Ethylene

Science Vol. 241, no. 4869, 26 August 1988, photo by Kurt Stepnitz, Michigan State

University

Ethylene: chemically

simple but functionally

complex

Air Ethylene

Arabidopsis

Ethylene (C2H4) is a

gaseous hormone with

diverse actions

Yoo et al., Trends in Plant Science (2009)

Ethylene responses in Arabidopsis

Lorenzo, O., Piqueras et al.,(2003) Plant Cell 15: 165-178

Rüžička, K., Ljung et al., (2007) Plant Cell 19: 2197-2212.

Inhibition of leaf

cell expansion Acceleration of leaf senescence

Ethylene-induced gene expression

Inhibition of root

elongation

http://www.plantcell.org/cgi/content/full/15/1/165http://www.plantcell.org/cgi/content/full/15/1/165http://www.plantcell.org/cgi/content/full/15/1/165http://www.plantcell.org/cgi/content/full/19/7/2197http://www.plantcell.org/cgi/content/full/19/7/2197http://www.plantcell.org/cgi/content/full/19/7/2197

Ethylene insensitive

Bleecker, A.B., Estelle, M.A., Somerville, C., and Kende, H. (1988). Science 241: 1086

Ethylene response – receptors and downstream

signaling

bri1

Wild-type

In wild-type plants, root

growth is inhibited by

elevated BR levels

Ethylene-regulated gene expression is

negatively regulated

In the absence of

ethylene, CTR binds the

receptor and prevents

transcription.

Ethylene binding to the

receptor releases CTR,

permitting transcription.

Receptor

active CTR

Ethylene

Benavente, L.M., and Alonso, J.M. (2006)

Cuo, H., and Ecker, J.R. (2004) Curr. Opin. Plant Biol.

- +

P

P

inactive CTR

ETHYLENE RESPONSE1 (ETR1) encodes

an ethylene receptor

ETR1 was the first protein to be unambiguously

identified as a phytohormone receptor (1993)

• ETR1 binds ethylene

• ETR1 is membrane localized (ER)

ETR1

histidine kinase receiver GAF ethylene binding

The GAF domain is sufficient to mediate

heteromerization

ERS1

EIN4

ETR2

ERS2

Subfamily I

(Type 1)

Subfamily II

(Type 2)

ETR1

histidine kinase receiver GAF ethylene binding

Arabidopsis ethylene receptor family

ETR1: ethylene response 1 ERS1: ethylene sensor 1 ERS2: ethylene sensor 2 ETR2: ethylene response 2 EIN4: ethylene insensitive 4

Type 1: contain a conserved histidine kinase domain Type 2: contain a degenerate histidine kinase domain

Two-component signal transduction

Skerker and Laub (2004) and Nature Reviews Microbiology 2: 325

The two components are sensor histidine kinases and response regulators

Multicomponent phosphorelays also exist

Signaling mechanisms of the ethylene receptors

and CTR1 in ER

The amino terminal sensor domains of the receptors contain a copper cofactor (Cu) that is needed for

ethylene binding and are

associated with the ER

membrane

Binds 1 copper ion per dimer

CTR1 interacts with the

histidine kinase domain of

the receptors, at a higher

affinity with type-1

members than with type-II

members

Guo and Ecker (2004) Current Opinion in Plant Biology 7: 40

CTR1

ctr1

Amino acid sequence of ETR1

1 MEVCNCIEPQ WPADELLMKY QYISDFFIAI AYFSIPLELI YFVKKSAVFP

51 YRWVLVQFGA FIVLCGATHL INLWTFTTHS RTVALVMTTA KVLTAVVSCA 101 TALMLVHIIP DLLSVKTREL FLKNKAAELD REMGLIRTQE ETGRHVRMLT 151 HEIRSTLDRH TILKTTLVEL GRTLALEECA LWMPTRTGLE LQLSYTLRHQ 201 HPVEYTVPIQ LPVINQVFGT SRAVKISPNS PVARLRPVSG KYMLGEVVAV 251 RVPLLHLSNF QINDWPELST KRYALMVLML PSDSARQWHV HELELVEVVA 301 DQVAVALSHA AILEESMRAR DLLMEQNVAL DLARREAETA IRARNDFLAV 351 MNHEMRTPMH AIIALSSLLQ ETELTPEQRL MVETILKSSN LLATLMNDVL 401 DLSRLEDGSL QLELGTFNLH TLFREVLNLI KPIAVVKKLP ITLNLAPDLP 451 EFVVGDEKRL MQIILNIVGN AVKFSKQGSI SVTALVTKSD TRAADFFVVP 501 TGSHFYLRVK VKDSGAGINP QDIPKIFTKF AQTQSLATRS SGGSGLGLAI 551 SKRFVNLMEG NIWIESDGLG KGCTAIFDVK LGISERSNES KQSGIPKVPA 601 IPRHSNFTGL KVLVMDENGV SRMVTKGLLV HLGCEVTTVS SNEECLRVVS 651 HEHKVVFMDV CMPGVENYQI ALRIHEKFTK QRHQRPLLVA LSGNTDKSTK 701 EKCMSFGLDG VLLKPVSLDN IRDVLSDLLE PRVLYEGM

