Download The Plant Cell Wall

The Plant Cell Wall
Why have a cell wall?
•  Plants have ‘discovered’ the ecological niche of
photoautotrophy.
•  To successfully compete for sunlight, plants need to
grow (high, fast, directed, adaptive).
•  To support growth, plants had to ‘invent’ construction
material that was dynamic and mechanically stable.
•  The cell wall is a complex molecular network of
carbohydrates and glycoproteins that is both.
Plant cell shape is defined by the cell wall
root growth movie
Plant cell shape is defined by the cell wall
Every plant cell is surrounded by a wall
primary cell wall (mechanical stress): cellulose,
hemicellulose, glycoproteins
primary cell wall (cell adhesion and separation): pectin, soluble
glycoproteins, enzymes, peptide
1mm
The cell wall is involved in every aspect of plant life
division
defence
expansion
secondary wall thickening
morphogenesis
Cell walls and plant health
•  The cell wall is the first line of defence against pathogens
•  The cell wall is actively degraded by many pathogens
•  An infected plant locally modifies its cell wall to defend itself
•  Cell walls are modified or newly made during parasitic and symbiotic
plant microbe interactions (e.g. nematode syncytia, arbuscular
mycorrhizal plant cell interface)
•  Genetic alterations of cell wall polymers can lead to systemic
pathogen resistance and enhanced tolerance of abiotic stress
(drought, cold etc.)
Cell wall molecular biology
•  Structure
•  Biosynthesis (biochemical and genetic perspective)
•  Map based cloning of biosynthetic genes
•  Cell biology or oriented cell wall deposition
•  Cell wall remodelling/loosening
•  Signal function of cell wall carbohydrates
Primary cell walls consist of interacting
networks carbohydrates
Cell wall fractions
Cellulose insoluble in aqueous buffers, Glc
Cell wall matrix polymers
Hemicellulose
neutral, soluble in alkali, Glc, Xyl,
Gal, L-Fuc, GlcA
Pectin
acidic, soluble with Ca2+-chelating
buffers, GalA, L-Rhm, L-Ara, Gal
Glycoproteins
partially water soluble, lipid-anchored
or bound to other polymers, Gal, L-Ara
L-Rhm, GlcA
Enzymes
mostly soluble
Lignin
insoluble, only secondary walls, polyphenolic
Heterogeneity of cell wall components
Plant cell wall carbohydrates contains ca. sixteen different
monosaccharides
also present: Aceric acid, L-galactose, N-acetyl-D-glucosamine, Kdo, Dha
Carbohydrates consist of chains of sugars linked in a specific way
Cellulose is a linear polymer
(1→4)β-D-glucan
Dozens of cellulose chains form paracrystalline microfibrils
Xyloglucan is the main hemicellulose in dicot primary cell walls
(1→4)β-D-glucan backbone with (1→6)α-D-xylose side chains
Glucuronoarabinoxylan is the main hemicellulose in dicot
secondary cell walls
(1→4)β-D-xylan backbone with (1→2)α-D-glucuronic acid and (1→2)α- Larabinose side chains
Pectin consists of three different polymers
Homogalacturonan (HGA)
methylesterified (1→4)α-D-galacturonan
Developmental variation of HGA esterification
highly esterified
relatively de-esterified
Rhamnogalacturonan I (RG I) contains different side
chains
(1→5)α-L-arabinose
(1→4)α-D-galactose
→2)α-D-rhamnose-(1→4)α-D-galacturonan
Rhamnogalacturonan II (RG II) has the most complex
known carbohydrate structure
Cell wall proteins contain hydroxyproline
•  Ara modification is predicted by the Ser(Pro)4 sequence
•  Extensins forms stiff rods.
•  Extensins can be oxidatively cross-linked by the action of cell wall peroxidases.
•  Extensin cross-linking might rigidify the cell wall after expansion has seized.
Arabinogalactan-proteins (AGPs) contain lipid anchors and
very complex carbohydrate modifications
Arabinogalactan-proteins (AGPs)
•  AG-modification predicted by Pro-Ala-Pro-Ala sequence.
