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 email@example.com,
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.
dPoplar Orthologs to PARVUS/AtGATL1 | 1041
protein into the secretory pathway. For PdGATL1.1, no consen-
sus was found among the prediction programs used. PSORT
predicts the presence of a cleavable N-terminal signal peptide
could be an integral membrane protein with one transmem-
brane domain. Similarly, the PARVUS/AtGATL1 protein is pre-
dicted to have a signal peptide sequence at the N-terminus by
Figure 1. Phylogenetic Tree and Multiple Sequence Alignments Were Constructed from the Deduced Amino Acid Sequences of the 10-
Member Arabidopsis thaliana GATL Family and the Orthologous Proteins from Populus trichocarpa and P. deltoides.
(A) The protein sequences from A. thaliana and P. trichocarpa were aligned using MAFFT v6.603 (Katoh et al., 2005) and the resulting
alignment was used to perform maximum likelihood phylogeny reconstruction using PhyML v2.4.4 (Guindon and Gascuel, 2003). P. tricho-
carpa GATL protein sequences are identified by their NCBI RefSeq accessions (www.ncbi.nlm.nih.gov/RefSeq/).
(B) Amino acid sequence alignment of PtGATL1.1, PtGATL1.2, PdGATL1.1, PdGATL1.2, and PARVUS/AtGATL1. Gaps (marked with dashes)
were introduced to maximize the sequence alignment. Identical and similar amino acid residues are shaded with black and gray, respec-
1042 | Kong et al.
dPoplar Orthologs to PARVUS/AtGATL1
PSORT, and no transmembrane domain is predicted for this
protein by TMHMM 2.0. However, SOSUI predict that PAR-
VUS/AtGATL1 has a 23-amino acid transmembrane domain
that includes the N-terminal amino acids 1 to 23. This would
leaveno cytosolicN-terminaldomain,whichwould beunusual
for a type II membrane. So it seems most likely that both PAR-
domain, but rather just an N-terminal signal peptide directing
the protein into the endomembrane secretory system. Consid-
ering the high amino acid similarity of PdGATL1.1 with these
two proteins, it is highly likely that PdGATL1.1 also has no
The PARVUS/GATL1 protein was previously suggested to be
localized in endoplasmic reticulum (ER) based on the localiza-
tion of a heterologously expressed enhanced yellow fluores-
cence protein (EYFP)-tagged PARVUS/GATL1 recombinant
protein in carrot protoplasts (Lee et al., 2007). To investigate
whether PdGATL1.1 and PdGATL1.2 have the same sub-cellular
localization as PARVUS/GATL1, we fused PdGATL1.1 and
PdGATL1.2 with EYFP at the C-terminus and transformed
the recombinant protein into Nicotiana benthamiana leaves.
Confocal microscopy was used to determine the sub-cellular
localization of recombinant PdGATL1.1 and PdGATL1.2. As
shown in Figure 3B, 3E, 3H, and 3K, EYFP-tagged PdGATL1.1
and PdGATL1.2 showed both punctuate and network-like
localization patterns in tobacco leaf epidermal cells. Co-
localization experiments (Figure 3C, 3F, 3I, and 3L) revealed
that these patterns overlap with those of both Gmct-ECFP,
an enhanced cyan fluorescent protein (ECFP)-tagged Golgi
marker (Saint-Jore-Dupas et al., 2006; Nelson et al., 2007),
and ECFP-WAK2-HDEL, an ER marker (Nelson et al., 2007)
(Figure 3A, 3D, 3G, and 3J).
PdGATL1.1 and PdGATL1.2 Rescue Arabidopsis parvus/
atgatl1 Mutant Phenotypes
To determine whether PdGATL1.1 and PdGATL1.2 have the
same function as PARVUS/AtGATL1, we attempted to comple-
ment the Arabidopsis parvus/gatl1mutant with the full-length
poplar genes. The open reading frames of the two P. deltoides
genes were cloned from xylem-derived cDNA fused with the
cauliflower mosaic virus (CaMV) 35S promoter, and introduced
individually into a heterozygous parvus/gatl1 mutant line of
Arabidopsis (homozygous parvus/gatl1 plants have very low
fertility (Lao et al., 2003; Shao et al., 2004; Lee et al., 2007),
necessitating the use of the heterozygous line). Transgenic
lines were tested for the presence of the PdGATL1.1 and
Figure 2. Expression Analysis of the PdGATL1.1 and PdGATL1.2
Genes by Quantitative Real-Time PCR.
