Assistant Professor, Biologygenetics.wustl.edu/bio5491/files/2015/01/Strader-Advanced-Genetics-2015.pdfThe history of ethylene • Ancient Egyptians - gas figs to stimulate ripening

Arabidopsis thaliana

Lucia Strader Assistant Professor, Biology

Arabidopsis as a genetic model • Easy to grow

• Small genome

• Short life cycle

• Self fertile

• Produces many progeny

• Easily transformed

E. coli

S. cerevisiae

C. elegans

Arabidopsis thaliana

D. melanogaster

Populus and Physcomitrella

Arabidopsis Genome Information

115 million basepairs

Arabidopsis as a developmental model

http://www.geochembio.com/biology/organisms/arabidopsis/ http://phytomorph.wisc.edu/results/gravitropism.php

• Photosynthesis • Amino acid biosynthesis • Secondary metabolites • etc.

Arabidopsis as a biochemical model

Arabidopsis as a disease model

PresenterPresentation NotesSame virulence factors affect Arabidopsis and mouse systems.

Indeed, 70% of genes implicated in cancer have Arabidopsis homologues, but only 41% have yeast homologues.

Arabidopsis Resources

• ~9000 Arabidopsis Research Labs worldwide • Fully sequenced genome • Centralized aggregation of information

(arabidopsis.org) • Library of insertion mutants and cDNA ($7 each) • 1000 genomes project • US Funding sources: DOE, USDA, NSF, NIH, HHMI,

DOD

Discoveries made in plants

• Mendel – heritable traits • Barbara McClintock – jumping genes • RNA-mediated silencing • Innate immunity and intracellular receptors • Protein degradation • Cryptochromes and the circadian clock • Binding of small molecules by SCF complexes • RUB1/Nedd8 conjugation

PresenterPresentation NotesTALensSmall RNAs and Dicer discovered in plants


• Mendel – heritable traits • Barbara McClintock – jumping genes • RNA-mediated silencing • Innate immunity and intracellular receptors • Protein degradation • Cryptochromes and the circadian clock • Binding of small molecules by SCF complexes • RUB1/Nedd8 conjugation

http://molbio.mgh.harvard.edu/sheenweb/pathogen.html

PresenterPresentation NotesNB-LRR proteins are the primary intracellular receptors of the plant immune system. First cloned in 1994. Orthologous and paralogous genes in animals are called NOD/CATERPILLAR. NOD2 was the first candidate gene cloned for Crohn’s disease by virtue of its homology to plant NB-LRRs.

In addition, the involvement of the chaperones HSP90, SGT1, and RAR1 were first found to be involved in NB-LRR regulation in plants.


• Mendel – heritable traits • Barbara McClintock – jumping genes • RNA-mediated silencing • Innate immunity and intracellular receptors • Protein degradation, RUB1/Nedd8 conjugation • Cryptochromes and the circadian clock • Binding of small molecules by SCF complexes • RUB1/Nedd8 conjugation

PresenterPresentation NotesNearly 20 years ago, screens for altered light responses in plants led to the discovery of DET, COP and FUS. Biochemical studies revealed that these proteins played roles in protein turnover. These proteins are part of a conserved complex called the COP9 signalosome, and is invovled in deneddylation, another process first discovered in plants. These studies allowed for understanding of p53 regulation in tumor suppression and CREB regulation.



PresenterPresentation NotesPlant cryptochromes (blue light receptors) were first discovered in 1993. These cryptochromes help regulate the circadian clock. In 1998, CRY homologues were found in mammalian systems and loss of CRY1 and CRY2 in mouse results in altered circadian periods.



PresenterPresentation NotesThe discovery of the E3 ubiquiting ligases, F-box proteins, acting as small molecule receptors was discovered by my colleague Mark Estelle.


• Mendel – heritable traits • Barbara McClintock – jumping genes • RNA-mediated silencing • Innate immunity and intracellular receptors • Protein degradation, RUB1/Nedd8 conjugation • Cryptochromes and the circadian clock • Binding of small molecules by SCF complexes • TILLING

Arabidopsis Mutagenesis and Mutant Screens

Seeds (M0)

Grow to the next generation Mutagenize Seeds (M1)

Seeds (M2)

M2 seeds are used in the mutant screen. Individuals displaying the phenotype of interest are moved to soil and grown to the next generation.

Recombination mapping

Bulk segregant analysis and WGS

Three questions in phytohormone research

• What are phytohormones? • What do phytohormones do? • How do they do it?

What are phytohormones?