•  hydrophobic C-terminus is replaced by cleavable glycolipid (GPI-anchor).
•  hundreds of different AGP like proteins exist in the cell wall.
•  AGPs have been implicated with many biological roles.
Summary cell wall structure/components
Cellulose
Cell wall matrix polymers
Hemicellulose
xyloglucan (XG)
glucurono(arabino)xylan
Pectin
homogalacturonan (HGA)
rhamnogalacturonan I (RGI)
rhamnogalacturonan II (RG II)
Glycoproteins
extensin
arabinogalactan-proteins (AGPs)
Enzymes
glycosyl hydrolases
esterases
peroxidases
...
The biosynthesis of complex carbohydrates depends on
the supply of activated monosaccharides
free sugar
activation
nucleotide
sugar
Interconversion
generates new
sugars
glycosyl
transferase
polymers and
conjugates
NDP-sugars are interconverted by
oxidoreductases and isomerases
UDP-D-Glc -4-epimerase
UDP-D-Glc
UDP-D-Gal
NAD+
UGD
NADH
UDP-D-GlcA -4-epimerase
UDP-D-GalA
UDP-D-GlcA
UXS
CO2
UDP-D-Xyl -4-epimerase
UDP-D-Xyl
UDP-L-Ara
Biosynthesis takes place at different cellular locations
How can the molecular machinery of cell wall
biosynthesis be determined?
•  biochemical approach
•  forward genetic approach
•  heterologous expression of candidate genes
•  systems biological approach
Biochemical-molecular dissection of cell wall
biosynthesis
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Reconstitute reaction in cell-free system
Acceptor and donor substrate
Detect product
Solubilize (!) and purify the activity
Isolate the enzyme
Sequence the peptide(s)
Clone the cDNA, gene
Example: Cloning of β-mannan synthase
•  In some plants hemicellulose acts as storage
polysaccharide analogous to starch
•  guar, fenugreek, locust beans, coconut:
galactomannan
•  tamarind: xyloglucan
•  galactomannan is used as food stabilizer (e.g. ice
cream)
Example: Cloning of β-mannan synthase
•  Reaction was assayed in microsomes of developing guar seeds by
detecting radioactive β-mannan using endomannanase
•  The enzyme was solubilized using digitonin
•  Peptides of a relatively pure fraction were partially sequenced
•  A new library of 15000 cDNA clones of the source tissue was made and
sequenced
•  The matching clones were assembled to obtain a full sequence
•  The gene was functionally expressed in soybean cells
•  The mannan-synthase belongs to the family of cellulose synthase-like
genes (Csl).
•  The work was done at Pioneer Dupont (Dhugga et al 2004 Science 303:
363ff)
Further examples of biochemical cloning
•  xyloglucan-specific fucosyl transferase (FUT1)
•  galactomannan-specific galactosyl transferase
•  homogalacturonan (HG)-specific galacturonosyl transferase (GAUT)
Molecular genetic dissection of cell wall
biosynthesis
•  Devise/perform a screen (based on hypothesis)
•  Characterize the mutant
•  Clone the gene
Example: Cloning of REB1, a gene required for normal roots and
arabinogalactan-protein (AGP) composition
2D electrophoresis of root AGP
REB1 (WT)
reb1
reb1 is a recessive single locus
X
P
↓
F1
↓
F2: 75%:25%
Single loci can be identified by mapping
ecotype L
X
ecotype C
↓
F1
↓
F2: 75%:25%
Single loci can be identified by mapping
{
:
≈ 50%
:
⇒ 100%
markers
Markers can detect differences in DNA sequencel/length at a defined
chromosomal locus (e.g. PCR fragments, restriction sites, single nucleotide
polymorphisms SNPs...)
For fine mapping, plants containing recombinations
close to the mutant locus are selected
•  DNA polymorphisms close to the locus have to be used / identified.
•  Thousands of mutant F2 plants are screened to find sufficiently close
recombinants.
Gene identification by direct sequencing, KO allele
selection and complementation.
•  Gene annotation databases for Arabidopsis:
http://atidb.org/ etc.
•  Mutant collections: SALK, SAIL ....
•  gDNA Clone collections: ABRC ...