Relative expression levels in all samples were normalized using
ubiquitin as a constitutively expressed internal control and the
PdGATL1.2 expression levels in root are set to 1. Data are the aver-
ages 6 SE of three biological replicates.
Figure 3. Sub-Cellular Localization of EYFP Tagged PdGATL1.1 and
EYFP-tagged PdGATL1.1 and PdGATL1.2 were transiently expressed
in leaf epidermal cells of Nicotiana benthamiana plants, and their
microscope. Scale bars represent 20 lm.
(A–C) Tobacco leaf epidermal cells expressing both PdGATL1.1–
EYFP and ECFP–WAK2–HDEL constructs. (A) localization of ECFP–
WAK2–HDEL ER marker protein (green). (B) localization of
PdGATL1.1–EYFP protein (red) in the same cell as in (A). (C) merged
image of (A) and (B), showing co-localization of PdGATL1.1–EYFP
and Gmct–ECFP constructs. (D) Localization of Gmct–ECFP Golgi
(red) in the same cell as in (D). (F) Merged image of (D) and (E),
showing co-localization of PdGATL1.1–EYFP and Gmct–ECFP.
(G–I) Tobacco leaf epidermal cells expressing both PdGATL1.2–EYFP
and ECFP-WAK2-HDEL constructs. (G) Localization of ECFP–WAK2–
HDEL ER marker protein (green). (H) Localization of PdGATL1.2–
EYFP protein (red) in the same cell as in (G). (I) Merged image of
(G) and (H), showing co-localization of PdGATL1.2–EYFP and
(J–L) Tobacco leaf epidermal cells expressing both PdGATL1.2–EYFP
and Gmct–ECFP constructs. (J) Localization of Gmct–ECFP Golgi
marker protein (green). (K) Localization of PdGATL1.2–EYFP pro-
tein (red) in the same cell as in (J). (L) Merged image of (J) and
(H), showing co-localization of PdGATL1.2–EYFP and Gmct–ECFP.
Kong et al.
dPoplar Orthologs to PARVUS/AtGATL1 | 1043
PdGATL1.2 transgenes in a homozygous parvus/gatl1 back-
ground. Using poplar gene-specific primers for transgene am-
plification, poplarmRNA expression
Arabidopsis lines was confirmed by RT–PCR (Figure 4B and
4C). The absence of PARVUS/AtGATL1 gene expression in the
transgenic lines was also confirmed using primers specific to
the wild-type (w.t.) Arabidopsis gene (Figure 4B and 4C).
Homozygous parvus/gatl1 mutants show dark-green leaves,
reduced plant stature, reduced size of all organs, including
leaves, floral organs, and fruits, and reduced fertility (Lao
et al., 2003; Shao et al., 2004; Lee et al., 2007). Expression of
either PdGATL1.1 or PdGATL1.2 in the parvus/gatl1 mutant res-
cued the morphological defects in the mutant. Indeed, the
morphology of the complemented plants is indistinguishable
from that of w.t. Arabidopsis plants (Figure 4A).
The parvus/gatl1 mutant also exhibits collapsed xylem ves-
sels and thinner secondary cell walls, which are largely due to
defects in xylan synthesis (Brown et al., 2007; Lee et al., 2007).
In parvus/gatl1 mutants, the xylose content is decreased by
about 47% compared to w.t. (Lee et al., 2007). To demonstrate
whether the morphological complementation by PdGATL1.1
and PdGATL1.2 could be correlated with a rescue of xylan syn-
thesis, xylem morphology and xylan immunolocalization were
compositions of the transgenic plants were determined. As
shown in Figure 5, the shapes of xylem vessels in either the
PdGATL1.1 or PdGATL1.2 complemented plants were essen-
tially indistinguishable from those of xylem vessels in w.t.
pression of either PdGATL1.1 or PdGATL1.2 in a parvus/gatl1
background restored the level of xylose to 86 and 80% of
w.t. levels, respectively (Figure 6).
