovementTargets for improvement in rice production fall into three categories
Biotic constraints- (pests and diseases)Weeds, Fungi (e.g. Blast), Bacteria (e.g. Blight), Viruses (e.g. Rice yellow mottle virus), Insects (e.g. Brown plant hopper), Nematodes (e.g. Cyst-knot nematode)
Abiotic constraints (adverse physical environment)Drought, Nutrient availability, Salinity Cold, Flooding
Yield and qualityPlant morphology, Photosynthetic efficiency, Nitrogen fixation, Carbon partitioning, Aroma
Genetics of improvement
Biotic constraints- Qualitative (complete resistance) Quantitative (partial resistance)
Abiotic constraints-Quantitative (mostly)
Yield and quality-Qualitative (aroma, partitioning)Quantitative (morphology, partitioning)Requires genetic engineering (photosynthesis, n. fixation)
Marker Assisted Selection
Useful when the gene(s) of interest is difficult to select for.
1. Recessive Genes
2. Multiple Genes for Disease Resistance
3. Quantitative traits
4. Large genotype x environment interaction
Molecular Maps
Molecular markers (especially RFLPs and SSRs) can be used to produce genetic maps because they represent an almost unlimited
number of alleles that can be followed in progeny of crosses.
R r
T t
or
Chromosomes with morphological marker alleles
RFLP1aRFLP2a
RFLP4a
RFLP3a
SSR1a
SSR2a
RFLP1b
RFLP2b
RFLP4b
RFLP3b
SSR1b
SSR2b
Chromosomes with molecular marker alleles
Molecular map of cross between rice varieties Azucena and Bala. Mapping population is an F6
51 cM
48 cM
54 cM
51 cM
54 cM
1 2 3 4 65
7 8 9 10 11 12
MOLECULAR MAPS CAN BE USED TO LOCATE GENES FOR USEFUL TRAITS (CHARACTERISTICS)
To locate useful genes on chromosomes by linkage mapping,
you need
1. A large mapping population (100 + individuals) derived from parental lines which differ in the characteristic or trait you are interested in.
2. Genotype the members of the population using molecular markers which are polymorphic between the parents (e.g. RFLPs, AFLPs, RAPDs)
3. Phenotype the members of the population for the trait making sure you asses each individual as accurately as possible
Azucena x Bala
F1 (self) 1 Individual
F2 F2 F2 F2 F2 (self) 205 individuals
F3 F3 F3 F3 F3 (self) 205 individuals
F4 F4 F4 F4 F4 (self) 205 individuals
F5 F5 F5 F5 F5 (self) 205 individuals
F6 F6 F6 F6 F6 205 families
Sin
gle
See
d D
ecen
tSeed multiplication
What is an F6 mapping population?
Making A Linkage Map Genotype No. of G320 RG2 C189 IndividualsA A A 47A A B 8A B A 5A B B 15B A A 19B B A 24B A B 3B B B 42 . Total 163
Recombinants between G320 and RG2 = 5 + 15 + 19 + 3 = 42 = 26%Recombinants between RG2 and C189 = 8 + 5 + 24 + 3 = 40 = 25%Recombinants between G320 and C189 = 8 + 15 + 19 + 24 = 66 = 40%
G1465
RG2
G44
G320
RZ141
R642
C189
Rice chromosome 11
Making a Linkage Map
A A AG320 RG2 C189
A A A
B B A
B B A 4785
1519243
42
Frequency of Genotype
Mapping a Qualitative Trait e.g. disease resistance
For a complete resistance gene, one parent is resistant, the other is susceptibleThe individuals in the segregating population are either resistant or susceptible.
