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Shaping the cell:General aspects Cellulose
synthesis
Thomas Rausch
Molecular Physiology Lab
HIP, Heidelberg
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One of the defining features of plants is a body plan based on thephysical properties of cell walls.
Structural analyses of the polysaccharide components, combined
with high resolution imaging, have provided the basis for much of
the current understanding of cell walls.
The application ofgenetic methods has begun to provide new
insights into
- how walls are made,
- how they are controlled, and
- how they function.
However, progress in integrating biophysical, developmental, andgenetic information into a useful model will require a system-based
approach.
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System-based approach- design principles
Body plan of a higher plant: made ofosmotic bricks.Each cell osmotically pressurized to between 0.1 and 3.0 MPa. The
pressure rigidifies the cells by creating tension in the cell walls.
Each cell is glued to adjacent cells by pectic polysaccharides thatnormally prevent sliding of the cells under large strains.
Cell walls also capable ofcontrolled modifications: cell expansion,
polarized growth.
Because each cell wall is attached to adjoining cell walls,
coordinated expansion is necessary.
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- system structure
Structural analysis of cell wall
polysaccharides has resulted in thecompilation ofaverage structures for the
major cell wall polysaccharides: Cellulose,
hemicellulose, pectins
Scale model of the
polysaccharides in anArabidopsis leaf cell
The amount of the various
polymers is shown based
approximately on their
ratio to the amount of
cellulose. The amount of
cellulose shown was
reduced, relative to a
living cell, for clarity.
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- Control, Synthesis, and Assembly
- System Dynamics
A simplified system diagramfor a primary cell wall
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Cellulose microfibrils:Insoluble cable-like structures,
composed of approx. 36 hydrogen-
bonded chains containing 500 to
14,000 -1,4-linked glucosemolecules.
Schematic model of cellulose synthesis. Cellulose
synthesis takes place in the plasma membrane.The plasma membrane is tightly appressed to the cell wall so that most of the cellulose
synthase is in or below the plane of the membrane, which minimizes friction as the enzyme
moves through the plasma membrane in response to elongation of the growing glucan chains
by addition of glucan moieties from cytoplasmic UDP-glucose. The
cellulose synthase complex is thought to contain as many as 36 CESA
proteins, only a subset of which are illustrated. That three types of CESAproteins are required to form a functional complex suggested that different types of CESA
proteins perform specific functions, such as interacting with the cortical microtubules.
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Diagrammatic representation of the CesA
proteins of higher plants with the observed
positions of various known mutations
Catalytic domain
A B
HVR
HVR
CesA domains in different colors:
- N-terminus domain (blue)
- zinc-binding domain (light orange)
- eight TMDs (orange)
The processive glycosyl transferase signature D1,D2,D3,QXXRW is shown in gray boxes.
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CesA proteins may be phosphorylated
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Genomic complexity of CesA genes- poplar predicted 17-18 genes (isoforms for
primary/secondary cell wall?)
- Arabidopsis predicted 10 genes
function of 6 AtCesA genes identified in
mutants:
- at least 3 AtCesA genes required for
primary cell wall (A1, A3, A6)
- at least 3 other AtCesA genes required for
secondary cell wall synthesis (A4, A7, A8)
However, precise composition of (hexameric)
rosette complex still unresolved (2007)
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Correspondence of mutations and genes
implicated in cellulose synthesis
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Putative coexpressor genes involved in the
cellulose biosynthesis of Arabidopsis during
secondary cell wall development (19 genes)
Derived from coexpression patterns on Affymetrix microarray
S ll
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S
S
Pathogen attack
or wounding
Sink cell
Source cell
phlo
em
CWI CIF
SS
vacuole
VI VIF
S S
G+F
G+Fmetabolism + signalling
S
G+F
G+F
CI
SUSY UDPG
starch
cellulose
HK
respiration
cytosol
CWI CIF
TP
chloroplast
cytosol
Sucrose
metabolism in
growing sinktissues
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Conclusions1. The structure of cellulose microfibrils implies that the synthesis of
cellulose involves the coordinate activity of approximately 36 active
sites. However, diversity of cellulose structure in various organismsimplies that the enzyme complex is modular
2. Cellulose is synthesized by a 30-nm-diameter rosette-shaped
plasma membrane complex with six visible subunits
3. The only known components of cellulose synthase are a family of
CESA proteins, but mutations in genes for a number of other
proteins indicate that many other proteins are involved in the overall
process
4. Recent evidence from live-cell imaging of cellulose synthase
indicates that microtubules exert a direct effect on the orientation of
cellulose deposition under some conditions, but the microtubules
are not required for oriented deposition of cellulose under other
conditions
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...continued
5. Cellulose synthase is posttranslationally regulated and isknown to be phosphorylated but the mechanisms that
regulate activity are not yet known
6. The genes for cellulose synthase are developmentally
regulated, but there is relatively little evidence for
environmental regulation of expression
7. Cellulose synthase belongs to the large GT-A family 2 of
glycosyltransferases, which includes chitin synthase, but
the reaction mechanism is unknown.
