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Research PAPER
RESEARCH paper

N-fertilization
In has different
Posidonia oceanica effects
cadmium on thechanges
induces growth, carbon
in DNA
and nitrogenand
methylation physiology,
chromatinand wood properties of slow- and
patterning
fast-growing Populus species
Maria Greco, Adriana Chiappetta, Leonardo Bruno and Maria Beatrice Bitonti*
Department of Ecology, University
Hong Li1, Mengchun Li 2 of Calabria,
, Jie Luo 2
, Xu CaoLaboratory
2 of Plant
, Long Qu 3 Cyto-physiology,
, Ying Gai 3 Ponte Pietro Bucci,
, Xiangning Jiang 3 I-87036 Arcavacata
, Tongxian Liu 1 di Rende,
, Hua Bai 4
,
Cosenza, Italy 4 4 5 2,5,
Dennis Janz , Andrea Polle , Changhui Peng and Zhi-Bin Luo *
*1  To whom correspondence should be addressed. E-mail: b.bitonti@unical.it
Key Laboratory of Applied Entomology, College of Plant Protection, Northwest Agriculture and Forestry University, Yangling, Shaanxi,
712100, PR China
Received
2  29 May 2011; Revised 8 July 2011; Accepted 18 August 2011
College of Life Sciences, Northwest Agriculture and Forestry University, Yangling, Shaanxi, 712100, PR China
3 
National Engineering Laboratory of Tree Breeding, College of Life Sciences and Biotechnology, Beijing Forestry University, Beijing

