Maize Cell Wall Database » Macroarray with Gene Specific Tags - Inra

incandescentnonBiotechnology

Dec 10, 2012 (4 years and 8 months ago)

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Approche
génomiques
pour comprendre
la structure et les
propriétés
des parois
lignocellulosiques
D. Goffner
M. Pichon, P. Ranocha, S. Guillaumie,
E. Pesquet, Y. Barrière
UMR CNRS-UPS 5546 «
Surfaces Cellulaires
et
Signalisation
chez les Végétaux
»
Pôle
de Biotechnologies Végétales
Castanet Tolosan
FRANCE
Identification of
new key
cell
wall
g
enes
through gene discovery programs
Systematic gene sequencing of suitable plant material
Global gene expression studies
Model cell systems
Model species
Crop species
Zinnia elegans
Zea
mays
Arabidopsis thaliana
Zinnia elegans
: an
in vitro
model system
to study secondary cell wall formation
T0
Mechanical
isolation
Plant
Plant
Hormones
Hormones
Day 4
Day 4
Cellulose
Secondary wall deposition
Programmed cell death

Lignin
Production of lignocellulosic
b
iomass in a test tube
Morphological, cytological events similar to what happens in the
plant
System well adapted to identification “lignocellulose” genes
Zinnia to Arabidopsis –
bridging the gap
between model systems
‘wood’
Arabidopsis
Zinnia
1. Hundreds of novel cell wall
genes identified
Functional analysis of Zinnia genes
Identification of:

closest Arabidopsis gene
for each Zinnia gene
2. Gene expression profiles determined

corresponding mutants
P
esquet
et
al, (2005)
Plant Physiol
The challenge of phenotyping
mutants
‘Medium throughput’
analyses:

growth characteristics under different conditions (
in vitro
/growth
chamber/greenhouse;
in vitro
light/dark

root morphology
in vitro

microscopy of vascular system of floral stems/cotyledons/leaves

β-glucuronidase
(GUS) activity
GUS activity in
vasc
ular system
in cotyledons

enzyme-mediated cell wall degradation in stems (Lusignan)

resistance to
Ralstonia
, a vascular bacterial pathogen
On selected candidates, cell wall-related analyses:
-
FTIR (Versailles)
-
Hemicelluloses (Nantes)
-
Lignins/phenolic
compounds (Grignon)
-
Mechanical properties ???
Arabidopsis mutants :
A diversity of cell wall-related phenotypes
Wild type
mutant
Vascular
bundle
Altered
cell
wall
autofluorescence
fibres
Hypolignified
fibres
N509.9
FT IR spectra
of
interfasci
cular
fibers
in 509.9 vs wt
Less lignin in mutant
More polysaccharides in mutant

fibers of mutant strongly resemble parenchyma cells”
Grégory
Mouille, Versailles
Altered mechanical properties in stems of N509.9
0
500
1000
CO
N586.9
N534.14
N509.9
E [MPa]
**
**
**
*
W
Modu
lus of elasticity
Collaboration L. Kohler, Freiburg, Germany
Maize cell wall genomics
Identification of
genes
that
determine
variations
in digestibility
of
silage
m
aize
Caffeic
acid
O-Methyltransferase
(COMT)
Candidate gene
approach
First demonstrated modification of lignin in
maize by genetic engineering
wt
AS COMT
2 GENE SOURCES
-
Keywords

‘Cell
wall’
and
‘Vascular
Patternin
g
-
Homologs
of
Zinnia ES
Ts
Genomic
approach
«
Maize
Cell
Wall
Database
»
Macroarray
with
Gene
Specific
Tags
750 GSTs
Maüle
staining of internodes
1.
Piquemal
et al, Plant Physiol
(2002)
2.
Pichon
e
t al., (2003) -
patent
3. Field trials with
AS-COMT
in 6 genetic
backgrounds-
Mol Breeding
2006
Publications
Maize cell wall genomics
Cell wall composition/structure = silage digestibility

