Two poplar methyl salicylate esterases display comparable biochemical properties but divergent expression patterns

Article abstract of DOI:10.1016/j.phytochem.2008.11.014

Two genes encoding proteins of 98% sequence identity that are highly homologous to tobacco methyl salicylate (MeSA) esterase (SABP2) were identified and cloned from poplar. Proteins encoded by these two genes displayed specific esterase activities towards MeSA to produce salicylic acid, and are named PtSABP2-1 and PtSABP2-2, respectively. Recombinant PtSABP2-1 and PtSABP2-2 exhibited apparent Km values of 68.2 ± 3.8 μM and 24.6 ± 1 μM with MeSA, respectively. Structural modeling using the three-dimensional structure of tobacco SABP2 as a template indicated that the active sites of PtSABP2-1 and PtSABP2-2 were highly similar to that of tobacco SABP2. Under normal growing conditions, PtSABP2-1 showed the highest level of expression in leaves and PtSABP2-2 was most highly expressed in roots. In leaf tissues of poplar plants under stress conditions, the expression of PtSABP2-1 was significantly down-regulated by two stress factors, whereas the expression of PtSABP2-2 was significantly up-regulated by four stress factors. The plausible mechanisms leading to these two highly homologous MeSA esterase genes involved in divergent biological processes in poplar are discussed.

Full text of DOI:10.1016/j.phytochem.2008.11.014

Phytochemistry 70 (2009) 32–39
Contents lists available at ScienceDirect
Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Two poplar methyl salicylate esterases display comparable biochemical
properties but divergent expression patterns
a
a
b
c
a
Nan Zhao , Ju Guan , Farhad Forouhar , Timothy J. Tschaplinski , Zong-Ming Cheng ,
b
a,
*
Liang Tong , Feng Chen
Department of Plant Sciences, University of Tennessee, 252 Ellington Plant Science Bldg., 2431 Joe Johnson Drive, Knoxville, TN 37996, USA
Department of Biological Sciences, Northeast Structural Genomics Consortium, Columbia University, New York, NY 10027, USA
Environmental Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831, USA
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 June 2008
Received in revised form 27 October 2008
Available online 10 January 2009
Two genes encoding proteins of 98% sequence identity that are highly homologous to tobacco methyl
salicylate (MeSA) esterase (SABP2) were identified and cloned from poplar. Proteins encoded by these
two genes displayed specific esterase activities towards MeSA to produce salicylic acid, and are named
PtSABP2-1 and PtSABP2-2, respectively. Recombinant PtSABP2-1 and PtSABP2-2 exhibited apparent
Km values of 68.2 ± 3.8
three-dimensional structure of tobacco SABP2 as a template indicated that the active sites of PtSABP2-
and PtSABP2-2 were highly similar to that of tobacco SABP2. Under normal growing conditions,
PtSABP2-1 showed the highest level of expression in leaves and PtSABP2-2 was most highly expressed
in roots. In leaf tissues of poplar plants under stress conditions, the expression of PtSABP2-1 was signif-
icantly down-regulated by two stress factors, whereas the expression of PtSABP2-2 was significantly up-
regulated by four stress factors. The plausible mechanisms leading to these two highly homologous MeSA
esterase genes involved in divergent biological processes in poplar are discussed.
Ó 2008 Elsevier Ltd. All rights reserved.
lM and 24.6 ± 1 lM with MeSA, respectively. Structural modeling using the
Keywords:
Black cottonwood
Populus trichocarpa
Methyl esterase
SABP2
Methyl salicylate
Salicylic acid
1
Gene family
Molecular modeling
1
. Introduction
2003). The gene encoding SABP2 was cloned and its deduced pro-
tein sequence showed high similarity to
a/b fold hydrolases
Plants respond to pathogen infection by activating a variety of
(Kumar and Klessig, 2003). A genetic study demonstrated that
SABP2 is a vital component of the SA (1) signaling pathway. When
SABP2 expression was silenced in tobacco, several SA-dependent
responses, including development of local resistance to tobacco
mosaic virus (TMV) and induction of PR gene expression, were im-
peded (Kumar and Klessig, 2003; Kumar et al., 2006). The three-
dimensional structure of SABP2 was recently determined, which
defense mechanisms (Dangl and Jones, 2001). At the infection site
and surrounding area, cells usually undergo programmed cell
death, which leads to the formation of necrotic lesions (Mittler
et al., 1996). In addition to local responses, plants frequently devel-
op resistance in other parts of the plant. This resistance, called sys-
temic acquired resistance (SAR), is broad-spectrum and long-
lasting (Ryals et al., 1996). Salicylic acid (SA) (1) (Fig. 1) is a critical
signal for activating both local and systemic defenses (Sticher et al.,
showed typical structural features of an a/b fold hydrolase super-
family (Forouhar et al., 2005). Further biochemical study demon-
strated that SABP2 has strong esterase activity using methyl
salicylate (MeSA) (2) as substrate (Forouhar et al., 2005). MeSA
(2) is an ubiquitous compound in plants. It is produced from SA
(1) by the action of SA methyltransferase (SAMT) (Ross et al.,
1999). The hydrophobic nature of MeSA (2) makes itself a diffusible
intercellular signal transducer. A recent study demonstrated that
MeSA (2) is a mobile signal for SAR in tobacco (Park et al., 2007).
