Plant Cell Tiss Organ Cult (2008) 93:303–310
DOI 10.1007/s11240-008-9377-x
ORIGINAL PAPER
Generation and characterization of transgenic poplar plants
overexpressing a cotton laccase gene
Ji Wang Æ Chenglong Wang Æ Mulan Zhu Æ Yang Yu Æ Yuebo Zhang Æ
Zhiming Wei
Received: 22 August 2007 / Accepted: 29 March 2008 / Published online: 11 April 2008
Ó Springer Science+Business Media B.V. 2008
Abstract Laccases are copper-containing glycoproteins, which are widespread in higher plants as
multigene families. To gain more insight in the
function of laccases in plants, especially potential
role in lignification, we produced transgenic poplar
plants overexpressing a cotton laccase cDNA
(GaLAC1) under the control of the cauliflower
mosaic virus 35S promoter. As compared with
untransformed control plants, transgenic plants
exhibited a 2.1- to 13.2-fold increased laccase
activity, whereas plant growth rate and morphological characters remained similar to control plants. A
2.1–19.6% increase in total lignin content of the
stem was found in transgenic plants. Moreover,
transgenic plants showed a dramatically accelerated
oxidation rate of phenolics, without obvious change
in total phenolic content. Our data suggested that
J. Wang C. Wang M. Zhu Y. Yu Z. Wei (&)
Shanghai Institute of Plant Physiology and Ecology,
Shanghai Institutes for Biological Sciences, Chinese
Academy of Sciences, Shanghai 200032, China
e-mail: zmwei@sippe.ac.cn
J. Wang C. Wang M. Zhu Y. Yu
Graduate School of the Chinese Academy of Sciences,
Shanghai 200032, China
Y. Zhang
Institute for Nutritional Sciences, Shanghai Institutes
for Biological Sciences, Chinese Academy of Sciences,
Shanghai 200032, China
GaLAC1 may participate in lignin synthesis and
phenolic metabolism in plants. The present work
provided a new genetic evidence for the involvement of plant laccases in lignification.
Keywords Laccase Lignin Phenolic metabolism Populus Transgenic poplars
Abbreviations
ABTS
2,2-Azinobis(3-ethylbenzothiazoline6-sulfonic acid)
AS
Acetosyringone
BAP
Benzylaminopurine
CaMV 35S Cauliflower mosaic virus 35S
promoter
promoter
IAA
Indole-3 acetic acid
PSK-a
Phytosulfokine-a
ZT
Zeatin
Introduction
Laccases (p-diphenol:O2 oxidoreductase, EC 1.10.3.2)
is a class of copper-containing, cell wall-localized
glycoproteins that can catalyze reduction of O2 to
water by oxidizing phenolic substrates. Laccases
have been found to be widely distributed in various
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organisms, especially in fungi. The fungus laccases
are believed to be involved in lignin degradation,
bioremediation, morphogenesis, pathogenicity as
well as pigment deposition (Mayer and Staples
2002). Although laccase was first discovered in plant
(Yoshida 1883), the precise physiological roles of
laccases in plants have been ambiguous. Some recent
works suggest that laccases may take part in some
important plant physiological processes, including
maintenance of cell wall structure and integrity
(Ranocha et al. 2002), biodegradation of allelopathic
substances and environmental pollutants (Wang et al.
2004), flavonoid metabolism (Pourcel et al. 2005),
responses to salt stress (Liang et al. 2006a), wound
healing (De Marco and Roubelakis-Angelakis 1997)
and iron metabolism (Hoopes and Dean 2004).
It is especially remarkable that laccase was the
first enzyme used to polymerize monolignols
(coniferyl alcohol) in vitro (Freudenberg et al.
1958), but evidences for its involvement in lignin
synthesis have been still impenetrable. Polymerizition experiments in vitro and immunolocalization
assays in vivo imply that laccases may play a role in
some aspects of secondary cell walls formation or
lignin polymerization (Freudenberg et al. 1958;
Sterjiades et al. 1992; Bao et al. 1993; LaFayette
et al. 1999). On the other hand, down-regulation of
laccase genes in L. tulipifera (Dean et al. 1998) and
poplar (Ranocha et al. 2002) as well as overexpression of laccase genes in L. tulipifera (Dean et al.
