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Caren Chang [email protected] Lab members enjoy finishing an experiment. The plant hormone ethylene. What does ethylene do? Is ethylene important? How can we study ethylene and use that knowledge to benefit humans?. Ethylene is a GAS!!!. Plants synthesize ethylene in response to stress.

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Caren chang carenc umd

Caren Chang

[email protected]

Lab members enjoy finishing an experiment


The plant hormone ethylene

The plant hormone ethylene

  • What does ethylene do?

  • Is ethylene important?

  • How can we study ethylene and use that knowledge to benefit humans?

Ethylene is a GAS!!!


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Plants synthesize ethylene in response to stress

Heat stress

Wounding

Drought stress

Flooding

Biotic stress

Cold stress

Osmotic stress

Mechanical stress

UV stress

Pathogen attack


Ethylene is also a pollutant in the environment

ETHYLENE is also a pollutant in the environment


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Ethylene responses

Developmental processes

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

Responses to abiotic and biotic stressAbscission 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

Historical background

  • Ethylene has been used (unwittingly) throughout history

Wood burning fires promote synchronous flowering in pineapple

Gashing promotes ripening in figs (4 days later)


Historical background1

Historical background

  • 1800s Illuminating gas caused detrimental effects


Historical background2

Historical background

  • 1901 Neljubov discovered that ethylene is the biologically active agent in illuminating gas, which was used to heat the greenhouse


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Wounding induces ethylene production

Ethylene causes senescence

Can block ethylene response using silver thiosulfate


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8 days in bag with apple slices

Controls, 8 days outside of bag

Apple slices inducing ripening of persimmons


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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


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Removal of external ethylene


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Global rice production increases are needed to meet demand by 2035


Ethylene rice and feeding billions

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

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

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


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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.


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Tank is filled to top

Complete submergence. The tank was completely filled with water on the first day of the treatment.


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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


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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.


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Long-term flooding vs. flash flooding


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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


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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

Flooding

SNORKEL1 & 2

proteins

Transcriptional response


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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

Flooding

SNORKEL1 & 2

Transcriptional response

Non-deepwater rice

Flooding

Non-deepwater rice does not have these genes!

No transcriptional response


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Localization of SNORKEL proteins to the plant nucleus using “protein fusions” to GFP

Yoko Hattori et al. (2009) Nature 460, 1026-1030


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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.


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Transcription factors turn on specific genes


Functions of gibberellic acid

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


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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.


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An actual field trial of the Sub1A gene in rice


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New Sub1 lines after 17 days submergence in the field at IRRI

IR64-Sub1

Samba-Sub1

IR49830 (Sub1)

Samba

Samba

IR64

IR42

IR49830 (Sub1)

IR42

IR64

IR49830 (Sub1)

IR64

IR64-Sub1

Samba

IR64-Sub1

Samba-Sub1

IR42

IR42

IR49830 (Sub1)

IR64-Sub1

IR49830 (Sub1)

Samba

Samba-Sub1

IR64


Drought tolerant varieties

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

Sahbhagi dhan in India

Sahod Ulan 1 in Philippines

Tarharra 1 in Nepal


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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


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Ethylene responses

Developmental processes

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

Responses to abiotic and biotic stressAbscission 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

Ethylene hormone signaling

  • What is “signaling”?

  • How is signaling studied?


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Signal transduction

Signal

?

plant cell

Response


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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.


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Plant growth, development, and survival depend on appropriate responses to a diverse array of constantly fluctuating external and internal signals


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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 asignal transduction pathway


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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

“Genetic Dissection” of the Ethylene Signaling Pathway(Question: What does this mean?)


How to genetically dissect a pathway

How to genetically dissect a pathway

  • Identify a phenotype that is specific to the process you are interested in

  • Design appropriate screen for isolating mutants based on this phenotype

  • Clone the corresponding gene by map-based cloning

  • Investigate the function of the corresponding protein at cell biological and biochemical levels


Arabidopsis thaliana

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.


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“Triple Response”

The seedling “triple response”

Arabidopsis thaliana

Pea seedlings

Neljubow (1901) Beih Bot Zentralbl 10, 128-139


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Bleecker et al. (1988) Science 241, 1086–1089

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.


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air

Ethylene-Response Mutants in Arabidopsis

Ethylene-insensitive mutants

etr1 etr2 ein4 (dominant)

ein2 ein3 ein5 (recessive)

ein6 ein7

C2H4

Constitutive-response mutants

ctr1 (recessive)

(eto1)


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*A genetic map of molecular markers on the chromosome allows one to clone any gene for which there is a mutant phenotype

Molecular markers provide

a link between genetic loci and physical DNA

Chang et al. (1988) PNAS 85: 6856-6860


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Generating a mapping population

mut

mut

X

Columbia (C)

Niederzenz (N)

heterozygous for mut

F1

Recombinant genotypes

F2

. . . . .

1

2

3

4

5

Mapping population


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Mapping population

Marker A

Marker B

Example of mapping with molecular markers


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C

Current model of the ethylene signaling pathway

Cu+

Golgi

C2H4

RAN1

N

Cu+

Lumen

ETR2

ETR1

EIN2

N

N

ER

Cu+

Cu+

N

ETP1/2

C

Degradation by

26S proteasome

-

CTR1

EIN3/EIL1

Cytoplasm

EBP1/2

Degradation by

26S proteasome

Nucleus

Ethylene Responsive Gene Expression


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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.


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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”

Lab: Screen for ethylene response mutants


Go over the lab and lab worksheet

Go over the lab and lab worksheet


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1 2

  • Which seedling was germinated in the presence of the plant hormone ethylene in the dark?

  • Seedling 1

  • Seedling 2


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No

ethylene

+

ethylene

  • Which of these seedlings is insensitive to the plant hormone ethylene?

  • Seedling 1

  • Seedling 2

  • Seedling 3

1 2 3


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How do research labs screen for mutants that are insensitive to ethylene?

Mutagenized seeds are plated on growth media that:

  • contains abscisic acid and is incubated in the dark

  • contains ACC and is incubated under lights in the growth chamber

  • contains ACC and is incubated in the dark

  • is incubated in the dark


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No

ethylene

+

ethylene

  • Which seedling is a “constitutive ethylene-response” mutant?

  • Seedling 1

  • Seedling 2

  • Seedling 3

  • Seedling 4

1 2 3 4


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How do research labs screen for mutants that have a constitutive response to ethylene?

Mutagenized seeds are plated on growth media that:

  • contains abscisic acid and is incubated in the dark

  • contains ACC and is incubated under lights in the growth chamber

  • contains ACC and is incubated in the dark

  • is incubated upside down in the dark


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Arabidopsis flower mutants


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