Molecular Plant • Volume 2 • Number 5 • Pages 1040–1050 • September 2009RESEARCH ARTICLE
Two Poplar Glycosyltransferase Genes, PdGATL1.1
and PdGATL1.2, Are Functional Orthologs to
PARVUS/AtGATL1 in Arabidopsis
Yingzhen Konga,b,2, Gongke Zhoua,b,2,3, Utku Avcia,b, Xiaogang Gua, Chelsea Jonesa, Yanbin Yinb,c,
Ying Xub,cand Michael G. Hahna,b,1
a Complex Carbohydrate Research Center, The University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA
b BioEnergy Science Center, The University of Georgia, 315 Riverbend Road, Athens, GA 30602, USA
c Computational System Biology Lab, Dept. of Biochemistry and Molecular Biology, and Institute of Bioinformatics, The University of Georgia, Athens, GA
the biosynthesis of xylanin woodyplants, wherethis polysaccharide is a majorcomponent of wood, is poorlyunderstood.
Here, we characterize two Populus genes, PdGATL1.1 and PdGATL1.2, the closest orthologs to the Arabidopsis PARVUS/
GATL1 gene, with respectto their gene expression in poplar,their sub-cellular localization, andtheir ability to complement
the parvus mutation in Arabidopsis. Overexpression of the two poplar genes in the parvus mutant rescued most of the
defects caused by the parvus mutation, including morphological changes, collapsed xylem, and altered cell wall mono-
saccharide composition. Quantitative RT–PCR showed that PdGATL1.1 is expressed most strongly in developing xylem of
poplar. In contrast, PdGATL1.2 is expressed much more uniformly in leaf, shoot tip, cortex, phloem, and xylem, and the
transcript level of PdGATL1.2 is much lowerthan that of PdGATL1.1 inall tissues examined. Sub-cellular localization experi-
ments showed that these two proteins are localized to both ER and Golgi in comparison with marker proteins resident to
these sub-cellular compartments. Our data indicate that PdGATL1.1 and PdGATL1.2 are functional orthologs of PARVUS/
GATL1 and can play a role in xylan synthesis, but may also have role(s) in the synthesis of other wall polymers.
Several genes in Arabidopsis, including PARVUS/AtGATL1, have been implicated in xylan synthesis. However,
Key words: Arabidopsis thaliana; poplar; xylan; glycosyltransferase.
Xylans are polymers with a linear backbone composed entirely
of b-D-Xyl residues connected through (1/4)-linkages that
are partially acetylated and sometimes substituted with glu-
curonic acid and 4-O-methyl glucuronic acid (glucuronoxylan,
GX), arabinose (arabinoxylan), or a combination of acidic and
neutral sugars (glucuronoarabinoxylan). Glucuronoxylans are
mass, composing, for example, 23% of the dry weight of
poplar wood (Simson and Timell, 1978). An understanding
of GX biosynthesis has implications in economically important
industries including biofuel production, where optimization
of the plant cell wall composition to overcome biomass recal-
citrance is a major goal of research (Bevan and Franssen,2006).
GX has typically been viewed as a polysaccharide whose syn-
thesis requires a xylan synthase for backbone formation and
one or more glycosyltransferases for the addition of side
chains. A number of glycosyltransferases have been identified
that appear to be involved in xylan synthesis in Arabidopsis,
including IRX8/AtGAUT12 (Persson et al., 2007; Pen ˜a et al.,
2007), IRX9 (Pen ˜a et al., 2007), IRX7/FRA8 (Pen ˜a et al., 2007;
Brown et al., 2007), PARVUS/AtGATL1 (Brown et al., 2007;
Lee et al., 2007), IRX14 (Brown et al., 2007), IRX10, and
IRX10-L (Brown et al., 2007; Wu et al., 2009). Plants carrying
1To whom correspondence should be addressed. E-mail firstname.lastname@example.org,
fax +01 706 542 4412, tel. +01 706 542 4457
2These authors contributed equally to this work.
3Current address: Qingdao Institute of Bioenergy and Bioprocess Technol-
ogy, No.189 Songling Road, Laoshan District, Qingdao
Republic of China.
ª The Author 2009. Published by the Molecular Plant Shanghai Editorial
Office in association with Oxford University Press on behalf of CSPP and
IPPE, SIBS, CAS.
