A Transcriptomic Network Underlies Microstructural

and Physiological Responses to Cadmium in

Populus 3 canescens 1[C][W]

Jiali He 2, Hong Li 2, Jie Luo, Chaofeng Ma, Shaojun Li, Long Qu, Ying Gai, Xiangning Jiang, Dennis Janz,
Andrea Polle, Melvin Tyree, and Zhi-Bin Luo*
College of Life Sciences and State Key Laboratory of Crop Stress Biology in Arid Areas (J.H., J.L., C.M., S.L.,
Z.-B.L.), Key Laboratory of Applied Entomology, College of Plant Protection (H.L.), and Key Laboratory of
Environment and Ecology in Western China, Ministry of Education, College of Forestry (M.T., Z.-B.L.),
Northwest A&F University, Yangling, Shaanxi 712100, China; National Engineering Laboratory of Tree
Breeding, College of Life Sciences and Biotechnology, Beijing Forestry University, Beijing 100083, China (L.Q.,
Y.G., X.J.); and Büsgen Institute, Department of Forest Botany and Tree Physiology, Georg-August University,
37077 Göttingen, Germany (D.J., A.P.)

Bark tissue of Populus 3 canescens can hyperaccumulate cadmium, but microstructural, transcriptomic, and physiological
response mechanisms are poorly understood. Histochemical assays, transmission electron microscopic observations, energy-
dispersive x-ray microanalysis, and transcriptomic and physiological analyses have been performed to enhance our
understanding of cadmium accumulation and detoxification in P. 3 canescens. Cadmium was allocated to the phloem of the
bark, and subcellular cadmium compartmentalization occurred mainly in vacuoles of phloem cells. Transcripts involved in
microstructural alteration, changes in nutrition and primary metabolism, and stimulation of stress responses showed significantly
differential expression in the bark of P. 3 canescens exposed to cadmium. About 48% of the differentially regulated transcripts
formed a coregulation network in which 43 hub genes played a central role both in cross talk among distinct biological processes
and in coordinating the transcriptomic regulation in the bark of P. 3 canescens in response to cadmium. The cadmium transcriptome
in the bark of P. 3 canescens was mirrored by physiological readouts. Cadmium accumulation led to decreased total nitrogen,
phosphorus, and calcium and increased sulfur in the bark. Cadmium inhibited photosynthesis, resulting in decreased
carbohydrate levels. Cadmium induced oxidative stress and antioxidants, including free proline, soluble phenolics, ascorbate,
and thiol compounds. These results suggest that orchestrated microstructural, transcriptomic, and physiological regulation may
sustain cadmium hyperaccumulation in P. 3 canescens bark and provide new insights into engineering woody plants for
phytoremediation.

Cadmium is a nonessential and highly toxic element
for plants. Cadmium in soil enters plants via trans-
porters for essential elements (e.g. calcium, iron, and
zinc) and is eventually taken up by the human body
through the food chain. Cadmium accumulation in
1 2010; Mendoza-Cózatl et al., 2011).
This work was supported by the Special Fund for Forest Science
and Technology Research in the Public Interest (grant no. 201204210),
the State Key Basic Research Development Program (grant no.
2012CB416902), the National Natural Science Foundation of China
(grant nos. 31070539, 31100481, and 31270647), the Program for
New Century Excellent Talents in University from the Ministry of
Education of China (grant no. NCET–08–0468), and the German Science
Foundation (grant no. INST 186/766–1 FUGG).
2
These authors contributed equally to the article.
* Corresponding author; e-mail luozbbill@163.com.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy de-
scribed in the Instructions for Authors (www.plantphysiol.org) is:
Zhi-Bin Luo (luozbbill@163.com).
[C]
Some figures in this article are displayed in color online but in
black and white in the print edition.
[W]
The online version of this article contains Web-only data.
www.plantphysiol.org/cgi/doi/10.1104/pp.113.215681
424 Plant PhysiologyÒ, May 2013, Vol. 162, pp. 424–439, www.plantphysiol.org Ó 2013 American Society of Plant Biologists. All Rights Reserved.
humans may cause prostate and lung cancers and
bone fractures (Bertin and Averbeck, 2006; Nawrot
et al., 2006). Thus, it is extremely important to reme-
diate cadmium-polluted soils. Phytoremediation is an
efficient and effective biotechnology to extract cadmium
from soil via harvestable portions of plants (Kramer,
There are numerous consequences of cadmium tox-
icity in plants. Soil cadmium enters plants via apo-
plastic and/or symplastic pathways. The cadmium
subsequently poisons tissues, causing changes in sub-
cellular structures and altering physiological and
molecular processes (Kramer, 2010; Mendoza-Cózatl
et al., 2011). Cadmium can modify cell wall structure
and degenerate cellular organelles such as chloroplasts
and mitochondria (Van Belleghem et al., 2007). Ionic
Cd (Cd2+) may compete with nutrient cations (e.g.
Ca2+, Fe2+, and Zn2+) for transporters, often leading to
decreases in these essential elements and to disrup-
tions in the ionic homeostasis of plants (Rodríguez-
Serrano et al., 2009). In particular, cadmium exposure
may provoke iron deficiency in plants due to compe-
tition for iron transporters between Cd2+ and Fe2+

(Besson-Bard et al., 2009; Wu et al., 2012). Repression
of photosynthesis is frequently observed in plants ex-
posed to cadmium due to damage of the photosyn-
thetic apparatus (Cunha et al., 2008). This can alter
carbohydrate concentrations throughout all plant tissues
(He et al., 2011). Cadmium in plants can indirectly in-
duce hydrogen peroxide (H2O2) and superoxide (O2c–),
giving rise to an oxidative burst (Rodríguez-Serrano
et al., 2009). Moreover, cadmium exposure can bring
about differential expression of genes involved both in
cadmium transport and in scavenging reactive oxygen
species (ROS) in plants (Küpper and Kochian, 2010).
Plants have evolved several strategies for cadmium
detoxification. First, the apoplast acts as a barrier for
cadmium entry because Cd2+ binds to polyuronic acids
and pectin in plant cell walls (Conn and Gilliham, 2010)
and stimulates increased lignification (Schützendübel
et al., 2001; Elobeid et al., 2012). Second, cadmium entry
into the symplast can be detoxified by cadmium che-
lates in cytosol and sequestration in vacuoles. Ionic
cadmium in cytosol can bind to reduced glutathione
(GSH) and phytochelatins (PCs), forming cadmium-
ligands that are subsequently transported to vacuoles,
where cadmium can be sequestered (Conn and Gilliham,
2010; Park et al., 2012). Finally, cadmium stress activates
several biochemical defenses, such as antioxidative en-
zymes and GSH synthesis, thus leading to reprogram-
ming of the root or leaf transcriptome (Herbette et al.,
2006; Mendoza-Cózatl et al., 2008; Rodríguez-Serrano
et al., 2009; Xu et al., 2012).
The above-mentioned processes of cadmium toxicity
and detoxification have been explored mostly in herba-
ceous plants such as Arabidopsis (Arabidopsis thaliana),
Arabidopsis halleri, and Thlaspi caerulescens (Kramer, 2010;
Mendoza-Cózatl et al., 2011). However, the limited bio-
mass of herbaceous plants constrains their application for
phytoremediation on a large scale. Poplars (Populus spp.)
have been proposed for phytoremediation because of
their rapid growth, deep root system, and relatively high
cadmium accumulation in some genotypes (Merkle, 2006;
He et al., 2013). The cadmium entering roots can be
transported to leaves along with the transpiration stream
and can be further translocated to the bark, where high
accumulation occurs in Populus 3 canescens grown in
hydroponics (He et al., 2011).
Bark contains phloem tissue, which plays a crucial
role in delivering nutrients to sink tissues (e.g. new
shoots and roots), thereby contributing to internal
cadmium allocation. Thus, it is essential to understand
cadmium toxicity and detoxification in the bark of P. 3
canescens to restrict recycling of cadmium to roots,
particularly in terms of using P. 3 canescens to reme-
diate cadmium-polluted soils. Although responses of
poplars to cadmium stress have been investigated at
the proteomic and physiological levels (Schützendübel
et al., 2002; Kieffer et al., 2008, 2009a, 2009b; Durand
et al., 2010; He et al., 2011), the microstructural, tran-
scriptomic, and physiological mechanisms underlying
cadmium toxicity and detoxification remain unknown
in the bark of poplars.
Plant Physiol. Vol. 162, 2013
The Cadmium Transcriptome of Poplar Bark
To elucidate cadmium distribution and transcriptomic
and physiological regulation mechanisms in the bark,
hybrid poplars (P. 3 canescens) were exposed to 0 or
200 mM CdSO4. These cadmium concentrations were
within the range (0–500 mM Cd2+) used in previous
studies (Ager et al., 2003; Wójcik and Tukiendorf, 2004;
Mendoza-Cózatl et al., 2011). The objective of this
paper is to address the following questions. (1) Where
is cadmium localized at the subcellular level, and is the
ultrastructure of the bark altered in response to cad-
mium exposure? (2) Are these alterations associated
with transcriptomic reprogramming? (3) Do these
changes lead to modifications of physiological pro-
cesses in the bark after cadmium exposure? The bark
tissue is the focus of this study, but root and leaf tis-
sues have been included in the microstructural and
physiological analyses because of the interconnection
of cadmium transport among these tissues. A study
addressing these questions will not only help us to
better understand the microstructural, transcriptomic,
and physiological regulation mechanisms of woody
plants in response to cadmium exposure, but also pro-
vide new insights into engineering woody plants for
phytoremediation.
RESULTS
Cadmium Results in Granular Deposition and Causes
Microstructural Changes
Plants of P. 3 canescens grown in sand cultures were
exposed to a cadmium concentration, which reduced
but did not abolish growth (Supplemental Table S1).
Under these conditions, the typical cadmium distri-
bution pattern was observed, with higher concentra-
tions in roots and lower concentrations in leaves and
bark (Fig. 1). Still, cadmium concentrations in all ana-
lyzed tissues were well above the threshold of 100 mg g–1
dry weight commonly defined for hyperaccumulation
Figure 1. Cadmium concentrations in the bark, root, and leaf tissues of
P. 3 canescens exposed to 0 (blank bars) or 200 mM (filled bars) CdSO4
for 20 d. Bars indicate means 6 SE (n = 6). Asterisks on the bars for the
same tissue indicate significant differences between treatments. [See
online article for color version of this figure.]
425
He et al.