Special glands Many parts of plant

Via blood to target May act where made

Active at low levels Active at low levels

Animal hormones Phytohormones

synthesis

transport

efficacy

response Very specific Less specific, often interact with other hormones

What do phytohormones do? Bioassays - Apply hormone and examine response

Screen for mutants altered in this response:

Auxotropic

Rescued by hormone application No response (aka insensitive, resistant)

Resistant in endogenous an bioassay response Constitutive response

Bioassay response even in the absence of hormone

What do phytohormones do? Bioassays - Apply hormone and examine response Screen for mutants altered in this response:

Defects reveal processes in which the hormone acts Defective genes reveal proteins acting in pathways for biosynthesis,

signaling, inactivation

Hormone inhibitors can reveal biosynthesis pathway (mutants) Hormone-responsive promoter-reporter fusions can reveal sites of

hormone action (mutants)

What do phytohormones do? Example 1 - Gibberellin

The history of gibberellin • “bakanae”: foolish seedling disease (rice)

– yellow slender leaves, elongated seedlings, stunted root – seriously lowers yield or killed plants

• 1898 - fungal infection identified as cause – now prevented by treating seeds with fungicide before sowing

• 1926 - cultured filtrates have similar response – fungus must secrete a factor that stimulates shoot elongation, inhibits

root elongation, inhibits chlorophyll (also in maize, oat)

• 1935 - “gibberellin” purified • 1950s - GAs found to be endogenous plant

hormones • current - 125 GAs identified in plants, fungi

Functions of gibberellin • Stimulates shoot elongation - cell division,

elongation • Stimulates bolting/flowering in long days • Breaks seed dormancy to induce germination • Stimulates enzyme production (α-amylase) in

germinating cereal grains to mobilize seed reserves

• Can delay senescence in leaves and citrus fruit

GA mutants - barley • grd2 – dwarf

– Recessive – GA-deficient – rescued by GAs

• = GA auxotroph • Sln1d – dwarf

– Dominant – Does not respond to GA

application • = GA response mutant (signaling) • sln1c – slender

– Recessive – Phenocopies GA application

• = constitutive response mutant (signaling)

Plant Cell, Vol. 14, S61-S80, May 2002

wild type

grd2 Sln1d

sln1c

grd2 mutant defect

grd2

X active

Plant Cell, Vol. 14, S61-S80, May 2002

What does grd2 tell us? • Auxotropic mutant

• Defective in biosynthesis -

elucidates/confirms pathway

• Distinguishes between hormone and precursors

• Reveals/confirms phytohormone role in development: – GA auxotroph is a dwarf – Therefore, GA is required for

shoot cell elongation

control 7 d, GA3 pea

control 100 pg GA3 rice

1 ng GA3

Biochemistry & Molecular Biology of Plants, Buchanan et al., eds., 2000, Chapter 17

What do phytohormones do? Example 2 - Ethylene

The history of ethylene • Ancient Egyptians - gas figs to stimulate ripening • Ancient Chinese - burn incense in closed rooms to

enhance pear ripening • 1864 - gas leaks from street lights led to stunted

growth, twisting, and abnormal stem thickening (triple response) and early senescence of nearby plants

• 1901 - Russian scientist showed that the active component was ethylene

• 1934 - plants synthesize ethylene • 1935 - ethylene proposed to be responsible for fruit

ripening as well as inhibition of vegetative tissues • 1980s – ethylene biosynthetic pathway elucidated

Ethylene functions • Inhibition of cell expansion • Pathogen response • Abiotic stress response • Formation of root hairs • Fruit ripening • Flower senescence and abscission • Seedling triple response

The triple response

• Dark-grown seedlings incubated with ethylene • exaggerated curvature of the apical hook • radial swelling of the hypocotyl • shortening of hypocotyl and root


Ethylene mutants • Exploits the

ethylene triple response to isolate mutants

• Use ethylene response mutants to elucidate the ethylene signaling pathway

Using the triple response to find mutants

• ein = ethylene insensitive • etr = ethylene resistant • Resistant in both air and ethylene • Ethylene acts to inhibit cell growth

Science 1999, 284:2148-2152

Using the triple response to find mutants • ctr = constitutive

triple response • eto = ethylene

overproducer

Plant Physiol. (1999) 119: 521-530

eto mutants vs. ctr mutants • eto mutants

overproduce ethylene

• eto mutants are sensitive to ethylene biosynthesis inhibitors (defect in biosynthesis)

• ctr mutants are not sensitive to inhibitors (defect in signaling)

Plant Physiol. (1999) 119: 521-530

So you have all these mutants ...

• How to order the mutants?