Downloaded from http://jxb.oxfordjournals.org/ by guest on October 30, 2012

100083, PR China
Abstract
4 
Büsgen-Institute, Department of Forest Botany and Tree Physiology, Georg-August University, Büsgenweg 2, D-37077 Göttingen,
Germany
In mammals, cadmium is widely considered as a non-genotoxic carcinogen acting through a methylation-dependent
5 
Key Laboratory
epigenetic of Environment
mechanism. Here,and
theEcology
effectsinof
Western China of Ministry
Cd treatment on the of Education,
DNA Collegepatten
methylation of Forestry, Northwest Agriculture
are examined together and
with
Forestry University, Yangling, Shaanxi, 712100, PR China
its effect on chromatin reconfiguration in Posidonia oceanica. DNA methylation level and pattern were analysed in
*  To whom
actively correspondence
growing organs,should
underbe addressed:
short- E-mail:
(6 h) and luozbbill@163.com
long- (2 d or 4 d) term and low (10 mM) and high (50 mM) doses of Cd,
through a Methylation-Sensitive Amplification Polymorphism technique and an immunocytological approach,
Received 23 JulyThe
respectively. 2012; Revised 28 August
expression of one2012; Accepted
member 3 September
of the 2012
CHROMOMETHYLASE (CMT) family, a DNA methyltransferase,
was also assessed by qRT-PCR. Nuclear chromatin ultrastructure was investigated by transmission electron
microscopy. Cd treatment induced a DNA hypermethylation, as well as an up-regulation of CMT, indicating that de
Abstract
novo methylation did indeed occur. Moreover, a high dose of Cd led to a progressive heterochromatinization of
interphase nuclei and apoptotic figures were also observed after long-term treatment. The data demonstrate that Cd
To investigate
perturbs how methylation
the DNA N-fertilization affects
status the growth,
through carbon and nitrogen
the involvement (N) physiology,
of a specific and woodSuch
methyltransferase. properties of pop-
changes are
lars with
linked to contrasting growth characteristics,
nuclear chromatin reconfiguration slow-growing (Populus
likely to establish popularis,
a new balancePp) of and fast-growing (P. alba×P. glan-
expressed/repressed chromatin.
dulosa, Pg)
Overall, the poplar saplings
data show were exposed
an epigenetic basistotodifferent N levels. underlying
the mechanism Above-ground biomass,
Cd toxicity in leaf area, photosynthetic rates
plants.
(A), instantaneous photosynthetic nitrogen use efficiency (PNUEi), chlorophyll and foliar sugar concentrations were
higher
Key in Pg 5-Methylcytosine-antibody,
words: than in Pp. Foliar nitrate cadmium-stress
reductase (NR)condition,
activitieschromatin
and root reconfiguration,
glutamate synthase (GOGAT) activities were
CHROMOMETHYLASE,
higher in Pg thanMethylation-
DNA-methylation, in Pp as were the N Amplification
Sensitive amount and Polymorphism
NUE of new shoots.
(MSAP),Lignin contents
Posidonia and(L.)
oceanica calorific
Delile. values of Pg wood
were less than that of Pp wood. N-fertilization reduced root biomass of Pg more than of Pp, but increased leaf bio-
mass, leaf area, A, and PNUEi of Pg more than of Pp. Among 13 genes involved in the transport of ammonium or nitrate
or in N assimilation, transcripts showed more pronounced changes to N-fertilization in Pg than in Pp. Increases in NR
activities and N contents due to N-fertilization were larger in Pg than in Pp. In both species, N-fertilization resulted in
Introduction
lower calorific values as well as shorter and wider vessel elements/fibres. These results suggest that growth, carbon
In
andthe Mediterranean
N physiology, coastal
and wood ecosystem,
properties the endemic
are more Although not
sensitive to increasing essential for
N availability plant growth,
in fast-growing in terrestrial
poplars than in
seagrass Posidonia oceanica (L.) Delile plays a relevant role plants, Cd is readily absorbed by roots
slow-growing ones, which is probably due to prioritized resource allocation to the leaves and accelerated N physi- and translocated into
by ensuring primary production, water oxygenation
ological processes in fast-growing poplars under higher N levels. and aerial organs while, in acquatic plants, it is directly taken up
provides niches for some animals, besides counteracting by leaves. In plants, Cd absorption induces complex changes
Key words:
coastal Amino
erosion acids,its
through ammonium
widespread transporter,
meadowsbioenergy,
(Ott, 1980;carbohydrates, gene biochemical
at the genetic, expression, nitrate transporter. levels which
and physiological
Piazzi et al., 1999; Alcoverro et al., 2001). There is also ultimately account for its toxicity (Valle and Ulmer, 1972;
considerable evidence that P. oceanica plants are able to Sanitz di Toppi and Gabrielli, 1999; Benavides et al., 2005;
absorb and accumulate metals from sediments (Sanchiz Weber et al., 2006; Liu et al., 2008). The most obvious
Introduction
et al., 1990; Pergent-Martini, 1998; Maserti et al., 2005) thus symptom of Cd toxicity is a reduction in plant growth due to
influencing metal bioavailability in the marine ecosystem. an inhibition of photosynthesis, respiration, and nitrogen
Poplar
For plantations
this reason, inthis
a short rotation
seagrass is coppicing system havetogreat
widely considered be metabolism, as et al.,
is limited (Finzi well as2007). Thus, N-fertilization
a reduction in water and is crucial
mineralto
apotential as a bioenergyspecies
metal bioindicator resource (Luo and
(Maserti Polle,
et al., 2009).
1988; Most uptake
Pergent ensure high biomass etproduction.
(Ouzonidou Previous studies indicate
al., 1997; Perfus-Barbeoch that
et al., 2000;
poplar
et al., plantations grow on
1995; Lafabrie et marginal landsCd
al., 2007). where N availability
is one high N et
of most Shukla levels stimulate
al., 2003; tree growth
Sobkowiak and biomass
and Deckert, 2003).production
widespread heavy metals in both terrestrial and marine At the genetic level, in both animals and plants, Cd
environments. can induce chromosomal aberrations, abnormalities in
© 2012 The Authors.
ª
This
2011is an Open
The Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
Author(s).
by-nc/2.0/uk/)
This is an Openwhich
Accesspermits
articleunrestricted
distributed non-commercial use,
under the terms of thedistribution, and reproduction
Creative Commons in Non-Commercial
Attribution any medium, provided the(http://creativecommons.org/licenses/by-
License original work is properly cited.
nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
6174  | Li et al.
(Luo et al., 2006), mainly because N-fertilization induces higher
photosynthetic rates, higher leaf chlorophyll concentrations,
larger leaves, and greater leaf area (Cooke et al., 2005). High
N supply also enhances above-ground biomass production, but
inhibits root growth, leading to decreases in the root:shoot ratio
(Novaes et al., 2009).
Ammonium ( NH4+) and nitrate ( NO3− ) are the two major
inorganic N sources for plants. Although abiotic (e.g. soil pH,
moisture) and biotic (e.g. mycorrhizal fungi) factors affect inor-
ganic N absorption by plants (Rennenberg et al., 2010; Luo
et al., 2011), the uptake of NH4+ and NO3− by woody plants is
largely determined by their specific transporters (Rennenberg
et al., 2010). The genome of Populus trichocarpa contains 14
putative ammonium transporters (AMTs) (Tuskan et al., 2006;
Couturier et al., 2007). BY contrast, full family members and
their functions of nitrate transporters (NRTs) have not been
investigated in Populus species. As a hint, the Arabidopsis
genome contains NRT1, NRT2, and NRT3 subfamilies and
some members such as AtNRT1;1, AtNRT1;2, AtNRT2;1, and
AtNRT3;1 have been functionally characterized (Tsay et al.,
2007; Kotur et al., 2012). Among the various putative inorganic
N transporters, members of the ammonium transporter family
(AMT1;2, AMT1;6, and AMT2;1) and nitrate transporter family
(NRT1;1, NRT1;2, NRT2;4B, NRT2;4C, NRT3.1A, NRT3.1B,
and NRT3.1C) might play crucial roles in the uptake of NH4+
and NO3− by Populus species (Dluzniewska et al., 2007; Ehlting
et al., 2007; Plett et al., 2010; Rennenberg et al., 2010). A few
studies have indicated that the above-mentioned AMTs are
highly expressed in poplar roots/leaves and that their transcript
levels respond to the internal N status of plants and external N
availability (Selle et al., 2005; Couturier et al., 2007; Ehlting
et al., 2007; Rennenberg et al., 2010). By contrast, no informa-
tion is currently available about the expression patterns of NRTs
in Populus species.
After uptake, NH4+ can be directly assimilated into amino
acids, whereas NO3− needs to be reduced to NH4+ involving
nitrate reductase (NR) before incorporation into amino acids
(Nunes-Nesi et al., 2010). NR is a critical enzyme involved in
the NO3− assimilation pathway and is substrate inducible in pop-
lar (Dluzniewska et al., 2007). After direct uptake or conversion
from NO3−, NH4+ is assimilated to glutamine and glutamate via
a pathway in which the enzyme glutamate synthase (GOGAT)
plays an essential role (Dluzniewska et al., 2007). The products
of the GOGAT pathway are required for biosynthesis of other
nitrogenous compounds (e.g. amino acids, proteins, chlorophylls,
and secondary metabolites) (Nunes-Nesi et al., 2010). Total N
and amino acid concentrations in plants are frequently found to
be elevated in response to higher N supply (Luo et al., 2008;
Rennenberg et al., 2010), but the influence of N-fertilization on
NUE remains controversial (Good et al., 2004; Finzi et al., 2007).
However, limited information is available about how slow- and
fast-growing poplar species respond to different N supply. One
possibility is that slow-growing poplars will accumulate N if
increased N cannot be used to stimulate growth, thus, decreasing
their nitrogen use efficiency (NUE).
With regard to bioenergy resources, not only the quantity but
also the quality of plant biomass is of interest because biomass
gains may be negatively offset by diminished quality (Canam
and Campbell, 2009). This is particularly important for woody
biomass because wood quality in terms of chemical and ana-
tomical properties may substantially affect the end use of this
material. For instance, both the lignin contents and the com-
position of syringyl and guaiacyl monomers can influence the
suitability of wood for bioethanol production (Studer et al.,
2011). N-fertilization can alter the lignin contents and compo-