1998), tobacco cells (LaFayette et al. 1999) had no
effect on the lignin content in transgenic plants. The
most recent evidence came from experiments carried
out in the Arabidopsis thaliana transparent testa10
(tt10) mutant. Pourcel et al. (2005) characterized a
new laccase-like enzyme, AtLAC15 (At5g48100),
which participated in oxidative polymerization of
flavonoids in Arabidopsis seed coat. Further chemical analysis performed by Liang et al. (2006b)
revealed that tt10 had an obvious accumulation of
proanthocyanidin and a reduction in extractable
lignin content in seeds.
To gain more insight in the function of laccases in
plants, especially potential role in lignification, we
introduced a chimeric construct of CaMV35:GaLAC1
into poplar (Populus deltoides). Molecular, biochemical, histochemical and biomass assays were
performed to evaluate the effect of overexpression
of GaLAC1 in transgenic poplar plants.
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Materials and methods
Plant transformation
The pBI121 vector plasmid carrying a cotton
(G. arboreum) laccase cDNA, GaLAC1, was constructed earlier by Wang et al. (2004) (Fig. 1). The
construction was introduced into A. tumefaciens strain
EHA105 and used in transformation experiments.
Petioles from in vitro micropropagated plantlets were
cut and pre-cultured on half-strength MS medium (1/
2 MS) supplemented with 2.0 mg l-1 ZT for 48 h. An
overnight culture of Agrobacterium (OD600 0.8–1.0)
was pelleted by centrifugation and resuspended in 1/
2 MS liquid medium supplemented with 10 g l-1
glucose, 0.02% (v/v) Silwet L-77 and 100 lM AS
(pH 5.3). The pre-cultured explants were soaked in
diluted Agrobacterium for 30 min and then co-cultured
on solid 1/2 MS medium (pH 5.3) with 2.0 mg l-1 ZT
and 100 lM AS for 3 days. After co-cultivation, the
explants were transferred to 1/2 MS medium supplemented with 1.0 mg l-1 ZT, 10 nM PSK-a
(Matsubayashi et al. 2004), 200 mg l-1 Timentin and
30 mg l-1 Kanamycin for shoot induction and selection. Adventitious buds were elongated and selected on
1/2 MS medium with 0.2 mg l-1 BAP, 0.2 mg l-1
IAA and the same antibiotics. Resistant shoots about
2 cm were cut off and rooted on hormone-free 1/2 MS
medium with 15 mg l-1 Kanamycin and 200 mg l-1
Timentin. Rooted shoots were screened for the presence of transgenes by the polymerase chain reaction
(PCR). The PCR-positive shoots were excised and
further propagated and selected on rooting medium
using axillary buds and then transferred to soil in a
greenhouse.
Molecular analysis
Genomic DNA was isolated and purified using the
Plant DNA Maxi Kit (Watson Biotechnologies Inc.,
Fig. 1 Schematic diagram of plasmid pBI121CaMV35:GaLAC1 for poplar transformation. LB, T-DNA left border; RB,
T-DNA right border; P nos, nopaline synthase promoter; npt II,
neomycin phosphotransferase II, T nos, nopaline synthase
terminator; P 35S, CaMV 35S promoter; GaLAC1, cotton
laccase cDNA
Plant Cell Tiss Organ Cult (2008) 93:303–310
Shanghai, China). The specific primers for GaLAC1
amplification were 50 -GCA CTT GGT TGG CGA
GGA GA-30 , 50 -TGA GAT TCG GGT TCA CAG
AGG T -30 . PCR reactions contained 50 ng of
genomic DNA, 0.5 lM of each primer, 200 lM
of a dNTP mixture, 19 PCR buffer and 1.25 units of
Taq DNA polymerase (Takara, Dalian, China). The
amplification reactions were initially denatured at
94°C for 10 min, then amplified with 30 cycles of
45 s at 94°C, 30 s at 60°C and 1 min at 72°C,
followed by a final extension of 10 min at 72°C.