(Milner and Kochian, 2008). To investigate the cellular
localization of cadmium in the bark compared with other
tissues, we conducted histochemical staining for cad-
mium (Fig. 2). In stem cross sections, strong cadmium-
dithizone staining was observed in the phloem, with
formation of distinct granules (Fig. 2B). Notably, such
granules were also observed on the surface (Fig. 2D) of
fine roots and in the apoplast of root cortical cells (Fig.
2F). In leaves, cadmium-dithizone staining was observed
mainly in leaf veins (Fig. 2H), in intercellular spaces of
mesophyll cells near veins (Fig. 2H), in petioles, and in
leaf trichomes (Supplemental Fig. S1). This suggests a
preferential accumulation of cadmium along the trans-
port path.
To corroborate cadmium accumulation in the gran-
ules, we studied the subcellular structures at higher
magnification with transmission electron microscopy
(TEM) and analyzed the elemental composition of the
granular deposits by energy-dispersive x-ray (EDX)
microanalysis (Figs. 3 and 4; Supplemental Figs. S2
and S3). Electron dense granules in vacuoles of phloem
cells were observed in the bark of cadmium-exposed
P. 3 canescens, whereas these granules were absent in
controls (Fig. 3, A and B). EDX analysis of these
granules in vacuoles of the phloem cells revealed strong
accumulation of cadmium (Fig. 4A; Supplemental Fig.
S2). These electron dense granules also contained sulfur
and other elements such as aluminum and copper
(Supplemental Fig. S2). Among these elements, a sig-
nificant correlation was observed between cadmium
and sulfur (Fig. 4B), indicating that cadmium may
chelate with sulfur-containing ligands in P. 3 canescens.
Cadmium accumulation in electron-dense granules was
also detected in the vacuoles of root and mesophyll cells
(Fig. 4A; for transmission electron microscopic micro-
graphs, see Supplemental Fig. S3).
Microscopic analysis of the phloem also revealed
that cadmium exposure altered the chloroplast struc-
ture (i.e. chloroplast grana were distorted in compan-
ion cells; Fig. 3, C–F). Furthermore, much fewer starch
grains per cell were observed in cadmium-exposed
plants (4.3 6 0.1) than in control plants (13.3 6 0.2,
P , 0.0001; Fig. 3, C–F). Moreover, compared with
control P. 3 canescens, mitochondria in companion

Figure 2. Cadmium localization in the bark (A

and B), root (C–F), and leaf tissues (G and H) of
P. 3 canescens exposed to 0 (A, C, E, and G) or
200 mM (B, D, F, and H) CdSO4 for 20 d. Arrows
point to precipitates of cadmium-dithizone. B,
Cadmium-dithizone precipitates in the bark. D,
Cadmium-dithizone precipitates in epidermal
cells of fine roots. F, Cadmium-dithizone precip-
itates in intercellular spaces, cell walls, and cy-
toplasm of cortical cells. H, Cadmium-dithizone
precipitates in leaf veins and in intercellular
spaces of mesophyll cells. col, Collenchyma; cor,
cortex; pf, phloem fiber; pr, phloem ray; ph,
phloem; cam, cambium; v, vessel; xf, xylem fiber;
xyl, xylem; cc, cortical cells; lv, leaf vein; m,
mesophyll cells. [See online article for color ver-
sion of this figure.]

426 Plant Physiol. Vol. 162, 2013


cells of the phloem appeared swollen in cadmium-
treated plants, and the cristae displayed an irregular
shape (Fig. 3, G and H). These results suggest that
cadmium toxicity may cause partial disorganization of
chloroplasts and mitochondria via membrane degra-
dation of subcellular structures in companion cells of
P. 3 canescens.
A Transcriptomic Coexpression Network Responds
to Cadmium in the Bark
To find out how cadmium accumulation altered
molecular functioning in bark tissue, we conducted
genome-wide transcriptional analyses of P. 3 canescens
bark using Affymetrix poplar genome arrays. After
cadmium exposure, 505 genes showed significant in-
creases in transcript abundance in P. 3 canescens bark,
Plant Physiol. Vol. 162, 2013
The Cadmium Transcriptome of Poplar Bark
Figure 3. Transmission electron microscopic
micrographs of granular deposits in vacuoles of
phloem cells (A and B) and companion cells (C–
H) in the bark of P. 3 canescens exposed to 0 (A,
C, E, and G) or 200 mM (B, D, F, and H) CdSO4 for
20 d. The insert in B highlights a granular deposit
in a vacuole. Arrows point to cadmium deposits.
Cadmium concentrations in granular deposits are
shown in Figure 4A. The EDX spectra of vacuoles
(A) and granular deposits in vacuoles (B) are
shown in Supplemental Figure S2. CW, Cell wall;
PM, plasma membrane; N, nucleus; V, vacuole;
Ch, chloroplast; Gr, granum; SG, starch grain; M,
mitochondrion; ER, endoplasmic reticulum.
whereas 366 genes showed significant decreases
(Supplemental Table S2). Among these differentially
regulated transcripts, 302 up-regulated and 223 down-
regulated transcripts were identified that had corre-
sponding annotations for homologs in Arabidopsis
(Supplemental Table S2). To further categorize the
functions of cadmium-responsive poplar genes, all
unique Arabidopsis gene identifiers (AGIs) were se-
lected. The 484 genes that were identified were further
analyzed in MapMan (Figs. 5 and 6; Supplemental
Table S3). An overview of functional categories
revealed that most of the cadmium-responsive genes
were involved in the regulation and processing of RNA
and proteins, as well as in signaling and stress (Fig. 5).
A closer look at the categories revealed that 20 of the
genes in the protein category belonged to the 26S pro-
teasome complex, which mediates ubiquitin-dependent
protein degradation (Supplemental Table S3). In the
427
He et al.

Figure 4. EDX analysis determined cadmium concentrations (A) and the correlation between cadmium and sulfur (B) in
granules of different cell compartments of P. 3 canescens exposed to 0 (blank bars) or 200 mM (filled bars) CdSO4 for 20 d. Bars
indicate means 6 SE (n = 3). Asterisks on bars for the same cell compartment indicate significant differences between treat-
ments. The data for correlation analysis were based on cadmium and sulfur concentrations in granules of all examined sub-
cellular compartments. Transmission electron microscopic micrographs of cadmium-deposited granules in subcellular compartments
of roots and leaves are shown in Supplemental Figure S3. pc.vac, Vacuoles of phloem cells; epi.ito, inside tonoplast of epidermal
cells; cc.cw, cell walls of cortical cells; cc.vac, vacuoles of cortical cells; mc.vac, vacuoles of mesophyll cells. [See online article for
color version of this figure.]

RNA regulation category, several transcription factors S3). In this cadmium-responsive network, 234 genes
involved in biotic stress, such as WRKY70/53, MYB94, were directly connected with each other via 1,254 edges,
ABI5, and AP2, were activated (Fig. 6A; Supplemental suggesting that the coexpressed genes are most likely
Table S3). In the signaling category, genes involved in coregulated.
biotic stress signaling were activated. This included, Forty-three of these coexpressed genes were strongly
for example, calmodulin-binding transcription activator interconnected, with each gene having more than 20
and Leu-rich repeat protein kinase family protein (Fig. edges (Fig. 7B). These genes were therefore defined as
6A; Supplemental Table S3). In the stress category, a hub genes. The hub genes were directly connected with
few genes encoding pathogenesis-related (PR)-proteins, each other via 308 edges, forming a highly interconnected
as well as genes implicated in cell wall metabolism such subnetwork (Fig. 7B; Supplemental Table S3). Highly
as glycosyl hydrolase9A4 and arabinogalactan protein6, connected hub genes have displayed a marked enrich-
were highly overexpressed (Fig. 6A; Supplemental ment for cross talk among essentially biological pro-
Table S3). These data suggest that cadmium induced cesses (Heyndrickx and Vandepoele, 2012). Consistently,
responses similar to those found after wounding and
that these responses may have also invoked activation
of cell wall metabolism. In addition, a number of genes
involved in primary and secondary metabolisms were
also significantly up-regulated (Fig. 6B). Overall, these
findings suggest that cadmium induces a tightly regu-
lated gene network for detoxification and metabolic
reprogramming.
To corroborate this idea, coexpression analysis was
conducted. Genes that cooperate in a shared function,
for example, in response to abiotic and biotic stressors,
are often expressed simultaneously (Wei et al., 2006).
By investigating how transcriptomic patterns vary in
concert across hundreds of microarray experiments,
new insights may be gained about the roles of coor-
dinated gene networks in plants (Wei et al., 2006;
Ficklin and Feltus, 2011; Osorio et al., 2012). To check
whether the differentially expressed genes were co-
regulated in the bark of cadmium-treated P. 3 can-
escens, genes with unique AGIs (Supplemental Table
S3) were used as query genes in the CressExpress da-
tabase version 2.0 (http://cressexpress.org/index.jsp),
which has analyzed correlations between genes on
486 arrays across various experimental conditions Figure 5. Number of genes assigned to each category by MapMan
(Wei et al., 2006). Out of 484 query genes, 234 genes analysis. Detailed information for each category is presented in
(approximately 48%) formed a cadmium-responsive Supplemental Table S3. [See online article for color version of this
coexpression network (Fig. 7A; Supplemental Table figure.]