sition (Pitre et al., 2007b; Luo and Polle, 2009). The caloric
value of wood, which is determined by its chemical composi-
tion (Zhou et al., 2011), is also responsive to N addition (Luo
and Polle, 2009). In addition, the anatomical properties of wood
(e.g. the dimensions of both vessel elements and fibre cells) are
sensitive to N availability (Luo et al., 2005; Pitre et al., 2007a;
Hacke et al., 2010). Few studies have addressed the impact of
N supply on wood properties in slow- and fast-growing poplar
species.
Little is known about the NUE of poplar species with dif-
ferent growth characteristics or how the trees balance growth
and carbon physiology in response to increasing N availability.
Downloaded from http://jxb.oxfordjournals.org/ by guest on October 30, 2012
Populus popularis is a slow-growing clone which is often found
on nutrient-deficient soils, whereas P. alba×P. glandulosa is a
fast-growing species which generally grows on relatively fer-
tile soils (Cao et al., 2012). Both of these poplar species may
be selected as woody bioenergy crops. To gain insights into
the physiological responses of poplars to N, Populus popularis
and P. alba×P. glandulosa were used to address the following
questions: (i) How does N availability in soil affect the growth
and carbon physiology of slow- and fast-growing poplars? (ii)
Do slow- and fast-growing poplars differ in N assimilation and
NUE? (iii) Are there N-induced differences in wood properties
between slow- and fast-growing poplars?
Materials and methods
Plant materials and nitrogen treatment
Cuttings (c. 15 cm in length, 2 cm in diameter, 1-year-old stem) of
Populus popularis (Pp) and P. alba×P. glandulosa (Pg) were obtained
from a tree-breeding programme (Cao et al., 2012). The cuttings
were rooted and planted in pots (10 L) filled with sandy soil, provid-
ing with modified Long-Ashton (LA) nutrient solution (Dluzniewska
et al., 2007). After cultivation for 8 weeks in a greenhouse, uniform
saplings were selected, transferred to a climate chamber (light intensity,
250 µmol m–2 s–1, 16/8 h day/night; day/night temperature, 25/20 °C;
relative humidity, 75%) and grown for 2 weeks. Plants were carefully
irrigated on the morning of every third day with 20 ml LA nutrient solu-
tion (Ehlting et al., 2007) and daily in the evening with 20 ml distilled
water avoiding runoff. Before N treatment, six plants from each species
were randomly harvested to determine diameter, height, and biomass.
For N treatment, 18 plants from each species were divided into three
groups (six plants in each group) and supplied with LA nutrient solution
containing, in addition, 0, 5 or 10 mM NH4NO3, respectively. At the
beginning of the N treatment, the apex of each plant was marked so that
the shoots formed during the N treatment could be distinguished. The N
treatment lasted for 7 weeks before harvest.
Analysis of growth and gas exchange
To analyse the growth characteristics of poplar plants, the height and
basal diameter of the stem of each plant were measured regularly.
 Responses of two contrasted poplar species to N-fertilization  |  6175
A slide caliper was used to determine the basal diameter of each plant c.
3 cm above the soil surface.
Before harvest, gas exchange of three mature leaves (leaf plastochron
index (LPI)=8–10) was determined for each plant. Net photosynthetic
rates (A), stomatal conductance (gs), and transpiration rates (E) were
determined with a portable photosynthesis system (Li-Cor-6400, Li-Cor
Inc, Lincoln, Nebraska, USA) and an attached LED light source (6400–
02) as described by He et al. (2011). The instantaneous photosynthetic
N use efficiency (PNUEi) was calculated as the net CO2 assimilation per
unit of N and time (µmol CO2 mg–1 N s–1) based on A, the specific leaf
area, and the N concentrations of leaves formed during the N treatment
(see below).
Harvesting
For each harvested plant, the shoot segment formed during the N treat-
ment (i.e. the shoot above the mark) and the segment below the mark
were separated into wood, bark, and leaves. The roots of each plant were
also harvested and the length and fresh weight were recorded. Leaves
formed during the N treatment were collected to analyse leaf area and
specific leaf area as described by Cao et al. (2012). Subsequently, the
samples were wrapped with tinfoil and immediately frozen in liquid
N. The frozen root, wood, bark, and leaf samples were ground into fine
powder in liquid N with a mortar and pestle and stored at –80 °C for
further analysis. Frozen powder (c. 50 mg) from each tissue was dried
at 60 °C to determine the fresh-to-dry-mass ratio which was used to
calculate the dry weight of each tissue. Subsamples from leaves and
stem wood formed during the N treatment period were also collected for
microscopic and calorific analysis.
Analysis of photosynthetic pigments, soluble sugars, and sugar
alcohols
The chlorophyll and carotenoid contents of leaves formed during the
N treatment were analysed spectrophotometrically according to the
method of He et al. (2011).
Fine powder (c. 50 mg) of fresh roots and leaves formed dur-
ing the N treatment was extracted with 500 µl of extraction solution
(methanol:chloroform:water, 12:5:3, by vol.). Subsequently, soluble
sugars and sugar alcohols were determined by gas chromatography-
mass spectrometry (GC-MS) as described previously (Luo et al.,
2009a, b).
Analysis of transcript levels of representative genes involved in
ammonium/nitrate transport or N assimilation
The frozen powder of roots or leaves formed during the N treatment
from two plants (c. 500 mg from each plant) in each treatment was
combined for total RNA extraction. Total RNA of the tissue was iso-
lated and purified with a plant RNA extraction kit (R6827, Omega
Bio-Tek, GA, USA). Aliquots of 1  µg total RNA were used for first
strand cDNA synthesis in a total volume of 20 µl, containing 0.5 µg
oligo d(T)18–primer and 200 U RevertAid Moloney murine leukemia
virus reverse transcriptase (K1621, Fermentas, Burlington, CA, USA)
according to the manufacturer’s instruction. The synthesized cDNA was
used for quantitative real time PCR as described by Luo et al. (2009a)
with minor modification. Quantitative PCR was performed using 10 µl
2× SYBR Green Premix Ex Taq II (DRR081A, Takara, Dalian, China),
0.5 µl cDNA, and 0.2 µM primer (see Supplementary Table S1 at JXB
online) in a CFX96 Real Time system (CFX96, Bio-Rad, Hercules, CA,
USA). The 18S rRNA was used as a reference gene (see Supplementary
Table S1 at JXB online). To ensure the specification, PCR products were
sequenced and aligned with homologues in other model plants (see
Supplementary Fig. S1 at JXB online). The efficiencies of all PCR reac-
tions were between 95% and 110% (see Supplementary Table S1 at JXB
online). PCR was performed in triplicate together with a dilution series
of the reference gene.
Analysis of nitrate reductase (NR) and glutamate synthase
(GOGAT) activities
NR and GOGAT activities were determined in roots and leaves formed
during the N treatment based on the methods of Ehlting et al. (2007) and
Lin and Kao (1996), respectively.
Determination of amino acids
Free amino acids were quantified in leaves formed during the N treat-
ment, but amino acids could not be measured in roots due to the shortage
of material. The extraction of free amino acids from leaves was adapted
after the method of Maro et al. (2011). About 50 µl of the extract was ana-
lysed by an Amino Acid Analyzer (121MB, Beckman, California, USA).
Determination of N concentrations, N amounts, and N use
efficiency (NUE)
To quantify N concentrations in different tissues of the two poplar spe-
cies, fine powder (c. 0.5 mg DW) from roots and shoots (wood, bark,
and leaves) below and above the mark for N treatment was weighed
into tin capsules and analysed by an element analyser (Euro EA 3000,
Elemental Analyzer, Milan, Italy) as described by Luo et al. (2011). The
total N amount of each tissue was estimated by multiplying the N con-
Downloaded from http://jxb.oxfordjournals.org/ by guest on October 30, 2012
centration in each tissue by the biomass of that tissue.
Nitrogen use efficiency (NUE) was defined according to Finzi et al.
(2007): NUE=biomass/N uptake
where biomass and N uptake are measured in units of dry matter (g
DW) production or N uptake (equivalent to N amount in plant) from
soil. The NUE of new shoots was calculated based on the dry weight of
new shoots formed during the N treatment and the total N amount of the
new shoots. Similarly, NUEs were estimated in shoots (above-ground
plant part) formed during the whole experimental phase and in plants
(roots and above-ground plant part).
Determination of lignin and calorific values in wood
The Klason lignin content was determined in stem-wood formed dur-
ing the N treatment according to the method of Luo et al. (2008). The
calorific value of wood formed during the N treatment was determined
based on the method of Luo and Polle (2009).
Analysis of wood anatomy
Wood samples (c. 1 cm above the mark) formed during the N treat-
ment were used for anatomical analysis. For scanning electron micros-
copy (SEM), wood samples were prepared according to the method of
Pitre et al. (2007a). The SEM observations were made at 13 kV using
a scanning electron microscope (JSM-6360LV, Japan Electron Optics
Laboratory Co. Ltd, Tokyo, Japan). The characteristics of vessel ele-
ments and fibres in wood formed during the N treatment were analysed
based on the method of Luo et al. (2005) with minor modifications
according to Cao et al. (2012).
Statistical analysis
Statistical tests were performed with Statgraphics (STN, St Louis, MO,
USA). The growth variables of the slow- and fast-growing poplar spe-
cies were compared by one-way ANOVA. To examine the effects of
species and N treatment on experimental variables, all variables were
analysed by two-way ANOVAs. Data were tested for normality prior
to statistical analysis. Differences between means were considered
significant when the P-value of the ANOVA F-test was less than 0.05.
The Ct values obtained after quantitative PCR were normalized and the
fold changes of transcripts were calculated as suggested by Luo et al.
(2009a). For principal component analysis (PCA), data were standard-
ized and subsequently computed by the command prcomp() in R (http://
www.r-project.org/).
6176  | Li et al.