Produced from SA (1) at the local infection site by the action of
SAMT, MeSA (2) is readily translocated through the phloem. In sys-
temic tissues, MeSA (2) is perceived by SABP2 and converted back
to SA (1), which is the active signal (Park et al., 2007).
1
997). After pathogen invasion, the endogenous levels of SA (1) in
the infection site and systemic tissues increase, which induces the
expression of defense-associated genes including those encoding
pathogenesis-related (PR) proteins (Lamb and Dixon, 1997). In re-
cent years, molecular genetic and biochemical studies led to iden-
tification of a number of key components of the SA (1) signaling
pathway (Durrant and Dong, 2004). The SA-binding protein 2
SABP2) is one of them.
SABP2 was originally identified from tobacco using a biochem-
ical approach. It is present in low abundance in tobacco cells and
can specifically bind SA (1) with high affinity (Kumar and Klessig,
(
The role of the SA-dependent signaling pathway in plant
defenses has been relatively well studied in a variety of annual spe-
cies, such as tobacco (Malamy et al., 1990), Arabidopsis (Cao et al.,
*
Corresponding author. Tel.: +1 865 974 8521; fax: +1 865 974 1947.
E-mail address: fengc@utk.edu (F. Chen).
0
031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.11.014

N. Zhao et al. / Phytochemistry 70 (2009) 32–39
33
O
1
2
994; Clarke et al., 2000) and rice (Chern et al., 2005; Iwai et al.,
007). In contrast, little is yet known about the role of the SA (1) sig-
COOH
COOCH3
naling pathway in plant defenses in perennial woody species. Such
species are long lived and therefore, more prone to attacks by patho-
gens during their life cycle (Rinaldi et al., 2007). SA (1) treatment can
induce expression of defense genes in trees such as pine (Davis et al.,
COOCH
3
OH
OH
2
1
3
6
O
2
002), suggesting that the SA-dependent signaling pathway is also
COOCH3
C O O H
important for defense responses in perennial woody species.
We have undertaken a project to study plant defense mecha-
nisms against biotic stresses in perennial woody species using Pop-
ulus as a model. There are about 40 species within the genus
Populus. Members of the Populus are among the fast-growing tree
species in the world. That is one important reason why Populus
has been considered a candidate as bioenergy crop. Because of its
small genome, relative ease of genetic manipulation, and rapid
growth, Populus has also been established as a model for forest tree
genetics and genomics (Jansson and Douglas, 2007). Black cotton-
wood (Populus trichocarpa, Torr. & Gray) is the first tree species
whose genome has been fully sequenced (Tuskan et al., 2006),
which provides unprecedented opportunities for the study of tree
biology and physiology. Despite the many advantages of using
poplar as a tree model as well as a bioenergy crop, poplar trees
are susceptible to many pathogens (Ostry and McNabb, 1985). Elu-
cidating the natural defense mechanisms of poplar is therefore
important for both basic and applied sciences. The current study
concerns the SA (1) signaling pathway in poplar.
C O OH
N
H
N
H
4
5
Fig. 1. Structures of compounds 1–6.
Full-length cDNAs of PtSABP2-1 and PtSABP2-2 were cloned via
RT–PCR from poplar leaf and root cDNAs, respectively. Both
PtSABP2-1 and PtSABP2-2 encode a protein of 263 amino acids.
Whereas PtSABP2-1 and PtSABP2-2 are 98% identical to each other,
their sequences are 77% identical to that of tobacco SABP2 (Fig. 2).
The putative pIs of PtSABP2-1, PtSABP2-2 and NtSABP2 are 5.01,
4.78 and 5.27, respectively.
2.2. PtSABP2-1 and PtSABP2-2 display MeSA esterase activity
Recombinant proteins of PtSABP2-1 and PtSABP2-2 were ex-
pressed in Escherichia coli from their corresponding full-length
cDNAs. In the vector pET100/D-TOPO, the full-length cDNAs of
PtSABP2-1 and PtSABP2-2 were fused to an N-terminal sequence
containing codons for six histidine residues. His-tagged PtSABP2-
1 and PtSABP2-2 were expressed in E. coli then purified using
Ni-NTA agarose to near electrophoretic homogeneity (see Supple-
mentary data).