428 Plant Physiol. Vol. 162, 2013

 Figure 6. Transcripts involved in stress (A) and metabolism (B) assigned by MapMan in the bark of P. 3 canescens exposed to 0
or 200 mM CdSO4 for 20 d. Positive fold change values (red) indicate up-regulation, whereas negative fold change values (blue)

Plant Physiol. Vol. 162, 2013 429

He et al.
10 out of 43 hub genes (approximately 23%) were in
the signaling category when testing the functional as-
signments of the genes in MapMan (Supplemental
Table S3). For instance, a putative wall-associated ki-
nase, a GDP dissociation inhibitor family protein/Rab
GTPase activator family protein, and a chloroplast
sensor kinase were among these 10 hub genes involved
in signaling (Fig. 7B; Supplemental Table S3). This
suggests that coordinated cadmium signaling can take
place across different subcellular compartments. Anal-
ysis of Gene Ontology (GO) term enrichment revealed
that the hub genes were significantly enriched in pro-
cesses related to transport, energy metabolism, photo-
synthesis, and isopentenyl diphosphate biosynthesis
(Fig. 7C; redundant GO terms not shown). Isopentenyl
diphosphate is the precursor for isoprenoids, which
are essential building blocks for photosynthetic pig-
ments, redox factors, hormones, feeding deterrents, and
phytoalexins (Davies, 2010).
Analysis of GO term enrichment indicated 85 GO
terms of all cadmium responsive genes and even 120
GO terms of the coexpressed genes for biological pro-
cesses, molecular functions, and cellular compartments
in which genes were strongly enriched (Supplemental
Table S4). This finding suggests that coexpression anal-
ysis removed functionally less interconnected genes. For
the coexpressed genes, some highly enriched, function-
ally different biological processes were biotic stimulus,
cell tip growth, negative regulation of defense response,
cell death, plant-type hypersensitive response, salicylic-
mediated signaling pathway, protein targeting to
membrane, developmental cell growth, plant-type cell
wall organization or biogenesis, and protein phospho-
rylation (Supplemental Table S4). More than one-half of
the coexpressed genes belonged to the primary metabolic
process category (GO:0044238; Supplemental Table S4).
Overall, these results indicate that the response to
cadmium involves strongly interlinked signaling and
transcriptional regulation events, which may trigger
transport processes for detoxification in vacuoles and
cell walls, activate biochemical defense reactions, and
influence subcellular microstructures.
The Cadmium Transcriptome Is Linked with Bark-Specific
Physiological Readouts
To identify key metabolic processes that were af-
fected by cadmium, we investigated the internal nutri-
ent status, carbohydrate metabolism, oxidative stress,
and antioxidants in P. 3 canescens (Supplemental Figs.
S4–S6; Supplemental Tables S5 and S6). Moreover, to
find out if the response of bark tissue to cadmium was
similar to that of root and leaf tissues, we compared the
Figure 6. (Continued.)
denote down-regulation. Color saturates at 5-fold change. Each square represents a differentially expressed transcript. [See
online article for color version of this figure.]
430
response patterns of nutrients, carbohydrates, and oxi-
dants and antioxidants using principal component
analysis (PCA; Fig. 8; Supplemental Table S7). The first
component, principal component 1 (PC1), of PCA for
nutrient status clearly separated bark, root, and leaf
tissues, accounting for 60% of the variation (Fig. 8A).
The second component, principal component 2 (PC2),
which accounted for 20% of the variation, separated the
cadmium effect from the controls in root and leaf tis-
sues; however, the separation was not as pronounced in
bark tissue (Fig. 8A). This observation indicates that
bark nutrient status was much less influenced by cad-
mium than other tissues were. The one exception was
phosphorus concentration, which strongly declined by
27% (Supplemental Table S5). Compared with the con-
trol, the bark of cadmium-exposed P. 3 canescens had
lower total nitrogen, phosphorus, and calcium concen-
trations, but higher sulfur concentrations. (Supplemental
Table S5). This is in line with the idea that differentially
expressed genes are involved in nutrient homeostasis,
such as nitrate transporters (NRT2;4 and NRT1;7),
phosphate transporters (PHT1;4 and PHT3;3), and a
calcium-binding elongation factor-hand family protein
(Supplemental Table S3).
The PCA of sugars and sugar alcohols in the bark,
root, and leaf tissues shows that PC1 also separated the
three tissues and accounted for 69% of the variation
(Fig. 8B). The PC2 displayed a clear cadmium effect in
all three tissue types, and Suc was the most important
contributor to PC2 (Fig. 8B; Supplemental Table S7).
The depletion of carbohydrates in the bark of cadmium-
exposed P. 3 canescens was corroborated by a reduction
in the number of starch grains (Fig. 3) and the over-
expression of two genes involved in Suc and starch
degradation (i.e. a chloroplast-targeted alkaline/neutral
invertase and a chloroplastidic phosphoglucan, water
dikinase; Fig. 6B; Supplemental Table S6). Stimula-
tion of cell wall metabolism and activation of energy-
requiring defenses may have contributed to the con-
sumption of carbohydrates. This can also be inferred
from increased transcript levels of enzymes involved
in anapleurotic reactions, lipid degradation, and the
citrate cycle. Furthermore, inhibited CO2 assimilation
in cadmium-exposed poplars (Supplemental Table S1)
may have resulted in lower Suc transport from leaves
to sink tissues. In agreement with decreased tran-
scripts involved in inositol and mannitol metabolism,
such as galactinol synthase1 and raffinose synthase5
(Fig. 6B; Supplemental Table S3), those sugar alcohols
were reduced in the bark of cadmium-exposed P. 3
canescens (Supplemental Table S6).
PCA of oxidants and antioxidants in the bark, root,
and leaf tissues shows that PC1 separated the tissues.
PC2 separated the cadmium effect in bark and leaf
Plant Physiol. Vol. 162, 2013

Figure 7. The coexpression network of significantly differentially
expressed genes (A), the subnetwork of hub genes (B), and the GO
Plant Physiol. Vol. 162, 2013
The Cadmium Transcriptome of Poplar Bark
tissue, but not in root tissue (Fig. 8C). In agreement
with overexpressed transcript level of the NADH-
ubiquinone dehydrogenase6 involved in O2 2 produc-
d
tion (Raczynska et al., 2006; Supplemental Table S3),
O2 2 concentrations were markedly higher in the bark
d
of cadmium-exposed P. 3 canescens (Supplemental Fig.
S4). In addition, overall activation of GSH metabolism
in the bark of cadmium-exposed P. 3 canescens was
also consistent with up-regulation of the glutathione
S-transferase involved in the conjugation of GSH
(Supplemental Fig. S6; Supplemental Table S3).
Overall, the PCA results indicate that nutrients have
a more distinct response pattern in bark than in either
root or leaf tissue. By contrast, the response pattern of
carbohydrates, oxidants, and antioxidants is similar in
both bark and leaf tissue.
DISCUSSION
Vacuolar Sequestration and Apoplastic Binding Are the
Microstructural Mechanisms for Cadmium Accumulation
and Detoxification
Soil cadmium enters root xylem vessels via both
apoplastic and symplastic pathways. The cadmium is
carried upward by the transpiration stream and fur-
ther transported to the bark through phloem loading
(He et al., 2011; Mendoza-Cózatl et al., 2011). Although
cadmium transport and accumulation are important
in the phloem, no information is currently available
about cadmium microlocalization in the bark (includ-
ing the phloem) of woody plants (Vollenweider et al.,
2006). In this study, cadmium microlocalization in
the bark of P. 3 canescens was analyzed using several
independent techniques (i.e. histochemical staining,
TEM observation, and EDX analysis; Figs. 1–4). These
data collectively confirm that P. 3 canescens can se-
quester cadmium in the phloem and highlight that the
subcellular compartmentalization of cadmium occurs
mainly in vacuoles.
Cadmium enters root cells via nutrient ion trans-
porters and is further transported in the form of free
ions in the xylem (Ueno et al., 2008). However, in the
phloem sap of herbaceous plants, cadmium is mainly
transported in the form of chelates with GSH and PCs
term enrichment of hub genes (C) in the bark of P. 3 canescens ex-
posed to 0 or 200 mM CdSO4 for 20 d. The network was generated by
setting a cutoff value of 0.15 for Kolmogorov-Smirnov quality-control
statistics and an r2 value of 0.36, which gives reliable results of
coexpressed genes in CressExpress as suggested by Wei et al. (2006).
An edge indicates the coexpression between two genes. Triangle nodes
represent genes from the category of RNA regulation, diamond nodes
represent genes from the signaling category, and circle nodes represent
genes from other categories. Red and blue nodes represent up- and
down-regulated genes, respectively. The presence of each node in the
network and the hub genes ($20 edges) are indicated in Supplemental
Table S3. [See online article for color version of this figure.]
431
He et al.
Figure 8. PCA plots of nutrients (A), soluble sugars and sugar alcohols
(B), and oxidants and antioxidants (C) in the bark, fine roots, and leaves
of P. 3 canescens exposed to 0 or 200 mM CdSO4. Pink indicates 0 mM
CdSO4, and blue indicates 200 mM CdSO4. PCA was conducted based
on data (both values were averaged in the same tissue with the same
treatment) presented in Supplemental Table S5 (A), Supplemental
Table S6 (B), and Supplemental Figures S4 to S6 (C). [See online article
for color version of this figure.]
(Mendoza-Cózatl et al., 2008). This may also be true in
poplars, as indicated by cadmium accumulation and
higher concentrations of thiols and sulfur in the bark
of cadmium-exposed P. 3 canescens. After transport,
cadmium-ligands reach vacuoles, and cadmium begins
to accumulate (Mendoza-Cózatl et al., 2011). The
432
accumulated cadmium may form cadmium granules,
which are displayed as electron-dense particles under
a transmission electron microscope (Van Belleghem
et al., 2007). We observed that this also occurs in bark,
suggesting massive plant internal cycling of cadmium.
Moreover, correlation between cadmium and sulfur
concentrations in cadmium granules indicates that
cadmium may be immobilized with sulfur-containing
compounds in vacuoles of cadmium-treated P. 3
canescens. Consistently, cadmium is found to be de-
posited with sulfur in Arabidopsis cells (Van Belleghem
et al., 2007). Transport and vacuolar sequestration of
cadmium-ligands (not free cadmium ions) in the phloem
can minimize the damage of cadmium ions to physio-
logically functional cells or subcellular organelles in the
phloem.
Although vacuolar sequestration of cadmium can
minimize toxicity, damage to subcellular structures
may occur because degeneration of chloroplasts, and
mitochondria were observed in the bark of cadmium-
exposed P. 3 canescens (Fig. 3). Structural changes such
as the reduction of grana will decrease photosynthetic
activity and thus result in reduced photosynthates
(McCarthy et al., 2001; Mysliwa-Kurdziel et al., 2004).
Actually, we observed fewer starch grains in com-
panion cells and reduced carbohydrate concentrations
in the bark of cadmium-exposed P. 3 canescens (Fig. 3;
Supplemental Table S6). Mitochondria are also often
injured by cadmium exposure in plant leaves (Lösch,
2004). In our study, the mitochondrial cristae of com-
panion cells of cadmium-exposed P. 3 canescens were
disorganized (Fig. 3). Cadmium-induced injury of the
membrane systems in chloroplasts and mitochondria
may give rise to lipid and protein degradation (see
below).
In fine roots and leaves of P. 3 canescens, cadmium
microlocalization was mainly confined to the apoplast
and vacuoles, suggesting that compartmentation may
play a critical role in cadmium accumulation and de-
toxification in woody plants. Cadmium precipitates
are mainly located at the plasma membrane in Arabi-
dopsis, (Van Belleghem et al., 2007), but not in pea
(Pisum sativum; Romero-Puertas et al., 2004) or in P. 3
canescens (this study). Cadmium deposition in the
vacuoles and cell walls of roots is typical for hyper-
accumulators such as A. halleri and T. caerulescens
(Vazquez et al., 1992; Küpper et al., 2000). P. 3 can-
escens can apparently utilize two strategies to maxi-
mize cadmium sequestration: (1) formation of an
apoplast barrier by binding cadmium ions with cell
walls and (2) symplastic sequestration by cadmium
deposition in vacuoles. As a result, cadmium retention
in roots may be maximized, reducing both cadmium
entry into the central cylinder and cadmium transport
to aerial parts. Once it reaches P. 3 canescens leaves,
cadmium can be deposited in vacuoles of the meso-
phyll cells as observed in A. halleri (Küpper et al., 2000;
Huguet et al., 2012). This suggests that P. 3 canescens
leaves also use vacuolar sequestration to reduce cad-
mium toxicity.
Plant Physiol. Vol. 162, 2013