Immunolocalization of xylan using the xylan-directed
monoclonal antibodies LM10 and LM11 was done to further
investigate whether the increased xylose content and the
complemented phenotype in the transgenic plants correlate
with the rescue of xylan synthesis in secondary cell walls.
but not to arabinoxylan and glucuronoarabinoxylan, whereas
LM11 interacts with both 4-O-methylglucuronoxylan and
Figure 4. Restoration of Plant Size in Arabidopsis parvus Plants by
Overexpression of the Poplar PdGATL1.1 and PdGATL1.2 Genes,
The results shown are representative of six independent transgenic
Arabidopsis lines. All transgenic plants were confirmed to have
a homozygous parvus background by PCR detection of the
T-DNA insertion and absence of an endogenous PARVUS gene com-
pared with the w.t. (upper two panels in (B) and (C)).
(A) The parvus mutant (right) has a short inflorescence stem and
a small rosette size, and overexpression of PdGATL1.1 and
PdGATL1.2, respectively, in parvusplants(middle)restoredthestem
height and rosette size to those of the w.t. (left).
(B) PCR detection of the PdGATL1.1 transgene and its transcript in
the transgenic parvus plants. The expression of the ACTIN gene was
used as an internal control.
(C) PCR detection of the PdGATL1.2 transgene and its transcript in
the transgenic parvus plants. The expression of the ACTIN gene was
used as an internal control. WT, wild-type.
Figure 5. Restoration of Secondary Wall Thickness of Vessels in the
Transgenic Arabidopsis parvus Plants Overexpressing the Poplar
PdGATL1.1 and PdGATL1.2 Genes, Respectively.
Stems and hypocotyls of 8-week-old plants were sectioned (250 nm
thick) and stained with toluidine blue for examination of vessels.
Arrows indicate collapsed vessels. ve, vessel. Images for each tissue
are taken at the same magnification. Bars = 50 lm.
(A–D) Transverse sections taken from the base of stems of w.t.,
PdGATL1.1-complemented parvus, PdGATL1.2-complemented par-
vus, and parvus, respectively.
parvus, and parvus, respectively.
1044 | Kong et al.
dPoplar Orthologs to PARVUS/AtGATL1
arabinoxylan (McCartney et al., 2005). Immunolabeling of
cross-sections of w.t. stems and hypocotyls with LM10 and
LM11 showed strong antibody binding to the walls of interfas-
cicular fibers and xylem cells (Figure 7), both of which have
abundant xylan in their secondary cell walls (York and O’Neill,
2008). Less labeling with these antibodies was observed in par-
vus/gatl1 stems, although the overall pattern of labeling was
unchanged from w.t. plants. In PdGATL1.1 or PdGATL1.2 com-
plemented parvus/gatl1, the levels of labeling with LM10 and
LM11 were restored to w.t. levels in the walls of both interfas-
cicular fibers and xylem cells (Figure 7). Control sections in
which the primary antibodies were omitted showed little, if
any, fluorescence (data not shown). These results show that
both PdGATL1.1 and PdGATL1.2 can perform a similar bio-
chemical function as PARVUS/GATL1 and largely complement
the xylan deficiency of the parvus mutant.
Two close homologs of the Arabidopsis thaliana PARVUS/
GATL1 gene, named GATL1.1 and GATL1.2, were identified
in a BLASTsearch of the Populus trichocarpa genome, and sub-
sequently cloned from the closely related P. deltoides. Their
predicted protein products share 82 and 81% overall amino
acid identity with PARVUS/AtGATL1, respectively.
PARVUS/AtGATL1 has been suggested to play a role in xylan
synthesis in Arabidopsis (Brown et al., 2007; Lee et al., 2007).
PdGATL1.1 or PdGATL1.2 can compensate all of the morpho-
logical changes and most of the cell wall defects caused by
the parvus mutation in Arabidopsis. It is interesting to note
that although the xylose deficiency in cell walls of the mutant
was only partially rescued by expression of the poplar ortho-
complemented plants are comparable with those observed in
w.t. plants. This phenomenon was also observed in PoGT43B
complemented irx9 plants, in which the xylose defect was par-
vessels was restored to w.t. level (Zhou et al., 2007). These
results provide strong evidence that these two poplar genes
are functional orthologs of PARVUS/AtGATL1 and suggest that
genes involved in secondary wall formation are, at least in
part, functionally conserved between the herbaceous plant,
Arabidopsis, and the woody species, poplar.