Purified PtSABP2-1 and PtSABP2-2 were tested for esterase
activity using MeSA as substrate. While the amounts of MeSA (2)
decreased, the amounts of SA (1) increased in the same reaction,
indicating that both PtSABP2-1 and PtSABP2-2 (not shown) cata-
lyzed the hydrolysis of the methyl group of MeSA (2) to form SA
(1). Both of PtSABP2-1 and PtSABP2-2 had the optimum pH at
7.0 (see Supplementary data). Under steady-state conditions,
Previous studies showed that some common characteristics of
the SA (1) signaling pathway are shared by poplar and some herba-
ceous plants. For example, SA (1) treatment can induce expression
of defense genes in both poplar (Koch et al., 2000) and tobacco
(Uknes et al., 1993). However, poplar also has distinct features.
For example, the basal levels of SA (1) in poplar are much higher
than those in tobacco and Arabidopsis (Koch et al., 2000), posing
a question on the uniqueness of the molecular mechanism of the
SA (1) signaling pathway in poplar. In this paper, we were particu-
larly interested in determining whether a key component of the SA
(1) signaling pathway, SABP2, is conserved in poplar. Gene cloning,
biochemical function characterization and expression analysis of
two SABP2 genes from black cottonwood are reported.
PtSABP2-1 exhibited an apparent Km value 68.2 ± 3.8
l
M with
MeSA and a Vmax value of 4.2 ± 0.04 pmol S (Fig. 3A). PtSABP2-2
had a Km value 24.6 ± 1 M with MeSA (2) and a Vmax value of
.35 ± 0.03 pmol S (Fig. 3B).
ꢀ1
2
. Results
l
ꢀ1
0
2.1. Identification and sequence analysis of putative poplar SABP2
genes
2.3. Substrate specificity analysis of PtSABP2-1 and PtSABP2-2
When the protein sequence of tobacco SABP2 (Kumar and
In in vitro enzyme assays, tobacco SABP2 displayed esterase
activity with multiple substrates, including MeSA (2), MeJA (3)
and MeIAA (4), with different specific activities (Forouhar et al.,
2005). For comparison, PtSABP2-1 and PtSABP2-2 were also ana-
lyzed for esterase activity with MeJA (3) and MeIAA (4). At
Klessig, 2003) was used to blast search the sequenced poplar gen-
ome (Tuskan et al., 2006) using e-10 as a cut-off E-value, 30 proteins
significantly homologous to SABP2 were identified. Two proteins,
estExt_fgenesh4_pm.C_LG_VII0354 and eugene3.00070971, are
most similar to tobacco SABP2 with an E-value lower than e-100.
The two proteins displayed specific MeSA esterase activity (see Sec-
tion 2). Thus, estExt_fgenesh4_pm.C_LG_VII0354 and eugene3.
10 lM and 100 lM concentrations of substrates, PtSABP2-1 and
PtSABP2-2 showed no or very low activity with MeJA (3) and MeI-
AA (4) (Fig. 4). At 1 mM concentration of substrates, PtSABP2-1 and
PtSABP2-2 showed significant activity with MeJA (3). However, the
esterase activity of PtSABP2-1 and PtSABP2-2 with MeJA (3) is only
9% and 14% of their corresponding activities with MeSA (2), respec-
tively (Fig. 4). These results indicate that the two poplar SABP2
proteins are highly specific for MeSA (2) among the three sub-
0
0070971 were named PtSABP2-1 and PtSABP2-2, respectively.
Both PtSABP2-1 and PtSABP2-2 are localized on chromosome
VII. They are about 34 kbs from each other. Both genes contain
two introns and three exons. Sequence comparison showed that
the first introns of the two genes are 100% identical and the second
introns 97% identical (data not shown). The promoter regions of
the two genes were also compared. Significant sequence similarity
was detected in the regions from start codon to about 300 bps up-
stream of the start codon. In contrast, the promoter regions be-
tween 300 and 800 bps upstream of the start codon of the two
genes share no significant sequence similarity (see Supplementary
data).
strates tested at both physiologically relevant (10
vant (1 mM) concentrations.
lM) and irrele-
2.4. Structural features of PtSABP2-1 and PtSABP2-2
The crystal structure of tobacco SABP2 has been experimentally
determined (Forouhar et al., 2005). Since tobacco SABP2 and the

3
4
N. Zhao et al. / Phytochemistry 70 (2009) 32–39
Fig. 2. Multiple sequence alignment of deduced amino acid sequences of PtSABP2-1 and PtSABP2-2 and the protein sequence of tobacco SABP2 (NtSABP2).
