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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
(Luo et al., 2006), mainly because N-fertilization induces higher gains may be negatively offset by diminished quality (Canam
photosynthetic rates, higher leaf chlorophyll concentrations, and Campbell, 2009). This is particularly important for woody
larger leaves, and greater leaf area (Cooke et al., 2005). High biomass because wood quality in terms of chemical and ana-
N supply also enhances above-ground biomass production, but tomical properties may substantially affect the end use of this
inhibits root growth, leading to decreases in the root:shoot ratio material. For instance, both the lignin contents and the com-
(Novaes et al., 2009). position of syringyl and guaiacyl monomers can influence the
Ammonium ( NH4+) and nitrate ( NO3− ) are the two major suitability of wood for bioethanol production (Studer et al.,
inorganic N sources for plants. Although abiotic (e.g. soil pH, 2011). N-fertilization can alter the lignin contents and compo-
moisture) and biotic (e.g. mycorrhizal fungi) factors affect inor- sition (Pitre et al., 2007b; Luo and Polle, 2009). The caloric
ganic N absorption by plants (Rennenberg et al., 2010; Luo value of wood, which is determined by its chemical composi-
et al., 2011), the uptake of NH4+ and NO3− by woody plants is tion (Zhou et al., 2011), is also responsive to N addition (Luo
largely determined by their specific transporters (Rennenberg and Polle, 2009). In addition, the anatomical properties of wood
et al., 2010). The genome of Populus trichocarpa contains 14 (e.g. the dimensions of both vessel elements and fibre cells) are
putative ammonium transporters (AMTs) (Tuskan et al., 2006; sensitive to N availability (Luo et al., 2005; Pitre et al., 2007a;
Couturier et al., 2007). BY contrast, full family members and Hacke et al., 2010). Few studies have addressed the impact of
their functions of nitrate transporters (NRTs) have not been N supply on wood properties in slow- and fast-growing poplar
investigated in Populus species. As a hint, the Arabidopsis species.
genome contains NRT1, NRT2, and NRT3 subfamilies and Little is known about the NUE of poplar species with dif-
some members such as AtNRT1;1, AtNRT1;2, AtNRT2;1, and ferent growth characteristics or how the trees balance growth
AtNRT3;1 have been functionally characterized (Tsay et al., and carbon physiology in response to increasing N availability.
A slide caliper was used to determine the basal diameter of each plant c. Analysis of nitrate reductase (NR) and glutamate synthase
3 cm above the soil surface. (GOGAT) activities
Before harvest, gas exchange of three mature leaves (leaf plastochron
NR and GOGAT activities were determined in roots and leaves formed
index (LPI)=8–10) was determined for each plant. Net photosynthetic
during the N treatment based on the methods of Ehlting et al. (2007) and
rates (A), stomatal conductance (gs), and transpiration rates (E) were
Lin and Kao (1996), respectively.
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 Determination of amino acids
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 Free amino acids were quantified in leaves formed during the N treat-
area, and the N concentrations of leaves formed during the N treatment ment, but amino acids could not be measured in roots due to the shortage
(see below). 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).
