Outline Ethylene hormone signaling Introduction to the ethylene hormone (effects, history, significance) Genetic dissection of the ethylene signaling pathway (this provides for the genetic engineering of many responses to ethylene) Addressing the food crisis: recent ethylene discoveries in rice Yesterday’s lecture did not go into what happens at the molecular and cellular level. Today, using Ethylene as an example of a signal and the very significant biotechnological aspects that can be manipulated.

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 Wounding Flooding Drought stress Biotic stress Heat stress Cold stress Oxidative stress Osmotic stress Mechanical stress UV stress Pathogen attack STRESS hormone It has been shown that ethylene is produced from essentially all parts of higher plants, including leaves, stems, roots, flowers, fruits, tubers, and seedlings. Elucidated 40-60 yrs ago Ethylene production is regulated by a variety of developmental and environmental factors. During the life of the plant, ethylene production is induced during certain stages of growth such as germination, ripening of fruits, abscission of leaves, and senescence of flowers. Ethylene production can also be induced by a variety of external aspects such as mechanical wounding, environmental stresses, and certain chemicals including auxin and other regulators Ethylene has it’s hands in everything

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 KNOWN AS STRESS HORMONE Pleiotropic effects. What is actually caused by the gas may depend on the tissue affected as well as environmental conditions. In flooding, root suffers from lack of oxygen, or anoxia, which leads to the synthesis of 1-Aminocyclopropane-1-carboxylic acid (ACC). ACC is transported upwards in the plant and then oxidized in leaves. The product, the ethylene causes epinasty of the leaves.

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 Plants around the beltway

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

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…”

Removal of external ethylene

Outline Ethylene hormone signaling Introduction to the ethylene hormone (effects, history, significance) Genetic dissection of the ethylene signaling pathway (this provides for the genetic engineering of many responses to ethylene) 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 The basic problem. Term coined in 1972. Gained popularity in 1980’s.

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.

Degradation by the 26S proteasome via SCFEBF1/2 Cu2+ N- -C Golgi RAN1 H H C=C = H H N RTE1 -C - N - ER ER/ Golgi Cu2+ Cu2+ Cu2+ N - ETR1 KD C - CTR1 - C Degradation by 26S proteasome Cytoplasm ETP1/2 ? N- EIN2 -C EIN5/XRN4 Nucleus EBF1/2 EIN3/EIL1 Degradation by the 26S proteasome via SCFEBF1/2 EBS ERF1 GCC C2H4 Responsive Gene Kendrick and Chang (2008) Curr. Opin. Plant Biol. 11: 479-485

How to genetically dissect a pathway/process Identify a phenotype that is specific to the process you are interested in Design appropriate screen for isolating mutants based on this phenotype Carry out genetic analysis of the mutant (e.g., epistasis) Clone the corresponding gene by map-based cloning 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. 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 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.

Ethylene-Response Mutants in Arabidopsis Ethylene-insensitive mutants etr1 etr2 ein4 (dominant) ein2 ein3 ein5 (recessive) ein6 ein7 The WT versions of these genes are “Positive Regulators” of ethylene response C2H4 Constitutive-response mutants air CTR1 is a “Negative Regulator” of responses 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 Landsberg Columbia hand-pollinate heterozygous for mut F1 self-pollinate 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

RAN1 RTE1/GR ETR1 CTR1 EIN2 EIN5/XRN4 Cu2+ RECEPTOR SUBFAMILIES N- -C 1 2 Golgi RAN1 H H C2H4-binding C=C = H H N RTE1/GR ER/ Golgi -C - GAF N - ER H H Cu2+ Cu2+ Cu2+ Histidine kinase Receiver N - ETR1 KD C - CTR1 Degradation by the 26S proteasome - C Cytoplasm ETP1/2 EIN2 ? N- -C EIN5/XRN4 Nucleus EBF1/2 EIN3/EIL1 Degradation by the 26S proteasome via SCFEBF1/2 EBS ERF1 GCC C2H4 Responsive Gene

Bleecker et al. (1988) Science 241, 1086–1089 Arabidopsis The tall etiolated seedling has a mutation in the ethylene receptor ETR1. The seedling cannot detect ethylene.

The mutant Arabidopsis etr1-1 gene has been transformed into other plants where it confers a high level degree of ethylene insensitivity The general pathway is CONSERVED. For exampie, receptors are conserved among plants Can also put mutations known from one plant into the gene of another plant 0, 3, and 8 days post polliniation (G,H, I) L detached flowers treated 16 hours with 1 ppm ethylene Wilkinson et al. (1997) Nature Biotech. 15: 444-448

Outline Ethylene hormone signaling Introduction to the ethylene hormone (effects, history, significance) Genetic dissection of the ethylene signaling pathway (this provides for the genetic engineering of many responses to ethylene) 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. internode Don’t have to search for mutants. Find the genes involved in flooding tolerance in the deepwater rice and transfer the genes to the high-yield varieties. HOWEVER, these deepwater varieties have low grain yield, unlike the high-yield varieties used for food.

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

Mapping the SNORKEL gene loci to the rice chromosomes 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

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 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.

Solving the problem 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.

Flood tolerant rice: Signaling from ethylene to another hormone GA, which controls elongation 1 billion per year lost to flooding 3 billion

Responses to Gibberellic Acid (GA) 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

Some uses of the GA hormone 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. Gibberellin induces growth in Thompson’s seedless grapes

Examples of signaling pathways that researchers are studying in plants using mutants Developmental regulatory pathways development 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)

Which one is the wild type? Face of Jesus in lower left??!!!

Lab Experiment: Ethylene mutant hunt “Triple Response”