Debarati Basu Cell Wall Seminar Outline What genes involved in cellulose biosynthesis Which compartment is involved in cellulose biosynthesis What are the protein components of the cellulose syntheses machinery and how they are coupled in the cytoskeleton ID: 703504
Download Presentation The PPT/PDF document "Current updates and evolving concepts on..." is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
Current updates and evolving concepts on Cellulose Biosynthesis in plants
Debarati Basu
Cell Wall SeminarSlide2
Outline
What genes involved in cellulose biosynthesis?
Which compartment is involved in cellulose biosynthesis?
What are the protein components of the cellulose syntheses machinery and how they are coupled in the cytoskeleton?
What is the first committed step in cellulose polymerization?
How trafficking of cellulose
synthase
occurs?
What are the approaches in studying cellulose
synthase
?Slide3
Aaron
H.
Liepman
,
Raymond
Wightman,
Naomi
Geshi
,
Simon R.
Turner
and
Henrik
Vibe
Scheller
(2010) Arabidopsis – a powerful model system for plant cell wall research
The Plant Journal
61
,
1107–1121.Slide4
The CESA proteins that make up the CSC responsible for
primary cell wall formation consist of CESA1
and CESA3
, together with some combination of
CESA2, CESA5
, CESA6, or
CESA9. ACesA2 and CesA5, which have been reported to bepartially redundant with CesA6, may compete for the same position in the enzyme complex Cellulose biosynthesis at the secondary wall requires CESA4, CESA7, and CESA8.
Illustration of the structure of CESA3, a typical CESA protein.
Chris Somerville (
2006)
Cellulose synthesis in higher plants
Annu. Rev. Cell Dev. Biol.
22
:53–78Slide5
Schematic model of cellulose synthesis
Chris Somerville (
2006)
Cellulose synthesis in higher plants
Annu. Rev. Cell Dev. Biol.
22
:53–78Slide6
Cellulose synthase complex
Plasma membrane bound complex.
A
rosette exhibits a typical diameter in
the range
25–30 nm and consists of 6 globular structures arranged with a six-fold symmetry.Eight transmembrane helices that anchor the proteins in the plasma membraneThe conserved D,D,D,QXXRW motif involved in the catalytic event.Other accessory proteins might be involved along with the microtubules.Slide7
Cellulose synthase complex parameters
EF Crowell, M
Gonneau
, Y-
DStierhof
, H Ho¨
fte and S vernhettes (2010) Regulated trafficking of cellulose synthases Current Opinion In Plant Biology 13: 1-6.Slide8
Figure 1. Hypothetical model for the biosynthesis of cellulose in higher plants.
Gea
Guerriero
, Johanna
Fugelstad
and Vincent Bulone (2010) What Do We Really Know about Cellulose Biosynthesis in Higher Plants? Journal of Integrative Plant Biology 52 161–175.Slide9
Cellulose synthase trafficking
Involvement of
cortical microtubules in the movement of the
cellulose
synthase
complex in the plasma membrane.Recent studies have shown SmaCCs/MASCs (small CESA compartments / microtubule associated cellulose synthase compartments ) are highly dynamic compartments that appear to play key roles both as intracellular stores of the CSC and in its delivery to the plasma membrane.Slide10
Overview of CSC intracellular trafficking
Raymond Wightman and Simon
Turner (2010) plant physiology
153
427–432.Slide11
Assembly of the CESA subunits
EF Crowell, M
Gonneau
, Y-
DStierhof
, H Ho¨
fte and S vernhettes (2010) Regulated trafficking of cellulose synthases Current Opinion In Plant Biology 13: 1-6.Slide12
Cellulose as cellulosic
biofuel
Lignin biosynthesisSlide13
Link between lignin and cellulose biosynthesis
The effects of
downregulation
of
Pt4CL1
expression on Pt4CL1 activity and lignin accumulation in transgenic aspen.