Quantitative RT–PCR analysis showed that PdGATL1.1 is
strongly expressed in poplar xylem cells. PARVUS was also
found to be highly expressed in interfascicular fibers and xy-
lem cells in Arabidopsis and has been implicated in the synthe-
sis of secondary wall xylan (Brown et al., 2007; Lee et al., 2007).
The observed expression patterns for PdGATL1.1 and AtGATL1
Figure 6. Monosaccharide Composition of Cell Walls Isolated from
Complemented with PdGATL1.2, and Wild-Type Plants.
Cell walls were prepared from stems of 8-week-old plants and their
glycosyl compositions determined as described in Methods. Data
are means (Mol %) 6 SE of analyses carried out on three biological
replicates and each replicate represents cell walls isolated from five
Figure 7. Immunofluorescent Labeling of Transverse Sections of
Wild-Type, parvus + PdGATL1.1, parvus + PdGATL1.2, and parvus
Stems and Hypocotyls.
Labeling was carried out on 250-nm-thick transverse sections taken
from stem (A-H) and hypocotyls (I-P) tissues of 7-week-old w.t. (col-
umn 1), parvus + PdGATL1.1 (column 2), parvus + PdGATL1.2 (col-
umn 3), and parvus (column 4) plants. Antibodies used for
labeling are indicated in the figure. Arrows indicate fibers and xy-
lem vessels. if, interfascicular fiber; xy, xylem; sx, secondary xylem.
Bars = 50 lm.
Kong et al.
dPoplar Orthologs to PARVUS/AtGATL1 | 1045
in poplar and Arabidopsis, respectively, are consistent with
a role for GATL1 in xylan synthesis. The similar expression pat-
terns further support the functional conservation of these
genes between Arabidopsis and Populus.
Although PdGATL1.1 and PdGATL1.2 share 93% identity in
amino acid sequence, the different expression patterns of
these two genes in Populus tissues suggest that they do not
play identical roles in cell wall biosynthesis in poplar.
PdGATL1.2 shows a much lower level of transcription than
PdGATL1.1 and its expression is fairly uniform in cortex,
phloem, xylem, shoot tip, and leaf, while PdGATL1.1 is highly
expressed only in xylem tissue. Such a differential pattern of ex-
pressionbetween genepairs iscommoninPopulus wood-form-
ing organs. About 14% of the duplicated genes were reported
to display differential expression in nodes and internodes of
Populus (Tuskan et al., 2006). According to the duplication–
degeneration–complementation (DDC) model proposed by
Force et al. (1999), most duplicated genes accumulate degener-
ative mutations for some time after the duplication event and
then undergo functional specialization by complementary
partitioning of the functions of the ancestral gene. In other
tary sub-functionalization of the progenitor gene’s functions
rather than through the evolution of new functions (Force
et al., 1999). The differences in gene expression patterns ob-
served for PdGATL1.1 and PdGATL1.2 suggest that this type
of sub-functionalization has occurred for these two poplar
genes, whereas, in Arabidopsis, all GATL1 functions are carried
out by a single gene.
bidopsis, and 40 times smaller than pine (Brunner et al.,
2004a). A total of 45 555 gene models have been predicted
in P. trichocarpa to date (Tuskan et al., 2006). Among the pre-
dicted gene models, 1603 are carbohydrate-active enzymes
(CAZymes; http://www.cazy.org/ (Cantarel et al., 2009)), which
is 1.6 times the number identified in the Arabidopsis genome.
Indeed, poplar has the largest number of CAZyme genes ob-
served among the fully sequenced plant genomes (Geisler-
Lee et al., 2006). It was also found that the number of lignin
biosynthesis-related genes in Populus is larger than in Arabi-
dopsis, and some of these genes occur in duplicate pairs rela-
tive to single copies in Arabidopsis (Tuskan et al., 2006),
suggesting a greater need for the expression of these genes
in poplar during the massive commitment to cell wall biosyn-
thesis that occurs during wood formation. Alternatively, it
could reflect pressure for sub-functionalization.