The Coexpression Network Plays a Central Role in
Transcriptomic Regulation Underlying the Microstructural
and Physiological Responses to Cadmium
Although transcriptional profiles have been studied
in cadmium-treated herbaceous plants, including
Arabidopsis, A. halleri, Solanum torvum, Solanum nig-
rum, and rice (Oryza sativa; Herbette et al., 2006; Weber
et al., 2006; Ogawa et al., 2009; Yamaguchi et al., 2010;
Romero-Puertas et al., 2012; Xu et al., 2012) and in
zinc-exposed Populus 3 euramericana (Di Baccio et al.,
2011), no information is available about transcriptomic
regulation underlying the microstructural responses to
cadmium in woody plants. Cadmium accumulation
in vacuoles of P. 3 canescens bark cells indicates that
some transporters for heavy metals may be involved
in this process. Vacuolar membrane-localized ATP-
binding cassette (ABC) transporters of yeast (Saccha-
romyces cerevisiae) and Arabidopsis (ABC C type)
are implicated in transport of cadmium-PCs and/or
cadmium-bis-glutathione (Ortiz et al., 1995; Li et al.,
1997; Park et al., 2012). In this study, increased tran-
script levels of two ABC transporters may contribute
to cadmium accumulation in P. 3 canescens bark.
These two P. 3 canescens ABC transporters belong to
B- and G-type members (Verrier et al., 2008), and their
function in cadmium transport remains to be studied.
A heavy metal transport/detoxification superfamily
protein and natural resistance-associated macrophage
protein1 are involved in cadmium transport in Arabi-
dopsis (Thomine et al., 2000; Wintz and Vulpe, 2002).
The differential expression of these genes may also
contribute to cadmium accumulation in P. 3 canescens
bark cells. PSII subunit P-1 is involved in the devel-
opment of normal thylakoid architecture (Yi et al.,
2009), and a NADH-ubiquinone oxidoreductase-
related gene affects the respiration process in mito-
chondria (Meyer et al., 2008); therefore, decreased
transcript levels of both genes are probably associated
with degeneration of chloroplasts and mitochondria in
P. 3 canescens bark cells. Furthermore, damage to
chloroplast grana and mitochondrial cristae in the bark
of cadmium-exposed P. 3 canescens is probably linked
with degradation of membrane lipids and proteins
caused by overexpression of a number of genes
(Supplemental Table S3). In fact, membrane proteoly-
sis is induced in pea and poplar leaves exposed to
cadmium (Romero-Puertas et al., 2002; Kieffer et al.,
2008, 2009b). These findings indicate that some tran-
scriptional changes may result from impaired metab-
olism due to cadmium toxicity in P. 3 canescens bark.
Overall, these data highlight that transcriptomic reg-
ulation is associated with cadmium transport and
vacuolar sequestration and with injury to chloroplasts
and mitochondria in the bark of cadmium-exposed
P. 3 canescens.
In the study, we have identified that transcriptomic
responses to cadmium are involved in internal nutrient
homeostasis, in carbohydrate metabolism, and in the
balance of oxidants and antioxidants in P. 3 canescens
Plant Physiol. Vol. 162, 2013
The Cadmium Transcriptome of Poplar Bark
bark. NRT2;4 is a plasma membrane nitrate trans-
porter that is expressed in shoot phloem in response to
external nitrogen concentrations (Kiba et al., 2012; Li
et al., 2012). Phosphate transporters (PHT1;4 and
PHT3;3) are involved in phosphorus signaling and
transport in Arabidopsis (Morcuende et al., 2007; Lei
et al., 2011). In P. 3 canescens bark, decreased expres-
sion of these genes may contribute to reductions
in nitrogen and phosphorus concentrations under
cadmium exposure. In cadmium-treated herbaceous
plants, decreased transcripts levels of genes involved
in photosynthesis and carbohydrate metabolism has
been observed (Mysliwa-Kurdziel et al., 2004; Herbette
et al., 2006; Weber et al., 2006; Ogawa et al., 2009;
Yamaguchi et al., 2010; Sandalio et al., 2012; Xu et al.,
2012). In cadmium-exposed Populus tremula leaves,
repression of essential proteins involved in the pho-
tosystem, chlorophyll biosynthesis, and carbohydrate
metabolism has been detected (Kieffer et al., 2008,
2009b). Because chloroplast-targeted alkaline/neutral
invertase and chloroplastidic phosphoglucanase are
involved in hydrolysis of Suc and starch, respectively
(Hejazi et al., 2008; Tamoi et al., 2010), activation of
both genes is in line with the changes in Suc and starch
that we observed in the bark of cadmium-treated P. 3
canescens. In addition, decreased transcript levels of
galactinol synthase1 and raffinose synthase5 may have
contributed to the reductions in inositol and mannitol
concentrations in the P. 3 canescens bark because both
genes are involved in inositol and mannitol metabo-
lism (Zuther et al., 2004; Nishizawa et al., 2008).
As a toxic heavy metal, cadmium can cause serious
stress to plants. The stimulation of stress responses at
the transcript level has often been reported in herba-
ceous plants (Herbette et al., 2006; Weber et al., 2006;
Ogawa et al., 2009; Yamaguchi et al., 2010). We
found a relatively high number of genes implicated
in biotic stress in the bark of cadmium-exposed P. 3
canescens. Proteomic analysis revealed overexpression
of pathogen-related proteins in cadmium-treated
P. tremula (Kieffer et al., 2008, 2009b). In agreement
with increased O2 2 radicals, cadmium-exposed poplars
d
exhibited an elevated transcript level of NADH-ubiquinone
dehydrogenase6, which has been shown to result in in-
creased oxidative stress (Raczynska et al., 2006). Thiols
are often depleted under cadmium stress (Sharma and
Dietz, 2009) and therefore may not have counter-
balanced elevated radical production (Schützendübel
and Polle, 2002).
Our data clearly demonstrate that transcriptomic
reprogramming underlies the microstructural and
physiological responses to cadmium accumulation and
detoxification in P. 3 canescens bark. Furthermore,
about one-half of the cadmium-responsive genes
formed a coexpression network, indicating that P. 3
canescens coordinates the transcriptomic regulation for
heavy metal stress. Recently, coregulation networks
of transcriptomes have been reported in rice (Zhang
et al., 2012), Arabidopsis (Bassel et al., 2011; Movahedi
et al., 2011), pepper (Capsicum annuum), and tomato
433
He et al.
(Solanum lycopersicum; Osorio et al., 2012), and maize
(Zea mays; Ficklin and Feltus, 2011). However, none of
these studies have addressed the coexpression of genes in
response to cadmium in plants. For the first time, we have
demonstrated the transcriptomic network underlying the
microstructural and physiological responses to cadmium
accumulation and detoxification in P. 3 canescens bark.
Moreover, GO term analysis indicates that hub genes
were enriched in fundamental processes such as transport
and photosynthesis. These data suggest that the highly
connected hub genes in the coexpression network play a
central role in cross talk among distinct biological pro-
cesses and in coordinating transcriptomic regulation in
the bark of cadmium-exposed P. 3 canescens.
Changed Nutrient and Carbohydrate Concentrations and
Shifted Homeostasis between ROS and the Antioxidants
Highlight the Physiological Regulation Mechanism
to Cadmium
The PCA of nutrients, carbohydrates, oxidants, and
antioxidants and the assessment data of these physi-
ological parameters in P. 3 canescens indicated that
P. 3 canescens possess a physiological regulation
mechanism to cadmium exposure. Previous studies
reported that nitrogen and phosphorus are involved in
detoxification of cadmium-exposed Arabidopsis (Van
Belleghem et al., 2007; Li et al., 2010; Mendoza-Cózatl
et al., 2011) and that decreased nitrogen and phos-
phorus are found in pea plants due to cadmium tox-
icity (Sandalio et al., 2001; Metwally et al., 2005). Bark
nitrogen and phosphorus concentrations were consis-
tently lower in cadmium-exposed P. 3 canescens than
in the control, probably due to repressed nitrogen
and phosphorus uptake. Sulfur-containing compounds
(e.g. GSH and PCs) are used as chelates for cadmium
(Herbette et al., 2006; Rodríguez-Serrano et al., 2009;
Gill et al., 2012). Sulfur accumulation in the bark of
cadmium-exposed P. 3 canescens is probably associ-
ated with accelerated biosynthesis of thiol compounds,
such as GSH, to detoxify cadmium. The finding of
decreases in calcium, iron, manganese, and zinc in
cadmium-treated P. 3 canescens is consistent with
previous studies (Sandalio et al., 2001; Metwally et al.,
2005; Solti et al., 2008; Rodríguez-Serrano et al., 2009)
and probably due to competition with cadmium ions
for transporters. Reduction in CO 2 assimilation is
probably due to defects in the photosynthetic appa-
ratus (McCarthy et al., 2001; Solti et al., 2008; Ivanova
et al., 2011), inactivation of photosynthesis-related
enzymes (Leon et al., 2002; He et al., 2011), decreases
in photosynthetic pigments or iron (Solti et al., 2008;
Besson-Bard et al., 2009; Wu et al., 2012), and differ-
ential expression of photosynthesis-related genes and
proteins (Kieffer et al., 2009a, 2009b; Durand et al.,
2010). Reduced CO 2 assimilation often leads to
changes in the concentrations of sugars and sugar al-
cohols in cadmium-treated plants (Devi et al., 2007;
Kieffer et al., 2009a; He et al., 2011). Decreases in Suc,
mannitol, inositol, and starch in P. 3 canescens bark are
434
in agreement with inhibited photosynthesis under
cadmium exposure. Accumulation of O2 2 in bark and
d
leaves and of H2O2 in leaves of cadmium-exposed P. 3
canescens suggests that oxidative stress occurs as ob-
served in previous studies (Schützendübel et al., 2002;
Romero-Puertas et al., 2004, 2012; Garnier et al., 2006;
Rodríguez-Serrano et al., 2006, 2009; He et al., 2011).
Oxidative burst is toxic for plant cells; therefore, excess
ROS must be scavenged in cadmium-treated P. 3
canescens. Enzymatic antioxidants are proposed to play
a role in ROS detoxification in cadmium-exposed
plants (Leon et al., 2002; Gratao et al., 2005; Rodríguez-
Serrano et al., 2006; Romero-Puertas et al., 2006, 2007;
Elobeid et al., 2012); however, the activities of enzymatic
antioxidants were unchanged or reduced by cadmium
exposure in our study, indicating that the role of
enzymatic antioxidants in ROS scavenging in cadmium-
treated P. 3 canescens was limited. Notably, other anti-
oxidants, such as free Pro, soluble phenolics, ascorbate,
GSH, oxidized glutathione (GSSG), and total thiols, are
involved in scavenging cadmium-induced O2 2 and
d
H2O2 (Sharma and Dietz, 2009). The induction of these
antioxidants in cadmium-treated P. 3 canescens implies
that these antioxidants play a pivotal role in scavenging
excess ROS. The induction of these antioxidants, par-
ticularly thiols, and their role in cadmium detoxification
have been documented in other plants (Vogel-Mikus
et al., 2010; Gaudet et al., 2011; Seth et al., 2012).
Overall, these data indicate that changes in internal
nutrients, carbohydrates, and antioxidants play a
role in physiological regulation of cadmium-exposed
P. 3 canescens.
In conclusion, P. 3 canescens bark tissue accumulated
cadmium about 2 times above the threshold of hyper-
accumulation. Cadmium was translocated to the phloem
in the bark, and subcellular cadmium compartmen-
talization in this tissue occurred mainly in vacuoles of
phloem cells. Degeneration of chloroplasts and mito-
chondria took place in companion cells. Microstructural
alterations, changes in nutrition and primary metabo-
lism, and stimulation of stress responses in cadmium-
exposed bark were mirrored by differential expression
of genes related to these processes. About one-half of
the differentially regulated transcripts formed a cor-
egulation network in which 43 hub genes may play a
central role both in cross talk among distinct biological
processes and in coordinating the transcriptomic regu-
lation in the bark of P. 3 canescens. In concert, these
results suggest that orchestrated microstructural, tran-
scriptomic, and physiological regulation can be essen-
tial for cadmium accumulation and detoxification in
P. 3 canescens and provide new insights into engi-
neering woody plants for phytoremediation.
MATERIALS AND METHODS
Cultivation of Plants and Cadmium Exposure
Plantlets of Populus 3 canescens (Populus tremula 3 Populus alba) were
produced by micropropagation (Leple et al., 1992) and cultivated in a climate
Plant Physiol. Vol. 162, 2013