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Tutorial (cellulose)
- How and where is cellulose synthesized? Which genes are involved?
- What are the individual roles of CesA genes?
- Explain the rosette structure of cellulose synthase!
- Describe the CesA protein structure (domains)!
- How is the cellulose synthase complex regulated?
- How does the cytoskeleton impact on cellulose synthesis?
- What are the phenotypes of mutations in CesA genes?
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Shaping the cell:Dynamic pectins and
role of pectin methylation status
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Synopsis
- Pectins: a highly dynamic, complex cell wall component
- Methylation status of homogalacturonan component
- The pectin methylesterase (PME) gene family
- Regulation of PMEs by inhibitory proteins (PMEIs)
- Characterization of two Arabidopsis PMEIs
- Processing oftype 1 PME: role ofPMEI-like prodomain
- Cloning ofPMEs and putative PMEIs expressed in
maize pollen
- Subcellular localization of putative PMEIs
- Pollen-expressed PMEs and PMEIs: How do they interact in vivo?
- Perspectives
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Schematic structure of plant cell wall
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Pectinsa highly dynamic, complex cell wall component
- localized in the apoplast
- contribute to the structural properties of the cell wall
- important for physiological processes such as
seed germination, fruit maturation etc.
- polymerized in the cis-Golgi- methyl-esterified in the medial Golgi
- substituted with side chains in the trans-Golgi
- secreted in highly methyl-esterified statusinto the cell wall
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Enzymes and substrates involved in
biosynthesis of homogalacturonan (HG)
S-adenosyl methionine & UDPGalA and their
transporters:
Import of necessary precursors into the Golgi
HG Galacturonosyl transferase (GAUT) & Pectin
Methyl-Transferase (PMT):
Supposed to act as hetero complex in polymerization
of fully methylesterified HG
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Current models of the pectin
network
Model A
Pectic backbone is an
extended chain with HG
and RG-I regions
(Visser and Voragen, 1996).
Model B/C/D
RG-I is decorated with neutral (AG-I, arabinan, possibly AG-II) and
HG/XGA side chains (Voragen et al., 2003):
B, only one kind of side chain is present,
C, sidechains are clustered randomly, orD, sidechains are arranged in a cluster-like
fashion.
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Ca++
- cross linking of deesterified HGA
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Pectins form complexes......A
Interaction through
Ca2+ bridges, more
then nine needed for
a stable connection
B
Borate-diol-esters
through RG-II
sidechains
C
Uronyl-esters through
trans-esterificationreactions
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Pectinases: Sites of attack
Methyl ester
GalA
Gal
Rha
Acetyl ester
PME
endoPGexoPG PLPLY RGH
HGAE
RGA
E
exoPG exopolygalacturonase
endoPGendopolygalacturonase
PLY pectate lyase
PL pectin lyase
PME pectin methylesteraseHGAE homogalacturonan acetylesterase
RGH rhamnogalacturonan hydrolase
RGL rhamnogalacturonan lyase
RGAE rhamnogalacturonan acetylesterase
RGL
The pectin methylesterase (PME) gene family
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The pectin methylesterase (PME) gene family
(inA. thaliana 67 annotated members!)
Domain structure ofA. thaliana type-I PME (CAB80040)
Signalpeptide
(aa 1-45)
pro-domain (aa 46-ca. 225)
similar to at5g46970
core domain (aa ca. 225-609)
similar to bacterial/fungal PMEs
Type-I PMEs: pre-pro-proteins
Type-II PMEs: no pro-domains
Pro-domains: sequence similarity with At-PMEI-RPs on chromosome 5
C-terminal domains of type-I PMEs and protein sequences of type-II
PMEs show sequence homology with bacterial and fungal PMEs
Outsourcing the post-translational regulation of PME:
From a regulatory pro-domain toan independent gene function, PMEI ?