Reaction products were separated by electrophoresis
on a 1.5% agarose gel.
Stable integration of the transgene into the genome
of transgenic plants was further confirmed by Southern blot analysis. Genomic DNA (40 lg) of
transformed and untransformed control plants was
digested with BamHI, electrophoretically separated
and transferred to a Hybond N+ nylon membrane
(Amersham, Buckinghamshire, UK) by standard
procedures. Hybridization was carried out with high
stringency hybridization conditions (Sambrook and
Russell 2001). The PCR-generated probes were
labeled with a-[32P]dCTP using a random primer
DNA labeling kit (Takara) according to the manufacturer’s instructions.
Laccase activity assay
Laccase enzyme extraction and activity assay was
performed using the method by Wang et al. (2004).
To a 1.5-ml mixture 30 ll of crude enzyme extract
was added to 1.4 ml reaction buffer (50 mM sodium
acetate, 100 lM CuCl2, pH 4.5). The reaction was
conducted at 30°C for 30 min and was terminated by
the addition of 70 ll of acetic acid. Laccase activity
was determined by monitoring the oxidation of
2 mg ml-1 ABTS at 420 nm.
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Lignin staining
For Wiesner staining, hand sections from the 7th
internodes were cut with a razor blade and stained
with phloroglucinol solution [1% (w/v) phloroglucinol in ethanol] for 5 min and acidified with 30%
HCl. All photographs were taken within 30 min of
staining.
Lignin quantification
For preparation of cell wall residues (CWR), the
11th–12th stem internodes of greenhouse-grown
poplars were homogenized in liquid nitrogen and
extracted in 50 ml of absolute ethanol for 48 h. The
extract was centrifuged at 44800g for 30 min and
dried overnight at room temperature. Total lignin
content in CWR was measured using the lignin
thioglycolic acid (LTGA) method (Bruce and West
1989; Lange et al. 1995). After derivatization with
thioglycolic acid, the relative total lignin contents of
CWR were expressed as the absorbance at 280 nm in
0.5 M NaOH.
Phenolics analysis
Estimation of laccase-mediated browning was done
using the method by Wang and Constabel (2004) with
some modifications. Briefly, leaf tissue (150 mg fresh
weight) was ground in 1 ml of distilled water (pH
4.5). The supernatant was collected by centrifugation
and incubated at 25°C for 18 h. The absorbance at
690 nm was recorded. To quantify total phenolic
content in leaf, we used the Folin–Ciocalteau method
as described by Wang and Constabel (2004).
Results
Biomass assays
Molecular confirmation of transgenic poplar
plants
Greenhouse-grown transgenic and control plants,
which ranged from 29 to 34 cm in height, were used
to biomass assays. Plants were bottom-watered every
other day. The heights of all plants were recorded
every 5 days. Plant height, above-ground (shoot) and
below-ground (root) biomass of these plants were
measured at the end of 30 days experimental periods.
Using our transformation protocol, a chimeric construct of CaMV35:GaLAC1 was introduced into
poplars by A. tumefaciens-mediated transformation.
A total of ten Kanamycin-resistant shoots were
regenerated, seven of which were able to root on
rooting medium. Five rooted transgenic shoots
showed the expected 402 bp fragments in the PCR
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Plant Cell Tiss Organ Cult (2008) 93:303–310
Fig. 2 Molecular confirmation of GaLAC1 in transgenic
poplar plants. (a) The representative PCR analysis for the
presence of GaLAC1 gene in putative transgenic poplar plants.
