Plant Cell Biol Modul3 Rausch SS10

8/6/2019 Plant Cell Biol Modul3 Rausch SS10

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Shaping the cell:General aspects Cellulose

synthesis

Thomas Rausch

Molecular Physiology Lab

HIP, Heidelberg

[email protected]


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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




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!

Plant Cell Biol Modul3 Rausch SS10

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