Results and discussion
Prioritized resource allocation to leaves in Pg but
not in Pp leads to differences in growth and carbon
physiology under N-fertilization
At a given N supply, there were inherent growth differences
that resulted in about 2-fold higher biomass in Pg than in Pp
(see Supplementary Fig. S2 at JXB online). Growth differences
were maintained when poplar plants were fertilized addition-
ally with either 5 or 10 mM NH4NO3 (see Supplementary Fig.
S3 and Supplementary Table S2 at JXB online). Growth differ-
ences may be associated with the stimulation of photosynthesis
and carbohydrate metabolism. Thus, both leaf gas exchange and
soluble sugar and sugar alcohol concentrations were measured
(see Supplementary Fig. S4 and Supplementary Table S2 at JXB
online). Principal component analysis (PCA) was performed on
growth, photosynthesis related parameters and carbohydrates
(Fig. 1; see Supplementary Table S3 at JXB online). Leaf area,
leaf dry weight, and PNUEi were the three most important con-
tributors to PC1, whereas trehalose and sucrose in roots and
that of Pp, resulting in increases in A, total carbohydrates, and
sugar alcohols (see Supplementary Fig. S4 at JXB online). These
results indicate that higher A in fast-growing poplars can result
in a higher supply of carbon assimilates for plants to develop
more and larger leaves, leading to higher leaf biomass and leaf
area. Actually, the atmospheric CO2 fixation capacity of a plant
depends on whole plant A and leaf area (Cooke et al., 2005; Zhao
et al., 2011; Zheng et al., 2011). Current data indicate that higher
leaf biomass, A, PNUEi, and larger leaf areas are critical traits
for Pg to be a highly productive bioenergy crop. Previous study
showed a positive correlation between stem wood formation and
whole-plant leaf area (Cooke et al., 2005), but this was not found
in the current study. The time scale of this experiment was lim-
ited. Therefore, the possibility cannot be excluded that the initial
trade-off between leaf and wood biomass will result in increased
wood formation in the long run.
The growth data suggest that slow- and fast-growing poplar
species have different strategies for biomass allocation under
increasing N availability. In Pg, resource allocation was prior-
itized to leaf production. Higher N supply might have caused an