The expected size of GaLAC1-specific products is 402 bp. (b)
Southern blot analysis of putative transgenic plants. Plasmid
and genomic DNA was digested with BamHI and hybridized
with an a-32P-labeled GaLAC1 probe. Lane M: Molecular
weight marker; Lane P: pBI121CaMV35:GaLAC1 plasmid
DNA (positive control); Lane WT: DNA from untransformed
plant (negative control); Lane 2.16A, 2.16H, YWA, YWB,
YWC: DNA from distinct transgenic lines
reaction. No amplification product was detected in
nontrangenic control (Fig. 2a). The PCR-positive
transformants were further propagated and selected
on rooting medium. Finally, five independent putative
transgenic lines were obtained. Genomic DNA of
transgenic and control plants was digested with
BamHI and hybridized with the PCR-generated
probes. As shown in Fig. 2b, the untransformed
control showed no band, whereas all transformed
lines displayed at least one band. Since there is a
single BamHI site in the plasmid pBI121CaMV35:GaLAC1 (Fig. 1), different hybridizing bands of
transformants reflects distinct integration events.
PCR and Southern blot analysis showed that the
LAC1 gene was successfully integration into the
genome of the transgenic plants.
transgenic line 2.16A and line 2.16H, 13.2- and 11.4fold higher than that in the control, respectively
(Fig. 3).
Phenotypes and plant growth rate of transgenic
poplar plants
The morphology of all transgenic lines was similar to
that of untransformed controls under greenhouse
conditions. Transgenic line 2.16A, which had the
highest laccase activity, and untransformed control
plants were used to further biomass assays. After a
30-day period of acclimation, the average biomass
(plant height, shoot weight, root weight and total
Laccase activity assay
To determine the effect of overexpression of LAC1 on
laccase activity in transgenic poplar plants, we
determined the total laccase activity of transgenic
and untransformed control plants. Overexpression of
GaLAC1 in transgenic plants caused a 2.1- to 13.2fold increase in laccase activity compared to control
plants. The highest laccase activity was measured in
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Fig. 3 Total laccase activities of transgenic (2.16A, 2.16H,
YWA, YWB, YWC) and untransformed plants (WT). Data are
presented as means ± SD of four independent experiments
Plant Cell Tiss Organ Cult (2008) 93:303–310
weight) and growth rate of transgenic line 2.16A did
not significantly differ from those of untransformed
control plants (Fig. 4a, b). These evidences implied
that GaLAC1 may not deal with the holistic growth
pattern of plants.
Lignin staining and quantification
For a preliminary evaluation of differences in lignin
deposition between transgenic and control plants,
transverse stem sections from transgenic line 2.16A
and control were stained with Wiesner reagent
(phloroglucinol–HCl). There were no distinct
Fig. 4 Biomass assays. (a) Height growth of transgenic line
2.16A and control (WT) within a 30-days period in greenhouse.
(b) shoot, root and total plant weight of transgenic line 2.16A
and control after a 30-days period in greenhouse. Measurements were made at day 5, 10, 15, 20, 25 and 30. Data are
presented as means ± SD of four different plantlets of the
control (WT) and transgenic line 2.16A
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differences in shape, size and general arrangement
of xylem cells between transgenic line 2.16A and
control, whereas stem sections from transgenic line
2.16A gave a higher intensity of phloroglucinol-HCl
staining (Fig. 5a, b). This qualitative results suggested that overexpression of GaLAC1 in transgenic
poplar plants may have some effects on total lignin
content in transformants. Quantitative determination
of total lignin content of stem cell wall residues
(CWR) were performed using the lignin thioglycolic
acid (LTGA) method. As shown in Fig. 6, total lignin
content in controls remained approximately constant.
By contrast, total lignin content in all tested transgenic lines was elevated in varying degrees. The
highest increase in total lignin content (19.6%) was
found in transgenic line 2.16A, which had the highest
laccase activity in all five transgenic lines examined.
Although significantly increased total lignin content
were only detected in transgenic line 2.16A, 2.16H
and YWC, detectable increase in total lignin content
were also observed in transgenic line YWA and
YWB. The overall correlation between increased
lignin content and enhanced laccase activity was
highly significant.
Fig. 6 Quantification of lignin. Total lignin content of stems
as measured by the LTGA method. Results were expressed as
the absorbance (280 nm) in 0.5 M NaOH. Data are presented
as means ± SD of five independent experiments
Fig. 5 Histochemical
staining of lignin.