chamber (day/night temperature, 25°C/18°C; relative air humidity, 50%–60%;
photoperiod, 14 h; photosynthetic photon flux, 150 mmol m–2 s–1). After
5 weeks, the rooted plantlets were transferred to 10-L plastic pots filled with
10 kg of sand. Plants were cultivated for 10 weeks in a greenhouse (day/night
temperature, 25°C /18°C; relative air humidity, 50%–60%; natural light) before
cadmium exposure. Nutrient solution (30 mL full-strength Hoagland solution)
was slowly added to the pots each morning. Water (30 mL) was added each
evening. Twenty-four plants with similar height were selected and divided
into two groups, with 12 plants in each group: one group for the cadmium
treatment (200 mM CdSO4) and the other for the control (0 mM CdSO4). Plants
were grown for 20 d in the absence or presence of cadmium, which was
supplied daily with the nutrient solution before harvest.
Gas Exchange Measurement and Harvest
Before harvest, three mature leaves (leaf plastochron index [Erickson and
Michelini, 1957] = 7–9) were selected from each plant for gas exchange mea-
surements. Net photosynthetic rate, stomatal conductance, and transpiration
rate were determined using a portable photosynthesis system (LiCor-6400) as
described previously (He et al., 2011).
After measuring photosynthesis, the root system of each plant was carefully
washed as suggested by Rauser (1987). Subsequently, the roots and above-
ground tissues of each plant were harvested. The aboveground tissues were
separated into bark, wood, and leaves. The samples were weighed (fresh
weight), wrapped with tinfoil, and immediately frozen in liquid nitrogen.
Frozen samples were ground into fine powder in liquid nitrogen with a mortar
and a pestle and stored at 280°C for further analysis. Fresh powder (ap-
proximately 30 mg) of each tissue from each plant was separately dried at
60°C to determine the fresh-to-dry mass ratio. This ratio was used to calculate
the dry weight (biomass) of each tissue. An equal amount of fine powder from
two plants in each treatment was combined and thoroughly mixed for bio-
chemical analysis. Fine powder of bark tissue from four plants in each treat-
ment was thoroughly mixed and used for RNA extraction. Subsamples of fine
roots, stems, petioles, and leaves were also harvested for anatomical and
histochemical analysis.
Analysis of Cadmium, Nutrient Elements,
and Foliar Pigments
Concentrations of cadmium and zinc were determined in roots, bark, and
leaves by flame atomic absorbance spectrometry (Hitachi 180-80) as described
by He et al. (2011). Total carbon and nitrogen in different tissues were ana-
lyzed with a carbon/nitrogen analyzer (Elemental Analyzer EA1108, Carlo
Erba Strumentazione) as described in Luo and Polle (2009). Mineral elements
were determined using an inductively coupled plasma-atomic emission
spectrometer (Spectroflame, Spectro Analytical Instruments) based on the
protocol described by Luo et al. (2009a). Chlorophyll and carotenoid contents
in leaves were determined spectrophotometrically as suggested by He et al.
(2013).
Cadmium Localization at Tissue and Subcellular Levels
To characterize cadmium localization at the tissue level, hand sections or
intact tissues of fresh samples of fine roots, stems, petioles, and leaves were
rinsed in deionized water, subsequently exposed to a staining solution (30 mg
diphenylthiocarbazone in 60 mL acetone, 20 mL water, 100 mL glacial acetic
acid) for 1 h, and then briefly rinsed in deionized water as suggested by
Clabeaux et al. (2011). Well-stained samples with cadmium-dithizone pre-
cipitates displaying red-black were photographed under a light microscope
(Eclipse E200, Nikon) with a CCD (DS-Fi1, Nikon) connected to a computer as
described by Cao et al. (2012).
To further characterize cadmium localization at the subcellular level,
samples were prepared for TEM as suggested by Gratao et al. (2009) with
minor modifications. Briefly, samples of fine roots, bark, and leaves were cut
into sections (approximately 1–2 mm3), fixed with 4% (v/v) glutaraldehyde in
0.1 M phosphate buffer (pH 6.8) at 4°C overnight, and subsequently washed
three times with phosphate buffer (0.1 M, pH 6.8). The samples were then fixed
with 1% (w/v) osmium tetroxide for 2 h and immersed in 0.1 M phosphate
buffer (pH 6.8) for 1 h. The sections were dehydrated in a graded ethanol
series (30%, 50%, 70%, 80%, 90%, and 100%, v/v) with 10- to 20-min intervals,
infiltrated, and embedded in Epon resin. Ultrathin (70 nm) were collected on
Plant Physiol. Vol. 162, 2013
The Cadmium Transcriptome of Poplar Bark
copper grids and stained with 2.5% (w/v) uranyl acetate followed by 0.1%
(w/v) lead citrate. Sections were photographed under a transmission electron
microscope (HT7700, Hitachi) at 75 kV.
To examine cadmium contents in the granular deposits, EDX microanalysis
was carried out on ultrathin sections (no staining with 2.5% [w/v] uranyl
acetate and 0.1% [w/v] lead citrate) of different tissues as proposed by Van
Belleghem et al. (2007). The transmission electron microscope was equipped
with an EDX detector (Genesis XM2, EDAX). The sections were analyzed at an
accelerating voltage of 80 kV. The EDX spectra with cadmium La (3.133 keV)
and sulfur Ka (2.308 keV) were recorded during an analysis period of 200 live
seconds (total spectrum collection time). Spectra analyses were conducted
using the SuperQuant program (EDAX). The measurement parameters (sec-
tion thickness, beam emission current, spot diameter, and tilt angle) were
constant throughout the analyses. The cadmium concentrations in granular
deposits were expressed as the ratio between the specific emission intensity of
cadmium above background and the nonspecific emission intensity of the
background (peak/background).
RNA Extraction and DNA Chip Hybridization
Fine powder of bark (approximately 500 mg) was used for RNA extraction
according to Chang et al. (1993) with minor modifications described pre-
viously (Luo et al., 2009a). The extraction buffer contained 2% (v/v)
b-mercaptoethanol and no spermidine. The RNA was purified (RNeasy
Mini Kit, Qiagen). The purity and integrity of the RNA were assessed
according to the Affymetrix GeneChip expression analysis protocol. Further
processing of the RNA and complementary RNA hybridization using the
Affymetrix poplar (Populus spp.) genome array were accomplished at the
microarray service facilities of the Shanghai Biotechnology Company. For
each treatment, three arrays were hybridized, thus yielding six arrays. Raw
data of the arrays are available at the ArrayExpress depository (http://
www.ebi.ac.uk/arrayexpress/; accession no. E-MEXP-3741).
Annotation, Functional Categorization, Coexpression,
and GO Enrichment Analysis
To obtain up-to-date annotations of differentially expressed genes, we per-
formed several BLAST searches as described by Janz et al. (2012). Briefly, the
Affymetrix GeneChip poplar genome array target sequences were used to blast
(BLASTn) against the Phytozome Populus trichocarpa version 2.2 transcript da-
tabase. The closest Arabidopsis (Arabidopsis thaliana) homolog (AGI identifica-
tion) of a P. 3 canescens gene was determined by a translated nucleotide BLAST
(BLASTx) of the coding sequence of the best P. trichocarpa hit against the Arab-
idopsis protein sequence data set. Annotations were taken from the latest release
of The Arabidopsis Information Resource genome, TAIR10.
To categorize differentially expressed genes based on their biological
functions, P. 3 canescens genes with unique AGIs (only the P. 3 canescens gene
with the lowest P value in Supplemental Table S2 was selected when multiple
P. 3 canescens genes corresponded to an AGI) were selected and submitted to
MapMan for analysis as suggested by Thimm et al. (2004). Subsequently, all
genes with functional categorization and subsets of genes assigned to cate-
gories of photosynthesis, stress, RNA regulation (mainly transcription factors),
signaling, and transport by MapMan were used for coexpression analysis
as described by Wei et al. (2006), using an online open resource (http://
cressexpress.org/index.jsp). Results of coexpression analysis were visualized
in Cytoscape version 2.8.3 as described by Shannon et al. (2003).
To perform GO term enrichment analyses for all cadmium-responsive
genes, coexpressed genes, and hub genes, AGIs of these genes were used
for singular enrichment analysis in the agriGO database (http://bioinfo.cau.
edu.cn/agriGO/analysis.php) as suggested by Du et al. (2010).
Validation of Microarray Analysis by Quantitative RT-PCR
Total RNA of bark was isolated and purified with a plant RNA extraction kit
(R6827, Omega Bio-Tek). Quantitative reverse transcription (RT)-PCR of seven
genes (for primer information, see Supplemental Table S8) was performed
according to Li et al. (2012). The reference gene was 18S ribosomal RNA
(Supplemental Table S8). To ensure the specification, PCR products were se-
quenced and aligned with the homologs in other model plants (Supplemental
Fig. S7). The correlation of gene expression was analyzed between the micro-
array and the quantitative RT-PCR data (Supplemental Fig. S8).
435
He et al.
Analysis of Soluble Sugars, Sugar Alcohols, and Starch
Soluble sugars and sugar alcohols were determined by gas chromatography-
mass spectrometry as described previously (Luo et al., 2009a, 2009b). Starch
concentrations (expressed as Glc equivalent) in samples were analyzed using the
anthrone method described by He et al. (2013). Absorption was determined
spectrophotometrically at 620 nm.
Determination of O2 2 and H2O2
d
Concentrations of O2d2 and H2O2 in samples were determined spectro-
photometrically at 530 and 410 nm, respectively, as described previously
(Martínez Domínguez et al., 2010; He et al., 2011). For O2d2 determination, fine
powder of fresh tissues (approximately 100 mg) was homogenized in 2 mL of
50 mM potassium phosphate buffer (pH 7.8) and centrifuged at 10,000g and
4°C for 10 min. The supernatant (1 mL) was mixed with 0.9 mL of 50 mM
potassium phosphate buffer (pH 7.8) and 0.1 mL of 10 mM hydroxylamine
hydrochloride. The reaction mixture was then incubated at 25°C for 20 min
before adding 1 mL of 17 mM P-aminobenzene sulfonic acid and 1 mL of 7 mM
a-naphthylamine. After further incubation at 25°C for 20 min, the absorbance
of the mixture was recorded spectrophotometrically at 530 nm.
To determine H2O2 concentrations, the fine powder of fresh tissue (ap-
proximately 60 mg) was extracted in 2 mL acetocaustin and centrifuged at
10,000g and 4°C for 10 min. The supernatant was collected. After adding
0.1 mL of 20% (v/v) TiCl4 and 0.2 mL of 25% (v/v) aqueous ammonia to the
supernatant, the mixture was immediately centrifuged again at 10,000g and
4°C for 10 min. The supernatant was then discarded, and the pellet was dis-
solved in 3 mL of 1 M H2SO4. The absorbance was recorded spectrophoto-
metrically at 410 nm.
Analysis of Enzyme Activities, Pro, and Soluble Phenolics
Soluble proteins were extracted and used for assays of enzyme activities as
described previously by He et al. (2011).
Free Pro was determined spectrophotometrically according to He et al.
(2011). The standard curve was generated using a series of diluted L-Pro so-
lutions (Amresco).
Soluble phenolics in samples were determined spectrophotometrically by
using the Folin-Ciocalteu reagent as reported by Luo et al. (2008). A standard
curve was prepared using a series of diluted catechin solutions (Sigma).
Analysis of Ascorbate, GSH, GSSG, and Total Thiols
Ascorbate was analyzed based on the protocol described by Chen et al.
(2011) with minor modifications. Plant samples (approximately 100 mg) were
extracted in 6% (w/v) TCA (prechilled on ice) and centrifuged at 12,000g and
4°C for 10 min. The supernatant was analyzed spectrophotometrically at
525 nm.
GSH and GSSG were analyzed using the 5,59-dithiobis(2-nitrobenzoic
acid)-glutathione reductase recycling procedure suggested by Chen et al.
(2011).
Total thiols were analyzed according to Tamás et al. (2008) with minor
modifications. Freeze-dried tissues (approximately 100 mg) were extracted in
1 mL of 20 mM EDTA on ice and centrifuged at 12,000g and 4°C for 10 min.
A 0.25-mL aliquot of the supernatant was mixed with 2.5 mL of Tris buffer
(0.2 M, pH 8.2) and 50 mL of 10 mM 5,59-dithiobis(2-nitrobenzoic acid). After
incubation at room temperature for 15 min, the absorbance of the reaction
mixture was determined spectrophotometrically at 412 nm.
Statistical Analysis
Statistical tests were performed with Statgraphics (STN). For experimental
variables, one-way ANOVA was applied with cadmium treatment as a factor.
Data were tested for normality prior to the statistical analysis. Differences
between means were considered significant when the P value of the ANOVA F
test was less than 0.05. For PCA, data were standardized and subsequently
computed by the command prcomp() in R (http://www.r-project.org/).
For microarray analysis, raw probe intensity values from the Affymetrix
CEL files generated at the microarray service facilities were analyzed in an SBC
Analysis System (http://sas.ebioservice.com) as described by Li et al. (2011),
which was developed based on algorithms in R package (http://www.
436
r-project.org/). Background correction and quantile normalization of raw data
were computed by the rma algorithm from the affy package (Irizarry et al.,
2003). Subsequently, differentially expressed genes (cadmium-treated versus
control group) were identified from background-corrected and normalized
data by one-way ANOVA with P values less than 0.05. Relative expression
ratios of selected genes for quantitative RT-PCR were determined using the
Relative Expression Software Tool (Pfaffl et al., 2002).
Supplemental Data
The following materials are available in the online version of this article.
Supplemental Figure S1. Cadmium localization in petioles and leaves.
Supplemental Figure S2. EDX spectra of granular deposits in vacuoles of
bark cells.
Supplemental Figure S3. Transmission electron microscopic micrographs.
Supplemental Figure S4. O2d2 and H2O2 in the bark, root, and leaf tissues.
Supplemental Figure S5. Activities of antioxidant enzymes.
Supplemental Figure S6. Nonenzymatic antioxidants.
Supplemental Figure S7. Alignments of genes for quantitative RT-PCR.
Supplemental Figure S8. Correlation of gene expression between the
microarray and quantitative RT-PCR data.
Supplemental Table S1. Photosynthesis and biomass.
Supplemental Table S2. Significantly differentially regulated transcripts.
Supplemental Table S3. Functional categories of genes and gene presence
in the coexpression network.
Supplemental Table S4. GO term enrichment analysis.
Supplemental Table S5. Total nitrogen, carbon, and nutrients.
Supplemental Table S6. Carbohydrate concentrations.
Supplemental Table S7. PCA of nutrients, carbohydrates, oxidants, and
antioxidants.
Supplemental Table S8. Primers used for quantitative RT-PCR.
ACKNOWLEDGMENTS
We thank Christine Kettner and Gisbert Langer-Kettner for nutrient
element analysis, William J. Gale for English correction, and anonymous
reviewers for their suggestions to improve the manuscript.
Received February 1, 2013; accepted March 22, 2013; published March 25,
2013.
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Supplementary Data
To Plant Physiology