Downloaded from http://jxb.oxfordjournals.org/ by guest on October 30, 2012

mannitol in leaves were the strongest contributors to PC2 (see
Supplementary Table S3 at JXB online). The PCA clearly sepa-
rated the slow-growing Pp from the fast-growing Pg (Fig. 1).
These data indicate that Pg has a stronger responsiveness to
N-fertilization than Pp has.
The growth and photosynthetic characteristics of Pp and Pg in
this experiment were consistent with results of Pp and Pg from
our previous field study (Cao et al., 2012), indicating that the
growth of Pp and Pg is stable across a variety of experimental
conditions. The leaf chlorophyll contents of Pg were higher than
expanded zone of leaf maturation where elevated cell division
and/or cell expansion can occur (Cooke et al., 2005). Such pro-
cesses were apparently more efficient in Pg than in Pp. Although
higher N levels resulted in increased photosynthetic rates in
both poplar species, the decreased foliar sucrose concentrations
suggest that N-fertilization may have resulted in an accelerated
export of sucrose to sink tissues and/or conversion to other car-
bohydrate forms. Consistent with the latter assumption, higher
concentrations of fructose, inositol, galactose, and manitol were
detected in leaves of fertilized Pg (see Supplementary Fig. S4 at

Fig. 1.  Principal component analysis plot of the first two principal components, depicting the relationship between slow-growing
P. popularis (Pp) and fast-growing P. alba×P. glandulosa (Pg) exposed to 0, 5 or 10 mM NH4NO3. The analysis was conducted using
data presented in Supplementary Table S2 and Supplementary Fig. S4 at JXB online for growth- and photosynthesis-related parameters
as well as soluble sugar and sugar alcohol concentrations. Data points are presented for 0 (white), 5 (grey), and 10 (black) mM NH4NO3.
(This figure is available in colour at JXB online.)
 Responses of two contrasted poplar species to N-fertilization  |  6177
JXB online). Taken together, these results indicate that prioritized
resource allocation to leaves in Pg but not in Pp may lead to
growth and carbon physiology differences under N-fertilization.
This is a crucial trait for a highly productive bioenergy crop.
Accelerated physiological processes of N uptake and
assimilation in Pg compared with Pp cause differences
in N physiology under N-fertilization
Nitrogen uptake and assimilation are essential growth-promoting
processes in plants. Based on previous studies in Populus species
(Selle et al., 2005; Couturier et al., 2007; Ehlting et al., 2007; Plett
et al., 2010) and our preliminary experiments, genes encoding
essential members of transporter families for ammonium (AMT1;2,
AMT1;6, and AMT2;1) and for nitrate (NRT1;1, NRT1;2, NRT2;4B,
NRT2;4C, NRT3;1A, NRT3;1B, and NRT3;1C) expressed in both
roots and leaves of Pp and Pg were selected for transcript analysis
by quantitative RT-PCR. In addition, genes encoding nitrate reduc-
tase (NR) and glutamate synthase (Fd-GOGAT, NADH-GOGAT)
were included since these genes play crucial roles in N assimila-
tion in poplar plants (Dluzniewska et al., 2007). Transcript analy-
sis and PCA showed that mRNA levels of the genes examined in
roots and leaves were different between Pp and Pg (Fig. 2; see
Supplementary Table S4 at JXB online). For all the transcript lev-
els analysed, PC1 and PC2 accounted for 37% and 23% of the var-
iation, respectively (see Supplementary Table S4 at JXB online).
Changes in transcript levels of NRT2;4C and NRT3;1A were the
two strongest contributing variables to PC1, whereas AMT1;6 and
NR were the strongest contributors to PC2 (see Supplementary
Table S4 at JXB online). Transcripts of most genes changed in
response to the addition of 5 and/or 10 mM NH4NO3 and, in most
cases, transcript levels of the genes examined in Pg were more
responsive to N addition than in Pp (Fig. 2; see Supplementary
Table S4 at JXB online). These results indicate that fast-growing
Pg takes up more N and/or needs to utilize the available N more
efficiently compared with slow-growing Pp.
Induction of AMT1;2 transcripts in roots and leaves of Pp
and Pg under a higher N supply is in agreement with previ-
ous studies (Selle et al., 2005; Couturier et al., 2007), indicat-
ing that AMT1;2 expression might be ammonium-inducible
and that AMT1;2 functions in ammonium transport in Populus
species. AMT1;6 transcripts gradually decreased in leaves of
P.×canescens during the N starvation period, which was closely
correlated with decreases in the asparagine pools (Couturier
et al., 2007). Consistently, AMT1;6 transcripts were induced in
roots of Pp and in both roots and leaves of Pg under the higher N
supply. This also corresponds well to increases in aspartic acid (a
close derivative of asparagine) pools (Fig. 2; see Supplementary
Fig. S5 at JXB online). AMT2;1 was highly expressed in leaves
of P.×canescens (Couturier et al., 2007), but no information is
available on the response of AMT2;1 transcript to external N
changes in poplar plants. The induction of AMT2;1 transcripts
in the roots of Pp and in both roots and leaves of Pg under the
higher N supply suggests that AMT2;1 may play a significant
role in ammonium transport in roots and leaves of Populus spe-
cies. These results indicate that the three AMTs analysed in this
study are involved in ammonium uptake and mobilization in
Populus species under the higher ammonium supply.
Nitrate uptake and mobilization in plants is mediated by
NRTs (Tsay et al., 2007; Wang et al., 2012). Using ‘nitrate trans-
porter’ as a search term in the genome database of P. trichocarpa
(Populus trichocarpa v2.2, http://www.phytozome.org/results.
php), 273 hits were found, indicating that the poplar genome
might contain a large family of NRTs. However, no informa-
tion is currently available about the functional characteristics
of any NRT in poplar. In Arabidopsis, NRT1, NRT2, and NRT3
contain 53, 7, and 2 subfamily members, respectively, and some
of these members have been functionally characterized (Tsay
et al., 2007; Wang et al., 2012). In Arabidopsis, AtNRT1;1
(At1G12110), AtNRT1;2 (At1G69850), AtNRT2;1 (At1G08090),
and AtNRT2;4 (At5G60770) are the closest homologues, respec-
tively, to NRT1;1, NRT1;2, NRT2;4C, and NRT2;4B of poplar
species in this study (see Supplementary Fig. S1 at JXB online).
In addition, AtNRT3;1 (At5G50200) is the closest homologue
to NRT3;1A, NRT3;1B, and NRT3;1C of our studied poplars
(see Supplementary Fig. S1 at JXB online). Consistent with the
induction of AtNRT1;1 transcript in roots by nitrate (Wang et al.,
2012, and references therein), increases in NRT1;1 mRNAs were
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detected in roots of Pp and Pg after adding 5 or 10 mM NH4NO3
(Fig. 2). In addition to root expression, AtNRT1;1 is expressed in
leaf guard cells, promoting stomatal opening in a nitrate-depend-
ent manner (Guo et al., 2003). NRT1;1 was also expressed in
leaves of Pp and Pg, but the lack of correlation between NRT1;1
expression and gs in both poplar species under different N levels
(Fig. 2; see Supplementary Table S2 at JXB online) indicates that
the function of NRT1;1 in regulating stomatal opening needs to
be further studied in poplar.
In contrast to the non-responsiveness of AtNRT1;2 mRNA
in roots to nitrate addition (Wang et al., 2012, and references
therein), repression of NRT1;2 mRNAs by 5 or 10 mM NH4NO3
supply in roots of Pp and Pg suggests that poplar plants may
modulate NO3− entry into roots partially through transcriptional
regulation of NRT1;2 under higher N availability. AtNRT2;1 is
a high-affinity transporter functioning in the micromole range
of substrate and it was induced by nitrate (Tsay et al., 2007;
Wang et al., 2012). Consistently, over-expression of NRT2;4C
transcripts was also observed in both roots and leaves of Pp
and Pg by 5 or 10 mM NH4NO3 supply. This substrate is in the
millimole range, which is used for low affinity transporters
(Fig. 2). AtNRT2;4 transcript is suppressed by nitrate or ammo-
nium in the micromole range (Wang et al., 2012, and references
therein). Down-regulation of NRT2;4B mRNAs was detected in
roots and leaves of Pp by 5 or 10 mM NH4NO3 and in roots of
Pg by 10 mM NH4NO3 in the low affinity range (Fig. 2). These
results imply that NRT2;1 and NRT2;4 between Arabidopsis
and Populus may have distinct substrate concentration ranges
for functioning or both of them may function as low affinity
transporters in poplars.
AtNRT3;1 (also known as AtNAR2;1) plays a role in nitrate trans-
port by regulating AtNRT2 genes and/or by forming a two-compo-
nent system for transport with AtNRT2s (Plett et al., 2010; Kotur
et al., 2012). Correlation analysis between NRT3;1 expression and
other genes analysed in Pp and Pg under various NH4NO3 levels
indicated significant relationships (RNRT3;1B=0.63, RNRT3;1C=0.64)
between NRT3;1B (NRT3;1C) and NRT2;4B. Interestingly, signifi-
cant correlations (RNRT1;2=0.91, RFd-GOGAT=0.95, RNADH-GOGAT=0.86)
6178  | Li et al.