doi: 10.1093/mp/ssp068, Advance Access publication 24 August 2009
Received 10 June 2009; accepted 18 July 2009
and decreased xylan and xylose content. However, these gly-
cosyltransferases appear to be involved in different aspects
of the biosynthetic process. For example, IRX9, IRX14, IRX10,
and IRX10-L (Pen ˜a et al., 2007; Brown et al., 2007; Wu et al.,
2009; Brown et al., 2009) appear to be involved in elongation
of the xylan backbone, whereas IRX7, IRX8/AtGAUT12, and
PARVUS/AtGATL1 (Persson et al., 2007; Pen ˜a et al., 2007; Lee
et al., 2007) appear to be involved in the synthesis of a galac-
turonic acid-containing tetramer that is located at the reduc-
ing end of xylan. This reducing-terminal tetrasaccharide
appears to play an important role during xylan synthesis, al-
though it is not known whether this oligosaccharide acts as
a primer or a terminator (York and O’Neill, 2008). The exact
role(s) of each of these proteins in the synthesis of xylan remain
unclear, in part, due to the absence of functional in vitro assays
of enzyme activity. Two of these proteins, IRX8/AtGAUT12 and
PARVUS/AtGATL1, are related by sequence to a functionally
characterized galacturonosyltransferase (AtGAUT1) (Sterling
et al., 2006), suggesting that they might be involved in the syn-
thesis of the xylan-terminal tetrasaccharide via the addition of
an a-linked GalA residue to the growing tetrasaccharide (Pers-
son et al., 2007; Pen ˜a et al., 2007). Another possibility is that
these two proteins are involved in the synthesis of a structure
example, a specific pectic polysaccharide (Mohnen, 2008).
Despite these advances in Arabidopsis, little is known about
the genes involved in wood formation in trees, which contain
xylan as a major hemicellulosic component (Ebringerova ´ et al.,
2005). Populus trichocarpa has been fully sequenced and a to-
tal of 45 555 gene models have been predicted (Tuskan et al.,
2006). The completion of the P. trichocarpa genome sequence
provides an opportunity to advance our knowledge of wood
formation. However, the scarcity of loss-of-function mutants
complicates the studyof
Arabidopsis has been suggested as a model system for the
study of secondary growth because this herbaceous species,
under specific growing conditions, can be induced to develop
features that exhibit many of the characteristics common to
secondary growth in tree species (Chaffey et al., 2002; Ko
and Han, 2004).
Recent studies have shown that PoGT43B and PoGT47C, the
poplar orthologs of IRX9 and IRX7/FRA8, respectively, are able
to rescue the xylan defects of irx9 and irx7/fra8 mutants in Ara-
bidopsis (Zhou et al., 2006, 2007). These findings indicate that
PoGT43B and PoGT47C are likely to be involved in xylan syn-
thesis during wood formation. These results also established
the feasibility of using Arabidopsis as a model plant in which
to study the functions of poplar glycosyltransferases that par-
ticipate in wood formation.
In this study, we report molecular and genetic characteriza-
tion of two poplar genes, PdGATL1.1 and PdGATL1.2, that are
orthologous to the Arabidopsis PARVUS/AtGATL1 gene. These
two poplar genes are highly expressed in developing wood
(Aspeborg et al., 2005), and are specifically up-regulated in sec-
andSundberg,2008)anddown-regulatedduring tension wood
formation (Andersson-Gunnera ˚s et al., 2006), which indicates
thatthey may playrolesinwoodformation.However, the exact
function(s) of the proteins encoded by these two genes are still
unclear. So, inorder togainfurtherinsight intothefunctionsof
these two poplar genes, we examined their expression patterns
ing proteins, and their ability to complement the parvus/gatl1
mutant in Arabidopsis.
Gene Structure and Expression Profiles of Poplar GATL1.1
Two poplar genes named GATL1.1 and GATL1.2 were identi-
fied from thePopulus trichocarpa
(www.jgi.doe.gov/poplar) on the basis of their sequence sim-
ilarity to the Arabidopsis PARVUS/AtGATL1 gene (Figure 1A).
The corresponding genes, PdGATL1.1 and PdGATL1.2, were
then cloned and sequenced from Populus deltoides xylem-
derived cDNA. Except for the P. trichocarpa sequences used
for generation of the phylogenetic tree, all the other sequen-
from Populus deltoides. PdGATL1.1 encodes a protein of 360
amino acids and PdGATL1.2 encodes a protein of 353 amino
acids. Pair-wise comparisons of the amino acid sequences
showed that these two proteins are highly similar, having
93% sequence identity with each other. Further, these two
poplar proteins have 82 and 81% identity, respectively, at
the amino acid level with PARVUS/AtGATL1 (Figure 1B).
The expression profiles of the two genes were examined by
quantitative real-time PCR using primers that were specific to
each gene. Cortex, phloem, xylem, shoot tip, leaf, and root tis-
sues were harvested from young Populus deltoides trees
grown in a greenhouse. Ubquitin was used as an internal con-
trol. As indicated in Figure 2, PdGATL1.1 and PdGATL1.2 are
pression levels are very different. PdGATL1.1 is highly expressed
in xylem compared to other tissues. PdGATL1.2 expression is
highest in xylem, but overall transcript levels are more uniform
in all tissues examined, except root and PdGATL1.2 transcript
lem, the expression level of PdGATL1.1 is 26 times that of
PdGATL1.1 and PdGATL1.2 Are Targeted to the Secretory
Predictions about the sub-cellular localization of the PdGATL1
proteins were made by subjecting the PdGATL1 amino acid
sequences to analyses using publicly available bioinformatics
packages, including SOSUI, TMHMM 2.0, and PSORT (see
Methods). PdGATL1.2 was predicted to have no transmem-
brane domain by all programs used, and is predicted by PSORT
to have a cleavable N-terminal signal peptide that directs the
Kong et al.
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dPoplar Orthologs to PARVUS/AtGATL1