Sub-cellular localization experiments showed that the
pathway, including ER and Golgi. Current models of cell wall
synthesis propose that non-cellulosic polysaccharides are syn-
thesized in the Golgi and transported to the cell wall in Golgi-
derived vesicles (Turner et al., 2007). For example, there is
volved in xylan synthesis in Arabidopsis, including IRX7, IRX8,
and IRX9, are localized to the Golgi (Zhong et al., 2005; Pen ˜a
et al., 2007), consistent with previous findings of the site for
xylan synthesis in French bean (Gregory et al., 2002). Our pres-
ent work showed that although PdGATL1.1 and PdGATL1.2 do
localize to the Golgi, they are also present in the ER. Although
a Golgi retention signal has not yet been identified, all glyco-
syltransferases characterized thus far have a similar domain
structure. They are all type II transmembrane proteins that
contain a short N-terminal cytosolic region, a single mem-
brane-spanning domain and a large luminal domain that con-
tains the catalytic site. In general, the membrane-spanning and
luminal domains appear to be most important for robust local-
ization of Golgi enzymes (Colley, 1997; Munro, 1998). Since no
transmembrane domain appears to be present in PdGATL1.1
withother membrane-anchoredcomponents inorder tobetar-
geted to the endomembrane system. In N. benthamiana cells,
because of the high level of expression of the PdGATL1.1 and
PdGATL1.2 genes such that there are insufficient amounts of
partner components available to form functional complexes
and thus some PdGATL1.1 and PdGATL1.2 are left in the ER.
Alternatively, overexpression of a protein might saturate a traf-
ficking pathway resulting in the accumulation of a fusion pro-
tein in an earlier endomembrane compartment (Sparkes et al.,
2006). However, we used bacterial inoculum levels that are on
studies (Hanton et al., 2005; Renna et al., 2005; Hanton et al.,
2009) to avoid pathway saturation, and the marker proteins
expressed under the same promoter showed no unexpected lo-
calization patterns in our studies.
Previous studies using carrot protoplasts as the heterolo-
gous expression system showed that the PARVUS/GATL1 pro-
tein in Arabidopsis is located only in the ER (Lee et al.,
2007). The differences in sub-cellular localization of GATL1
proteins in these two studies might be due to the absence
of partnering components in carrot protoplasts, given that
no cell wall is present in protoplasts, whereassuch partners ap-
pear available in tobacco leaf cells that do have walls. Direct
would resolve uncertainties about the sub-cellular localization
of GATL1, although this approach may be problematic, given
the low expression levels of most cell wall-related glycosyl-
transferases. Furthermore, the high degree of sequence simi-
larity amongst GATL proteins may preclude the generation of
act with GATL1 to form synthetic complexes will be necessary
to more clearly explain how the GATL1 protein is retained
within the endomembrane compartment, rather than being
transportedtothe apoplast, as would be expectedfor proteins
that carry no targeting signals (Rojo and Denecke, 2008).
are functional orthologs of AtGATL1 and that the correspond-
Further, we have provided evidence that the two poplar genes
1046 | Kong et al.
dPoplar Orthologs to PARVUS/AtGATL1
have become functionally specialized since the duplication
event that gave rise to these two paralogous genes. Lastly,
the expression pattern observed for PdGATL1.1 is consistent
with an involvement of this gene in the synthesis of secondary
wall polysaccharides (e.g. xylan) that occurs in developing xy-
lem. The results of this study provide further evidence that Ara-
bidopsis is a suitable experimental system for functional studies
of genes involved in wood formation in trees.
Bioinformatic Analyses of GATL Protein Sequences
Protein sequences in Populus trichocarpa similar to PARVUS/
AtGATL1 were identified by a BLAST search (http://blast.
ncbi.nlm.nih.gov/Blast.cgi) of the P. trichocarpa genome
(www.jgi.doe.gov/poplar). The P. trichocarpa sequences iden-
tified from the BLAST search were aligned with all 10 AtGATL
protein sequences using MAFFT v6.603 (Katoh et al., 2005)
and the resulting alignment was used to perform maximum
likelihood phylogeny reconstruction using PhyML v2.4.4
(Guindon and Gascuel, 2003). PhyML analyses were conducted
with the JTT model, 1000 replicates of bootstrap analyses,
ofaminoacids tobe invariableduringthe evolution),fourrate
categories (assign each site a probability to belong to given
evolutionary rate categories), estimated gamma distribution
parameter, and optimized starting BIONJ tree. The resulting
tree was visualized using Treeview (Page, 2009) and the figure
generated using PowerPoint?.