with their high sequence conservation. In the active site region,
4
.5
3
PtSABP2-1 has only one substitution with respect to tobacco
SABP2: G212 of tobacco SABP2 is replaced by A215 in PtSABP2-1
A
3
2
1
0
(Fig. 5A). The extra methyl group in PtSABP2-1 resulted from the
G to A change is not expected to affect the binding of SA, in agree-
ment with the kinetic studies. PtSABP2-2 has two substitutions:
G212 and L181 of tobacco SABP2 are respectively replaced by
A215 and M184 in PtSABP2-2 (Fig. 5B). The introduction of a bulk-
ier Met side chain in PtSABP2-2 probably has an impact on the ob-
served lower Km value of this enzyme (Fig. 3).
.5
2
.5
1
Km = 68.2± 3.8 µM
-
1
2.5. Expression analysis of PtSABP2-1 and PtSABP2-2
Vmax = 4.2 ± 0.04 pmol s
.5
0
RT–PCR analysis was performed to determine the expression
patterns of PtSABP2-1 and PtSABP2-2. Gene specific primers were
designed to discriminate the transcripts of the two genes
0
0.05
0.1
0.15
0.2
-
0.5
-
1
1
/[MeSA] (µM )
(Fig. 6A). Total RNA was isolated from young leaves, old leaves,
stems, and roots of one year-old poplar trees and used for semi-
quantitative RT–PCR analysis. Gene expression analysis (Fig. 6B)
showed that PtSABP2-1 had the highest level of expression in
leaves and a low level of expression in stems and roots. In contrast,
PtSABP2-2 had the highest level of expression in roots, a moderate
level of expression in stems and a low level of expression in leaves.
To understand the potential roles of PtSABP2-1 and PtSABP2-2 in
plant defense, expression of the two genes under stress conditions
was also analyzed. Poplar plants were either physically injured or
treated with SA (1), MeJA (3), or a fungal elicitor alamethicin.
Leaves of the treated poplar plants and appropriate control plants
were collected for extraction of RNAs, which were used RT-PCR
analysis. The expression of PtSABP2-1 (1) was not significantly af-
fected by the stress factors tested, except for 2 h wounding and
SA (1) treatment for 24 h, which significantly suppressed the
expression of PtSABP2-1 (Fig. 6C). In contrast, the expression of
PtSABP2-2 was significantly up-regulated by a number of treat-
ments, including 24 h wounding, 2 h SA (1) treatment, 4 h MeJA
2
1
1
1
1
1
0
8
6
4
2
0
8
6
4
2
0
B
Km = 24.6± 1 µM
-1
Vmax = 0.35 ± 0.03 pmol s
-0.05
0
0.05
0.1
0.15
0.2
-
1
1
/[MeSA] (µM )
Fig. 3. Biochemical properties of PtSABP2-1 and PtSABP2-2. Steady-state kinetics of
PtSABP2-1 and PtSABP2-2 was measured using MeSA (2) as substrate. Examples of
the Lineweaver–Burk plot for PtSABP2-1 (A) and PtSABP2-2 (B) were shown.
(3) treatment and 2 h alamethicin treatment. Treatments by
wounding, SA (1), and MeJA (3) at other time points did not signif-
icantly affect the expression of PtSABP2-2 (Fig. 6C).
two poplar SABP2s possess 77% sequence identity, reliable three-
dimensional models of PtSABP2-1 and PtSABP2-2, including their
active sites, were constructed using tobacco SABP2 structure as a
template (Fig. 5). The overall structures of PtSABP2-1 and
PtSABP2-2 are very similar to that of tobacco SABP2, consistent
3. Discussion
SAR has been shown to be activated by a vascular-mobile signal
that moves throughout the plant from the infected leaves (Durrant
and Dong, 2004). The nature of the mobile signal for SAR, however,

N. Zhao et al. / Phytochemistry 70 (2009) 32–39
35
A 1
00
B100
8
0
80
60
6
0
0
4
40
20
0
2
0
0
1
0 µM
100 µM
1mM
10 µM
100 µM
1 mM
Fig. 4. Substrate specificity of PtSABP2-1 and PtSABP2-2. Relative methyl esterase activity of PtSABP2-1 (A) and PtSABP2-2 (B) was measured with MeSA (2), MeIAA (4), and
MeJA (3) at three different concentrations (10 M, 100 M, and 1 mM). The activity with MeSA (2) at each of the substrate concentrations was set at 100%. The activity ratios
between MeSA (2) and the other substrates are indicated.
