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