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Glycosyltransferase Research Projects:
Characterization of Mur3 paralogs:
This project entails the identification of
putative glycosyltransferases which are involved in plant cell
wall biosynthesis. 
When mur3 was isolated from an EMS mutagenized population, it
showed a 50% reduction in L-fucose based on total cell wall
composition. The mur3 mutation has recently been shown to eliminate
galactosylation of the third xylose residue within the XXXG
core structure of xyloglucan whereas galactosylation of the
second xylose residue is markedly enhanced. Because of the substrate
specificity of XyG fucosyltransferase, mur3 plants lack the
entire α-L-fucosyl-(1→2)-β-D-galactosyl-(1→2) side chain that is normally attached
to the XXXG core structure of most XyGs. Positional cloning
of the MUR3 gene revealed that it encodes a XyG galactosyltransferase
specific for the third xylose residue within the XXXG repeat
unit, a finding that is consistent with the altered XyG structure
of mur3 plants. Nevertheless the present of large quantities
of XLXG in mur3 xyloglucan indicates there is another galactosyltransferase
which catalyze the galactosylation of the center xylosyl residue
of XXXG oligomers.
Searching bioinformatic database revealed that MUR3 belongs to
a multigene family of putative glycosyltransferases encompassing
ten closely related members and 28 more distantly related coding
regions. RT-PCR and GUS:promoter based transcriptional analysis
will help to determine the possible gene function by studying
sugar composition of cell walls from appropriate plant tissues.
To analyze the function of these putative glycosyltransferases
in plant cell wall synthesis, we exploit reverse genetic approach
by knockout gene function with T-DNA insertion. Several T-DNA
mutant of MUR3 family members have been obtained from the Salk Institute and Wisconsin
Knockout Facility. Analysis of these mutants for their
monosaccharide composition by gas chromatography aditol acetate
will provide us more information about these putative glycosyltransferases.
Further analysis of individual cell wall matrix components (pectins,
hemicellulose and AGPs) is necessary to specify the function for
each of these family members.
Authors:
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Li X, Cordero I, Caplan J, Molhoj M, Reiter WD. |
Reprint: |
| Title: |
"Molecular analysis of 10 coding regions from Arabidopsis that are homologous to the MUR3 xyloglucan galactosyltransferase." |
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| Journal: |
Plant Physiol. 2004 Mar;134(3):940-50 |
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Identification and characterization of putative Arabinosyltransferase:
The cell wall is a critical component of all
higher plants and is composed of pectins, hemicelluloses, and
cellulose. For most dicot and non-graminaceous monocot plants,
the principal hemicellulose is xyloglucan (XyG), which has a (1→4)-
β-D-glucan backbone similar to cellulose. In most XyGs, three
consecutive glucose residues are substituted by D-xylose in an
α-(1→6)-linkage
leaving a fourth glucosyl residue unbranched. This structure is
called XXXG in standard nomenclature. Recently we demonstrated
that the MUR3 gene product of Arabidopsis thaliana attaches
D-galactose to the third xylose residue within the XXXG repeat
unit leading to the formation of XXLG building blocks. Solanaceous
plants have a XyG structure that is different from other dicots
primarily because of the absence of the first xylose residue,
and the substitution of L-arabinose for D-galactose during modification
of the XyG core structure. We are interested in the identification
of arabinosyltransferases in plant cell wall synthesis, since
these enzymes are involved in the synthesis of a multitude of
arabinosylated glycans including rhamnogalacturonans I and II,
arabinogalactan-proteins, hydroxyproline-rich glycoproteins, and,
in the case of solanaceous plants, the synthesis of arabino-xyloglucan.
No genes encoding arabinosyltransferases have been identified thus far; however, we have been intrigued by the observation that the database of expressed sequence tags (dbEST) contains sequences from a tomato gene (LeGT1) that is predicted to encode a protein with a very high degree of sequence similarity to Arabidopsis MUR3 even though tomato XyG is arabinosylated rather then galactosylated. Based on these results, we speculate that LeGT1 represents a glycosyltransferase catalyzing the arabinosylation of the XyG core structure. To test this hypothesis, we are in the process of expressing LeGT1 in Pichia pastoris for enzyme assays, and of transforming the Arabidopsis mur3 mutant with LeGT1 to determine if this will lead to the arabinosylation of XyG in Arabidopsis. Experiments are also underway to express the MUR3 gene in tomato, since this may lead to interesting alterations in XyG structure in an important crop plant.
