Transcript Ethylene 2013

Caren Chang
[email protected]
Lab members enjoy finishing an experiment
The plant hormone ethylene
1. What does ethylene do?
2. Is ethylene important?
Ethylene is a GAS!!!
3. How can we study ethylene and use
that knowledge to benefit humans?
Plants synthesize ethylene in response to stress
Wounding
Heat stress
Drought stress
Cold stress
Osmotic stress
Mechanical stress
UV stress
Pathogen attack
Biotic stress
Flooding
ETHYLENE is also a
pollutant in the
environment
Ethylene responses
Developmental processes
Responses to abiotic and biotic stress
Fruit ripening - ethylene is essential
Promotion of seed germination
Root initiation
Bud dormancy release
Inhibition/promotion of flowering
Sex shifts in flowers
Senescence of leaves, flowers
Abscission of leaves, flowers, fruits
Epinasty of leaves
Inhibition/promotion of cell division/elongation
Altered geotropism in roots, stems
Induction of phytoalexins/disease resistance
Aerenchyma formation
Historical background
• Ethylene has been used (unwittingly)
throughout history
Gashing promotes ripening
in figs (4 days later)
Wood burning fires
promote synchronous
flowering in pineapple
Historical background
• 1800s Illuminating gas caused detrimental
effects
Historical background
• 1901 Neljubov discovered that ethylene is the
biologically active agent in illuminating gas, which
was used to heat the greenhouse
Wounding induces
ethylene production
Can block ethylene response
using silver thiosulfate
Ethylene causes senescence
Apple slices inducing ripening of persimmons
8 days in
bag with
apple
slices
Controls,
8 days
outside
of bag
Ethylene has far-reaching consequences for agriculture and
horticulture
Transport and storage of
fruits and vegetables
requires ethylene control
Flood-tolerant rice created by
expression of ethylene response
factor genes
“One bad apple spoils
the whole bunch…”
Therefore, we would like to manipulate the
biosynthesis and/or responses to ethylene
Removal of external ethylene
Global rice production increases are needed
to meet demand by 2035
Million tons milled rice
600
Additional rice needed:
114 million tons by 2035
550
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400
2010 global rice production
350
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Asia
Africa
Americas
Rest of world
Ethylene, rice, and feeding billions
• Half the world's population eats rice as a
staple. In Asia, about 3 billion people depend
on rice to survive. The demand for food is
increasing as the population increases.
Rice is two-thirds of the diet
of subsistence farmers in
India and Bangladesh. When
rice crops suffer, millions
starve (e.g., the great floods
of 1974).
The problem
• A quarter of the world's rice grows in
areas prone to flooding.
• Rice plants normally grow well in
standing water. However, most will die if
they are completely underwater for more
than 5-7 days, due to the lack of oxygen,
carbon dioxide and sunlight.
• Annual flooding costs rice farmers in
South and South-East Asia more than $1
billion dollars (U.S. equivalent) each year
in addition to reducing the food supply!
Solution: Nature has already designed
two types of flood-tolerant rice
a. Escape strategy:
There are deepwater rice cultivars
that have evolved and adapted to
long-term flooding by acquiring the
ability to elongate their internodes,
which have hollow structures and
function as “snorkels” to allow gas
exchange with the atmosphere, and
thus prevent drowning.
internode
Deepwater conditions. Plants were submerged in water up to 70%
of the plant height, and the water level was then increased by 10 cm
every day until the tank was full.
Tank is filled to top
Complete submergence. The tank was completely filled
with water on the first day of the treatment.
This elongated
deepwater rice
plant in Thailand
was preserved
after flooding
occurred and
shows the typical
flooding height.
White bar = 1
meter.
http://www.nature.com/nature/jo
urnal/v460/n7258/suppinfo/natur
e08258.html
b. Quiescent strategy:
A few rice cultivars, known as submergence
tolerant lowland rice, have adapted to areas
where flash flooding is common by learning how
to “hold their breath”. These cultivars can
survive under water for up to 2 weeks.
These cultivars do NOT use elongation as an
escape strategy. Instead, they become
quiescent and stay submerged, conserving
energy so that they can produce new leaves
when the flooding subsides. For example, they
increase anaerobic respiration.
Long-term flooding vs. flash flooding
WHAT GENES ARE RESPONSIBLE? Discovery of the
SNORKEL genes
Water
level
- Taichung65 (T65) is a non-deepwater rice
- C9285 is a deepwater rice
- NIL-12 is the progeny of a cross that transferred the key
portion of chromosome 12 into T65
The researchers found that the SNORKEL genes belong
to the ERF (Ethylene Response Factor) type of
transcription factors, which are induced by ethylene.