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Fig. 2.  Fold changes of transcripts involved in ammonium (A),nitrate (B, C), transport or N assimilation (D) in roots and leaves of slow-
growing P. popularis (Pp) and fast-growing P. alba×P. glandulosa (Pg) exposed to 0, 5 or 10 mM NH4NO3 (N). Bars indicate fold changes
±SE (n=3). For each gene, the expression level was set to 1 in roots of Pp exposed to 0 mM NH4NO3 and, subsequently, fold changes of
transcripts were calculated in roots and leaves of both poplar species. The insert in (C) shows induced mRNA levels of NRT3;1B. (This
figure is available in colour at JXB online.)
 Responses of two contrasted poplar species to N-fertilization  |  6179

also existed between the expression of NRT3;1C and NRT1;2 and
between NRT3;1B and Fd-GOGAT (NADH-GOGAT) in both Pp
and Pg. These correlations indicate that NRT3;1B and NRT3;1C
may play a pivotal role in the regulation of nitrate uptake and N
assimilation in poplar plants.
The expression and enzyme activities of NR and GOGAT
are dependent on substrate and on the flux of inorganic N into
organic compounds, which is crucial in N assimilation in pop-
lars (Dluzniewska et al., 2007). Although no species difference
was detected at the transcript levels of NR between Pp and Pg
(Fig. 2), higher N induced greater NR activities in Pg roots
than in Pp roots (Fig. 3). At the transcript level, the greater
responsiveness of NADH-GOGAT to changes in N (Fig. 2) indi-
cates that this isoform may play a more important role than
Fd-GOGAT in both Pp and Pg. Higher activities of GOGAT in
Pg roots than in Pp roots and suppression of GOGAT activities
in Pp leaves but not in Pg leaves by higher N levels were found
(Fig. 3). Lack of correlations between transcript abundance of
NR/GOGAT and enzyme activity of NR/GOGAT implies that
multiple levels of regulation after transcription occur to modu-
late glutamate synthesis in Pp and Pg under applied N levels.
These results suggest that Pg may require higher NR and/or
GOGAT activities than Pp does to efficiently utilize absorbed
nitrate and/or ammonium.
Both ammonium and nitrate have to be converted into amino
acids after their uptake in plants. Thus, amino acid concentra-
tions may be affected by ammonium and/or nitrate supply. In
most cases, higher N supply enhanced the total and individual
amino acid concentrations in both species and the increases
were greater in Pg than in Pp (see Supplementary Fig. S5 at JXB
online). Higher amino acid levels in Pg leaves than in Pp leaves
indicate that more N in Pg is channelled to the production of pre-
cursors of protein biosynthesis to provide basic compounds for
rapid biomass production (Nunes-Nesi et al., 2010).

