Lab members enjoy finishing an experiment Caren Chang carenc@umd.edu 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!!! What’s a hormone?

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

ETHYLENE is also a pollutant in the environment

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

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

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

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

Removal of external ethylene

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.

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

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 1 billion per year lost to flooding 3 billion

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

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

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!

Localization of SNORKEL proteins to the plant nucleus using “protein fusions” to GFP Yoko Hattori et al. (2009) Nature 460, 1026-1030

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.

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

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

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.

An actual field trial of the Sub1A gene in rice

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

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 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 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

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?

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 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

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

The seedling “triple response” Arabidopsis thaliana “Triple Response” Pea seedlings Neljubow (1901) Beih Bot Zentralbl 10, 128-139

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.

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)

*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

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

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

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

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.

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 2

Which of these seedlings is insensitive to the plant hormone ethylene? No ethylene + ethylene 1 2 3

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

Which seedling is a “constitutive ethylene-response” mutant? No ethylene + ethylene 1 2 3 4

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

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