•  general Arabidopsis portal: TAIR http://www.arabidopsis.org/
•  Before the Arabidopsis genome was completely sequenced the work took
two years of one postdoc. (e.g. Seifert et al. 2002 Current Biol. 12:1840ff).
•  Nowadays map based cloning takes one MSc student 3 to 6 months and is
offered commercially.
ATIdb
REB1 encodes an enzyme required for UDPGal biosynthesis
AGP
UDP-D-Glc -4-epimerase
UDP-D-Glc
UDP-D-Gal
pectin
NAD+
UGD
hemicell.
NADH
UDP-D-GlcA -4-epimerase
UDP-D-GalA
UDP-D-GlcA
UXS
CO2
UDP-D-Xyl -4-epimerase
UDP-D-Xyl
UDP-L-Ara
Biochemical-molecular dissection of cell wall
biosynthesis. Pros and cons?
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Advantages
access to mechanism
addition of cofactors etc.
direct response
kinetic observation
you know what you get
•  Disadvantages
•  obtaining substrates
•  setting up the assay
conditions
•  identifying source containing
high activity
•  availability of material
•  instability of isolated enzyme
•  sometimes no cDNA library
available
•  system disrupted
•  artificial conditions
•  no in vivo function
Molecular genetic dissection of cell wall
biosynthesis pros and cons?
•  Advantages
•  many screens possible
•  system intact
•  in vivo function
•  map-based cloning is
straightforward
•  Because of phenotype the
functionality of modified versions
can be assayed in vivo.
•  epistasis analysis
•  multiple KO mutants
•  Disadvantages
•  some screens are
cumbersome
•  no mechanism
•  abiotic stress
•  compensation
•  genetic redundancy
•  lethality
•  you don t know what you
will end up with
Genetic screens that have elucidated cell
wall biosynthesis/function
•  root morphology
•  xylem morphology
•  cell wall carbohydrate composition
Some root morphology mutants
•  Genes required for primary cell wall (growing)
•  root swelling
•  root hair deficient
•  things falling apart
•  salt overly sensitive
•  cobra, procuste, korrigan, quasimodo, kojak ...
xylem/fibre morphology mutants
•  Genes required for thickening cell wall (secondary)
•  irregular xylem: IRX
•  fragile fibre: FRA
Most CesA genes were isolated by Arabidopsis genetics
Three CesA genes form the catalytic core of plant
cellulose synthase
Primary cell walls:
CesA1, -3, -6
Secondary cell walls:
CesA4, -7, -8
Two different sets CesA isoforms are necessary for primary and for secondary cell wall formation
CesA multimers might form ‘rosette’ strucutres at the
the plasma membrane
Several Arabidopsis genes involved in cellulose
synthesis are unclear in their function
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KORRIGAN (3 genes): membrane bound β-1-4 glucanase
COBRA (11 genes): GPI-anchored, novel
KOBITO (3 genes): PM-localized, novel
POM POM1: endochitinase (?)
TBR1 (several genes): PM-localized, novel
•  The difficulty to annotate biochemical functions to these gene
products highlights the limitation of forward genetics.
•  It is doubtful whether the novel genes would have been found
otherwise.
•  Forward genetics has the potential of discovering not only
new genes but also new processes.
Carbohydrate compositional mutants: The MUR genes
•  collect leaves
•  extract proteins lipids etc. → crude cell walls
•  hydrolyze poly- to monosaccharides
•  derivatize for gas chromatography (GC)
•  quantify on GC + mass spectroscopy (MS)
•  11 mutant loci identified from 5000
mutagenized plants
•  6 cloned
•  nucleotide sugar metabolism (2)
•  glycosyl transferases (3)
•  novel (1)
Systems biology approach to functional
gene identification
• 
Systems biology is the study of the interactions between the components of a
biological system, and how these interactions give rise to the function and
behaviour of that system (for example, the enzymes and metabolites in a
metabolic pathway)."
Systems biology approach to functional
gene identification
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Systems biology is the study of the interactions between the components of a
biological system, and how these interactions give rise to the function and
behaviour of that system (for example, the enzymes and metabolites in a
metabolic pathway)."