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Fig. 3.  Nitrate reductase (NR, µM NO32− h–1 mg–1 protein) and glutamate synthase (GOGAT, nkat g–1 protein) activities in fine roots
and leaves of slow-growing P. popularis (Pp) and fast-growing P. alba×P. glandulosa (Pg) exposed to 0, 5 or 10 mM NH4NO3 (N). Bars
indicate means ±SE (n=6). Different letters on the bars indicate significant difference. P-values of the ANOVAs of species, N treatment
and their interaction are indicated: *P < 0.05; **P < 0.01; ns, not significant.
6180  | Li et al.
Higher total N in roots, greater increases in the
total N of bark and leaves, and higher NUE of Pg in
comparison with Pp demonstrate differences in N
physiology
To investigate how plant N status was affected by differences
in growth physiology and responsiveness to N-fertilization,
N concentrations and amounts were quantified in Pp and Pg
(Fig. 4; see Supplementary Table S5 at JXB online). In the 0 mM
NH4NO3 treatment, N concentration was significantly higher
in Pg roots than in Pp roots, but similar results were not found
in wood, bark, and leaves (Fig. 4A–D). The addition of 5 mM
NH4NO3 increased root and bark N concentrations in both spe-
cies compared with the controls (Fig. 4A) and greater increases
in bark N concentrations were found in Pg than in Pp (Fig. 4C).
Supply of 10 mM NH4NO3 enhanced N concentrations in the
roots of both species and in the bark and leaves of Pg compared
with the controls (Fig. 4A, 4C, 4D). There were huge differ-
ences in above- and below-ground N partitioning between Pg
and Pp (Fig. 4E–H; see Supplementary Table S5 at JXB online).
Specifically, Pp showed strong increases in the amounts of N
allocated to wood, bark, and leaves in response to the addition
of 5 mM NH4NO3 and further increments in response to 10 mM
NH4NO3 addition (Fig. 4F–H). By contrast, the amounts of N
allocated to wood and bark were not so pronounced in ferti-
lized Pg and the N amount in Pg roots even showed a strong
decrease in response to N fertilization (Fig. 4E–H). The amount
of N in leaves was much higher in Pg than in Pp (Fig. 4E–H).
The greater allocation of N to leaves of fast-growing Pg under
higher N levels again demonstrates prioritized resource alloca-
tion to leaves of Pg, rendering Pg be more effective on N utiliza-
tion since more N is allocated to the photosynthetic tissue. This
contention is supported by higher PNUEi in Pg than in Pp under
elevated N levels (see Supplementary Table S2 at JXB online).
In unfertilized poplars, NUE was markedly higher in new
shoots of Pg than of Pp (Fig. 5A). N-fertilization decreased NUE
in new shoots of both poplar species, but under 5 mM NH4NO3
condition, NUE was higher in new shoots of Pg than of Pp
(Fig. 5A). Shoot NUE was similar in both poplar species under
the control condition (Fig. 5B). The addition of N reduced shoot
NUEs of both species, but under the addition of 5 mM NH4NO3,
shoot NUE of Pg was higher than of Pp (Fig. 5B). NUE in plants
was similar in both species under 0 mM NH4NO3 conditions
(Fig. 5C). Higher N supply decreased NUE in plants of Pp by
18–25% and of Pg by 6–15%, respectively, compared with the
controls (Fig. 5C).
Although NUE is an important trait that is often evaluated for
agricultural crops (Good et al., 2004; Ju et al., 2009), little infor-
mation is available about the NUE of woody bioenergy crops
such as poplars (Garnett et al., 2009). Over-application of N fer-
tilizers to agricultural land leads to serious environmental threats
including the production of the greenhouse gas N2O and nitrate
leaching to ground water (Good et al., 2004). Therefore, it is
extremely important to select poplars with high NUEs. The NUE
of both poplar species in this study decreased as the supply of
N increased, suggesting that N-fertilization application to poplar
stands should be carefully managed. Under 5 mM NH4NO3 addi-
tion, the higher NUEs in Pg than in Pp imply that fast-growing
poplars are more N economic than slow-growing poplars in
terms of biomass production.
N-fertilization induced differences in wood properties
of Pp and Pg
The chemical and anatomical properties are important aspects of
wood utilization. Therefore, these properties were characterized
(Figs 6, 7; see Supplementary Fig. S6 at JXB online). The lignin
content of Pg was lower than that of Pp (Fig. 6). Consistently,
previous studies suggest that lignin concentrations are lower in
fast-growing species than in slow-growing species (Novaes et al.,
2009, 2010). Lignin biosynthesis requires a large amount of car-
bon which becomes structurally fixed and unavailable for other
processes (Luo et al., 2006). Our data indicate that lower lignin
concentrations in Pg are likely to be due to more carbon being
channelled to active metabolites. N-fertilization tended to reduce
lignin contents in wood of both species (Fig. 6), which is consistent
with previous studies (Novaes et al., 2009; Pitre et al., 2007a, b).
Lower lignin contents in the wood of N-fertilized poplars are
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advantageous for bioethanol production (Studer et al., 2011) and
are probably associated with the formation of a G-layer that is
almost devoid of lignin (Pitre et al., 2007b). The calorific value
was c. 8% lower in Pg wood than in Pp wood (Fig. 6). The 5 mM
NH4NO3 addition resulted in a reduction in calorific values by
0.9 kJ g–1 DW in Pp wood and by 1.3 kJ g–1 DW in Pg wood,
respectively, compared with the controls (Fig. 6). Furthermore,
the addition of 10 mM NH4NO3 led to reduces in calorific values
by 1.6 kJ g–1 DW in Pp wood and by 1.9 kJ g–1 DW in Pg wood,
respectively, compared with the controls (Fig. 6). The calorific
value of wood is positively correlated with lignin contents in
wood cell walls (Amthor, 2003). Thus, the lower calorific val-
ues may be ascribed to reduction in lignin in Pg wood compared
with Pp wood and in N-fertilized poplars compared with unfer-
tilized ones. N-fertilization increased biomass production but
had a negative effect on the heating values of Pg and Pp wood.
Using biomass and calorific values of wood formed during the N
treatment, wood heating values were calculated as 6.7, 15.3, and
14.4 kJ for Pp and 11.5, 14.8, and 13.8 kJ for Pg in the presence
of 0, 5, and 10 mM NH4NO3, respectively. These data reveal a
substantial trade-off with respect to wood energetic use after fer-
tilization and indicate that 5 mM NH4NO3 addition may result in
optimal energy gain in both poplar species.
Transverse sections of wood in both poplar species were
viewed with a scanning electron microscope (Fig. 7). These pho-
tographs suggest that wall thickness of fibre cells was greater
in Pp wood than in Pg wood under N-fertilization conditions,
although no differences in wall thickness of fibre cells were
observed under the control conditions (Fig. 7). The cell walls of
Pp wood were thicker in the N-fertilized treatment than in the
control, indicating that higher N supply stimulated biosynthesis
of fibre cell walls in Pp wood (Fig. 7A–C). This increase resulted
from the formation of a G-layer in the fibre lumina of Pp wood
under N addition (Fig. 7B, 7C). A stimulation of G-layer forma-
tion under higher N supply has been observed in other poplar
species and is associated with N-induced changes in secondary
wall chemical composition and gene expression (Pitre et al.,
2010). The dimensions of vessel elements and fibre cells were
 Responses of two contrasted poplar species to N-fertilization  |  6181