A transcriptomic network underlies microstructural and physiological responses

to cadmium in Populus × canescens
Jiali He§, Hong Li§, Jie Luo, Chaofeng Ma, Shaojun Li, Long Qu, Ying Gai,
Xiangning Jiang, Dennis Janz, Andrea Polle, Melvin Tyree, and Zhi-Bin Luo*
* Corresponding author; e-mail luozbbill@163.com

1
Table S1 CO2 assimilation rate (A, µmol CO2 m-2 s-1), stomatal conductance (gs, mol H2O m−2 s−1), transpiration rate (E, mmol H2O m-2 s-1) and
photosynthetic pigments (mg g-1 DW) in leaves and biomass (g DW) of P. × canescens exposed to 0 or 200 µM CdSO4 for 20 days. Data
indicate means ± SE (n = 6). Different letters behind the values in the same column indicate significant difference between the treatments.
P-values of the ANOVAs of CdSO4 (Cd) were also shown. Chl a: chlorophyll a; Chl b: chlorophyll b; Chl (a+b): sum of chlorophyll a and b; Car:
carotenoid.

Cd (µM) A gs E Chl（a+b） Car

0 15.4 ± 0.6 b 0.376 ± 0.028 b 6.9 ± 0.3 b 10.6 ± 0.3 a 1.6 ± 0.1 a
200 8.5 ± 1.5 a 0.197 ± 0.054 a 3.5 ± 0.8 a 8.9 ± 0.5 b 1.4 ± 0.1 a
P-values 0.0019 0.0149 0.0016 0.0455 0.2927

During Cd exposure
Cd (µM) Root biomass Bark biomass Leaf biomass Root biomass Bark biomass Leaf biomass Height Leaf number
increment increment increment increment (cm) increment
0 1.55 ± 0.05 b 0.62 ± 0.04 a 3.20 ± 0.01 b no data 0.29 ± 0.02 a 1.34 ± 0.08 b 32.50 ± 1.44 a 11.50 ± 0.43 a
200 1.06 ± 0.07 a 0.56 ± 0.04 a 3.14 ± 0.01 a no data 0.22 ± 0.03 a 1.18 ± 0.07 a 27.97 ± 2.07 a 9.83 ± 0.51 a
P-values 0.0045 0.3173 0.0105 no data 0.1394 0.0186 0.1464 0.3146

2
Table S2 Significantly differentially regulated transcripts in the bark of P. × canescens exposed to 0 or 200 μM CdSO4 for 20 days. It is available
in the online version because it is too big to be included here.