Harvesting
For each harvested plant, the shoot segment formed during the N treat- Determination of N concentrations, N amounts, and N use
ment (i.e. the shoot above the mark) and the segment below the mark efficiency (NUE)
were separated into wood, bark, and leaves. The roots of each plant were
also harvested and the length and fresh weight were recorded. Leaves To quantify N concentrations in different tissues of the two poplar spe-
formed during the N treatment were collected to analyse leaf area and cies, fine powder (c. 0.5 mg DW) from roots and shoots (wood, bark,
specific leaf area as described by Cao et al. (2012). Subsequently, the and leaves) below and above the mark for N treatment was weighed
samples were wrapped with tinfoil and immediately frozen in liquid into tin capsules and analysed by an element analyser (Euro EA 3000,
N. The frozen root, wood, bark, and leaf samples were ground into fine 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-
Results and discussion that of Pp, resulting in increases in A, total carbohydrates, and
sugar alcohols (see Supplementary Fig. S4 at JXB online). These
Prioritized resource allocation to leaves in Pg but results indicate that higher A in fast-growing poplars can result
not in Pp leads to differences in growth and carbon in a higher supply of carbon assimilates for plants to develop
physiology under N-fertilization more and larger leaves, leading to higher leaf biomass and leaf
At a given N supply, there were inherent growth differences area. Actually, the atmospheric CO2 fixation capacity of a plant
that resulted in about 2-fold higher biomass in Pg than in Pp depends on whole plant A and leaf area (Cooke et al., 2005; Zhao
(see Supplementary Fig. S2 at JXB online). Growth differences et al., 2011; Zheng et al., 2011). Current data indicate that higher
were maintained when poplar plants were fertilized addition- leaf biomass, A, PNUEi, and larger leaf areas are critical traits
ally with either 5 or 10 mM NH4NO3 (see Supplementary Fig. for Pg to be a highly productive bioenergy crop. Previous study
S3 and Supplementary Table S2 at JXB online). Growth differ- showed a positive correlation between stem wood formation and
ences may be associated with the stimulation of photosynthesis whole-plant leaf area (Cooke et al., 2005), but this was not found
and carbohydrate metabolism. Thus, both leaf gas exchange and in the current study. The time scale of this experiment was lim-
soluble sugar and sugar alcohol concentrations were measured ited. Therefore, the possibility cannot be excluded that the initial
(see Supplementary Fig. S4 and Supplementary Table S2 at JXB trade-off between leaf and wood biomass will result in increased
online). Principal component analysis (PCA) was performed on wood formation in the long run.
growth, photosynthesis related parameters and carbohydrates The growth data suggest that slow- and fast-growing poplar
(Fig. 1; see Supplementary Table S3 at JXB online). Leaf area, species have different strategies for biomass allocation under
leaf dry weight, and PNUEi were the three most important con- increasing N availability. In Pg, resource allocation was prior-
tributors to PC1, whereas trehalose and sucrose in roots and itized to leaf production. Higher N supply might have caused an
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 Nitrate uptake and mobilization in plants is mediated by
resource allocation to leaves in Pg but not in Pp may lead to NRTs (Tsay et al., 2007; Wang et al., 2012). Using ‘nitrate trans-
growth and carbon physiology differences under N-fertilization. porter’ as a search term in the genome database of P. trichocarpa
This is a crucial trait for a highly productive bioenergy crop. (Populus trichocarpa v2.2, http://www.phytozome.org/results.
php), 273 hits were found, indicating that the poplar genome
Accelerated physiological processes of N uptake and might contain a large family of NRTs. However, no informa-
assimilation in Pg compared with Pp cause differences tion is currently available about the functional characteristics
in N physiology under N-fertilization of any NRT in poplar. In Arabidopsis, NRT1, NRT2, and NRT3
contain 53, 7, and 2 subfamily members, respectively, and some
Nitrogen uptake and assimilation are essential growth-promoting of these members have been functionally characterized (Tsay
processes in plants. Based on previous studies in Populus species et al., 2007; Wang et al., 2012). In Arabidopsis, AtNRT1;1
(Selle et al., 2005; Couturier et al., 2007; Ehlting et al., 2007; Plett (At1G12110), AtNRT1;2 (At1G69850), AtNRT2;1 (At1G08090),
et al., 2010) and our preliminary experiments, genes encoding and AtNRT2;4 (At5G60770) are the closest homologues, respec-
essential members of transporter families for ammonium (AMT1;2, tively, to NRT1;1, NRT1;2, NRT2;4C, and NRT2;4B of poplar
AMT1;6, and AMT2;1) and for nitrate (NRT1;1, NRT1;2, NRT2;4B, species in this study (see Supplementary Fig. S1 at JXB online).
