Outline Ethylene hormone

signaling 1. Introduction to the ethylene hormone

(effects, history, significance)

2. Genetic dissection of the ethylene signaling pathway (this provides for the genetic engineering of many responses to ethylene)

2. Addressing the food crisis: recent ethylene discoveries in rice

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

ETHYLENE is a gaseous plant hormone.

Ethylene Biosynthesis

Cold stressOxidative stressOsmotic stressMechanical stressUV stressPathogen attack

Biotic stressFlooding

WoundingHeat stress

Drought stress

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

• Illuminating gas caused detrimental effects

Historical background

• 1901 Neljubov - ethylene is the biologically active agent in illuminating gas

• 1934 Gane - ethylene is produced by plants

Apple slices inducing ripening of persimmons

8 days in bag with apple slices

Controls, 8 days outside of bag

Wounding induces ethylene production Ethylene causes senescence

Can block ethylene receptors with silver thiosulfate

“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

Removal of external ethylene

Outline Ethylene hormone

signaling 1. Introduction to the ethylene hormone

(effects, history, significance)

2. Genetic dissection of the ethylene signaling pathway (this provides for the genetic engineering of many responses to ethylene)

2. Addressing the food crisis: recent ethylene discoveries in rice

“Genetic Dissection” of the Ethylene Signaling

Pathway

(Question: What does this mean?)

Signal transduction

Response

Signal

plant cell

?

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 an ordered sequence of biochemical reactions or other responses within the cell, resulting in a signal transduction pathway

QUESTIONS

WHAT CONSTITUTES AN UNDERSTANDING OF SIGNALING PATHWAYS?

HOW CAN RESEARCHERS ELUCIDATE SIGNALING PATHWAYS?

Frequency of Signal Transduction Publications 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 reviews.

RAN1Golgi

EBF1/2

EIN2

C2H4 Responsive Gene

CTR1

ETR1

ER

Nucleus

GCC

Degradation by 26S proteasome

C

--

C

EBS

-CN-

EIN5/XRN4

-C

N-

ERF1

HHH

C=C =H

Cytoplasm

EIN3/EIL1

N-

KD

Degradation by the 26S proteasome via

SCFEBF1/2

N-

Cu2+ER/

Golgi

N

RTE1

-C

-

?

Cu2+

Cu2+Cu2+

Kendrick and Chang (2008) Curr. Opin. Plant Biol. 11: 479-485

ETP1/2

How to genetically dissect a pathway/process

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. Carry out genetic analysis of the mutant (e.g., epistasis)

4. Clone the corresponding gene by map-based cloning

5. Investigate function 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 and 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

The WT versions of these genes are “Positive Regulators” of ethylene response

CTR1 is a “NegativeRegulator” of responses

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

Landsberg Columbia

F1

F2

1 2 3 4 5 . . . . .

mutmut

heterozygous for mut

Recombinant genotypes

Generating a mapping population

Mapping population

self-pollinate

hand-pollinate

Example of mapping with molecular markers

Mapping population

Marker B

Marker A

RAN1Golgi

EBF1/2

EIN2

C2H4 Responsive Gene

CTR1

ETR1

ER

Nucleus

GCC

Degradation by the 26S proteasome

C

--

C

EBS

-CN-

EIN5/XRN4

C2H4-binding

GAF

Histidine kinase

Receiver

-C

N-

ERF1

HHH

C=C =H

Cytoplasm

EIN3/EIL1

N-

KD

Degradation by the 26S proteasome via

SCFEBF1/2

N-

RECEPTOR SUBFAMILIES

Cu2+

N

RTE1/GRER/

Golgi-C

-

1 2

HH

?

Cu2+

Cu2+Cu2+

ETP1/2

The tall etiolated seedling has a mutation in the ethylene receptor ETR1. The seedling cannot detect ethylene.

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

Arabidopsis

The mutant Arabidopsis etr1-1 gene has been transformed into other plants where it confers a high level degree of ethylene insensitivity

Wilkinson et al. (1997) Nature Biotech. 15: 444-448

Outline Ethylene hormone

signaling 1. Introduction to the ethylene hormone

(effects, history, significance)

2. Genetic dissection of the ethylene signaling pathway (this provides for the genetic engineering of many responses to ethylene)

2. Addressing the food crisis: recent ethylene discoveries in rice

Ethylene, rice, and feeding millions

• 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 4 days, due to 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.

Flood-tolerant rice exists in nature

• There are deepwater rice cultivars that have evolved and adapted to constant 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.

• HOWEVER, these deepwater varieties have low grain yield, unlike the high-yield varieties used for food.

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

Water level

Mapping the SNORKEL gene loci to the rice chromosomes

- 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

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

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

The researchers found that the SNORKEL genes belong to the ERF (Ethylene Response Factor) family 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!

Long-term flooding vs. flash flooding

• A few rice cultivars 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. They become quiescent and stay submerged, avoiding the energy consumption that is involved in elongation. For example, they increase anaerobic respiration.

• The gene controlling this response, named SUB1, was identified and cloned in 2006. Like the SNORKEL genes, it is also a member of the ERF gene family.

• 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.

• Now transferring these genes to 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

Flood tolerant rice:Signaling from ethylene to another hormone GA, which controls elongation

• 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

Responses to Gibberellic Acid (GA)

Some uses of the GA hormone

Gibberellin induces growth in Thompson’s seedless grapes

• During germination, the storage starches are converted to simple sugars for use in seedling development. The “malting” of barley seeds in beer production is the process of using GA to induce enzymes in seed germination causing conversion of starches to sugars. Germination is then stopped by heating and the sugars are fermented.

• GA induces seedlessness in grapes, while increasing fruit size.

Developmental regulatory pathwaysdevelopment of embryo, flower, leaf, root,

trichome root apical meristem formation shoot apical meristem formation

polarity and cytoskeletal rearrangement

Specific cell fate determination and differentiation(xylem and phloem specification, root patterning)

Abiotic stress response pathways(salt, drought, heat, cold, metals, vernalization etc.)

Plant hormone signal transduction(auxin, ethylene, cytokinin, gibberellin, abscisic acid, brassinosteroid, jasmonic acid)

Examples of signaling pathways that researchers are studying in plants using mutants

Which one is the wild type?

Lab Experiment: Ethylene mutant hunt

“Triple Response”