GAF domain

Transmembrane domain

Histidine kinase domain

Response regulatory

Position 4 : C S : Prevents dimerization but ethylene binding (position 6) Position 31: A V in etr1-3; ethylene insensitivity Position 62: I F in ert1-4; ethylene insensitivity Position 65: C Y or S in ert1-1; no copper binding and ethylene insensitivity Position 69: H A in ert1-1; no copper binding and ethylene insensitivity

The ethylene-binding domain

NH2

ETR1

histidine kinase receiver GAF ethylene binding

Rodríguez et al., (1999). A copper cofactor for the ethylene receptor ETR1 from Arabidopsis. Science 283:

Arabidopsis ethylene receptors resemble

hybrid histidine kinases

The GAF domain is sufficient to

mediate heteromerization, a secondary

structure motif

Many signaling components were identified genetically

ctr1

ein2 ein3 ein5 ein6

Constitutive-response mutants

Ethylene-insensitive mutants

etr1 etr2 ein4

air

C2H4

Ethylene-insensitive –

no triple response in ethylene

Constitutive response –

triple response in air

“Triple response” of etiolated dicotyledonous seedling

- Inhibition of hypocotyl and root cell elongation

- Radial swelling of the hypocotyl

- Exaggerated curvature of the apical hook

CTR1 is a negative regulator of

ethylene signaling

Kieber et al., (1993) Cell 72: 427

Air

Ethylene

Wild type ctr1

The ctr1 mutant has a constitutive

triple response

The genetic pathway of ethylene signaling

CTR1

ETR1 ERS1 ETR2 EIN4 ERS2

EIN2 EIN3 EIN5 EIN6

responses to ethylene

C2H4

(constitutive)

Receptor

family

CTR1 is a serine/threonine

protein kinase that resembles

animal Raf kinases and is

predicted to act in a MAPK

cascade

Ethylene acts as a negative regulator of the

signaling pathway

Receptor

active CTR

Ethylene - +

P

P

inactive CTR

This means that….

The signal pathway is turned on in the absence of ethylene and is shut

down when ethylene is present.

As a result of this negative regulation, mutations in the ethylene receptor are

perceived as dominant gain-of-function mutation.

Perception of Brassinosteroids by the Extracellular

Domain of the Receptor Kinase BRI1

He et al, (2000) Science 288: 2360-2363

An assay was developed to study plant receptor kinase activation and

signaling mechanisms. The extracellular leucine-rich repeat (LRR) and

transmembrane domains of the Arabidopsis receptor kinase BRI1, which is

implicated in brassinosteroid signaling, were fused to the serine/threonine

kinase domain of XA21, the rice disease resistance receptor. The chimeric

receptor initiates plant defense responses in rice cells upon treatment with

brassinosteroids. These results, which indicate that the extracellular domain

of BRI1 perceives brassinosteroids, suggest a general signaling mechanism

for the LRR receptor kinases of plants. This system should allow the

discovery of ligands for the LRR kinases, the largest group of plant receptor

kinases.

A) The XA21 and BRI1 protein

structures are labeled in white and

gray, respectively, with signal

peptides indicated in dark gray.

These chimeras were constructed

by in vitro mutagenesis and driven

by the cauliflower mosaic virus

35S promoter in rice cells

B) Northern hybridization shows

mRNA accumulation of each

chimeric gene, with a 1.3-kb DNA

fragment of the Xa21 kinase

domain as a probe.

C) Western blot shows the expression

of BRI1-XA21 chimeric proteins.

Schematic diagram of chimeric receptor kinases NRG1, NRG2, and

NRG3 and mutant controls NRG1mL and NRG1mK

He et al, (2000) Science 288: 2360-2363

http://www.sciencemag.org/content/288/5475/2360/F2.large.jpg

(A)Cell suspensions: NRG1-30, NRG1-34,

NRG1mL, NRG1mK, and wild-type

Taipei 309 were treated with 2 μM BL

for 24 hours. Cell death was assayed as

described in Fig. 1A.

(B) NRG1 and control cell lines were

treated for 30 min with 2 μM BL for

H2O2 production assay, with gray bars

for treatment and open bars for

nontreatment.