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Fig. 4.  N concentrations and N amounts in fine roots (A, E), wood (B, F), bark (C, G), and leaves (D, H) of slow-growing P. popularis (Pp)
and fast-growing P. alba×P. glandulosa (Pg) exposed to 0, 5 or 10 mM NH4NO3 (N). Bars indicate means ±SE (n=6). Different letters on
the bars indicate significant difference. P-values of the ANOVAs of species, N treatment and their interaction are indicated: *P < 05; **:
P < 0.01; ***P < 0.001; ****P < 0.0001; ns, not significant.
6182  | Li et al.

characterized in both poplar species (see Supplementary Fig. S6

at JXB online). Under control conditions, vessel elements were
wider and longer in Pg wood than in Pp wood. Higher N levels
decreased the length but increased the width of vessel elements
and fibres of both species. Similar changes in the dimensions
of vessel and fibre cells due to N-fertilization were reported in
P.×euramericana (Luo et al., 2005) and P. trichocarpa×deltoides
(Pitre et al., 2007a). One explanation is that the supply of N may
induce changes in cell elongation and expansion (Cooke et al.,
2003; Pitre et al., 2010; Plavcova et al., 2012).
In conclusion, above-ground biomass, leaf area, A, and PNUEi
were higher in fast-growing Pg than in slow-growing Pp. Leaf
gluctose, fructose, inositol, and galactose concentrations were also
higher in Pg than in Pp. In both species, N-fertilization decreased
root length and root biomass and increased leaf biomass, leaf area,
A, and PNUEi and these changes were greater for Pg than for Pp.
Among 13 genes involved in ammonium or nitrate transport or N
assimilation, transcripts of most genes displayed greater respon-
siveness to N addition in Pg than in Pp. Leaf NR activity and root
GOGAT activity were higher in Pg than in Pp as were N amounts

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and NUEs in new shoots and in shoots. N-fertilization increased
NR activities, N concentrations, and N amounts in both species,
but the increases were larger in Pg than in Pp. Lignin contents
and calorific values were lower in Pg wood than in Pp wood, but
the vessel elements were longer and wider in Pg wood than in
Pp wood. N-fertilization not only reduced the calorific values of
wood in both species, but also shortened and widened vessel ele-
ments and fibres. The supply of 5 mM NH4NO3 may result in opti-
mal energy gain in both poplar species. These results suggest that
growth, carbon, and N physiology, and wood chemical properties
are more sensitive to increasing N availability in fast-growing
poplars than in slow-growing ones. This is probably due to prior-
itized resource allocation to leaves and accelerated N physiologi-
cal processes in the fast-growing species under higher N levels.

Supplementary data
Supplementary data can be found at JXB online.
Supplementary Table S1. Primers used for qRT-PCR.
Supplementary Table S2. Growth and photosynthetic
characteristics.
Supplementary Table S3. PCA of growth, photosynthesis and
carbohydrates.
Supplementary Table S4. PCA of transcript changes of
representative genes.
Fig. 5.  N use efficiency (NUE, g DW g–1 N) in new shoots (A)
Supplementary Table S5. N concentrations and amounts.
formed during the N treatment, in shoots (B) formed during the
Supplementary Fig. S1. Alignments of examined genes at the
whole experimental phase and in plants (C) of slow-growing
cDNA and amino acid levels.
P. popularis (Pp) and fast-growing P. alba×P. glandulosa (Pg)
Supplementary Fig. S2. Growth characteristics of both poplar
exposed to 0, 5 or 10 mM NH4NO3 (N). Bars indicate means
species before the N treatment.
±SE (n=6). Different letters on the bars indicate significant
Supplementary Fig. S3. Root to shoot biomass ratios.
difference. P-values of the ANOVAs of species, N treatment and
Supplementary Fig. S4. Soluble sugars and sugar alcohols.
their interaction are indicated: *P < 0.05; **P < 0.01; ***P < 0.001;
Supplementary Fig. S5. Amino acids.
****P < 0.0001; ns, not significant.
Supplementary Fig. S6. Characteristics of vessel elements and
fibres.
 Responses of two contrasted poplar species to N-fertilization  |  6183

Fig. 6.  Lignin contents and calorific values in stem wood of slow-growing P. popularis (Pp) and fast-growing P. alba × P. glandulosa
(Pg) exposed to 0, 5 or 10 mM NH4NO3 (N). Bars indicate means ±SE (n=6). Different letters on the bars indicate significant difference.
P-values of the ANOVAs of species, N treatment and their interaction are indicated: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns,

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not significant.

Fig. 7.  Scanning electronic microscope photographs of secondary xylem from stem wood of slow-growing P. popularis (Pp) and fast-
growing P. alba×P. glandulosa (Pg) exposed to 0, 5 or 10 mM NH4NO3 (N). (A, B, C) Pp; (D, E, F) Pg. (A, D) 0 mM; (B, E) 5 mM; (C, F)
10 mM. Representative sections are presented. The magnification is indicated by the scale bars on the panels.

Acknowledgements of China (Grant No. 20090204110027), and the Fundamental

This work was financially supported by the National Key
Basic Research Program of China (973 Program, Grant No.
2012CB416902), the National Natural Science Foundation of
China (Grant No. 31070539, 31100481), the Program for New
Century Excellent Talents in University from the Ministry of
Education of China (Grant No. NCET-08-0468), the Fok Ying
Tung Education Foundation (Grant No. 121026), the Specialized
Research Fund for the Doctoral Program of Higher Education
Research Funds for the Central Universities of China (Grant No.
QN2009063). The work in the laboratory of Professor Xiangning
Jiang was supported by the 12th 5-Year ‘863’ Project (Grant
No. 2011AA100203) and work in the laboratory of Professor
Andrea Polle by the European Commission within the Seventh
Framework Program for Project EnergyPoplar (Grant No. FP7-
211917). Dr William J Gale is sincerely thanked for the English
correction.
6184  | Li et al.
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