Transcript Cellulose biosynthesis *old and new concepts

Current updates and evolving
concepts on Cellulose
Biosynthesis in plants
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?
• What is the first committed step in cellulose
polymerization?
• How trafficking of cellulose synthase occurs?
• What are the approaches in studying cellulose
synthase?
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.
• 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. 22:53–78
Schematic model of cellulose synthesis
Chris Somerville (2006) Cellulose synthesis in higher plants Annu. Rev. Cell Dev. Biol. 22:53–78
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 membrane
• The conserved D,D,D,QXXRW motif involved in
the catalytic event.
• Other accessory proteins might be involved along
with the microtubules.
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.
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.
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.
Overview of CSC intracellular trafficking
Raymond Wightman and Simon Turner (2010) plant physiology 153 427–432.
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.
Cellulose as cellulosic biofuel
Lignin biosynthesis
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 in
transgenic trees Nature Biotechnology 17 808812.
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.
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.
Sucrose synthase as a component of the
catalytic unit of Cellulose synthase
A
B
C
(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.
Sucrose synthase as a component of the
catalytic unit of Cellulose synthase
D
E
(D) Ultracel YM-3 membrane-retained products in (C) after treatment with β -1,4 or β -1,3glucanase. (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.
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
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.
Methods in studying cellulose
biosynthesis
• Genetic approaches
Reverse genetics and mutant analysis
• Microscopy
• Live cell imaging
• GC-MS- cellulose content analysis and linkage
analysis.
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.
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.
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.
Identification of a cellulose synthaseassociated 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.
Fig. 1. Identification of CSI1. (A) Schematic representation of CESA and CSI1 proteins. (B)
CSI1 interacts with three primary CESA proteins in yeast.
Interactive model of CSI
Truncated coexpression network for primary wall cellulose-related genes using the AraGenNet
Phylogeny of CSI protein in land plants.
Full-length CSI-like sequences were identified in GenBank using BLASTP and aligned
using ClustalW.
GUS assay
Promoter GUS analysis
of CSI1::GUS (D, F, and
H) and CESA3::GUS (E,
G, and I
Expression pattern of the CSI1 genes
assessed through GUS Assay
Mutant Analysis
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 analysis
C
F
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 wildtype plants and csi1 mutants: (Left to Right) Arabidopsis thaliana Columbia (Col-0), csi1-3,
and csi1-6 mutants. (F) Cellulose content estimation.
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.
CSI1 is localized to CESA-like particles in dark-grown hypocotyls cells.
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
Localization of GFP
(G–I) Localization of GFP-CESA3 (G),
RFP-CSI1 (H), and
merge (I)
YFP-CESA6 dynamics are altered in csi1-3 mutants
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.
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.mov
Polarized light analysis of csi1 mutants.
Polarized-light micrographs of (A) wild-type and (B) csi1-1-mutant roots. (C) Quantification of
retardance and azimuth
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.