Full genome sequence soon available

Insertional
mutants

Microarray
chips

Amenable to genetic transformation
But, little cell wall knowledge available.
Differences in gene expression/ regulatory mechanisms
predicted
based on differences in monocot/dicot
cell wall
composition and structure
MAIZEWALL
a
maize cell wall database
Home Page
Project description
Gene catalogue
Searc
h
(keywords, blast, accession number….)
Latest update :
May 2, 2006
Expression data
Coordinat
ors: Deborah Goffner, Magalie
Pichon, Yves Barrière
General information
contact
MAIZEWALL :
a complete bioinformatic
analysis
and gene expression data
repertory
of cell wall biosynthesis and
assembly in maize
Maize god 3 Yum kaax-
courtesy of Robert Elliot
http://www.polebio.scsv.ups-tlse.fr/MAIZEWALL
Guillaumie
et al., (2007)
Plant Physiol
.
Maize
cell
wall
gene
profiling
with
a cell
wall
macroarray
Different
organs,
develo
pmental
stages
Complete
cell
wall
transcriptome
fingerprint
in maize
Inbred
lines
of
varying
digestibility
Correlate
transcriptome
profile with
digestibility
Reservoir of genes
involved in the regulation
of
cell wall
structure/digestibility
Lignin
mutants
transformants
Co-regulation
of genes
within
the
lignin
pathway
or other
cell
wall
pathways?
Guillaumie
et al.,
Plant Physiol
., 2007
Guillaumie
et al.,
Planta
in press
Putative preferred routes of lignin synthesis in maize
Phenylalanine
Cinnamic
acid
p-Coumaric
acid
p-Coumar
oyl
CoA
Tyro
sine
PAL
C4H
TAL
++
++
-
+
++
++
+++
++
++
++
++
++
++++
+++
++
-
-
+
-
-
-
-
-
-
-
+++
+
+++
+++
+++
+++
+++++
+++
+++
+++
-
+
++
++
-
+++
++
+++
++
++
-
+
-
+
-
++
++
-
+
++
++
+
++
++
+++
4CL
p-Coumaraldehyde
CCR
-
-
-
-
-
+
-
-
-
-
+
+
-
+
-
-
-
-
-
-
++
++
+++
++
++
+
-
-
-
+
-
-
-
-
-
+++
+
+++
+++
++
p-Coumaryl
alcohol
CAD
++
+
+++
++
++
++
++
+
+
-
-
+
-
-
-
++
+
-
+
++
++
++
+
+
+++
-
-
+
+
-
H-units
Caffeic
acid?
Ferulic
acid
5-hydroxyferulic acid
Sinapic
acid
Caffeoyl-CoA
Feruloyl-CoA
5-hydroxyferuloyl-CoA
Sinapoyl-CoA
4CL
4CL
4CL
4CL
Caffeyl
aldehyde
Coniferaldehyde
5-hydroxyconiferaldehyde
Sinapaldehyde
CCR
CCR
CCR
CCR
Caffeyl
alcohol
Coniferyl
alcohol
5-hydroxyconiferyl alcohol
Sinapyl
alcohol
G-units
S-units
CAD
CAD
CAD
CAD
CCoAOMT
F5H
++
+
+++
+++
+++
-
+
-
+
-
++
++
+
+
+
+
++
+
+
+
+
-
+
+
+
COMT
COMT
F5H
+++++
+++++
+++++
+++++
+++++
HC
T C3H HCT
HCT
p-coumaroyl
shikim
ic
acid
HCT
p-coum
aroyl
quinic
acid
HCT
Caffeoyl
shikimic
acid
Caffeoyl
quinic
acid
HCT
C3
H
C3H
+++++
+++++
+++++
+++++
+++++
+++++
+++
+++++
+++++
+++++
-
+++
-
-
-
?
?
?
COM
T
COM
T
COM
T
COMT?
COMT
F5H
?
F5H?
COMT?
R YS L IN6 IN1
C4H
4Cl
CCR
CCoAOMT
CCoAOMT
CAD
F5H
COMT
C3H
HCT
PAL
+++
+
++++
++
+++++
++
++++
++
+++-
-
*
Conclusions

Optimizing plant biomass is a realis
tic goal for i
mproving downstream uses

Shuttling back and forth between model systems is a viable approach
for identifying genes in secondary wall formation
Genomic approaches multiply the potential ways cell walls can be
modified
and allow us to identify new ways for controlling wall structure

A major challeng
e is establishing the link ‘gene –
phenotype’.
Identifying subtle phenotypes (perhaps the most interesting) requires
sophisticated technologies (spectroscopy, metabolic profiling….)
Acknowledgments
Sabine Guillaumie
Edouard Pesquet
INRA, Versailles
Philippe Ranocha
Amandine Freydier
Lise Jouanin
Cao Trung
Grégory Mouille
Ricardo Chavez
Alain Jauneau
Magalie
Pichon
INRA, Lusignan
LIPM Toulouse
Yves Barrière
Yves Marco

Génoplante
programme