l
l
Fig. 5. Active sites of PtSABP2-1 and PtSABP2-2. (A) Stereo-view representation of the active site of PtSABP2-1; the SA (light grey) and the side chains of the catalytic triads,
S84, D213, and H241 (all in magenta) are shown as ball-and-stick models. A215 (magenta) is labeled in black so as to distinguish it from the corresponding residue G212 of
tobacco SABP2, which is depicted in green ball; and (B) stereo-view representation of the active site of PtSABP2-2. The two substitutions, A215 and M184, in the active site of
PtSABP2-2, corresponding to G213 and L181 (both shown in green) of tobacco SABP2, are shown in ball-and-stick models and labeled in black.
has been a subject of controversy. SA (1) has been demonstrated to
be essential for the activation of SAR (Gaffney et al., 1993). SA (1)
itself, however, has been disassociated as the mobile signal
showed that poplar, a woody perennial species, contains two func-
tional SABP2 genes. PtSABP2-1 and PtSABP2-2 were identified from
the poplar genome based on their high sequence similarity to to-
bacco SABP2 (Fig. 2). In in vitro biochemical studies, like tobacco
SABP2, both PtSABP2-1 and PtSABP2-2 displayed highly specific
esterase activity towards MeSA (2) (Figs. 3 and 4), supporting that
MeSA (2) is the in vivo substrate. Whether PtSABP2-1 and
PtSABP2-2 accept substrates other than MeSA (2) in planta, how-
ever, remains to be determined. The Km value of tobacco SABP2
(Vernooij et al., 1994). MeSA, the methyl ester of SA (1), was re-
cently demonstrated to be a mobile signal for SAR in tobacco (Park
et al., 2007). With MeSA (2) being the inactive mobile signal, it is
critical that MeSA (2) is converted back to SA (1) at the site of ac-
tion. This reaction is catalyzed by MeSA esterase. Prior to this
study, the only MeSA esterase that has been functionally character-
ized is SABP2 from tobacco (Forouhar et al., 2005). Here, we
was reported to be 8.6 lM (Forouhar et al., 2005). The Km values

3
6
N. Zhao et al. / Phytochemistry 70 (2009) 32–39
et al., 1997), and elicitor treatment (Chen et al., 2003a). MeSA (2)
produced under such stress conditions has been suggested to have
a number of biological roles. For example, insect-induced volatile
MeSA (2) has been suggested to be involved in attracting natural
enemies of the feeding insects (Dicke et al., 1990). Virus-induced
air-borne MeSA (2) has been suggested to act as a signal to activate
defense responses in nearby healthy plants (Shulaev et al., 1997).
In all these cases, if SA (1) is the active signal, then inactive MeSA
(2) would need to be converted back to SA (1). If true, it would sug-
gest that SABP2 has multiple biological functions in addition to its
role in SAR, which is supported by induction of expression of pop-
lar SABP2 genes by multiple stress factors (Fig. 6).
The presence of two MeSA esterase genes with a same biochem-
ical function but seemingly divergent biological roles is intriguing.
It is tempting to speculate that this reflects the specific nature of
natural defenses of perennial woody species. Due to their long gen-
eration time, perennial woody species can not match the evolu-
tionary rates of microbial pathogens that go through several
generations every year (Rinaldi et al., 2007). Therefore, positive
adaptation of defense mechanism may be necessary for perennial
woody species to survive. Overrepresentation of defense-related
genes, such as NBS-LRR in the first sequenced tree genome, sup-
ports this notion (Tuskan et al., 2006). The presence of two SABP2
genes with divergent expression patterns may provide poplar trees
an advantage in defense against pathogens.
A
PtSABP2-1
PtSABP2-2
YL OL
S
R
B
PtSABP2-1
PtSABP2-2
Ubiquitin
Wounding Tween
SA
EtOH MeJA Meth Ala
C
Ctr 2h 24h 2h 24h 2h 24h 40m 4h 40m 4h 2h
2h
PtSABP2-1
*
*
PtSABP2-2
Ubiquitin
*
*
*
*
Fig. 6. Expression analysis of PtSABP2-1 and PtSABP2-2. (A) Plasmids PPtSABP2-1
and PPtSABP2-2 containing full-length cDNAs of PtSABP2-1 and PtSABP2-2, respec-
tively, were used as templates in PCRs with gene-specific primers for PtSABP2-1
and PtSABP2-2; (B) tissue-specific expression of PtSABP2-1 and PtSABP2-2. Young
leaves (YL), old leaves (OL), stems (S), and roots (R) were colleted from one year-old
poplar trees grown in the greenhouse; and (C) expression of PtSABP2-1 and
PtSABP2-2 in leaves of poplar plants that were either physically injured (wounding,
h and 24 h), or treated with one of the following chemicals: salicylic acid (SA) (1),
methyl jasmonate (MeJA) (3) and alamethicin (Ala). Treatments with 0.1% tween
Tween), ethanol (EtOH) and methanol (Meth) were used as controls for SA (1),
2
(
MeJA (3) and Ala treatments, respectively. Bands marked with a asterisk (ꢁ) indicate
that the gene showed significant difference in expression levels in treated vs.
control plants (P < 0.05). In both (B) and (C), PCR with primers for Ubiquitin was
used to judge equality of the concentration of cDNA templates in different samples.