Phloroglucinol–HCl
staining of transverse stem
sections of control (a) and
transgenic line 2.16A (b)
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308
Content and oxidation rate of phenolics
To investigate the role of laccase gene in plant
phenolic metabolism, we performed quantitative
assays of phenolics and browning reaction rate in
transgenic and control leaf. Absorbance (690 nm) of
fresh leaf extracts did not differ between transgenic
and control plants. After incubation at 25°C for 18 h,
leaf extracts from transformants exhibited a strong
browning, with a obvious increase in A690. In
contrast, untransformed plants showed a weak
browning (Fig. 7a, b). As shown in Fig. 7c, the total
phenolic content in transgenic plants was similar to
that in control plants. Thus, rapid autonomous
browning in transgenic plants ought to be caused by
increased oxidation rate rather than accumulation of
phenolics. Interestingly, the total phenolic content in
Fig. 7 Browning and total phenolic content of leaf extracts
from transgenic and control plants (WT). (a) Representative
photograph illustrating autonomous browning of leaf extracts
from transgenic line 2.16A and control plant after incubation at
25°C for 18 h. (b) Absorbance (690 nm) of leaf extracts
measured before and after incubation at 25°C for 18 h. (c)
Total phenolic content of leaf. Data are presented as
means ± SD of four experiments
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distinct transgenic lines was not influenced by their
varying laccase activity.
Discussion
Laccases can be roughly divided into two major
groups, plant laccases and fungal laccases. Although
laccase was first discovered in plant, most of our
knowledge concerning laccase has come from fungi
so far (Mayer and Staples 2002; Gavnholt and Larsen
2002). A major reason for this is the absence of a
practical plant model system for the study of laccase
function in plant. In preliminary reports, sense
construction of laccase was introduced into suspension culture cells of L. tulipifera or tobacco.
Transgenic cells turned brown and the laccase
activity usually declined to background levels within
several months. Increased laccase activity in transgenic cells may be deleterious to the normal growth
of suspension-cultured cells (Dean et al. 1998;
LaFayette et al. 1999). Laccase genes exist as
multigene families in plants. Moreover, the relatively
low substrate specificity of laccases may result in that
laccases have overlapping function with other oxidases in plants (Dean and Eriksson 1994; Mayer and
Staples 2002). Therefore, potential functional compensation caused by divergent laccase and/or other
oxidase genes, may prevent us from better understanding the functions of laccase in plants by
antisense strategy (Ranocha et al. 2002).
Due to rapid growth, modest genome size, relative
facile transformation and recently sequenced genome,
Populus has been a widely used model plant, not only
in commercial use but also in fundamental plant
physiology research (Herschbach and Kopriva 2002;
Brunner et al. 2004; Tuskan et al. 2006). Compared
with other model plants, such as Arabidopsis, tobacco
and rice, Populus has profuse xylem. Therefore,
transgenic poplar plants may be the optimal system
to investigating the function of laccase in plants. By
Agrobacterium-mediated genetic transformation, we
generated transgenic poplar plants with overexpression
of cotton laccase GaLAC1. Transgenic plants showed a
stable increased (2.1- to 13.2-fold) laccase activity,
whereas plant growth rate and morphological characters remained similar to control plants. Our work
provided a useful system to investigate the function of
laccases in plants.
Plant Cell Tiss Organ Cult (2008) 93:303–310
The possibility that laccases could catalyze the
polymerization of monolignols in higher plants was
first raised by Freudenberg et al. (1958). Biochemical
and cellular evidence implied that laccases may be
involved in lignin polymerization (Freudenberg
et al. 1958, Sterjiades et al. 1992, Bao et al. 1993,
LaFayette et al. 1999). Mutation of Arabidopsis
laccase gene AtLAC15 resulted in a 30% reduction
in extractable lignin content in seeds (Liang et al.
2006b). Our data showed that overexpressing
GaLAC1 caused a 2.1–19.6% increase in lignin
content in transformants. By contrast, overexpression
of Liriodendron laccase gene LtLAC in L. tulipifera
(Dean et al. 1998) and tobacco cells (LaFayette et al.