• Epistasis • Make the double

mutants • Signaling pathway

--> Phenotype of the later mutant is visible

Plant Physiol. (1999) 119: 521-530

Epistasis with ethylene mutants

etr1 = ein1 Etiolated ein2 Etiolated ein3 Etiolated

Genotype Ethylene phenotype

Wild type Triple response

eto1 Triple response ctr1 Triple response

etr1 eto1 Etiolated ein2 eto1 Etiolated ein3 eto1 Etiolated

ETR1, EIN2, and EIN3 act after ETO1


Wild type etr1 = ein1 Etiolated

ein2 Etiolated ein3 Etiolated

Triple response

eto1 Triple response ctr1 Triple response

etr1 ctr1 Triple response ein2 ctr1 Etiolated ein3 ctr1 Etiolated

CTR1 acts after ETR1 EIN2 and EIN3 act after CTR1

Epistasis with ethylene mutants

How to order ein2 and ein3? • Epistasis doesn’t work when mutants act the same


Wild type ein2 Etiolated ein3 Etiolated

Triple response

ein2 ein3 Etiolated OE EIN3 Triple response

OE EIN3 ein2 Triple response

EIN3 acts after EIN2

Molecular characterization can support epistasis

• EIN3 encodes a transcription factor that turns on ethylene induced genes, secondary transcription factors

• Transcription is last step in most pathways - makes sense that mutant is at the end

Genes Dev, 1998, 132:3703

Molecular characterization doesn’t always tell you what you want to know

• EIN2 shows similarity to a disease-related family of metal-ion transporters

• The strongest mutant!

Science. (1999) 284:2148-52

Recessive mutants reveal negative and postive regulation

ethylene

ETO1

ETR1

CTR1

EIN2

EIN3

response constitutive response

EIN3

EIN2

CTR1 X

constitutive response

CTR1

EIN2

EIN3

ETO1 X

EIN3 X

ethylene resistant

EIN2 X

ACC synthase inhibitor

Raf-like kinase

Membrane protein

Transcription factor

Membrane-bound His kinase

But, etr1 alleles are dominant

ethylene

ETR1

CTR1

response

ethylene

ETR1

CTR1

response

Dominant etr1 mutant is resistant to ethylene What is the interaction between ethylene, ETR1, and responses?

Dominant ETR1 - can’t bind ethylene,

always turns on CTR1?

Dominant ETR1 - signaling defect,

can’t turn off CTR1?

Why are all original etr1 alleles dominant?

• Loss-of-function mutant is dead – Essential gene

• Loss-of-function mutant has no phenotype – Redundant genes

ETR1 encodes a His kinase

• 5 members can homodimerize or heterodimerize • Have different C-terminal structural domains • 3 dominant mutants identified defective in

receptors


Identify recessive, loss-of-function alleles

ethylene

ETR1

CTR1

response

ethylene

ETR1

CTR1

response

Dominant ETR1 - can’t bind ethylene,

always turns on CTR1?

Dominant ETR1 - signaling defect,

can’t turn off CTR1?

Dominant etr1 mutant is resistant to ethylene

ETR1 X ETR1 X

ethylene resistant


Receptor loss-of-function mutants are ~wild type

Cell 1998, 94:261271

Triple and quadruple LOF mutants resemble ctr1

• Proteins are functionally redundant Cell 1998, 94:261271

wt ctr1 etr1-6 etr2-3 ein4-4

etr1-6 etr2-3 ein4-4

etr1-6 etr2-3 ein4-4 ers2-3

ctr1 wt

Loss of ethylene receptors leads to constitutive response

ethylene

ETR1

CTR1

response

Gain-of-function etr1 mutant shown to bind less ethylene Therefore, always turns on CTR1 ethylene insensitive phenotype

Ethylene acts to inhibit an otherwise active receptor

ETR1 X


A Schematic Model for Ethylene Signal Transduction and the MAPK Pathway in Ethylene Biosynthesis.Ethylene gas is perceived by the ER-integrated receptor proteins including

ETR1, ETR2, ERS1, ERS2, and EIN4 (Bleecker et al., 1988; Hua and Meyerowitz, 1998; Sakai et al., 1998; Voet-van-Vormizeele and Groth, 2008).

Zhao Q , and Guo H Mol. Plant 2011;4:626-634

© The Author 2011. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPP and IPPE, SIBS, CAS.

