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