 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


Hormones, physical environment, pathogens

On the plasmamembrane, or internal

Ca 2+ , G-proteins, Inositol Phosphate

Protein kinases or phosphatases
Transcription factors

Stomatal closure
Change in growth direction

2+
2+
Kin
Plasma membrane
Phos
Nuclear membrane
TF
DNA

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

A dimer of a 1200 amino acid protein with several domains and 2 molecules of a chromophore.
Chromophore
660 nm
730 nm
Pr Pfr

• The two variations of the phytochrome are photoreversible
•The P r to P fr 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 P is the form the plant synthesizes, to P fr r
, which in the presence of sunlight
 P fr 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 P is the form the plant synthesizes, to P fr r
, which in the presence of sunlight
 P fr 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
 In darkness, the Phytochrome ratio shifts towards P r to P r in some plants, and also because P fr synthesized as P r
, because P fr is converted is degraded and new pigment is
 Role of Phytochrome may be to synchronize the biological clock by signaling when the sun sets and rises

Membrane
Pr
Pfr
Ca 2+ /CaM
Calmodulin
G a
G protein a subunit
Guanylate cyclase
Cyclic guanidine cGMP monophosphate
CAB, PS II
ATPase
Rubisco
FNR
PS I
Cyt b/f
CHS

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 -159 -131 +1
IV III II I
Unit 1
5’-CCTTATTCCACGTGGCCATCCGGTGGTGGCCGTCCCTCCAACCTAACCTCCCTTG-3’ bZIP
Myb
Transcription
Factors

 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.

 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

 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
(not a complete list)
Auxin
Cytokinin
Gibberellin
Abscisic Acid
Ethylene
Response
Abscission suppression; apical dominance; cell elongation; fruit ripening; tropism; xylem differentiation
Bud activation; cell division; fruit and embryo development; prevents leaf senescence
Stem elongation; pollen tube growth; dormancy breaking
Initiation of dormancy; response to stress; stomatal closure
Fruit ripening and abscission; initiation of root hairs; wounding responses

ABA is involved in two distinct processes
1/ Control of seed development and germination
2/ Stress responses of the mature plant
DROUGHT
IN SALINITY
COLD
A suite of stress response genes are turned on
The signal transduction pathway is still poorly understood but certain common regulatory elements have been found in the promoters of ABA responsive genes.
CH
3
CH
3
CH
3
O
OH
CH
3
COOH

Promoter studies of ABA responsive elements in Barley
Section of the upstream region of a barley ABA responsive gene
CCGGCTGCCCGCCACGTACACGCCAAGCACCCGGTGCCATTGCCACCGG
-104 -56
Minimal promoter
(Shen and Ho 1997)
Reporter gene (GUS)
ABA responsiveness
GUS activity in the presence of ABA related to no ABA
1x
38x
24x
55x
87x

GCC ACGT ACANNNNNNNNNNNNNNNNNNNNTGCCACCGG--------
ACGCGTCCTC CCT ACGT GGC -----------------------------------

 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 resistance vertical resistance
Highly specific (race
specific)
Involves evolutionary genetic interaction (arms race) between host and one species of pathogen.
QUALITATIVE
Partial Resistance horizontal resistance
Not specific- confers resistance to a range of pathogens
QUANTITATIVE

Complete resistance Partial resistance
Frequency %
20
10
0
40
30
1 2 3 4 5 6 7 8 9 10
Disease severity class
Frequency %
30
25
20
15
10
5
0
1 2 3 4 5 6 7 8 9 10
Disease severity class

Plant has resistance gene Pathogen has virulence (a) and avirulence
(A) genes
A a
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.

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
N
N
N
N
Membrane anchor site
Serine/threonine protein kinase domain
Signal peptide
Leucine-rich repeat
C Pto tomato; bacterial resistance
C Xa21 rice; bacterial resistance
C
Hs1 sugar beet; nematode resistance.
Cf9, Cf2 tomato; fungal resistance
L6 flax; fungal resistance
C
C
RPS2, RMP1 Arabidopsis; bacterial res.
N tomato; viral resistance
Prf tomato; bacterial resistance
Trans-membrane domain
Conserved motif
Leucine zipper domain
DNA binding site

Leucine-rich receptor
Transmembrane domain
Kinase
Elicitor
Cell Wall
Signal transduction
([Ca 2+ ], gene expression)
Plant Cell

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
3. trigger/cause host cell death
Oxidative
Burst
Evidence is accumulating that host cell also undergo changes in gene expression which lead to cell death

Inducer inoculation
Local acquired resistance
3 days to months, then inoculate
SAR- long-term resistance to a range of pathogens throughout plant caused by inoculation with inducer inoculum
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 [Ca 2+ ].
When transformed in to tobacco, this gene can be used to study the role of
[Ca 2+ ] in signal transduction
Aequorin
Transient increase in luminescence of tobacco plant challenged with fungal elicitor.
Ca 2+ involved in pathogen recognition
Time
Tobacco
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.
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
O
2
.-
H
2
O
2
Superoxide Dismutase
Ascorbate peroxidase
H
2
O
MDHA Ascorbate
DHA
Dehydroascorbate reductase
GSSG GSH
Glutathione reductase NADP +
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.
NADPH

Transgenic plants to identify gene function through over expression e.g. over-expression of antioxidant proteins
Gene Construct Host
Superoxide Dismutase
Plant Phenotype
Chloroplastic Tobacco No protection from MV or O
3
Reduced MV damage and photoinhibition
Reduced MV damage by no protection of photoinhibition
Tomato No protection from photoinhibition
Potato Reduced MV damage
Alfalfa Reduced aciflurofen, freezing and drought damage
Mitochondrial
Cytosolic
Tobacco Reduced MV damage in the dark
Alfalfa Reduced freezing and drought damage
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 SO
2 damage, not O
Poplar Reduced photoinhibition
3
E. coli in cytosol Tobacco Reduced MV damage
Pea Tobacco Reduced O
3 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.
Result- tomatoes don’t soften so quickly- FLAVR SAVR TOMATO
Untransformed Sense mRNA Sense and anti-sense mRNAs hybridise in the cytoplasm and cause large
Anti-sense mRNA reductions in expression
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 (NaN
3
)
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