Download Ethylene and Sub1

Ethylene
A gaseous growth regulator
What does signalling mean in biology?
‘nongenomic’,
‘cellular’
‘nongenomic’,
‘cellular’
‘genomic’
‘Growth inhibiting’ and stress regulators
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Ethylene (C2H2)
Abscissic acid (ABA)
Jasmonate (JA)
Salicylic acid (SA)
‘Growth promoting’ regulators
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Auxin (IAA)
Cytokinin (CK)
Gibberellins (GA)
Brassinolide
Ethylene
H
H
C
C
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H
Ethylene has many Biological effects
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Inhibition of root and hypocotyl elongation
Formation of hypocotyl hook
Floral induction
Anther development and dehiscence
Senescence
Abscission
Fruit ripening
Pathogen response
Abiotic stresses
modulation of carbohydrate metabolism
Ethylene acts over a wide concentration range
• The lowest level of ethylene causing an effect on seedling
growth is 0.001 ppm (1nl/L)
• During fruit ripening 100ppm (100µl/L) are produced
• Some genes respond to ethylene between 0.1 and 1000 ppm
• This wide ‘detection range’ requires a highly sensitive and
adaptive signalling system.
• This requires molecular mechanisms of signal amplification and
attenuation.
Genetic dissection of signalling
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Devise mutant screen
Characterize the mutant
Clone the gene
Place gene in interaction networks (genetic, genomic,
biochemical, cellular)
• Integrate genotype phenotype relations
The triple response provides a simple assay for C2H4 response
•shorter hypocotyl
•fatter hypocotyl
•exaggerated apical hook
Mutants defective in the triple response provided the raw material
to study the mechanism of ethylene signalling
etr1
Pathway for ethylene signal transduction based on genetic analysis
ETR: ethylene response
ERS: ethylene responsive sensor
EIN: ethylene insensitive
CTR: constitutive triple response
EIL: EIN3-like
ERF1: ethylene response factor
ETO: ethylene over-producer
HLS: hookless
Genetic analysis: Interpretation of double
mutants
• A ‘genetic pathway’ is based on the interactions between gene
products that are inferred from the phenotype of mutant combinations
• If the double mutant ab looks exactly like the single mutant b, b is said
to suppress a.
• If locus b suppresses a, B is called epistatic to A. Epistasis suggests
that A and B act in the same pathway, B acting downstream of B.
• If a b looks like a combination A and B are thought to act in separate
pathways.
• Genetic analysis does not provide information on the biochemical
mechanism but contributes to the formation of hypotheses.
EIN2 and several transcription factors (TFs) act
downstream the kinase cascade
• EIN2 is a key positive regulator of ethylene, CK
and ABA signalling homologous to metal-ion
transporters.
• EIN3 and EILs activate the transcription of
ethylene response factor (ERF1). Rapid ubiquitindependent proteolysis of EIN3 is slowed down at
higher ethylene levels.
• ERF1 and related TFs regulate ethylene
responsive genes.
• Kinase cascade and transcriptional cascade
amplify low signals.
• Increased receptor induction attenuates high
signals (receptors are negative regulators).
The proteasome and ubiquitin
• Ubiquitin tagged proteins are targeted for proteolysis
• The proteasome constitutes 1% of total protein
• Proteins are degraded at the expense of ATP
• This is another example how a lot a energy is ‘sacrificed’ to achieve a
high level of control
Ubiquitin dependent protein degradation
The SCF ligase complex in protein degradation
•The F-box component determines substrate specificity.
•The F-box proteins EBF1 and -2 recruit ubiquitinated EIN3 into the
SCFEBF1/EBF2 complex leading to its contiuous destruction in the absence
of ethylene.
•Ethylene prevent EIN3 ubiquitination by activating EIN2 and ubiquitin
dependent destruction of EBF1/2.
Summary ethylene
• Ethylene controls many processes related to stress and “ageing”
• Ethylene binds to ER localized receptors (ETR1 + four more)
• This suppresses the cytoplasmic negative regulator CTR, on top
of a MAP kinase cascade.
• EIN2 is an important positive regulator of unknown activity.
• EIN3 is the key transcription factor that activates ethylene
responsive transcription factors (ERFs).
• EIN3 is usually recruited for degradation by the SCFEBF complex.
• Ethylene signalling prevents EIN3 degradation thereby allowing it
to activate transcription.
• The protein EBF1 that marks out EIN3 for ubiquitin dependent
destruction is also controlled by ubiquitin dependent destruction.
Flooding
• after 1 week of submergence most rice cultivars die
• a major constraint to cultivation of rice in South Asia and
Southeast Asia
• ca $1.000.000.000/a lost due to flooding
Flooding response: Hypoxia
• hypoxic (cellular O2 deficiency) and anoxic (negligible
respiratory vs. fermentative ATP production) conditions
• morphological adaptations: adv. roots, aerenchyma,
internodal stem elongation [rice], lenticels,
pneumatophores, mitochondrial morphology
• physiological adaptations: slower metabolism and
growth
Aerenchyma
Lenticels and pneumatophores
Mitochondrial changes
aerobic
anaerobic
Metabolic adaptations to anoxia
self-disposal
survival
Growth, reproduction
• Hypoxia generates two important metabolic bottlenecks:
• Because O2 is lacking for respiration:
• ATP production is too low
• NADH/NAD+ is too high
• During periods of anoxic stress, growth needs to be precisely
controlled to ensure survival!
Paper Xu et al
Nature 2006 442:705ff
The Sub1 locus
tolerant
intolerant
Presence of the strongly ethylene-responsive allele
Sub1A-1 correlates with tolerance
Some tolerant varieties from geographically distant regions
share identical Sub1 haplotype - this indicates that this trait
was introduced by transporting tolerant varieties over
1000km and subsequently introgressed.
Transformation with Sub1A-1 of intolerant varieties
makes them tolerant and shorter
Introgression of Sub1 from tolerant haplotype into intolerant
variety by marker assisted selection confers submergence
tolerance
How does Sub1 work?
A simple tool
The Sub1 locus, introgressed from a tolerant into an intolerant background,
confers tolerance.
A simple tool
• While the intolerant plant elongates, the tolerant plants stops elongating
to protect its resources.
• Expansins are less strongly induced in the tolerant genotype.
A simple tool
• In the tolerant plant expression of glycolytic enzymes is prolonged.
• This could protect the plant from accumulation of toxic acetaldehyde and
maintain energy supply for a longer period.
A simple tool
•The Sub1 locus, makes the plant more economic with storage
carbohydrates.
•Degrading enzymes are less rapidly induced in the tolerant genotype.
In response to ethylene, Sub1 genes regulate transcription of
important enzymes
Submergence tolerant rice
• tolerant cultivars can survive complete submergence
10-14d
• crossed: tolerant FR13A x intolerant M-202 to
produce mapping population
• map Sub1 locus to 0.06cM (182kb) region
• locus contains cluster of 2 or 3 ethylene response
factors (ERFs) and 10 more putative genes
• ERFs respond to submergence
Key authors for Sub1
• Dave Mackill: Intl. Rice Research Inst. Manila, marker
assisted breeding
• Julia Bailey-Serres: UC Riverside, hypoxic stress
biology
• Pam Ronald: UC Davies, rice genetics, signal
transduction
Authors