NRT2;4C, NRT3;1A, NRT3;1B, and NRT3;1C) expressed in both In addition, AtNRT3;1 (At5G50200) is the closest homologue
roots and leaves of Pp and Pg were selected for transcript analysis to NRT3;1A, NRT3;1B, and NRT3;1C of our studied poplars
by quantitative RT-PCR. In addition, genes encoding nitrate reduc- (see Supplementary Fig. S1 at JXB online). Consistent with the
tase (NR) and glutamate synthase (Fd-GOGAT, NADH-GOGAT) induction of AtNRT1;1 transcript in roots by nitrate (Wang et al.,
were included since these genes play crucial roles in N assimila- 2012, and references therein), increases in NRT1;1 mRNAs were
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 (Fig. 3). Lack of correlations between transcript abundance of
between NRT3;1B and Fd-GOGAT (NADH-GOGAT) in both Pp NR/GOGAT and enzyme activity of NR/GOGAT implies that
and Pg. These correlations indicate that NRT3;1B and NRT3;1C multiple levels of regulation after transcription occur to modu-
may play a pivotal role in the regulation of nitrate uptake and N late glutamate synthesis in Pp and Pg under applied N levels.
assimilation in poplar plants. These results suggest that Pg may require higher NR and/or
The expression and enzyme activities of NR and GOGAT GOGAT activities than Pp does to efficiently utilize absorbed
are dependent on substrate and on the flux of inorganic N into nitrate and/or ammonium.
organic compounds, which is crucial in N assimilation in pop- Both ammonium and nitrate have to be converted into amino
lars (Dluzniewska et al., 2007). Although no species difference acids after their uptake in plants. Thus, amino acid concentra-
was detected at the transcript levels of NR between Pp and Pg tions may be affected by ammonium and/or nitrate supply. In
(Fig. 2), higher N induced greater NR activities in Pg roots most cases, higher N supply enhanced the total and individual
than in Pp roots (Fig. 3). At the transcript level, the greater amino acid concentrations in both species and the increases
responsiveness of NADH-GOGAT to changes in N (Fig. 2) indi- were greater in Pg than in Pp (see Supplementary Fig. S5 at JXB
cates that this isoform may play a more important role than online). Higher amino acid levels in Pg leaves than in Pp leaves
Fd-GOGAT in both Pp and Pg. Higher activities of GOGAT in indicate that more N in Pg is channelled to the production of pre-
Pg roots than in Pp roots and suppression of GOGAT activities cursors of protein biosynthesis to provide basic compounds for
in Pp leaves but not in Pg leaves by higher N levels were found rapid biomass production (Nunes-Nesi et al., 2010).
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 poplars are more N economic than slow-growing poplars in
total N of bark and leaves, and higher NUE of Pg in terms of biomass production.
comparison with Pp demonstrate differences in N
physiology N-fertilization induced differences in wood properties
To investigate how plant N status was affected by differences of Pp and Pg
in growth physiology and responsiveness to N-fertilization, The chemical and anatomical properties are important aspects of
N concentrations and amounts were quantified in Pp and Pg wood utilization. Therefore, these properties were characterized
(Fig. 4; see Supplementary Table S5 at JXB online). In the 0 mM (Figs 6, 7; see Supplementary Fig. S6 at JXB online). The lignin
NH4NO3 treatment, N concentration was significantly higher content of Pg was lower than that of Pp (Fig. 6). Consistently,
in Pg roots than in Pp roots, but similar results were not found previous studies suggest that lignin concentrations are lower in
in wood, bark, and leaves (Fig. 4A–D). The addition of 5 mM fast-growing species than in slow-growing species (Novaes et al.,
NH4NO3 increased root and bark N concentrations in both spe- 2009, 2010). Lignin biosynthesis requires a large amount of car-
cies compared with the controls (Fig. 4A) and greater increases bon which becomes structurally fixed and unavailable for other
in bark N concentrations were found in Pg than in Pp (Fig. 4C). processes (Luo et al., 2006). Our data indicate that lower lignin
Supply of 10 mM NH4NO3 enhanced N concentrations in the concentrations in Pg are likely to be due to more carbon being
roots of both species and in the bark and leaves of Pg compared channelled to active metabolites. N-fertilization tended to reduce
with the controls (Fig. 4A, 4C, 4D). There were huge differ- lignin contents in wood of both species (Fig. 6), which is consistent
ences in above- and below-ground N partitioning between Pg with previous studies (Novaes et al., 2009; Pitre et al., 2007a, b).
and Pp (Fig. 4E–H; see Supplementary Table S5 at JXB online). Lower lignin contents in the wood of N-fertilized poplars are
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.
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,
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.
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