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Caren Changcarenc@umd.edu
Lab members enjoy finishing an experiment
The plant hormone ethylene
1. What does ethylene do?
2. Is ethylene important?
3. How can we study ethylene and use that knowledge to benefit humans?
Ethylene is a GAS!!!
Plants synthesize ethylene in response to stress
Cold stressOsmotic stressMechanical stressUV stressPathogen attack
Biotic stressFlooding
WoundingHeat stress
Drought stress
ETHYLENE is also a pollutant in the environment
Ethylene responses
Developmental processesFruit ripening - ethylene is essential Promotion of seed germinationRoot initiationBud dormancy release Inhibition/promotion of floweringSex shifts in flowers Senescence of leaves, flowers
Responses to abiotic and biotic stress Abscission of leaves, flowers, fruitsEpinasty of leaves Inhibition/promotion of cell division/elongationAltered geotropism in roots, stems Induction of phytoalexins/disease resistanceAerenchyma 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 Ethylene causes senescence
Can block ethylene response using silver thiosulfate
Apple slices inducing ripening of persimmons
8 days in bag with apple slices
Controls, 8 days outside of bag
“One bad apple spoils the whole bunch…”
Transport and storage of fruits and vegetables requires ethylene control
Ethylene has far-reaching consequences for agriculture and horticulture
Flood-tolerant rice created by expression of ethylene response factor genes
Therefore, we would like to manipulate the biosynthesis and/or responses to ethylene
Removal of external ethylene
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Asia Africa Americas Rest of world
Million tons milled rice
2010 global rice production
Additional rice needed: 114 million tons by 2035
Global rice production increases are needed to meet demand by 2035
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.
Complete submergence. The tank was completely filled with water on the first day of the treatment.
Tank is filled to top
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/journal/v460/n7258/suppinfo/nature08258.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
Water level
WHAT GENES ARE RESPONSIBLE? Discovery of the SNORKEL genes
- 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
Transcriptional response
SNORKEL1 & 2proteins
Flooding
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
Non-deepwater rice
Transcriptional response
No transcriptional response
SNORKEL1 & 2
Flooding
Flooding
Non-deepwater rice does not have these genes!
Localization of SNORKEL proteins to the plant nucleus using “protein fusions” to GFP
Yoko Hattori et al. (2009) Nature 460, 1026-1030
• 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.
SUBMERGENCE1 GENE (SUB1) – Quiescent strategy
Transcription factors turn on specific genes
• 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
Functions of Gibberellic Acid
• 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.
Solving the problem
• These deepwater varieties have low grain yield, unlike the high-yield varieties that are used for food.
An actual field trial of the Sub1A gene in rice
New Sub1 lines after 17 days submergence in the field at
IRRI
Samba-Sub1
Samba
Samba-Sub1IR64-Sub1
IR49830 (Sub1)
IR64
IR42
IR64
IR64-Sub1
Samba-Sub1
IR49830 (Sub1)
Samba
IR64
IR64-Sub1IR49830 (Sub1)
IR42
IR64-Sub1
IR64
IR49830 (Sub1)
IR49830 (Sub1)
IR42
Samba
IR42
Samba
Drought tolerant varietiesSix drought tolerant
varieties released during 2009-11Yield advantage of 0.8-1.2 tons/ha under moderate to severe drought, but with no penalty under non-stress conditions Sahbhagi dhan in India
Tarharra 1 in Nepal 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 processesFruit ripening - ethylene is essential Promotion of seed germinationRoot initiationBud dormancy release Inhibition/promotion of floweringSex shifts in flowers Senescence of leaves, flowers
Responses to abiotic and biotic stress Abscission of leaves, flowers, fruitsEpinasty of leaves Inhibition/promotion of cell division/elongationAltered geotropism in roots, stems Induction of phytoalexins/disease resistanceAerenchyma formation
Ethylene hormone signaling
1. What is “signaling”?
2. How is signaling studied?
Signal transduction
Response
Signal
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
Example of signaling pathway activated by an extracellular signal
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
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.
Pea seedlings
Neljubow (1901) Beih Bot Zentralbl 10, 128-139
The seedling “triple response”
“Triple Response”
Arabidopsis thaliana
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
ctr1 (recessive)
(eto1)
ein2 ein3 ein5 (recessive)ein6 ein7
Constitutive-response mutants
Ethylene-insensitive mutants
etr1 etr2 ein4 (dominant)
Ethylene-Response Mutants in Arabidopsis
air
C2H4
Molecular markers provide a link between genetic loci and physical DNA
*A genetic map of molecular markers on the chromosome allows one to clone any gene for which there is a mutant phenotype
Chang et al. (1988) PNAS 85: 6856-6860
X
Niederzenz (N) Columbia (C)
F1
F2
1 2 3 4 5 . . . . .
mutmut
heterozygous for mut
Recombinant genotypes
Generating a mapping population
Mapping population
Example of mapping with molecular markers
Mapping population
Marker B
Marker A
Ethylene Responsive Gene Expression
CTR1
ETR1
Nucleus
-
C
Cytoplasm
ER
EIN2
N
Lumen
C
EIN3/EIL1
ETR2
ETP1/2
Degradation by 26S proteasome
Current model of the ethylene signaling pathway
EBP1/2
Degradation by 26S proteasome
RAN1
Golgi
N
C2H4N
N
Cu+
Cu+
Cu+
Cu+
What can we do with this information?
The tall etiolated seedling has a mutation in the ethylene receptor ETR1. The seedling cannot detect ethylene.
Arabidopsis
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.Seedling 12.Seedling 2
1 2
• Which of these seedlings is insensitive to the plant hormone ethylene?
1.Seedling 12.Seedling 23.Seedling 3
1 2 3
Noethylene
+ ethylene
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 dark2. contains ACC and is incubated under lights in the
growth chamber3. contains ACC and is incubated in the dark4. is incubated in the dark
• Which seedling is a “constitutive ethylene-response” mutant?
1.Seedling 12.Seedling 23.Seedling 34.Seedling 4
1 2 3 4
Noethylene
+ ethylene
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 dark2. contains ACC and is incubated under lights in the
growth chamber3. contains ACC and is incubated in the dark4. is incubated upside down in the dark
Arabidopsis flower mutants
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