2. Lab members enjoy finishing an experiment
Caren Chang
Lab members enjoy finishing an experiment

3. 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!!!
What’s a hormone?

5. Plants synthesize ethylene in response to stress
Wounding
Flooding
Drought stress
Biotic stress
Heat stress
Cold stress
Osmotic stress
Mechanical stress
UV stress
Pathogen attack
Ethylene has it’s hands in everything

6. ETHYLENE is also a pollutant in the environment

7. 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 stress Abscission 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

8. 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)

9. Historical background
1800s Illuminating gas caused detrimental effects
Plants around the beltway

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

11. Wounding induces ethylene production
Ethylene causes senescence
Can block ethylene response using silver thiosulfate

12. Apple slices inducing ripening of persimmons
8 days in bag with apple slices
Controls, 8 days outside of bag

13. 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

14. Removal of external ethylene

16. Global rice production increases are needed to meet demand by 2035

18. 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).

19. 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!

20. 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

21. 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.

22. Tank is filled to top
Complete submergence. The tank was completely filled with water on the first day of the treatment.

23. This elongated deepwater rice plant in Thailand was preserved after flooding occurred and shows the typical flooding height. White bar = 1 meter.

24. 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.

25. Long-term flooding vs. flash flooding
1 billion per year lost to flooding
3 billion

26. 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

27. 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 & 2
proteins
Flooding

29. 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
Non-deepwater rice does not have these genes!

30. Localization of SNORKEL proteins to the plant nucleus using “protein fusions” to GFP
Yoko Hattori et al. (2009) Nature 460,

31. SUBMERGENCE1 GENE (SUB1) – Quiescent strategy
Identified and cloned in 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.

32. Transcription factors turn on specific genes
1 billion per year lost to flooding
3 billion

33. 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

34. 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.

36. An actual field trial of the Sub1A gene in rice

37. New Sub1 lines after 17 days submergence in the field at IRRI
IR64-Sub1
Samba-Sub1
IR49830 (Sub1)
Samba
IR64
Samba
IR42
IR42
IR49830 (Sub1)
IR64
IR49830 (Sub1)
IR64
IR64-Sub1
Samba
IR64-Sub1
Samba-Sub1
IR42
IR42
IR49830 (Sub1)
IR64-Sub1
IR49830 (Sub1)
Samba
Samba-Sub1
IR64 37

38. Drought tolerant varieties
Six drought tolerant varieties released during
Yield advantage of 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

39. 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

40. 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 stress Abscission 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

41. Ethylene hormone signaling
What is “signaling”?
How is signaling studied?
How to get the genes that nature did not already manipulate so that we can manipulate them?

42. Signal transduction
Response
Signal
plant cell ?

43. 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.

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

45. 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

46. WHAT CONSTITUTES AN UNDERSTANDING OF SIGNALING PATHWAYS?
HOW CAN RESEARCHERS ELUCIDATE SIGNALING PATHWAYS?

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

48. 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
You will be doing 2 aspects of this in the lab today and next week

49. 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.
Attracted geneticists. Previously crop plants - slow genetics, large genomes, require growth space

50. The seedling “triple response”
Arabidopsis thaliana
“Triple Response”
Pea seedlings
Neljubow (1901) Beih Bot Zentralbl 10,

51. 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.

52. Ethylene-Response Mutants in Arabidopsis
Ethylene-insensitive mutants
etr1 etr2 ein4 (dominant)
ein2 ein3 ein5 (recessive)
ein6 ein7
C2H4
Constitutive-response mutants
air
ctr1 (recessive)
(eto1)

53. *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:

54. Generating a mapping population
mut
mut X
Niederzenz (N)
Columbia (C)
heterozygous for mut F1
Recombinant genotypes F2 1 2 3 4 5
Mapping population

55. Mapping population Marker A Marker B
Example of mapping with molecular markers
Mapping population
Marker B
Marker A

56. Ethylene Responsive Gene Expression
Current model of the ethylene signaling pathway
Cu+
Golgi
RAN1
C2H4 N
Cu+
Lumen
ETR2
ETR1
EIN2 N N ER
Cu+
Cu+ N
ETP1/2 C
Degradation by
26S proteasome -
CTR1 C
Cytoplasm
EIN3/EIL1
EBP1/2
Degradation by
26S proteasome
Nucleus
Ethylene Responsive Gene Expression

57. 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.

58. 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:

59. Lab: Screen for ethylene response mutants
“Triple Response”

60. Go over the lab and lab worksheet

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

62. Which of these seedlings is insensitive to the plant hormone ethylene?No
ethylene +
ethylene

63. Mutagenized seeds are plated on growth media that:
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

64. Which seedling is a “constitutive ethylene-response” mutant?No
ethylene +
ethylene

65. Mutagenized seeds are plated on growth media that:
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

66. Arabidopsis flower mutants
Face of Jesus in lower left??!!!