Nt-VIF96
Bootstrap1 T-DNA transformed Arabidopsis lines
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Nt-CIF
at1g47960
at3g17130
at2g31430
at3g55680
at1g48020
at3g17220
Ac-PMEI
at5g64620
at3g12880
at5g50070
at5g46970
at5g46940
at5g46960
at5g46980
At1g23350
ZmPMEI-RP1
Zm-PMEI-RP2
Zm-PMEI-RP3
Zm-PMEI-RP4
100
86
82
69
97
96
100
98
99
98
100
95
100
100
1
1
2
1
Arabidopsis knock out facility [AKF], Madison
available within GABI
available within GABI
available within GABI
available within GABI
T DNA transformed Arabidopsis lines
Arabidopsis PMEI protein
family, including invertase
invertase inhibitors
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Group 1 (light
blue circles) and
Group 2 (darkblue circles)PMEs are showntogether withPMEIs
(redcircles).
Microarray data
and cluster
analysis was
realized using
Genevestigator
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Genomic complexity of target
enzyme families:CWI, VI and PME
6 putative CWI genes (3 as ESTs)
2 putative VI genes (2 as ESTs)CAZy Family Glycoside Hydrolase Family 32Known Activities invertase (EC 3.2.1.26); and others*
and a total of
67 putative PME genes (type-I & -II)CAZy Family Carbohydrate Esterase Family 8
Known Activities pectin methylesterase (EC 3.1.1.11.)*
*Coutinho, P.M. & Henrissat, B. (1999) Carbohydrate-Active Enzymes server atURL: http:afmb.cnrs-mrs.fr/~cazy/CAZY/index.html
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Processing of type I PectinMethylesterase in the Golgi Apparatus:
Prerequisite for extracellular targeting
P ti
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Rhamnogalacturonan I
Rhamnogalacturonan II Homogalacturonan (HG)
Synthesized in the Golgi
Secreted highly methylesterified forms distinct domains defined
by degree and pattern of
methylesterification de-methylated patches form
Ca2+-crosslinked gels
cell-wall stiffening
Pectins
P ti th l t (PME)
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Pectin methylesterase (PME)
Cell wall loosening Cell wall rigidificationMicheli 2001, TIPS
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HG properties affect plant growth and developmentvgd1
Jiang et al., 2005, The Plant Cell
VANGUARD1 (VGD1) is necessary for normal pollen tube development
WT WTvgd1
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HG properties affect plant growth and development
35S:VGD1
35S:VGD1
WT
WT 35S:VGD1
2F4
Low Methyl
JIM7
High Methyl
Immunohistochemical staining with pectin antibodies
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PMEs and Inhibitors in the Arabidopsis genome
Type II PMEs 22 ORFs
PME Inhibitors (PMEIs) 39 ORFs
Type I PMEs 44 ORFs
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Analysis of PME linker region
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Proteolytic Processing in two type I PMEs
VGD1proPME-1
unprocessed
intermediatemature
unprocessed
intermediate
mature
S b ll l l li ti f VGD1
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Subcellular localization of VGD1:
GFP and proPME-1:GFP
proPME-1 M3:GFPVGD1:GFP proPME-1:GFP
72 hpi
total cell wall total cell wall total cell wall
48 hpi
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Intracellular PME fluorescence overlaps with
Golgi-Marker GONST1
GONST1:mRFP
VGD1:GFP
proPME-1M3:GFP
Bars = 10 M
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Targeting is mediated exclusively by theN-terminal region of PMEs
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Inhibitory role of the pro region ?
