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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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slide2

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!!!

slide5

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

slide7

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
slide11

Wounding induces ethylene production

Ethylene causes senescence

Can block ethylene response using silver thiosulfate

slide12

8 days in bag with apple slices

Controls, 8 days outside of bag

Apple slices inducing ripening of persimmons

slide13

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

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

slide21

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.

slide22

Tank is filled to top

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

slide23

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

slide24

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.

slide26

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

slide27

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

slide29

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

slide30

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

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

slide31

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.
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
slide34

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.
slide37

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

slide39

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

slide40

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?
slide42

Signal transduction

Signal

?

plant cell

Response

slide43

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.

slide44

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

slide45

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

slide46
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.
slide50

“Triple Response”

The seedling “triple response”

Arabidopsis thaliana

Pea seedlings

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

slide51

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.

slide52

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)

slide53

*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

slide54

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

slide55

Mapping population

Marker A

Marker B

Example of mapping with molecular markers

slide56

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

slide57

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.

slide58

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

slide61

1 2

  • Which seedling was germinated in the presence of the plant hormone ethylene in the dark?
  • Seedling 1
  • Seedling 2
slide62

No

ethylene

+

ethylene

  • Which of these seedlings is insensitive to the plant hormone ethylene?
  • Seedling 1
  • Seedling 2
  • Seedling 3

1 2 3

slide63

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
slide64

No

ethylene

+

ethylene

  • Which seedling is a “constitutive ethylene-response” mutant?
  • Seedling 1
  • Seedling 2
  • Seedling 3
  • Seedling 4

1 2 3 4

slide65

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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