Gene regulation and expression in plants- overviewPlant development and the environment

Signal transduction- a general viewRegulation of plant genes and transcription factors

Light regulation in plants and Phytochrome Light regulated elementsPlant growth regulators

Abscisic acid (ABA) and ABA-responsive genes

Gene Regulation

Gene Regulation

Regulation of gene expression in plants is essentially the same as in animals.

But, the hormones (plant growth regulators) in plants are very different. Also, plant development is profoundly influenced by environment.

Therefore, this lecture deals with gene regulation in response to1/ light,2/ plant growth regulators using absisic acid as an example

Plants undergo chronological changes in morphology and therefore require developmentally regulated gene expression.

Plants have organs (not so many as higher animals) and therefore require organ specific gene expression.

Plants cannot move their entire body and must therefore respond to changes in the environment. They therefore require environmentally regulated gene expression.

Plants and gene expression

Plant Development and the EnvironmentENVIRONMENTAL STIMULI

Light- intensity, direction, durationGravityTouch

TemperatureWater

Pathogen infection

COMPONENTS OF ENVIROMENTALLY

REGULATED BEHAVOIUR

PerceptionSignal Transduction

Response

RESPONSES TO THE ENVIRONMENT

Nastic ResponsesTropic Responses

Morphogenic ResponsesLocalised Cellular ResponsesSystemic Cellular Responses

Nastic Responses- (greek nastos = ‘pressed closed’) typically a non-growth response that is not orientated with regard to the stimulus e.g. closing flower at night, stomatal closure.

Tropic Responses- (greek trope = ‘turn’)typically a growth response that is orientated with regard to the stimulus e.g. gravitropism, phototropism.

Morphogenic Responses- a response which results in fundamental change in plant metabolism or form e.g. photomorphogenesis.

Localised Cellular Responses- small scale changes in cell metabolism e.g. hypersensitive response to pathogens.

Systemic Cellular Responses- whole plant changes in cell metabolism e.g. systematic acquired resistance

Plant Development and the Environment

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

Regulation of a Plant Gene

I I

TCS (transcription start site ATG)

TATA boxCAAT/AGGA boxDistal elements

and enhancers

-1000 -74 bp -54 to -50bp to -16 bp

Polyadenylationsignal

I

Promoter

Transcribed untranslated regions

Coding sequence (exons)

Introns

Stop codonTAA,TAG,TGA

Regulation of transcription:Transcription factors Bind to RNA

polymerase and effect the rate of transcription

TATA box Coding region

Transcription initiation

RNA Polymerase

Transcription factors

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

bZIPMyb

?

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 ABA1x38x24x55x87x

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

Product67%

Pathogens 12%

Pests 11%

Weeds 10%

Where does our food go?

The proportion of total production lost due to biotic constraints

1967

Product58%

Weeds 13%

Pathogens 13%

Pests 16%

1988-90

We are engaged in a continuous struggle to control weeds, pests and diseases

Some important pest and pathogens of plants

PathogensFungiBacteriaViruses

PestsNematodesInsectsVertebrates (not fish!)

Specific moleculardefense mechanisms

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 %

05

1015202530

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 explainedAn 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

Similarity with animals1. Resistance/avirulence gene interaction

is analogous to animal antibodies- involves protein-protein binding is highly specific

2. Oxidative burst is analogous to neutrophil action

3. Programmed cell death is common to both plants and animals

4. Systemic acquired resistance is like immunity

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 RFLP1a

RFLP2a

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

RG2G44

G320RZ141

R642

C189

Rice chromosome 11

Making a Linkage MapA A AG320 RG2 C189A A A

B B A

B B A 4785

151924

342

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.

0102030405060

% o

f Ind

ivid

uals

0 1 2 3 4 5 6 7 8 9Disease Severity Class

Segregation of disease resistance in population

G1465

RG2

G44

G320RZ141

R64211

C189

Blast resistance gene0

20

40

60

80

100

Parents G320 RG2 C189

Genotype at RFLP

% o

f Ind

ivid

uals

Not

In

fect

ed AB

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

F 1 Sele

cted

BC

1

Non

-sel

ecte

d B

C1

Azu

cena

Kal

inga

III

F 1 Sele

cted

BC

1

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 ResistanceSe

lect

able

mar

kers

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

Max

. Roo

t Len

gth

(mm

)

A

B

Mapping a Quantitative Traite.g. rooting depth

G1465

RG2

G44

G320RZ141

R64211

C189

Root length gene

0

10

20

30

40

% o

f Ind

ivid

uals

200 250 300 350 400 450 500 550 600 650 Max. Root Length Class (mm)

300350400450500550600

Parents G320 RG2 C189

Genotype at RFLP

Max

. Roo

t Len

gth

(mm

)

A

B

Mapping a Quantitative Traite.g. rooting depth

0

10

20

30

40

% o

f Ind

ivid

uals

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

Lum

ines

cenc

e

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

afte

r col

d sh

ock

rela

tive

to

cont

rol

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

activ

ityTime

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 barleySection 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