1999), as well as down-regulation of L. tulipifera
LtLAC (Dean et al. 1998) and P. trichocarpa LAC3,
LAC 90 and LAC110 (Ranocha et al. 2002) had no
effect on the lignin content in transgenic plants.
Peroxidases are also considered to be involved in
polymerization of monolignols and similar confused
results were also found in transgenic plants expressing peroxidase genes (Lagrimini 1991, Lagrimini
et al. 1997; Mansouri et al. 1999; Elfstrand et al.
2002). There were some possible reasons which may
account for these phenomenon. First, the phylogenetic analysis shows that laccase-like multicopper
oxidase (LMCO) is comprised with six distinct
groups. Members from the same group generally
have highly conserved structure and possible similar
functions (McCaig et al. 2005). LtLAC and
PtLAC110 are members from group 1; PtLAC3 and
PtLAC90 belongs to group 2 and group 3, respectively; AtLAC15 and GaLAC1 come from group 4. It
is possible that not all groups are involved in
lignification. Therefore, not all genetic manipulation
of plant laccase gene had effect on the lignin content
in transgenic plants. Moreover, plant laccases are
diversely expressed in different tissues, at different
developmental stages and under different environmental conditions (Hoopes et al. 1995; McCaig et al.
2005; Caparrós-Ruiz et al. 2006). Therefore, various
control patterns of plant laccases may result in that
different laccase genes participated in different
physiology process. Alternatively, some laccases
may be involved in lignification with divergent
function, but only certain members of them seem to
be concerned with lignin content.
In plants, browning is usually caused by the
oxidation of phenolic compounds. Besides laccases,
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catechol oxidase, tyrosinase, peroxidases are able to
oxidize phenolics to quinones. Unstable quinones
trend to form brown polymers (Guyot et al. 1996).
Transgenic PPO-overexpressing tomato and poplar
plants showed a strong browning but a similar total
phenolic content as compared with untransformed
controls (Li and Steffens 2002; Wang and Constabel
2004). In previous reports, transgenic L. tulipifera or
tobacco cell with overexpressed laccase gene markedly turned brown, although the total phenolic
content in transformants was not determined (Dean
et al. 1998; LaFayette et al. 1999). Wang et al.
(2004) found that overexpression GaLAC1 in Arabidopsis did not significantly alter overall phenolic
synthesis. Together with our data, transgenic evidences suggested that GaLAC1 may mainly impact
oxidation rate rather than content of phenolics. On the
other side, transgenic poplars underexpressing
P. trichocarpa LAC3 displayed a 2–3 fold increase
in total soluble phenolic content (Ranocha et al.
2002). While Arabidopsis mutant tt10, caused by the
mutant of AtLAC15 (At5g48100), had a 59% increase
in soluble proanthocyanidin or condensed tannin
compared with wild-type seeds (Liang et al. 2006b).
These evidences implied that plant laccases may play
diverse roles in plant secondary metabolism.
In summary, we generated and characterized
transgenic poplar plants overexpressing GaLAC1.
Our results suggested that GaLAC1 may be involved
in lignification and phenolic metabolism in plants.
The present work provided a new genetic evidence
for the involvement of plant laccases in lignification.
Laccase can protect the fungi from the toxic
phytoalexins and environmental toxicants. In plants,
laccases may participate in degradation of allelopathics/pollutants, wound healing and responses to salt
stress. Additionly, plant laccases also are likely
involved in phenolic/flavonoid metabolism and lignin
biosynthesis, which are considered to play indirect
roles in defense to herbivore or pathogen. Therefore,
better understanding of laccase function facilitates
generating engineered organisms with resistance to
environmental stress or industrial pollutants.
Acknowledgments We sincerely thank to Dr. Xiaoya Chen
for providing GaLAC1 gene, Dr. Bin Luo for his help with the
experiments, Dr. Yoshikatsu Matsubayashi for providing PSKa, Dr. Jeffrey Dean for providing his electronic version article,
Dr. Zhangqun Ye for providing antibiotic and Dr. Longcheng
Li for critically reading of the manuscript.
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