(C) Cell lines were treated with 2 μM BL

for 0 to 24 hours. Transcript

accumulation of defense-related

genesRCH10, PAL, and OsCatB was

determined by Northern blotting

(D)RNA levels were estimated as in Fig.

1D. Cell lines are NRG1-30 (▪), NRG1-34 (•), NRG1mL (□), NRG1mK (○), and Taipei 309 (▵).

BL induces cell death, oxidative burst, and defense pathway

activation in NRG1 cell lines

He et al, (2000) Science 288: 2360-2363

http://www.sciencemag.org/content/288/5475/2360/F3.large.jpg

Cells were treated with 0 to 4 μM BL. RNA

was extracted 6 hours after treatment, and

transcript levels were determined (24). Cell

lines are NRG1-30 (▪), NRG1-34 (•), NRG1mL (□), NRG1mK (○), and Taipei 309 (▵).

BL dose response for RCH10 induction in NRG1 cell lines

He et al, (2000) Science 288: 2360-2363

http://www.sciencemag.org/content/288/5475/2360/F4.large.jpg

Osakabe et al., (2013) 64:445-458

Overview of plant receptor-like kinases (RLKs)

and their functions

The model of ERECTA, BRI1, BAK1, and FLS signalling pathways.

Osakabe Y et al. J. Exp. Bot. 2013;64:445-458

Guo H et al. PNAS 2009;106:7648-7653

HERK1, THE1, and FER are related receptor-like kinases

induced by BRs

Guo H et al. PNAS 2009;106:7648-7653

(A) Expression of HERK1, THE1, and FER is induced by BL treatment in 10-day-old seedlings and 4-week-old adult plants.

(B) HERK1, THE1, and FER are up-regulated in bes1-D and down-regulated in bri1. Ten-day-old seedlings were used to prepare RNA for qRT-PCR as described in A.

(C–E) HERK1 cellular localization: A HERK1-GFP (C) or BES1-D-GFP

(D) construct was introduced into protoplasts or transgenic plants

(E). HERK1 is mostly localized at the plasma membrane (C and E), whereas BES1-D is primarily in the nucleus

(F) Autophosphorylation was detected by phosphorimaging, and the proteins were detected by SYPRO RUBY staining. (G–I) Expression patterns of HERK1, THE1, and FER in 10-day-old seedlings (G and H) or 3-week-old adult leaves (I) as revealed by GUS reporter gene. (Scale bars: C and D, 10 μm; G and H, 2.5 mm; I, 10 mm.)

Transcription factor (TF)

Yin et al., (2002)109 : 181–191

http://www.sciencedirect.com/science/article/pii/S0092867402007213#gr4

HERK1, THE1 and FER are required for cell elongation

Guo H et al. PNAS 2009;106:7648-7653

(A) BR responses of the herk1, the1, and fer mutants at the seedling stage.

(B) Shoot phenotypes of 24-day-old adult plants. (Scale bar: 10 mm.)

(C) The leaves of the WT, herk1 the1 double mutants, and fer1 mutants, showing the reduced lengths in leaf blades and leaf petioles. (Scale bar: 10 mm.)

(D) Quantification of petiole lengths of the 6th leaf in WT and mutants.

(E and F) The herk1 the1 double mutant plants have reduced cell elongation. The petioles of the WT (E) and herk1 the1 double mutant (F) plants were fixed, stained with toluidine blue, and embedded. Longitudinal sections were examined under a bright-field light microscope and photographed.

HERK1/THE1/FER and BR pathways affect independent genes with some overlap.

Guo H et al. PNAS 2009;106:7648-7653

Yin et al., (2002)109 : 181–191

Braam et al., (1997)

Braam et al., (1997)

Braam et al., (1997)

Braam et al., (1997)

The Plant Cell Wall

Cosgrove (2000). Nature 407, 321-326.

All land plants make a primary cell wall that is remarkably similar

in structure to, but distinct from, that of most algae, fungi, bacteria

and other groups with cell walls.

Cellulose microfibrils are synthesized by large complexes in the

plasma membrane. Newly secreted cellulose then associates with

matrix glycans (hemicelluloses and pectins) which are synthesized

in the Golgi apparatus and delivered to the wall by secretory vesicles.

The cellulose microfibril is a thin ribbon. It consists of an ordered

array of many parallel chains of an unbranched glucose polymer

(1,4-&upbeta;-glucan).

Hemicelluloses typically are branched polysaccharides

characterized by a strong tendency to bind to cellulose, whereas

pectins are generally acidic polysaccharides with a strong tendency

to form ionic gels. A small amount of structural protein is also

found on the wall, but its function is uncertain.

a diagram of cell-wall assembly

Acid Growth hypothesis

This is dependent on the growth hormone auxin.