4
. Concluding remarks
The mechanism that leads to functional divergence of PtSABP2-
and PtSABP2-2 is also intriguing. The two SABP2s are highly
1
homologous to each other, suggesting they are the consequence
of gene duplication. Gene duplication may result from whole gen-
ome duplication, segmental duplication of chromosomes, or local
duplication caused by unequal crossover (Zhang, 2003; Yang
et al., 2006). PtSABP2-1 and PtSABP2-2 are localized on chromo-
some VII and are 30 kbs from each other. They are therefore likely
the consequence of local gene duplication. The high sequence sim-
ilarity between the two genes, even in introns, implies that the
duplication event occurred relatively recently. The promoter re-
gions of the two genes, however, show relatively high degree of
divergence, suggesting that promoter divergence is probably a ma-
jor determinant of functional divergence of PtSABP2-1 and
PtSABP2-2.
of PtSABP2-1 and PtSABP2-2 are about 10 times and three times
higher than that of tobacco SABP2. It will be interesting to deter-
mine whether the endogenous levels of MeSA (2) in poplar tissues
under stresses are higher than that in tobacco tissues under stres-
ses. The overall three-dimensional structures and active sites of the
three proteins are highly conserved (Fig. 5). Despite their similar
biochemical properties, the two poplar SABP2 genes display diver-
gent expression patterns. They showed different tissue-specificity
under normal growing conditions and different responses to stress
factors (Fig. 6), suggesting that these two genes have divergent bio-
logical roles.
Studying the potential involvement of SABP2s in poplar-patho-
gen interactions will be important for elucidation of the biological
roles of poplar SABP2s, especially in the context of a SA metabolism
that is somehow different from that in tobacco. Whereas the SA (1)
levels in healthy leaves of tobacco are low, in healthy tissues of
poplar they are high (Koch et al., 2000; Morse et al., 2007). In pop-
lar, overexpressing a bacterial SA hydroxylase gene nahG in poplar
did not cause a significant reduction of the SA (1) levels (Morse
et al., 2007), which is different from the study with tobacco
5. Experimental
5.1. Plant material and chemicals
The female black cottonwood clone ‘Nisqually-1’, which was
used for whole genome sequencing (Tuskan et al., 2006), was used
for gene cloning and expression analysis in this study. The tissues
used for gene expression analysis at normal growing conditions,
including young leaves, old leaves, stems, and roots, were collected
from one year-old poplar trees grown in the greenhouse. Leaf tis-
sues used for gene expression analysis under stress conditions were
collected from poplar plants at the eight-leaf stage that were vege-
tatively propagated grown on MS medium. Poplar plants were
either physically injured or treated with several chemicals, includ-
ing SA (1), methyl jasmonate (MeJA) (3) or a fungal elicitor alame-
thicin. For physical injury, leaves were cut with a sterile razor blade
to produce three lateral incisions on each side of the midvein and
the wounded leaves were collected at 2 h and 24 h after wounding.
For SA (1) treatment, poplar plants were sprayed with either 5 mM
SA (1) solution in 0.1% Tween (pH 7.0) or 0.1% Tween only as con-
trol. Leaves were collected at 2 h and 24 h after initiation of the
(Delaney et al., 1994). How PtSABP2-1 and PtSABP2-2 function in
poplar with respect to SA metabolism as compared with that in to-
bacco remains to be determined. Poplar mosaic virus (PopMV) is
the best characterized tree virus (Cooper, 1993). The infection
dynamics of PopMV has been investigated using different geno-
types of Populus (Smith and Campbell, 2004). The poplar-PopMV
system, therefore, presents a good model for determining whether
PtSABP2s function in SAR in this perennial woody species. Such
study will provide further evidence on whether SABP2-dependent
SAR mediated by SA (1) is a conserved defense pathway.