Example: transcriptional co-regulation of a metabolic pathway dedicated to
secondary cell wall formation."
Relative transcript abundance for >22000 Arabidopsis genes has been analysed
in hundreds of different stress and developmental conditions, tissues, cell types
etc."
Pairwise comparison of transcript abundance identifies co-regulated genes."
Transcriptional co-regulation is a hint to involvement in the same biological
process."
This hypothesis can be tested by reverse genetics using a large collection of TDNA or transposon tagged Arabidopsis mutants, or by gene silencing."
Example: Genes co-regulated with secondary cell
wall specific CesA genes
Primary cell walls:
CesA1, -3, -6
Secondary cell walls:
CesA4, -7, -8
Two different sets CesA isoforms are necessary for primary and for secondary cell wall formation
Pairwise comparison
http://affymetrix.arabidopsis.info/narrays/twogenescatter.pl
•  RSW1 = CesA1 (primary walls)
•  PRC1 = CesA6 (primary walls)
•  IRX3 = CesA7 (secondary walls)
•  IRX5 = CesA4 (secondary walls)
CSB.DB: Comprehensive systems biology database
IRX7 co-regulated genes
Mutant Phenotype
IRX6
IRX5
IRX8
IRX12
IRX1
No
IRX9
nd
nd
nd
No
nd
No
nd
nd
No
IRX ?
The molecular biology of cell wall biosynthesis today
•  Most nucleotide sugar interconversion enzymes have been cloned.
•  Cellulose synthase requires three isoforms of CesA genes.
•  Many genes that are essential for cellulose biosynthesis (e.g. COBRA,
KORRIGAN, POMPOM) are presently lacking a clearly defined biochemical
function.
•  β-mannan synthase is encoded by CslA genes.
•  Xyloglucan fucose and galactose side chains are attached by the MUR2 and
MUR3, glycosyltransferases.
•  Several ‘hot candidate genes’ for a functioning in matrix polymer synthesis
from forward and reverse genetics and biochemistry.
•  There are still huge gaps in our knowledge.
•  The database for carbohydrate active enzymes (CAZY) contains a
disproportionate number of plant genes. Most of unknown biochemical and
biological function.
Cellulose microfibrils (CMF) are aligned perpendicular
to the growth direction
Cell wall deposition during elongation growth is
highly anisotropic
Sugimoto ea. 2000
Anisotropy of load-bearing cell wall polymers is a
precondition of anisotropic expansion
turgor pressure during growth: 10 bar
tensile strain on 0.1µm thick primary wall: 5000 bar
Two molecular problems of anisotropic expansion:
•  How is anisotropic deposition controlled/achieved?
•  How can mechanically stable polymers yield to turgor
pressure in a controlled manner?
ften also microtubules are seen aligned perpendicular to the growth
direction
Do microtubules control the oriented movement
of cellulose synthase?
Two molecular problems of anisotropic expansion:
•  How is anisotropic deposition controlled/acheived?
•  How can mechanically stable polymers yield to turgor
pressure in a controlled manner?
How is cell wall loosening controlled?
How is cell wall loosening controlled?
•  XET: xyloglucan endo-transglycosylases
•  expansin: topoisomerase (?)
•  (1→4)β-glucanases
•  non-enzymatic loosening by hydroxyl
radicals (OH•).
Enzymes involved in fruit softening
•  pectin hydrolases
•  pectate lyases
•  pectin esterases
•  expansin
•  glucanases
Cell separation is locally controlled
•  The action of pectin hydrolases, - lyases and esterases
has to be restricted to small area at the cell corners to allow
tissue growth but prevent that things are falling apart.
Control of cell wall anisotropy
•  Morphogenesis depends on spatial anisotropy of load bearing
cell wall polymers esp. cellulose.
•  cellulose microfibrils are aligned perpendicular to the growth
axis.
•  Oriented movements of cellulose synthase partially depend on
microtubules but the are additional mechanisms.
•  Controlled cell wall creep is mediated by enzymes acting on the
cross-linking glucans (e.g. expansin, XET).
•  The important problem of local cell separation is elusive