The lower pH, in turn, activates growth-specific enzymes that hydrolyze the bonds holding the cellulose microfibrils to xyloglucan. The cleavage of these bonds results in the loosening of the cell wall. Causes uptake of water – which leads to a passive increase in cell size.

Auxin activates a plasma membrane proton pump, which acidifies the cell wall. Experimentally, H-ions have the same effect as auxin – so lowering the pH is a good substitute.

From: Biochemistry and Molecular Biology of plants Cosgrove (2000). Nature 407, 321-326.

Wall-loosening enzymes

Expansins: Break hydrogen bonds between cellulose and xyloglucan.

They are the only proteins shown to the active expansion of cell walls in vitro. They are always present in growing tissue of all

plant cell material.

When expansins are added to a heat-inactivated stem sample at acidic pH, cell wall extension is restored.

From: Biochemistry and Molecular Biology of plants Cosgrove (2000). Nature 407, 321-326.

Phylogenetic tree of rice alpha- and beta-expansins

Cosgrove (2000). Nature 407, 321-326.

A model of expansin's wall-loosening action

Cellulose microfibrils are connected to each other by glycans (thin yellow and red strands) that can stick to the microfibril surface and to each other. The expansin protein (blue) is hypothesized to disrupt the bonding of the glycans to the microfibril surface (a) or to each other (b). Under the mechanical stress arising from turgor, expansin action results in a displacement of the wall polymers (c) and slippage in the points of polymer adhesion (compare a and c).

Cosgrove (2000). Nature 407, 321-326.

Schematic diagram of how expansin might induce cell wall enlargement by breaking the non-covalent bonding

(short bridges) between cellulose (large rod) and

hemicellulose (curved line).

The presumptive binding domain (‘CBD’, sketched as a tail)

is hypothesized to anchor expansin to the cellulose surface,

while the putative catalytic domain (‘cat dom.’) would be

able to interact with hemicellulose at the microfibril surface

or in the matrix between microfibrils.

The dotted arrow indicates the direction of expansin motion,

which would be driven by release of mechanical strain

energy in the wall polymers. The hemicellulose bonding to

the cellulose is reversible (short bridges, broken lines) and

results in an inchworm-like motion of the hemicellulose

and a stress relaxation of the wall.

Expansive growth of plant cell walls

Cosgrove (2000)

Predicted structure of expansin protein

Cosgrove (2000). Nature 407, 321-326.

Geitmann and Ortega (2009) 41:467-478

Mechanics of a composite material with parallel

arrangement of the fiber component

Overview of cell wall expansion patterns resulting in

various cellular geometries

Geitmann and Ortega (2009) 41:467-478

http://www.google.co.kr/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&docid=fUSRAkUIm2wzIM&tbnid=-1A5dgROzSIKSM:&ved=0CAUQjRw&url=http://www.saburchill.com/chapters/chap0069.html&ei=DONEU-CuLMfSkwXaqIHQAg&bvm=bv.64507335,d.dGI&psig=AFQjCNEhM_qlzX9LXH7kuv4bfBjhQqBH0g&ust=1397109846428043http://www.google.co.kr/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&docid=OwZS1kLzzofkpM&tbnid=c6bT-visMHppiM:&ved=0CAUQjRw&url=http://en.wikipedia.org/wiki/Tomato&ei=TONEU5j5LMqekQWTrIHADg&bvm=bv.64507335,d.dGI&psig=AFQjCNGFshLwo0IZIXqzcpF9YmPYbjiyLw&ust=1397109945985169

Kohorn (2001) 13:529-533.

WAKs; cell wall associated kinases

Kohorn (2001) 13:529-533.

GRP : glycine rich protein

an a-expansin gene Le-EXP1 was found to be expressed

specifically in the later stages of tomato fruit ripening and

its expression was also stimulated by the ripening hormone

ethylene.

A role in fruit softening

strawberry and cantaloupe, a-expansin mRNAs are also

expressed in late stages of ripening and so this may be a common

feature of fruit softening. Wall protein extracts from several kinds

of ripening fruit possess significant expansin activity.

http://www.google.co.kr/url?sa=i&rct=j&q=&esrc=s&source=images&cd=&cad=rja&uact=8&docid=hyDMAWhMCrgo1M&tbnid=OHUw4vaOMGDPeM:&ved=0CAUQjRw&url=http://www.aaas.org/news/science-breeding-tomatoes-look-pretty-sacrifices-their-sweetness&ei=ahNFU6bsB8fEkQWV4ICYCw&bvm=bv.64507335,d.dGI&psig=AFQjCNHCX6z7HKyt5jpTXxdmCJqnip4x-Q&ust=1397122260832344