PresenterPresentation NotesA Schematic Model for Ethylene Signal Transduction and the MAPK Pathway in Ethylene Biosynthesis.Ethylene gas is perceived by the ER-integrated receptor proteins including ETR1, ETR2, ERS1, ERS2, and EIN4 (Bleecker et al., 1988; Hua and Meyerowitz, 1998; Sakai et al., 1998; Voet-van-Vormizeele and Groth, 2008). A Golgi-localized protein RAN1 (RESPONSIVE-TO-ANTAGONIST 1) is a P-type ATPase copper transporter that delivers the copper ion to the receptors to facilitate ethylene binding (Woeste and Kieber, 2000). RTE1 (REVERSION-TO-ETHYLENE SENSITIVITY 1), another membrane-located protein, promotes the transition of ETR1 from active to inactive state likely through modulating the action of ETR1 N-terminus (Dong et al., 2008; Resnick et al., 2008). In normal growth conditions in which the ethylene level is low, the unoccupied receptors remain in the active state and associate with CTR1, which, in turn, represses the downstream signaling pathway. When plants encounter stress conditions, the MAPK cascade composed of MKK4/5/9 and MPK3/6 can be activated, which then phosphorylates ACS2/6. The phosphorylated ACS2/6 become stabilized and consequently enhance the production of ethylene (Liu and Zhang, 2004). Upon binding by ethylene, the receptor complexes disassociate, and CTR1 released from ER membrane is somehow inactivated (Kieber et al., 1993; Clark et al., 1998; Huang et al., 2003). Therefore, the downstream ethylene signaling pathway including EIN2 is de-repressed (Alonso et al., 1999; Bisson et al., 2009). EIN2 is a short half-life protein targeted by SCFETP1/2 for degradation. Ethylene promotes the accumulation of EIN2 probably by down-regulating the level of ETP1/2 protein through an unknown mechanism (Qiao et al., 2009). In the nucleus, two transcription factors (EIN3 and EIL1) are both necessary and sufficient for the activation of ethylene-regulated gene expression and diverse responses (Chao et al., 1997; Solano et al., 1998; Alonso et al., 2003). EIN3 and EIL1 are also short-lived proteins that are targeted by SCFEBF1/2 for degradation (Guo and Ecker, 2003; Potuschak et al., 2003; Gagne et al., 2004). The ethylene signal is transmitted via the action of EIN2 to stabilize EIN3/EIL1, probably by promoting the proteasomal degradation of EBF1/2 proteins (An et al., 2010). EBF2 is a direct target gene of EIN3, which activates EBF2 transcription to form a negative feedback loop (Konishi and Yanagisawa, 2008). EBF1/2 mRNAs are subjected to negative regulation mediated by a 5'→3' exoribonuclease EIN5 (XRN4) (Olmedo et al., 2006; Potuschak et al., 2006). EIN3/EIL1 also directly regulate the expression of a diverse array of genes including ERF1 (ETHYLENE RESPONSE FACTOR 1), PORA, PORB, FLS2, and SID2, which initiate various interplays between ethylene and other signals, such as light and innate immunity (Chen et al., 2009; Zhong et al., 2009; Boutrot et al., 2010).The symbol ‘?’ represents an unknown factor or element. Arrows and T-bars represent positive and negative effects, respectively. Solid lines indicate effects that occur through direct interaction, whereas dotted lines indicate effects that have yet to be shown via direct interaction.

Arabidopsis thalianaArabidopsis as a genetic modelHIVE. coliS. cerevisiaeC. elegansArabidopsis thalianaD. melanogasterPopulus and PhyscomitrellaMaizeHumanRiceWheatArabidopsis Genome InformationArabidopsis as a developmental modelArabidopsis as a biochemical modelArabidopsis as a disease modelArabidopsis ResourcesDiscoveries made in plantsDiscoveries made in plantsDiscoveries made in plantsDiscoveries made in plantsDiscoveries made in plantsDiscoveries made in plantsArabidopsis Mutagenesis and Mutant ScreensRecombination mappingSlide Number 27Bulk segregant analysis and WGSThree questions in phytohormone researchWhat do phytohormones do?What do phytohormones do?What do phytohormones do?�Example 1 - GibberellinThe history of gibberellinFunctions of gibberellinGA mutants - barleygrd2 mutant defectWhat does grd2 tell us?What do phytohormones do?�Example 2 - EthyleneThe history of ethyleneEthylene functionsThe triple responseEthylene mutantsUsing the triple response to find mutantsUsing the triple response to find mutantseto mutants vs. ctr mutantsSo you have all these mutants ...Epistasis with ethylene mutantsEpistasis with ethylene mutantsHow to order ein2 and ein3?Molecular characterization can support epistasisMolecular characterization doesn’t always tell you what you want to knowRecessive mutants reveal negative and postive regulationBut, etr1 alleles are dominantWhy are all original etr1 alleles dominant?ETR1 encodes a His kinaseIdentify recessive, loss-of-function allelesReceptor loss-of-function mutants are ~wild type Triple and quadruple LOF mutants resemble ctr1 Loss of ethylene receptors leads to constitutive responseSlide Number 67

Documents

Assistant Professor, Biologygenetics.wustl.edu/bio5491/files/2015/01/Strader-Advanced-Genetics-2015.pdfThe history of ethylene • Ancient Egyptians - gas figs to stimulate ripening