Table S3 Functional categories of significantly differentially regulated genes assigned by MapMan and gene presence in the network in the bark
of P. × canescens exposed to 0 or 200 μM CdSO4 for 20 days. It is available in the online version because it is too big to be included here.

Table S4 GO term enrichment analyses of all Cd responsive genes (Column A-L), co-expressed genes (Column N-Y) and hub genes (Column
AA-AL) in the bark of P. × canescens exposed to 0 or 200 μM CdSO4 for 20 days. It is available in the online version because it is too big to be
included here.

3
Table S5 Total nitrogen (N), carbon (C) and nutrients (μmol g-1 DW) in the bark, roots and leaves of P. × canescens exposed to 0 or 200 µM
CdSO4 for 20 days. Data indicate means ± SE (n = 6). Different letters behind the values in the same column for the same tissue indicate
significant difference between the treatments. It is available in the online version because it is too big to be included here.

Cd
Tissues Total N (%) Total C (%) P S K Ca
(µM)
0 1.51 ± 0.05 b 43.8 ± 0.1 a 103.58 ± 4.75 b 46.16 ± 0.68 a 576.32 ± 145.62 a 289.51 ± 2.65 b
Bark
200 1.45 ± 0.09 a 44.2 ± 0.1 a 75.56 ± 2.49 a 50.67 ± 0.19 b 532.37 ± 162.23 a 270.40 ± 2.21 a
0 1.83 ± 0.17 a 37.0 ± 0.6 a 75.22 ± 6.96 a 119.02 ± 5.82 a 243.35 ± 22.20 a 513.08 ± 24.62 a
Roots
200 2.12 ± 0.19 a 38.6 ± 0.6 a 81.43 ± 5.00 a 189.46 ± 1.46 b 247.30 ± 12.37 a 566.10 ± 1.71 a
0 3.67 ± 0.14 b 46.4 ± 0.2 b 100.18 ± 2.72 b 105.23 ± 2.91 a 608.89 ± 28.10 a 329.65 ± 9.80 a
Leaves
200 3.04 ± 0.07 a 44.7 ± 0.1 a 83.59 ± 4.23 a 98.54 ± 6.20 a 616.14 ± 9.29 a 325.67 ± 5.42 a

Cd
Tissues Total N (%) Mg Mn Fe Zn Al
(µM)
0 1.51 ± 0.05 b 2.82 ± 0.77 a 0.41 ± 0.11 a 1.13 ± 0.31 a 2.93 ± 0.51 a 2.51 ± 0.21 a
Bark
200 1.45 ± 0.09 a 2.85 ± 0.84 a 0.44 ± 0.14 a 1.18 ± 0.32 a 2.79 ± 0.58 a 3.14 ± 0.21 a
0 1.83 ± 0.17 a 96.13 ± 1.93 a 5.88 ± 0.59 b 52.49 ± 1.71 a 6.89 ± 0.71 b 114.57 ± 12.89 b
Roots
200 2.12 ± 0.19 a 127.83 ± 14.78 a 3.34 ± 0.10 a 52.57 ± 2.74 a 3.44 ± 0.01 a 79.27 ± 3.30 a
0 3.67 ± 0.14 b 299.02 ±7.32 a 2.54 ± 0.15 a 8.02 ± 0.91 b 2.51 ± 0.09 b 11.67 ± 0.70 a
Leaves
200 3.04 ± 0.07 a 315.61 ± 20.32 a 2.24 ± 0.02 a 4.97 ± 0.14 a 1.96 ± 0.13 a 11.13 ± 1.63 a

4
Table S6 Soluble sugars and sugar alcohols (nmol g-1 DW) and starch (mg g-1 DW) in the bark, roots and leaves of P. × canescens exposed to 0
or 200 µM CdSO4 for 20 days. Data indicate means ± SE (n = 6). Different letters behind the values in the same column for the same tissue.

Cd Trehalose
Tissues Sucrose (× 103) Glucose (× 103) Galactose Mannitol Inositol (× 103) Xylose Rhamnose
(µm) (× 103)

0 10.62 ± 2.75 b 5.48 ± 0.32 b 0.58 ± 0.03 a 990.12 ± 9.21 b 79.19 ± 3.86 b 23.21 ± 1.35 b 72.05 ± 6.34 a 10.77 ± 0.84 a
Bark
200 1.26 ± 0.39 a 1.41 ± 0.30 a 0.57 ± 0.02 a 282.2 ± 12.18 a 51.24 ± 5.59 a 18.71 ± 0.16 a 77.01 ± 17.83 a 10.77 ± 1.11 a
0 8.61 ± 0.58 b 75.47 ± 6.04 b 11.70 ± 3.19 a 20.0 ± 2.58 a 60.00 ± 4.53 a 0.60 ± 0.17 a 0.00 ± 0.00 a 3.23 ± 0.32 b
Roots
200 1.99 ± 0.18 a 30.64 ± 7.09 a 21.30 ± 0.15 b 23.3 ± 2.25 a 102.80 ± 14.12 b 2.60 ± 0.18 b 6.17 ± 1.04 b 0.00 ± 0.00 a
0 22.18 ± 6.76 b 14.50 ± 0.22 b 0.72 ± 0.09 a 1370.4 ± 6.61 b 214.77 ± 9.54 a 37.01 ± 0.36 b 176.74 ± 38.12 a 9.39 ± 0.30 a
Leaves
200 2.17 ± 0.03 a 1.07 ± 0.07 a 1.16 ± 0.11 b 862.3 ± 2.59 a 500.28 ± 14.24 b 32.48 ± 1.11 a 137.18 ± 21.84 a 9.32 ± 2.10 a

Cd
Tissues Fucose Arabinose 2-Deoxy-Ribitol Arabitol Xylitol Sorbitol Galactitol Starch
(µm)

0 57.24 ± 7.14 a 3.31 ± 0.20 b 5.11 ± 0.94 a 87.08 ± 8.40 b 51.07 ± 6.05 a 37.15 ± 1.88 b 322.08 ± 16.48 b 20.47 ± 0.94 b
Bark
200 54.48 ± 0.10 a 2.10 ± 0.05 a 9.25 ± 0.20 b 43.59 ± 1.75 a 55.36 ± 16.91 a 24.62 ± 0.98 a 177.66 ± 1.33 a 17.52 ± 0.42 a
0 33.67 ± 8.38 b 0.88 ± 0.03 a 1.40 ± 0.00 b 22.27 ± 11.29 a 0.00 ± 0.00 a 0.00 ± 0.00 a 0.00 ± 0.00 a 32.73 ± 1.62 a
Roots
200 0.00 ± 0.00 a 1.26 ± 0.12 b 0.77 ± 0.00 a 22.42 ± 11.71 a 5.87 ± 0.70 b 2.75 ± 0.86 b 12.64 ± 1.79 b 39.34 ± 1.51 b
0 118.70 ± 24.51 b 8.34 ± 1.101 a 33.14 ± 0.40 b 157.91 ± 5.23 b 48.79 ± 2.22 a 46.40 ± 8.65 a 613.87 ± 41.43 b 8.22 ± 0.30 a
Leaves
200 48.35 ± 3.18 a 6.85 ± 1.14 a 21.41 ± 3.73 a 86.73 ± 9.69 a 38.52 ± 6.89 a 32.58 ± 6.24 a 439.27 ± 25.95 a 10.37 ± 0.45 b

5
Table S7 Principal component analysis (PCA) of nutrients, soluble sugars and sugar
alcohols, and oxidants and antioxidants in the bark, fine roots and leaves of P. ×
canescens exposed to 0 or 200 μM CdSO4.

Variables PC1 PC2 PC3

Nutrients
Total N 0.11 0.63 0.04
Total C 0.35 0.14 -0.11
P 0.20 0.16 -0.57
S -0.29 0.37 0.30
K 0.31 0.11 -0.28
Ca -0.37 0.17 0.03
Mg 0.21 0.53 0.06
Mn -0.32 0.27 -0.27
Fe -0.38 0.12 -0.04
Zn -0.29 -0.08 -0.63
Al -0.35 0.06 -0.10
Proportion of variation (%) 60.1 20.0 7.9
Sugars and sugar alcohols
Sucrose 0.15 0.54 -0.26
Trehalose -0.20 0.47 0.13
Glucose 0.26 -0.30 0.15
Galactose 0.29 0.05 -0.10
Xylose 0.27 -0.05 0.09
Rhamnose 0.21 -0.27 -0.31
Fucose 0.24 0.28 -0.23
Arabinose 0.26 0.13 0.23
Erythritol 0.27 0.19 0.29
2-Deoxy-Ribitol 0.27 0.15 0.18
Arabitol 0.28 0.20 -0.05
Xylitol 0.21 -0.28 -0.34
Inositol 0.30 -0.10 0.01
Mannitol 0.15 -0.15 0.63
Sorbitol 0.28 -0.04 -0.20
Galactitol 0.30 0.07 0.03
Proportion of variation (%) 68.9 11.3 10.0
Oxidants and antioxidants
O2•- -0.19 -0.46 0.24
H2O2 -0.32 -0.11 -0.36
Free proline 0.20 -0.17 -0.74
Soluble phenolics -0.33 -0.19 -0.06
ASC -0.35 0.13 -0.25
GSH -0.31 0.29 -0.18
GSSG -0.36 -0.12 0.03
T-SH -0.31 0.27 -0.21
GPX 0.36 0.01 -0.17
CAT -0.17 0.49 0.09
APX 0.33 0.18 -0.24
GR 0.09 0.50 0.16
Proportion of variation (%) 59.2 25.0 8.7

6
Table S8 Primers used for qRT-PCR.
Gene
symbol Affymetrix ID Gene model Gene name Closest AGI Primer-Forward Primer-Reverse