Arabidopsis plants were grown on soil in controlled-environ-
ment cabinets under a 16-h light/8-h dark cycle at 19?C during
the light period and 15?C during the dark period. The light in-
tensity was 120 lmol m?2s?1, and the relative humidity was
maintained at 70%. Populus deltoides saplings were obtained
from Arborgen LLC (Summerville, SC) and were grown on soil
in a greenhouse and watered every day and fertilized every
2 months. Approximately 1-year-old plants were used to col-
lect different tissues (see below).
Screening of Homozygous Plants with T-DNA Insertion
Identification of homozygous plants with T-DNA insertions in
At1g19300 (parvus-3, Salk_045368) was performed as de-
scribed by Brown et al. (2007). Briefly, T-DNA insertions were
identified using the flanking primers (LP and RP) generated by
the SIGnal T-DNA verification primer design website (http://
T-DNA left border LBa1 (5#-GCGTGGACCGCTTGCTGCAACT-3’)
and LBb1 (5#-TCAAACAGGATTTTCGCCTGCT-3’).
and primersfrom the
Quantitative Real-Time PCR
Cortex, developing phloem, and developing xylem were
harvested from the bottom stems by sequential peeling essen-
tially as described (Suzuki et al., 2006). In addition, young
shoot tips (first to third internodes), leaves (from the fourth
to the sixth internode), and fine root tissues were also har-
vested. All tissues were frozen immediately in liquid nitrogen
and stored at –80?C until use. Total RNA was isolated using
RNeasy plant mini kits (Qiagen) and then treated with RN-
ase-free DNase (Qiagen) to remove contaminating genomic
DNA. 1 lg of total RNA was reverse-transcribed using Super-
script?III Reverse Transcriptase (Invitrogen). Quantitative
real-time PCR (qRT–PCR) was performed in 20 ll of reaction
mixture, composed of 1 ll of a given cDNA, 10 ll master
mix IQ? SYBR?Green Supermix (Bio-Rad), 0.4 ll each primers
(10 lM), and 8.2 ll nuclease-free water. Each reaction was re-
peated three times using cDNAs generated from indepen-
dently isolatedRNA preparations.
performed using an iCycler iQ System (Bio-Rad) under the fol-
lowing conditions: initial polymerase activation: 95?C, 4 min;
then 45 cycles of 10 s at 95?C and 30 s at 55.2?C.
follows: PdGATL1.1 (forward, 5#-GGAGTGGATGGAACTACAGA-
GAGTCTATTCCA-3’). In addition, two primers (forward, 5#-
GTTGATTTTTGCTGGGAAGC-3’; reverse, 5#-GATCTTGGCCTTCA-
CGTTGT-3’) to amplify the ubiquitin (BU879229) (Brunner
for quantification. A melting curve was produced at the end of
every experiment to ensure that only single products were
the products on an agrose gel to ensure that only a single band
et al., 2001). The expression level of ubiquitin was measured
and used as a point of reference, being a housekeeping gene
(Rajinikanth et al., 2007).
Mutant Complementation Analysis
The PdGATL1.1 and PdGATL1.2 cDNAs were isolated by RT–PCR
from Populus deltoides developing xylem cDNAs. Gene-specific
primers were designed based on the Populus trichocarpa
sequences (5#-CTAGCCACCTATTTTTAATTTCCC-3’ and 5#- CGAT-
CCTTGAAACTTGACATC-3’ for PdGATL1.1, and 5#-CTCCATAGCC
AAGAGCTACATGTC-3’ and 5#-CACTAACATCTTCGAATCCATGA-
GAG-3’ for PdGATL1.2). The amplified fragments were ligated
into T-easy vector and sequenced using a dye-based cycle se-
quencing kit (Applied Biosystems, Foster City, CA, USA). The
sequences obtained were used for designing the primers used
for cloning of the open reading frames that is described below.