Cell Wall Genomics:
We are working with the University of Wisconsin, Purdue, and the National Renewable Energy Research Laboratory to create a public resource for all genes involved in cell wall biogenesis, modification and turnover. Many genes have already been listed on public web sites such as:
Carbohydrate Active enzymes (CAZy)
WallBioNet
Glucan Synthases
Expansins
XTH
In addition, we are identifying putative type II glycosyltransferases by searching through all proteins with an N-terminal transmembrane domains as predicted by THMM and Kyte-Doolittle plots. Each protein meeting our search criterion is autonomously submitted to an NCBI BLAST search using a PERL program. The results are compared to other searches and interesting proteins are annotated and grouped into families. These genes are added to the list of mutants that are being made and characterized by our partners in this project.
Other wall related genes such as HRGP's and AGP's and PRP's are being identified by BLAST and web searches. For more information visit Cell Wall Genomics at Purdue which will host all of the compiled genes and information. Thus far, there are over 945 genes on our master list and more genes will be added as they are uncovered.
The MUR3 gene of Arabidopsis thaliana encodes a xyloglucan galactosyltransferase:
Xyloglucans are the principal glycans that interlace
cellulose microfibrils in most flowering plants. The mur3 mutant of Arabidopsis thaliana contains a severely altered
structure of this polysaccharide because of the absence of a conserved
α-L-fucosyl-(1→2)-β-D-galactosyl
side chain and excessive galactosylation at an alternative xylose
residue.
Despite this severe structural alteration, mur3
plants were phenotypically normal and exhibited tensile strengths
in their inflorescence stems comparable to those of wild-type
plants. The MUR3 gene was positionally cloned and shown
to encode a xyloglucan galactosyltransferase acting specifically
on the third xylose residue within the XXXG core structure of
xyloglucan. MUR3 belongs to a large family of type II membrane
proteins that is evolutionarily conserved among higher plants.
The enzyme shows sequence similarities to the glucuronosyltransferase
domain of exostosins, a class of animal glycosyltransferases that
catalyze the synthesis of heparan sulfate, a glycosaminoglycan
with numerous roles in cell differentiation and development. This
suggests that components of the plant cell wall and of the animal
extracellular matrix are synthesized by evolutionarily related
enzymes even though the structures of the corresponding polysaccharides
are entirely different from each other.
Authors:
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Madson M, Dunand C, Li X, Verma R, Vanzin GF, Caplan J, Shoue DA, Carpita NC, Reiter WD. |
Reprint: |
| Title: |
"The MUR3 gene of Arabidopsis encodes a xyloglucan galactosyltransferase that is evolutionarily related to animal exostosins." |
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| Journal: |
Plant Cell. 2003 Jul;15(7):1662-70 |
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The mur2 mutant lacks fucosylated xyloglucan:
Cell walls of the Arabidopsis mutant mur2 contain
less than 2% of the wild-type amount of fucosylated xyloglucan
because of a point mutation in the fucosyltransferase AtFUT1.
The mur2 mutation eliminates xyloglucan fucosylation in all major
plant organs, indicating that Arabidopsis thaliana fucosyltransferase
1 (AtFUT1) accounts for all of the xyloglucan fucosyltransferase
activity in Arabidopsis. Despite this alteration in structure,
mur2 plants show a normal growth habit and wall strength. In contrast,
Arabidopsis mur1 mutants that are defective in the de novo synthesis
of L-fucose exhibit a dwarfed growth habit and decreased wall
strength [Reiter, W. D., Chapple, C. & Somerville, C. R. (1993)
Science 261, 1032-1035]. Because the mur1 mutation affects several
cell wall polysaccharides, whereas the mur2 mutation is specific
to xyloglucan, the phenotypes of mur1 plants appear to be caused
by structural changes in fucosylated pectic components such as
rhamnogalacturonan-II. The normal growth habit and wall strength
of mur2 plants casts doubt on hypotheses regarding roles of xyloglucan
fucosylation in facilitating xyloglucan-cellulose interactions
or in modulating growth regulator activity.
Authors:
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Vanzin GF, Madson M, Carpita NC, Raikhel NV, Keegstra K, Reiter WD. |
Reprint: |
| Title: |
"The mur2 mutant of Arabidopsis thaliana lacks fucosylated xyloglucan
because of a lesion in fucosyltransferase AtFUT1." |
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| Journal: |
Proc Natl Acad Sci U S A 2002 Mar 5;99(5):3340-5 |
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