SPX2 PtpAffx.163910.1.S1_at POPTR_0006s06880 SPX domain gene 2 At2g26660 5’-CTGAAGGTGGTGGAGCAGATAGC-3’ 5’-CTTTGCCTTTGCCACACTATCTTG-3’

NB-ARC
domain-containing
NDDRP PtpAffx.225724.1.S1_x_at POPTR_0017s04690 disease resistance protein At3g14470 5’-TCTTGATAGCAGGTTGTCCCG-3’ 5’-GGTCTTCGCAGCCAGCAGTA-3’
glutathione S-transferase
GSTU22 PtpAffx.3457.1.A1_at POPTR_0011s14380 TAU 22 At1g78340 5'-GCATAAGGACTGGAGAGAGCAA-3' 5'-AGAACCAAAGGGACGAACG-3'

P80146 PtpAffx.80146.1.S1_at NA Unknown Protein NA 5'-ATAACGGAGGGGTGTGGATG-3' 5'-CACGTGTAGCTGATCTTTGGTTG-3'

Ribosomal protein
PGY1 PtpAffx.1126.3.A1_at POPTR_0004s21290 L1p/L10e family At2g27530 5'-TGTGCATGGGTGTTGCTGT-3' 5'-GCTTAGTAGAGGCGGACTGGTG-3'
NAC domain containing
NAC044 PtpAffx.151346.1.S1_at POPTR_0001s35490 protein 44 At3g01600 5'-GCTTATGCTACTGGTCAACG-3' 5'-CCCAGATGGTACTGATGC-3'
Pyridoxal phosphate
(PLP)-dependent
transferases superfamily
PLP PtpAffx.215435.1.S1_at POPTR_0017s14070 protein At1g80360 5’-TCAGCACCTTGCTCTCTATTCCT-3' 5'-ATTGCTCCTTCTCCACCCTTC-3’

a 18S rRNA 5’-AGAAACGGCTACCACATCCAA-3' 5’-CCAGACTTGCCCTCCAATGG-3'

a: From Junghans U, Polle A, Düchting P, Weiler E, Kuhlman B, Gruber F, Teichmann T (2006) Adaptation to high salinity in poplar involves
changes in xylem anatomy and auxin physiology. Plant Cell Environ 29: 1519–1531

7
Fig. S1 Cd localization in petioles (A-F) and leaves (G-J) of P. × canescens exposed
to 0 (A,C,E,G,I) or 200 µM (B,D,F,H,J) CdSO4 for 20 days. Arrows point to
precipitates of Cd-dithizone. B: Cd-dithizone precipitates in collenchyma cells; D:
Cd-dithizone precipitates in intercellular space of parenchyma cells; F: Cd-dithizone
precipitates in vascular tissue; H and J: Cd-dithizone precipitates in leaf trichomes.

G H

200 μm 200 μm

I J

200 μm 200 μm

8
Fig. S2 The EDX spectra of vacuoles (A) and granular deposits (B) in vacuoles of
bark cells of P. × canescens exposed to 0 (A) or 200 µM (B) CdSO4 for 20 days. The
EDX spectra A and B correspond to TEM micrographs presented in Figure 3A and 3B,
respectively.

A B

9
Fig. S3 Transmission electron microscopic micrographs of root epidermal cells (A),
root cortical cells (B, C) and mesophyll cells (D) of P. × canescens exposed to 200
µM CdSO4 for 20 days. B is derived from the dashed area of C. The inserts highlight
deposited-Cd granules on inside-tonoplast (A) and in vacuoles (C), respectively.
Arrows point to Cd deposits. Cd concentrations in granular deposits are shown in
Figure 4A. CW: cell wall; M: mitochondrion; ER: endoplasmic reticulum; V: vacuole;
N: nucleus; Nue: nucleolus; P: plastoglobulus; SG: starch grain. Ch: chloroplast; Os:
osmophilic plastoglobulus.

V CW

2 μm

CW

E
R
M

1 μm

C CW
M
V

Nue
N

S
G
2 μm

N Os

M
V
SG Ch
2 μm

10
Fig. S4 O2•- and H2O2 in the bark, root and leaf tissues of P. × canescens exposed to 0
or 200 µM CdSO4 for 20 days. Bars indicate means ± SE (n = 6). Asterisks on the bars
for the same tissue indicate significant difference between the treatments.

18
bark root leaf A bark root leaf B
15 200

H2O2 (μmol g DW)

∗ ∗
O2 (μmol g DW)

12

-1
160
-1

9 ∗
120
6
._

80
3

40
0 200 0 200 0 200 0 200 0 200 0 200

Cd (μM)

11
Fig. S5 Activities of guaiacol peroxidase (GPX), catalase (CAT), ascorbate peroxidase
(APX) and glutathione reductase (GR) in the bark, roots and leaves of P. × canescens
exposed to 0 or 200 µM CdSO4 for 20 days. Bars indicate means ± SE (n = 6).
Asterisks on the bars for the same tissue indicate significant difference between the
treatments.

9000
bark root leaf A bark root leaf B 120
GPX (nkat g protein)

7500

CAT (nkat g protein)

∗ 80
6000
∗ 40
4000
-1

-1
∗ 6
3000

2000 4
∗
1000 ∗ 2

0 0
4500 C D 750

GR (nkat g protein)
APX (nkat g protein)

3000 ∗
600
1500
450

-1
800
-1

∗
600 ∗
300
∗
400
∗
150
200
0 0
0 200 0 200 0 200 0 200 0 200 0 200
Cd (μM) Cd (μM)

12
Fig. S6 Free proline, soluble phenolics, ascorbate (ASC), reduced- (GSH) and
oxidized- (GSSG) glutathione, and total thiols (T-SH) in the bark, root and leaf tissues
of P. × canescens exposed to 0 or 200 µM CdSO4 for 20 days. Bars indicate means ±
SE (n = 6). Asterisks on the bars for the same tissue indicate significant difference
between the treatments.

3.5
bark root leaf A bark root leaf B 250
3.0
∗ ∗

Soluble phenolics
(μmol g DW)

∗ 200
Free proline

(mg g DW)
2.5 ∗
∗
-1

-1
150
2.0

1.5 100

1.0 50
30
C D 500
∗ ∗
400
20

(nmol g DW)
(μmol g DW)

∗ 300

GSH
-1
ASC
-1

∗
6 ∗ 150
100
3
50
0 0
250 E ∗
F 200

200 ∗
(μmol g DW)
(nmol g DW)

150 ∗ 160
T-SH
-1
GSSG
-1

∗ 90
100

∗ 60
50 ∗
30
0 0
0 200 0 200 0 200 0 200 0 200 0 200

Cd (μM)

13
Fig. S7 Alignments of SPX domain gene 2 (SPX2; Affymetrix ID:
PtpAffx.163910.1.S1_at), NB-ARC domain-containing disease resistance protein
(NDDRP; Affymetrix ID: PtpAffx.225724.1.S1_x_at), glutathione S-transferase TAU
22 (GSTU22; Affymetrix ID: PtpAffx.3457.1.A1_at), Ribosomal protein L1p/L10e
family (PGY1; Affymetrix ID: PtpAffx.1126.3.A1_at ), NAC domain containing
protein 44 (NAC044; Affymetrix ID: PtpAffx.151346.1.S1_at) and Pyridoxal
phosphate (PLP)-dependent transferases superfamily protein (PLP; Affymetrix ID:
PtpAffx.215435.1.S1_at) at the cDNA and amino acid levels among P.× canescens
(Pc), P. trichocarpa (Pt) and A. thaliana (At).
 

 
Alignments at the cDNA level, P. trichocarpa as a reference
Accession number: PtSPX2: XM_002308045; AtSPX2: NM_128223
Identity for SPX2: Pc: 151/154*100 % = 98.7%; At: 111/154*100 % = 72.1%

 
Alignments at the amino acid level, P. trichocarpa as a reference
Identity for SPX2: Pc: 51/51*100 % = 100.0%; At: 44/51*100 % = 86.3%
‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 

14
Alignments at the cDNA level, P. trichocarpa as a reference
Accession number: PtNDDRP: XM_002328003; AtNDDRP: NM_112307
Identity for NDDRP: Pc: 201/205*100 % = 98.0%; At: 107/205*100 % = 52.2%

 
Alignments at the amino acid level, P. trichocarpa as a reference
Identity for NDDRP: Pc: 63/67*100 % = 94.0%; At: 28/67*100 % = 41.8%
‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 

 
Alignments at the cDNA level, P. trichocarpa as a reference
Accession number: PtGSTU22: XM_002317570; AtGSTU22: NM_106482
Identity for GSTU22: Pc: 118/124*100 % = 95.2%; At: 79/124*100 % = 63.7%

 
Alignments at the amino acid level, P. trichocarpa as a reference
Identity for GSTU22: Pc: 38/40*100 % = 95.0%; At: 29/40*100 % = 72.5%
‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 

 
Alignments at the cDNA level, P. trichocarpa as a reference
Accession number: PtPGY1: XM_002328216; AtPGY1: NM_128313
Identity for PGY1: Pc: 161/166*100 % = 97.0%; At: 130/166*100 % = 78.3%
15
  
Alignments at the amino acid level, P. trichocarpa as a reference
Identity for PGY1: Pc: 54/54*100 % = 100.0%; At: 47/54*100 % = 87.0%
‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 

 
Alignments at the cDNA level, P. trichocarpa as a reference
Accession number: PtNAC044: XM_002298514; AtNAC044: NM_111026
Identity for NAC044: Pc: 211/216*100 % = 97.7%; At: 152/216*100 % = 70.4%

 
Alignments at the amino acid level, P. trichocarpa as a reference
Identity for NAC044: Pc: 70/71*100 % = 98.6%; At: 58/71*100 % = 81.7%
‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐‐ 

 
Alignments at the cDNA level, P. trichocarpa as a reference
Accession number: PtPLP: XM_002329230; AtPLP: NM_106685
Identity for PLP: Pc: 138/144*100 % = 95.8%; At: 91/144*100 % = 63.2%

 
16
Alignments at the amino acid level, P. trichocarpa as a reference
Identity for PLP: Pc: 45/47*100 % = 95.7%; At: 34/47*100 % = 72.3%
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
17
Fig. S8 Relative expressions of mRNA levels of seven genes analyzed by arrays and
qRT-PCR, respectively, in the bark of P. × canescens exposed to 0 or 200 µM CdSO4
for 20 days.

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

18