In addition to its involvement in SAR, MeSA (2), the substrate of
SABP2, has been implicated in a number of other biological/ecolog-
ical processes. MeSA (2) is often emitted as a volatile compound
from the plants that are being challenged by stress factors, includ-
ing insect feeding (Chen et al., 2003a), virus infection (Shulaev

N. Zhao et al. / Phytochemistry 70 (2009) 32–39
37
treatment. For MeJA (3) treatment, poplar plants were placed in a
L glass jar containing a cotton tip applied with 1 l MeJA (3) dis-
solved in EtOH (200 l) ethanol. Poplar plants placed in another 1 L
glass jar containing only EtOH (200 l) were used as control. The
glass jar was quickly sealed. Leaves were collected after 40 min
and 4 h after initiation of the treatment. For alamethicin treatment,
leaves were cut-off from the base of the petiole and the detached
leaves then placed apright in a small glass beaker containing
5.5. MeSA esterase activity assay
1
l
l
In a single assay, the reaction was performed in 90
l
l volume
l
containing 50 mM Tris–HCl, pH 7.5, 4
600 M MeSA (2) at 25 °C. The individual assays were terminated
by addition of 10 l 2 N HCl at one of the following time points:
0 h, 1 h, 2 h, and 4 h after initiation of the reaction. Then individual
assays were divided into two halves. The first half (50 l) was ex-
tracted with EtOH (100 l) and the organic phase analyzed for the
lg purified enzyme, and
l
l
l
1
0 ml of 5
a 5 mg/ml stock solution in 100% MeOH). As a control, detached
leaves were submerged in 0.1% MeOH in H O. Only the petiole of
l
g/ml alamethicin (dissolved 1000-fold in water from
l
amount of MeSA (2) using GC–MS as previously described (Zhao
et al., 2008). The second half was used for identification and quan-
2
each leaf was submerged in the solution. The glass beaker was then
sealed with Saran wrap and placed in a growth chamber. Leaves
were collected 2 h after the treatment. Two replicates were
performed for each treatment. All chemicals were purchased from
Sigma–Aldrich (St. Louis, MO, USA).
tification of SA (1). Aliquots (50
tively transferred to scintillation vials and dried down in a He
stream. The dried samples were dissolved in 500 L of silylation–
grade CH CN followed by addition of 500 L N-methyl-N-trimeth-
ylsilyltrifluoroacetamide (MSTFA) with 1% trimethylchlorosilane
TMCS) (Pierce Chemical Co., Rockford, IL, USA), and then heated
for 1 h at 70 °C to generate trimethylsilyl (TMS) derivatives. After
days, 1 L aliquots were injected into a ThermoFisher DSQII
GC–MS, fitted with an Rtx-5MS (crosslinked 5% PH ME Siloxane)
30 m ꢂ 0.25 mm ꢂ 0.25 m film thickness capillary column (Res-
ll) of crude assays were quantita-
l
3
l
(
5.2. Database search and sequence analysis
2
l
To identify putative poplar SABP2-like esterase genes, the pro-
tein sequence of tobacco SABP2 (accession: AY485932) was used
as a query to search the genome sequence database of poplar
l
tek, Bellefonte, PA, USA). The standard quadrupole GC–MS was
operated in electron impact (70 eV) ionization mode, with six
full-spectrum (70–650 Da) scans per second. Gas (helium) flow
was set at 1.1 mL/min with the injection port configured in the
splitless mode. The injection port and detector temperatures were
set to 220 °C and 300 °C, respectively. The initial oven temperature
was held at 50 °C for 2 min and was programmed to increase at
20 °C/min to 325 °C and held for another 11.25 min, before cycling
back to the initial conditions. The SA (1) peak was quantified by
extracting 267 m/z to minimize integration of co-eluting metabo-
lites. Peaks were quantified by area integration and the concentra-
tions were derived from an external calibration curve of amount of
SA (1) injected versus peak area integration of the extracted m/z.
Final values are an average of three independent measurements.
(http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html)
using
the BlastP algorithm (Altschul et al., 1990). Two poplar genes
encoding proteins with the highest level of sequence similarities
(
77%) to tobacco SABP2 were chosen for further analysis.
Nucleic acid and protein sequence alignments were made using
the ClustalX program (Thompson et al., 1997), and displayed using
GeneDoc (http://www.psc.edu/biomed/genedoc/).
5.3. Cloning full-length cDNA of PtSABP2-1 and PtSABP2-2
Total RNA was extracted from the leaf and root tissues using the
RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) and DNA contam-
ination was removed with an on-column DNase (Qiagen, Valencia,
CA, USA) treatment. Total RNA (1.5
lg) was reverse-transcribed
into first-strand cDNA in a 15 L reaction volume using the first-
l
5.6. Determination of relative specific activity of PtSABP2-1 and
PtSABP2-2 with three substrates
strand cDNA synthesis Kit (Amersham Biosciences, Piscataway,
NJ, USA) as previously described (Chen et al., 2003b). PtSABP2-1
full-length cDNA was amplified with the leaf cDNA using the for-
A two-step radiochemical esterase assay was performed to
determine the substrate specificity of PtSABP2-1 and PtSABP2-2
following a protocol previously reported (Forouhar et al., 2005).