Deepwater rice
Floodin
g
SNORKEL1 & 2
proteins
Transcriptional
response
The researchers found that the SNORKEL genes belong
to the ERF (Ethylene Response Factor) type of
transcription factors, which are induced by ethylene.
Deepwater rice
Floodin
g
SNORKEL1 & 2
Transcriptional
response
Non-deepwater rice
Floodin Non-deepwater
g
rice does not
have these
genes!
No transcriptional
response
Localization of SNORKEL proteins to the plant nucleus
using “protein fusions” to GFP
Yoko Hattori et al. (2009) Nature 460, 1026-1030
SUBMERGENCE1 GENE (SUB1) – Quiescent strategy
•
Identified and cloned in 2006. Like the SNORKEL genes,
it is also an ethylene response transcription factor (ERF)
• When plants are under water, ethylene accumulates in the
plant. The ethylene then induces expression of these ERF
genes. SNORKEL1 and SNORKEL2 trigger remarkable
internode elongation via the hormone gibberellin. In
contrast, SUB1A inhibits internode elongation.
Transcription factors turn on specific genes
Functions of Gibberellic Acid
•
•
•
•
•
•
•
•
Cell enlargement and cell
divisions in sub-apical
meristems
Growth in stems, fruits, and
leaves
Stem and leaf expansion
Fruit development and
expansion
Stimulation of flowering
Cell divisions in some tissues
Dormancy and senescence
Seed germination
Solving the problem
• These deepwater varieties have low grain yield,
unlike the high-yield varieties that are used for
food.
• So these genes are being genetically crossed into
the high-yield cultivars.
• These “engineered” strains will be able to resist
floods that destroy vast tracts of rice fields each
year, preventing starvation and offering hope to
hundreds of millions of people who make their living
from rice farming.
An actual field trial of the Sub1A gene in rice
New Sub1 lines after 17 days
submergence in the field at IRRI
IR64-Sub1
Samba-Sub1
Samba
IR49830 (Sub1)
Samba
IR64
IR42
IR42
IR64
IR49830 (Sub1)
IR49830 (Sub1)
IR64-Sub1
IR64
Samba
Samba-Sub1
IR64-Sub1
IR42
IR49830 (Sub1)
IR42
IR64-Sub1
Samba
Samba-Sub1
IR64
IR49830 (Sub1)
Drought tolerant varieties
Six drought tolerant varieties
released during 2009-11
Yield advantage of 0.8-1.2
tons/ha under moderate to
severe drought, but with no
penalty under non-stress
conditions
Tarharra 1 in Nepal
Sahbhagi dhan in India
Sahod Ulan 1 in Philippines
Nature devised the Snorkel and Submergence
genes to control flooding tolerance in rice.
But what about the genes involved in many other
ethylene responses (such as fruit ripening,
senescence, abscission, etc)?
Obtaining basic molecular knowledge of ethylene
biology allows for genetic engineering of many
responses to ethylene
Ethylene responses
Developmental processes
Responses to abiotic and biotic stress
Fruit ripening - ethylene is essential
Promotion of seed germination
Root initiation
Bud dormancy release
Inhibition/promotion of flowering
Sex shifts in flowers
Senescence of leaves, flowers
Abscission of leaves, flowers, fruits
Epinasty of leaves
Inhibition/promotion of cell division/elongation
Altered geotropism in roots, stems
Induction of phytoalexins/disease resistance
Aerenchyma formation
Ethylene hormone signaling
1. What is “signaling”?
2. How is signaling studied?
Signal transduction
Signal
?
Response
plant cell
Frequency of “Signal Transduction”
research papers in the past 30 years
The total number of papers published per year since 1977 containing the
term “signal transduction” in their title or abstract. These figures are from
analysis of papers in the MEDLINE database. The total published since Jan
1, 1977-Dec 31, 2007 is 48,377, of which 11,211 are review articles.
Plant growth, development, and survival depend on
appropriate responses to a diverse array of constantly
fluctuating external and internal signals
Signal transduction the process by which a cell
converts one kind of signal or
stimulus into another.
Signal transduction processes
typically involve a sequence of
biochemical reactions or other
responses within the cell,
resulting in a signal
transduction pathway
Example of signaling pathway activated by an extracellular signal
WHAT CONSTITUTES
AN UNDERSTANDING
OF SIGNALING
PATHWAYS?