For complementation analysis, the plant transformation
vector pCAMBIA (CAMBIA) was used for the generation of
transformed plants and the full-length PdGATL1.1 and
PdGATL1.2 open reading frames were PCR-amplified from
the fragments sequenced above, respectively, with PCR
primers as follows: PdGATL1.1 (PdGATL1.1OV-F) 5#-TATATGG-
TACCCTAGCCACCTATTTTTAATTTCCC-3’ and (PdGATL1.1OV-R)
Kong et al.
dPoplar Orthologs to PARVUS/AtGATL1 | 1047
CATGTC-3’ and (PdGATL1.2OV-R) 5#-TTTACTCTAGACACTAA-
CATCTTCGAATCCATGAGAG-3#. The DNA fragments were
each inserted between the CaMV 35S promoter and the nopa-
line synthase 3#-terminator in separate pCAMBIA vectors.
The constructs were introduced into Arabidopsis parvus/gatl1
mutant plants by using the Agrobacterium-mediated floral
dip method (Clough and Bent, 1998). Transgenic plants were
selected on hygromycin, and homozygous lines were identi-
fied by PCR and the first generation of transgenic plants
was used for morphological and anatomical analyses.
Topological Analysis of GATL1 Proteins
PdGATL1proteins: SOSUI Classificationand Secondary Structure
Prediction of Membrane Proteins (http://bp.nuap.nagoya-
u.ac.jp/sosui/), TMHMM server version 2.0 (www.cbs.dtu.dk/
services/TMHMM/), and PSORT (http://psort.ims.u-tokyo.ac.jp/
Sub-Cellular Localization of PdGATL1.1 and PdGATL1.2
The PdGATL1.1 and PdGATL1.2 coding regions were cloned in-
frame with an enhanced yellow fluorescent protein (EYFP)
gene under the control of the 35S promoter in a pCAMBIA-
based binary vector (Pattathil et al., 2005) to generate the fu-
sion constructs 35S-PdGATL1.1-EYFP and 35S-PdGATL1.2-EYFP.
The PdGATL1.1 coding region was amplified with the primers
TCCG-3’) and PdGATL1.1-R (5#-AATTAGTCGACCAAGAATC-
CAAGGCGAATGGAGTC-3’) and the PdGATL1.2 coding region
was amplified with the primers PdGATL1.2-F (5#-AATTATCAT-
ing xylem-derived cDNA as the template. The PdGATL1 con-
structs were sequenced and then transformed individually
were individually co-transformed into fully expanded leaf of
Nicotiana benthamiana plants (;8-week-old seedlings grown
at 22?C) together with the enhanced cyan fluorescent protein
(ECFP)-tagged markers specific to either the Golgi or the endo-
plasmic reticulum (ER) compartments. Golgi localization was
based on the cytoplasmic tail and transmembrane domain
(first 49 aa) of soybean a-1,2-mannosidase I (GmMan1)
The ER marker, ECFP–WAK2–HDEL, was created by combining
the signal peptide of Arabidopsis thaliana wall-associated ki-
nase 2 (AtWAK2) (He et al., 1999) at the N-terminus of the fu-
sion protein and the ER retention signal His–Asp–Glu–Leu
(HDEL) atitsC-terminus (Nelsonetal.,2007).Co-transformation
was done as described previously (Johansen and Carrington,
2001), using three different bacterial densities (OD600 = 0.05,
0.3, and 3) as suggested by the Brandizzi group (Hanton et al.,
2005; Renna et al., 2005; Hanton et al., 2009). The reported
localizationresultsarefrom the plants withthelowest bacterial
innoculum, but similar localization patterns were observed for
all three bacterial densities. Three or four days post-infection,
the injectedarea(;2.5 cmradius) was cut and subsequentlyex-
SP2 confocal microscope (Leica Microsystems, Heidelberg,
Germany). For the co-localization studies, excitation lines of
an argon ion laser of 458 nm for CFP and 514 nm for EYFP were
used alternately with line switching using the line-sequencing
scanning facility of the microscope. Fluorescence was detected
using a 475–525-nm bandpass filter for CFP and a 530–590-nm
bandpass filter for EYFP. Images were saved and processed with
Adobe Photoshop version 7.0 (Adobe Systems, San Jose, CA).