0
10
20
30
40
50
60
% o
f In
div
idu
als
0 1 2 3 4 5 6 7 8 9Disease Severity Class
Segregation of disease resistance in population
G1465
RG2
G44
G320
RZ141
R64211
C189
Blast resistance gene0
20
40
60
80
100
Parents G320 RG2 C189
Genotype at RFLP
% o
f In
div
idu
als
No
t In
fec
ted
A
B
Mapping a Qualitative Trait
0%0%
80%87%37%
100%0%
100%
Disease resistant individuals for each genotype
Marker Assisted Selection for Aroma in Rice
The variety Azucena is aromatic (i.e. it smells pleasant and it’s seeds smell and taste pleasant)
Therefore Azucena rice fetches a higher price
The aroma gene is recessive. Therefore, it can’t be followed in backcross breeding.
The gene for aroma has been mapped to chromosome 8
Kalinga III is a popular variety in Eastern India but it is not aromatic.
The aroma gene of Azucena has been crossed into Kalinga III by selection for RFLPs linked to the aroma gene
Azu
cena
Kal
inga
III
F1 Sel
ecte
d B
C1
Non
-sel
ecte
d B
C1
Azu
cena
Kal
inga
III
F1 Sel
ecte
d B
C1
Non
-sel
ecte
d B
C1
Marker Assisted SelectionUsing molecular markers as selection criteria rather than the gene you want to transfer
R2676
G1073
Chromosome 8
Aroma gene flanked by G1073 and R2676
Marker Assisted Selection in Disease Resistance
Resistance genes can be selected for by screening with the disease. So, conventional breeding can produce resistant varieties.
But, resistance genes break-down. The disease organism mutates to overcome them (in 2-3 years).
If there were several resistance genes, the disease organism would take very much longer to overcome all resistance genes (in fact it is virtually impossible).
But, you can’t select for say 3 resistance genes conventionally- you can’t tell the difference between 1 gene and 2 or 3 by phenotype.
But if you select for markers linked to the resistance genes, you can introduce multiple resistance genes.
Marker Assisted Selection in Disease ResistanceS
elec
tabl
e m
arke
rs
Multiple crosses followed by backcrossingwith selection for markers at every stage
Elite variety with multiple resistance genes
Elite variety Donor1 Donor 2 Donor 3
300350400450500550600
Parents G320 RG2 C189
Genotype at RFLP
Ma
x. R
oo
t L
en
gth
(m
m)
A
B
Mapping a Quantitative Traite.g. rooting depth
G1465
RG2
G44
G320
RZ141
R64211
C189
Root length gene
0
10
20
30
40
% o
f In
div
idu
als
200 250 300 350 400 450 500 550 600 650 Max. Root Length Class (mm)
300350400450500550600
Parents G320 RG2 C189
Genotype at RFLP
Ma
x. R
oo
t L
en
gth
(m
m)
A
B
Mapping a Quantitative Traite.g. rooting depth
0
10
20
30
40
% o
f In
div
idu
als
200 250 300 350 400 450 500 550 600 650 Max. Root Length Class (mm)
Difference between parents is 360 mm
Difference between genotype classes at RG2 is 50 mm
This locus accounts for 16% of the difference
Quantitative trait loci (QTLs) and Marker Assisted Selection
QTLs (the location of a gene contributing to a quantitatively variable trait) are difficult to select for conventionally;it is very difficult to identify individuals with the QTL from those without because its effect is small.
Marker assisted selection can be used once markers at the QTL have been found.
Multiple QTLs can be combined for greater effect.