HOW CAN
RESEARCHERS
ELUCIDATE
SIGNALING
PATHWAYS?
“Genetic Dissection” of the
Ethylene Signaling Pathway
(Question: What does this mean?)
How to genetically dissect a pathway
1. Identify a phenotype that is specific to the
process you are interested in
2. Design appropriate screen for isolating mutants
based on this phenotype
3. Clone the corresponding gene by map-based
cloning
4. Investigate the function of the corresponding
protein at cell biological and biochemical levels
Arabidopsis thaliana
•
The life cycle is short--about 6 weeks
from germination to seed maturation.
•
Seed production is prolific and the
plant is easily cultivated in restricted space.
•
Self-fertilizing, but can also be out-crossed
by hand.
•
Relatively small genome (1.5 MB), completely sequenced
•
Extensive genetic and physical maps of all 5 chromosomes
•
A large number of mutant lines and genomic resources is
available - Mutants are available in nearly every gene
•
Genetic transformation is simple using Agrobacterium
tumefaciens
•
Extensive databases for gene expression analyses,
multinational projects, etc.
The seedling “triple response”
Arabidopsis thaliana
Pea seedlings
Neljubow (1901) Beih Bot Zentralbl 10,
128-139
“Triple
Response”
Seeds are mutagenized
in the lab, then
screened for mutants in
the ethylene signaling
pathway, based on the
“triple response”
phenotype.
The mutants that we
discover correspond to
mutated genes.
Bleecker et al. (1988) Science
241, 1086–1089
Ethylene-Response Mutants in Arabidopsis
Ethylene-insensitive mutants
etr1 etr2 ein4 (dominant)
ein2 ein3 ein5 (recessive)
ein6 ein7
C2H4
Constitutive-response mutants
ctr1 (recessive)
air
(eto1)
Molecular markers provide
a link between genetic loci
and physical DNA
Chang et al. (1988) PNAS 85: 6856-6860
*A genetic map
of molecular
markers on the
chromosome
allows one to
clone any gene
for which there is
a mutant
phenotype
Generating a mapping population
mut
mut
X
Columbia (C)
Niederzenz (N)
heterozygous for mut
F1
Recombinant genotypes
F2
1
2
3
4
Mapping population
5
.....
Example of mapping with molecular markers
Mapping population
Marker A
Marker B
Current model of the
ethylene signaling pathway
Cu+
Golgi
C2H4
RAN1
Cu+
Lumen
ETR2
ER
ETR1 EIN2
Cu+
Cu+
ETP1/2
Degradation by
26S proteasome
CTR1
C
Cytoplasm
EIN3/EIL1
Ethylene Responsive Gene
Expression
EBP1/2
Degradation by
26S proteasome
Arabidopsis
What can we do with
this information?
The tall etiolated
seedling has a
mutation in the
ethylene receptor
ETR1. The seedling
cannot detect
ethylene.
The mutant
Arabidopsis gene
(etr1-1) has been
transformed into
other plants where
it confers a high
level of ethylene
insensitivity
Wilkinson et al. (1997)
Nature Biotech. 15: 444-448
Lab: Screen for ethylene response mutants
“Triple
Response”
Go over the lab and lab worksheet
• Which seedling was germinated in the
presence of the plant hormone ethylene in
the dark?
1
2
1. Seedling 1
2. Seedling 2
• Which of these seedlings is insensitive to the
plant hormone ethylene?
1. Seedling 1
2. Seedling 2
3. Seedling 3
No
ethylene
1
2
+
ethylene
3
How do research labs screen for mutants
that are insensitive to ethylene?
Mutagenized seeds are plated on growth
media that:
1. contains abscisic acid and is incubated in the dark
2. contains ACC and is incubated under lights in the growth
chamber
3. contains ACC and is incubated in the dark
4. is incubated in the dark
• Which seedling is a “constitutive ethyleneresponse” mutant?
1. Seedling 1
2. Seedling 2
3. Seedling 3
4. Seedling 4
No
+
ethylene ethylene
1 2 3 4
How do research labs screen for mutants
that have a constitutive response to
ethylene?
Mutagenized seeds are plated on growth
media that:
1. contains abscisic acid and is incubated in the dark
2. contains ACC and is incubated under lights in the growth
chamber
3. contains ACC and is incubated in the dark
4. is incubated upside down in the dark
Arabidopsis flower mutants