0
50
100
150
200
250
300
secGFP proPME-1 proPME-1 M3
P
MEactivity[%]
n = 12
In vitro activity assay with recombinantpro region protein
Extracts from plants expressing proPME-1and proPME-1M3 (unprocessed mutant)
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Mammalian Site-1 protease (SKI-1) cleavesRRLL-type motifs
AtS1P has recently been characterized by the Howell
lab (Liu et al., 2007, TPJ; Liu et al., 2008, TPC)
Involved in bZIP TF release from ER membrane
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AtS1P colocalizes with VGD protein
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Summary
- Type I PME processing occurs at two basic motifs
- Processing occurs inside of the cell, presumably in the Golgi Apparatus
- Unprocessed protein is retained in the Golgi and only fully mature protein
is secreted into the extracellular space
- The N-terminus is sufficient to mediate cellular targeting/retention
- The pro region has only weak inhibitory capacity
- AtS1P, a subtilisin-like protease can form a complex with PMEs in
the Golgi
PME activities can be
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PME activities can be
regulated by changes in cell wall pH
degree of pectin methylation
concentration of methanol
various cations
phytohormones (auxin, ABA, GA3, ethylene)
constitutive/differential gene expression
post-translational silencing by inhibitor proteins
Example of hormonal effect (ethylene) on pectin turnover:
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Pectin turnover induces ethylene formation via oligogalacturonide signalling,
but ethylene negatively feeds back on pectin turnover by inhibiting PMEexpression
hydrolysis of demethylated pectin by polygalacturonidase
PME mRNA
PME activity
pectin demethylation
release of oligogalacturonide (DP4-6)
increased ethylene synthesis via induction of ACC oxidase
repression of PME transcription
ethylene
PMEI
Structural Basis for the
I t ti b t
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Interaction between
Pectin Methylesteraseand a Specific Inhibitor
Protein
Di Matteo et al. 2005 Plant Cell
Structural Insights into
the Target Specificity of
Plant Invertase andPectin Methylesterase
Inhibitory Proteins
Hothorn et al. 2004 Plant Cell
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PME-PMEI interactions:
A crucial factor for plant development impinging
on plant cell shape?a. spatial gradients of cell wall structure,
signalling and/or wall extensibility
b. precisely determined temporal PME activitypatterns
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Basic assumptions
PME activity has a dual function:
Change ofbiophysical cell wall properties & generation ofsignals
(H+
/Ca2+
ratio, oligosaccharides, ascorbic acid precursor, WAK?)
PMEIs and PMEs are (also) ligands and receptors, respectively
PMEs and PMEIs have co-evolved to achieve specificity
Binding constants forprodomains of type I-PMEs are weak:
after release from catalytic PME core no PMEI function in vivo
Corresponding PMEIs and PMEs) are secreted by
neighbouring/communicating cells (cell groups)
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A secretes PMEI
- diffuses from A, following exponential gradient
- is transported in extracellular water film
B secretes type I/II PME
- diffuses from B, following exponential gradient
- removal of prodomain upon arrival in cell wall (?)
- prodomain NOT active in cell wall
- enzymatic deesterification
- immediate change in cell wall biophysics
- activation of polygalacturonidase
- cellular signals: change of H+/Ca2+ ratio synthesis of ascorbic acid precursor release of oligosaccharides activation of WAK?
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Pollen as a model system to study PMEand PMEI function
Co-expression of PME and PMEI
in pollen:
Do they interact, and if yes, is it part ofPME regulation?
Model for pollen tube growth
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Model for pollen tube growth
Lord (2000)TIPS
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Two Arabidopsis thaliana PMEI isoforms are exclusivelyexpressed in pollen
(microarray data indicate that several PME and PMEI isoforms are expressed
inArabidopsis pollen)
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Localization of PMEIrp1::YFP
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Localization ofPMEIrp1::YFP
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Transient expression of ZmPMEI 2 or ZmPMEI3 impairs
pollen tube germination and expansion
Transformed pollen identified via co-expression with cytosolic YFP-construct
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0.03U/ml
0.06U/ml
mock
In the presence of orange peel PME (Sigma)
the germination of pollen is inhibited
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A Hypothetical Model of
Pectin Modification in the
Pollen TubeGroup I PMEs and PMEIs travel from theGolgi apparatus to an annulus-shaped zone
just below the extreme apex, where they are
secreted. It is presently unclear whether PMEI
and PME form a complex upon arrival in thecell wall (1) or are already associated in the
secretory vesicles (2).
At the supapical region of the pollen tube,
PMEI is internalized via clathrin-mediatedendocytosis following dissociation of the
complex in response to unknown cues. As a
result, PME is free to perform de-
methylesterification of the pectin in the shank
of the pollen tube.
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Tutorial (pectins)
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- How and where are pectins synthesized?
- Of which components are pectins made and how do they interact?
- Why does PME activity change the biophysical properties of pectin?
- How does PME activity impact on signaling processes?- Pectin formes complexes with ions, other wall polymers: How?
- How are pectins degraded?
- Explain the difference between type I and type II PMEs!- Which hypothesis exist for the role of the prodomain?
- Where and how is the prodomain removed?
- Which role play pectins and pectin esterification during pollen tube growth?
- Speculate on the role of the large PME and PMEI protein families in plants!