51 cM
48 cM
54 cM
51 cM
54 cM
1 2 3 4 65
7 8 9 10 11 12
Azucena QTLs targeted in the Marker Assisted Selection to improve the root system of Kallinga III
Genetic transformationsAgrobacterium transformations
Direct transfer methods for transformation Transformation cassettes
From transformed cells to plantsThe use of transformed plants in research
MutantsTransposon
Transposon and T-DNA tagging
Genetic Engineering
Genomic DNATi Plasmid(tumor inducing)
T-DNA(transfer)
Foreign DNAT-DNA(transfer)
Restrict and ligate together
Re-introduce recombinant DNA
Genetic Engineering of Plants- Agrobacterium transformation- The bacteria Agrobacterium tumefaciens causes galls or tumors on plants
Infect plant with recombinant agrobacterium
Whole T-DNA transferred randomly into plant chromosome
Grow up transformed plants from single cells
Agrobacterium transformation 2
All involve getting DNA directly across the plasma membrane
“GENETIC ENGINEERING” without AGROBACTERIUM
Shock of protoplasts
Micro-injection
Biolistics
Transformation constructs or cassettes
•Genes of interest•Promoter•Selectable (marker) gene
Gene of interest
Promotere.g. CauliflowerMosaic Virus 35S RNA gene promoter(CAM 35S)
Selectable marker-genee.g. antibiotic resistance or herbicide resistance
T-DNA T-DNA
Allows transgenic cells to be selected from non-transgenic
From transformed cells to plants
Plant cells are grown as a callus of undifferentiated cells on agar plates
transformation
After transformation, cells grown on selective media (e.g. containing antibiotic)
Untransformed cells die
selection
Transfer to tube with hormones
Cells containing transgenes grow
into plantlets
Transgenic plants as a research tool for non-genetic studiese.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+].
Aequorin
Tobacco
When transformed in to tobacco, this gene can be used to study the role of [Ca2+] in signal transduction
Transient increase in luminescence of tobacco plant challenged with fungal elicitor.Ca2+ involved in pathogen recognition
Lu
min
esce
nce
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.
•Transformation of tobacco (which lacks the enzyme) with the enzyme from Arabidopsis, increases fatty acid unsaturation.
Gro
wth
aft
er c
old
shoc
k re
lati
ve to
co
ntro
l
Untransformed
Transformed
-3fatty acid desaturase transformation confers cold tolerance, confirming that unsaturation is important.
Transgenic plants to identify gene function through over expressione.g. over-expression of antioxidant proteins
O2.-
H2O2
H2O MDHA Ascorbate
DHA
GSSG GSH
NADP+
NADPH
Superoxide Dismutase
Ascorbate peroxidase
Glutathione reductase
Dehydroascorbate reductase
The Halliwell-Asada pathway 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 expressione.g. over-expression of antioxidant proteins
Gene Construct Host Plant PhenotypeSuperoxide Dismutase Chloroplastic Tobacco No protection from MV or O3
Reduced MV damage and photoinhibitionReduced MV damage by no protection of photoinhibition
Tomato No protection from photoinhibitionPotato Reduced MV damageAlfalfa Reduced aciflurofen, freezing and drought damage
Mitochondrial Tobacco Reduced MV damage in the darkAlfalfa Reduced freezing and drought damage
Cytosolic Potato Reduced MV damage
Ascorbate Peroxidase Cytosoloc Tobacco Reduced MV damage and photoinhibition Chloroplastic Tobacco Reduced MV damage and photoinhibition
Glutathione Reductase E. coli in c.plast Tobacco Reduced MV and SO2 damage, not O3
Poplar Reduced photoinhibition E. coli in cytosol Tobacco Reduced MV damage
Pea Tobacco Reduced O3 damage, variable with MV
MV = methyl viologen = paraquat Allen et al. 1997
Po
lyga
lact
urin
ase
acti
vity
Time
Untransformed
Transgenic Plants to identifying gene function through gene repression
e.g. polygalacturinase and fruit ripening in tomato
Sense mRNA
Anti-sense mRNA
Sense and anti-sense mRNAs hybridise in
the cytoplasm and cause large
reductions in expression
•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.
Transformed
Result- tomatoes don’t soften so quickly- FLAVR SAVR TOMATO
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 geneCCGGCTGCCCGCCACGTACACGCCAAGCACCCGGTGCCATTGCCACCGG-104 -56
Minimal promoter
Reporter gene (GUS)
(Shen and Ho 1997)
ABA responsivenessGUS activity in the presence of ABA related to no ABA
1x38x24x55x87x
Mutants and Plant Genetics
DNA damage- X and Gamma rays, sodium azide (NaN3)
Transposons and T-DNA tagging
The Ac transposable element of maize
Cis-determinants for excision
11-bp inverted repeats
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
LigatePCR amplify using primers homologous to and facing out of insert