0
0
ward primer 5 -CACCATGGTAGAGACCAAGAATCAGA-3 and the re-
0
0
verse primer 5 -CAAACCACTATAAGTAAGGGTGCTGA-3 . PtSABP2-2
full-length cDNA was amplified with the root cDNA using the for-
The reaction of the first step was performed with a 40
containing 50 mM Tris–HCl, pH 7.5, one of the three substrates
(MeSA, MeJA and methyl indole-3-acetate (MeIAA)) at 10 M,
100 M or 1 mM concentrations and 0.5 g purified PtSABP2-1 or
ll volume
0
0
ward primer 5 -CACCATGGTAGAGACCAAGAATCAGG-3 and the re-
0
0
verse primer 5 -TATAACTAAGGGTGCTGATAAGGACA-3 . PCR was
l
set as follows: 94 °C for 2 min followed by 30 cycles at 94 °C for
l
l
3
0 s, 60 °C for 45 s and 72 °C for 1 min 30 s, and a final extension
PtSABP2-2 for 30 min at 25 °C. After that, the samples were boiled
at 72 °C for 10 min. The PCR product was separated on 1.0% agrose
gel. The target band was sliced from the gel and purified using QIA-
quick Gel Extraction Kit (Qiagen, Valencia, CA, USA). The PCR prod-
uct was cloned into pET100/D-TOPO vector using the protocol
recommended by the vendor (Invitrogen, Carlsband, CA, USA). The
cloned cDNAs in pET100/D-TOPO vector were fully sequenced.
for 5 min to stop the reaction and denature the enzyme. The sec-
14
ond step reaction started with the addition of 3
syl- -methionine (SAM) with a specific activity of 51.4 mCi/mmol
(Perkin Elmer, Boston, MA, USA), 120 M SAM and purified meth-
lM
C-adeno-
L
l
yltransferase (SAMT from Arabidopsis (Chen et al., 2003a), indole-
3-acetic acid (5) methyltransferase from poplar (Zhao et al., 2007),
or jasmonic acid (6) methyltransferase from Arabidopsis (Seo et al.,
2001)). The reaction was allowed to proceed for 30 min at 25 °C.
Radiolabeled products were extracted and radioactivity counted
using a liquid scintillation counter (Beckman Coulter, Fullerton,
CA, USA) as previously described (D’Auria et al., 2002). Three inde-
pendent assays were performed for each substrate.
5.4. Purification from E. coli-expressed recombinant proteins
To express PtSABP2-1 and PtSABP2-2, the corresponding pro-
tein expression constructs were transformed into E. coli strain
BL21 (DE3) CodonPlus (Stratagene, La Jolla, CA, USA). Protein
expression was induced by IPTG for 18 h at 22 °C and cells were
lysed by sonication. His-tagged PtSABP2-1 and PtSABP2-2 proteins
were purified from E. coli cell lysate using Ni-NTA agarose follow-
ing the manufacturer’s instruction (Invitrogen, Carlsband, CA,
USA). The purity of the protein was verified by SDS–PAGE and
the concentration of the protein was determined by the Bradford
assay (Bradford, 1976).
5.7. pH optimum for PtSABP2-1 and PtSABP2-2 activities
Both PtSABP2-1 and PtSABP2-2 activities were determined in
50 mM Bis–Tris propane buffer for the pH range across 6.0–10.0
using the radiochemical assay described above. Data presented
are the average of three independent assays.

3
8
N. Zhao et al. / Phytochemistry 70 (2009) 32–39
5.8. Determination of kinetic parameters of PtSABP2-1 and PtSABP2-2
Acknowledgements
The increase in reaction rate with increasing concentrations of
We are grateful to Byung-Guk Kang for providing poplar plants
for gene expression analysis. This research was supported in part
by the DOE Office Biological and Environmental Research – Gen-
ome to Life Program through the BioEnergy Science Center (BESC),
and by the Tennessee Agricultural Experiment Station.
MeSA (2) was evaluated with the radiochemical assay described
above and was found to obey Michaelis–Mention kinetics. Appro-
priate enzyme concentrations and incubation times were deter-
mined in time-course assays so that the reaction velocity was
linear during the assay period. To determine the Km for MeSA
(2), the concentrations of MeSA (2) were independently varied in
the range from 5 M to 100 M. Lineweaver–Burk plots were made
l
l
Appendix A. Supplementary data
to obtain apparent Km values and maximum velocity values, as
previously described (Chen et al., 2003a). Final values are an aver-
age of three independent measurements.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.phytochem.2008.11.014.
5.9. Molecular modeling
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