Wen-Jing Hu, Scott A. Harding, Jrhau
Lung, Jacqueline L. Popko, John Ralph, Douglas D. Stokke, Chung-Jui
Tsai, and Vincent L. Chiang (1999) Repression of lignin biosynthesis promotes cellulose accumulation and growth intransgenic trees Nature Biotechnology 17 808-812.Slide14
Enhanced growth in transgenic aspen
Wen
-Jing
Hu
, Scott A. Harding,
Jrhau
Lung, Jacqueline L. Popko, John Ralph, Douglas D. Stokke, Chung-Jui Tsai, and Vincent L. Chiang (1999) Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees Nature Biotechnology 17 808-812.Slide15
Sucrose metabolism and cellulose biosynthesis
Haigler
, C.H., M.
Ivanova-Datcheva
, P. S. Hogan, V. V.
Salnikov, S. Hwang, L. K. Martin, and Delmer, D.P. (2001) Carbon partitioning to cellulose synthesis. Plant Molecular Biology 47: 29-51.Slide16
Sucrose synthase
as a component of the catalytic unit of Cellulose
synthase
(A) The deduced amino acid sequence of sucrose
synthase
in
Azuki bean.(B) immune blotting using antibodies raised against mung bean sucrose synthase. (C) Cellulose synthesis by immunoprecipitate preparation after incubation with UDP-glucose.
A
B
CSlide17
(D)
Ultracel
YM-3 membrane-retained products in (C) after treatment with
β -1,4
or
β -1,3-
glucanase. (E) Membrane-retained cellulose using either UDP-[ 14 C]glucose (UDPG) or [ 14 C]sucrose plus UDP (sucrose/UDP) as substrates determined by incorporated glucose.D
ESatoshi Fujii
, Takahisa Hayashi and Koichi Mizuno (2010) Sucrose Synthase is an Integral Component of the Cellulose Synthesis Machinery Plant Cell Physiol.
51
: 294–301
Sucrose
synthase
as a component of the catalytic unit of Cellulose
synthaseSlide18
SG serves as primer for elongation of β
-1,4-glucan chains
Liangcai
Peng
, Yasushi Kawagoe, Pat Hogan, and Deborah
Delmer (2002 )Sitosterol-β-glucoside as Primer for Cellulose Synthesis in Plants 295 147 – 150.Slide19
Methods in studying cellulose biosynthesis
Genetic approaches
Reverse genetics and mutant analysis
Microscopy
Live cell imaging
GC-MS- cellulose content analysis and linkage analysis. Slide20
Approaches in studying cellulose biosynthesis
Live cell imaging
Live cell imaging of the
CSC. The
images are taken of
YFP-CESA6 fusion
within the epidermis of a cotyledon petiole cell (A) and pavement cells (B) and characteristic ring-like
appearance in Golgi (C).Raymond Wightman and Simon
Turner (2010) plant physiology 153 427–432.Slide21
Unanswered Questions
Process
of assembly of individual
β
-(
1→4)-
glucan chains as microfibrils is poorly understood.The direct physical association of sucrose synthase with the cellulose synthase machinery has not yet been demonstrated.The role of sitosterol- β
-glucoside as a primer for cellulose biosynthesis remains to be firmly demonstrated
in vivo.The mechanism of translocation of the cellulose chains across the plasma membrane has yet to be elucidated.Slide22
References
Aaron H.
Liepman
, Raymond Wightman, Naomi
Geshi
, Simon R. Turner and
Henrik Vibe Scheller (2010) Arabidopsis – a powerful model system for plant cell wall research The Plant Journal
61, 1107–1121.
EF Crowell, M Gonneau, Y-
DStierhof
, H Ho¨
fte
and S
vernhettes
(2010) Regulated trafficking of cellulose
synthases
Current Opinion In Plant Biology
13
: 1-6.
Gea
Guerriero
, Johanna
Fugelstad
and Vincent
Bulone
What Do We Really Know about
Cellulose Biosynthesis
in Higher Plants
?
Journal of Integrative Plant Biology 2010,
52
: 161–175.
Haigler
, C.H., M.
Ivanova-Datcheva
, P. S. Hogan, V. V.
Salnikov
, S. Hwang, L. K. Martin, and
Delmer
, D.P. (2001) Carbon partitioning to cellulose synthesis.
Plant Molecular Biology
47:
29-51.
Raymond
Wightman and Simon
Turner (2010)
Trafficking of the Plant Cellulose
Synthase
Complex Plant Physiology,
153
:427-432
.