Microscopic Investigations of Stem and Hypocotyl
Stems and hypocotyls from 8-week-old transgenic Arabidopsis
plants were fixed in 2.5% glutaraldehyde buffered with
0.05 M phosphate buffer (pH 6.8) at 4?C for 24 h. After fixa-
tion, the tissues were dehydrated using an ethanol series
and embedded in LR White resin (Ted Pella Inc., Redding,
CA) as described (Freshour et al., 1996). Sections (250 nm thick)
were cut using a Reichert-Jung Ultracut E ultra microtome
(Reichert-Jung, Wien, Austria) and stained with toluidine blue
for light microscopy, carried out on an Eclipse80i microscope
(Nikon, Melville, NY). Images were captured with a Nikon
DS-Ril camera head (Nikon, Melville, NY) using NIS-Elements
Basic Research software and images were assembled using
Adobe Photoshop 7.0.
Immunolocalization of Xylan
For detection of xylan, sections (250 nm thick) of stems and
hypocotyls prepared as described above were incubated
with the LM10 and LM11 monoclonal antibodies, which recog-
nize glucuronoxylan (PlantProbes;
(McCartney et al., 2005), and then with 1:100 diluted goat
antibodies (Invitrogen). Negative controls were carried out
in the absence of primary antibody. The fluorescence-labeled
sections were observed using the Nikon Eclipse80i microscope
equipped with epifluorescence optics and photographed at
identical exposure times for all sections as described above.
Cell Wall Isolation and Extraction
Cell walls were prepared as alcohol-insoluble residues as
described previously (Persson et al., 2007). In brief, stems from
10-week-old Arabidopsis plants were harvested on ice, flash-
frozen in liquid nitrogen, and ground to a fine powder with
a mortar and pestle. The ground material was extracted over
a sintered glass funnel (Kimax 60M) under vacuum sequen-
tially with 2 vol. of 100 mL of ice-cold 80% (v/v) ethanol,
100% ethanol, chloroform:methanol (1:1; v/v), and 100% ace-
tone. Starch was removed from the walls by treatment with
Type-I porcine a-amylase (Sigma-Aldrich; 47 units/100 mg cell
wall) in 100 mM ammonium formate for 48 h at 25?C with ro-
tation. De-starched walls werecentrifuged, washed twice with
1048 | Kong et al.
dPoplar Orthologs to PARVUS/AtGATL1
sterile water, twice with 100% acetone, and air dried. Sugar
composition analyseswerecarried out on threeindependently
prepared cell wall preparations as described below.
Each sample (1–2 mg) was hydrolyzed with 2 M TFA contain-
ing 20 lg of myo-inositol (internal standard) for 90 min at
120?C, after which the acid was removed by drying under
an air stream and the samples washed twice with isopropanol.
Reduction was performed by incubation for 2 h in sodium bo-
rohydride (10 mg ml?1) dissolved in 1 M ammonium hydrox-
ide. Samples were neutralized with glacial acetic acid, and
methanol:acetic acid (9:1, v/v) was added before drying the
samples and washing them with methanol. O-acetylation
was performed by adding acetic anhydride and concentrated
TFA for 10 min at 50?C. The samples were washed with isopro-
panol and dried (repeated twice). Sodium carbonate (0.2 M)
and dichloromethane were added, and the samples were vor-
texed and centrifuged before removing the aqueous phase.
Water was added, and the samples were vortexed and briefly
centrifuged. The aqueous layer was removed, and the proce-
sample was concentrated to 100 ll. The alditol acetates were
separated by gas-liquid chromatography on an Agilent 6890N
(Wilmington, DE, USA) equipped with a 30 m 3 0.25 mm (i.d.)
silica capillary column DB 225 (Alltech Assoc., Deerfield, IL,
USA). The temperature was held at 80?C for 1 min upon in-
jection, then programmed from 80 to 170?C at 25?C min?1,
then to 240?C at 5?C min?1, with a 6-min hold at the upper
Sequence data for PdGATL1.1 and PdGATL1.2 described in this
article can be found in the GenBank data library under acces-
sion numbers GQ464114 and GQ464115.
This work was supported by the US Department of Energy (BioEn-
ergy Science Center and grant DE-PS02-06ER64304). The BioEnergy
Science Center is a US Department of Energy Bioenergy Research
search in the DOE Office of Science.
The authors thank Arborgen LLC (Summerville, SC) for providing
saplings of Populus deltoides, and Malcolm O’Neill (CCRC) for ad-
vice on glycosyl composition analyses. No conflict of interest
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