Satoshi
Fujii
,
Takahisa
Hayashi and Koichi Mizuno (2010) Sucrose
Synthase
is an Integral Component of the Cellulose Synthesis Machinery Plant Cell Physiol.
51
: 294–301.
Somerville
C (2006) Cellulose synthesis in higher plants.
Annu
Rev Cell Dev
Biol
22
: 53–78.
Wen
-Jing
Hu
, Scott A. Harding,
Jrhau
Lung, Jacqueline L.
Popko
, John Ralph, Douglas D.
Stokke
, Chung-
Jui
Tsai, and Vincent L. Chiang (1999) Repression of lignin biosynthesis promotes cellulose accumulation and growth in transgenic trees Nature Biotechnology
17
808-812.Slide23
Identification of a cellulose synthase
-associated protein required for cellulose biosynthesis
Ying
Gu
,
Nick
Kaplinsky, Martin Bringmann, Alex Cobb, Andrew Carrolla, Arun Sampathkumar, Tobias I. Baskin,
Staffan Persson, and Chris R. Somerville. PNAS 2010 107(29):12866-12871. Slide24
Fig. 1. Identification of CSI1
.
(A) Schematic representation of CESA and CSI1 proteins. (B) CSI1 interacts with three primary CESA proteins in yeast.Slide25
Interactive model of CSI
Truncated
coexpression
network for primary wall cellulose-related genes using the
AraGenNetSlide26
Phylogeny of CSI protein in land plants.
Full-length CSI-like sequences were identified in
GenBank
using BLASTP and aligned using
ClustalW
.Slide27
GUS assay
Promoter GUS analysis of CSI1::GUS (D, F, and H) and CESA3::GUS (E, G, and ISlide28
Expression pattern of the CSI1 genes assessed through GUS AssaySlide29
Fig. 2.
Schematic representation of six T-DNA insertion sites in csi1. Morphology of 4-d-old dark grown seedlings: (Left to Right) Col-0 (wild-type) and csi1-1, csi1-2, csi1-3, csi1-4, csi1-5, and csi1-6 mutants.
Mutant AnalysisSlide30
Mutant analysis
C
D
Determination of
Hypocotyl
length (C) and growth rate (D) of dark-grown wild-type (Col-0) plants and csi1-3, csi1-6, and prc1-1 mutants. (E) SEM of dark-grown hypocotyls in wild-type plants and csi1 mutants: (Left to Right) Arabidopsis thaliana Columbia (Col-0), csi1-3, and csi1-6 mutants. (F) Cellulose content estimation.
FSlide31
Morphology of csi1 mutants.
(A) RT-PCR analysis of CSI1 mRNA expression in various transfer DNA (T-DNA) insertion lines. (B-K) Phenotypic analysis of the mutants.Slide32
Fig. 3
.
(A–D) Optical sections of epidermal cells in 3-d-old dark-grown hypocotyls expressing RFP-CSI1 (A and C) and YFP-CESA6 (B and D).
(E) Plot of RFP-CSI1 particle velocity vs. (F) Histogram of measured RFP-CSI1 particle velocities
CSI1 is localized to CESA-like particles in dark-grown hypocotyls cells.Slide33Slide34
Localization of GFP
(G–I) Localization of GFP-CESA3 (G), RFP-CSI1 (H), and
merge (I)Slide35
Fig. 4
.
YFP-CESA6 localization in dark-grown hypocotyls cells is shown in wild-type plants (A and B) and csi1-3 mutants (C and D).
(E) Histogram of measured particle velocities.
YFP-CESA6 dynamics are altered in csi1-3 mutantsSlide36
Movie
http://www.pnas.org/content/suppl/2010/07/01/1007092107.DCSupplemental/
sm01
http://www.pnas.org/content/suppl/2010/07/01/1007092107.DCSupplemental/sm02.movSlide37
Polarized light analysis of csi1 mutants.
Polarized-light micrographs of (A) wild-type and (B) csi1-1-mutant roots. (C) Quantification of
retardance
and azimuthSlide38
Conclusion
The CSI1 protein is the first non-CESA protein associated
with primary CESA complexes not in the secondary cell wall.
CSI1
colocalizes
with primary CESA
complexes, and csi1 mutations affect the distribution and movement of CESA complexes.The csi1 mutations appeared to decrease the degree to which cellulose microfibrils are coaligned.