Environmental Pollution 170 (2012) 124e130

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High nitrogen and elevated [CO2] effects on the growth, defense and
photosynthetic performance of two eucalypt species
Eka Novriyanti a, Makoto Watanabe b, Mitsutoshi Kitao c, Hajime Utsugi c, Akira Uemura c,
Takayoshi Koike b, *
a
Research Institute of Fiber Technology for Plantation Forest, FORDA, Ministry of Forestry, Kotak Pos 4/BKN, Bangkinang 28401, Riau, Indonesia
b
Silviculture & Forest Ecological Studies, Faculty of Agriculture, Hokkaido Univ., North 9, West 9, Kita-Ku, Sapporo 060-8589, Hokkaido, Japan
c
Forestry and Forest Product Research Institute, Sapporo 062-8516, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Atmospheric nitrogen deposition and [CO2] are increasing and represent environmental problems.
Received 10 January 2012 Planting fast-growing species is prospering to moderate these environmental impacts by fixing CO2.
Received in revised form Therefore, we examined the responses of growth, photosynthesis, and defense chemical in leaves of
3 June 2012
Eucalyptus urophylla (U) and the hybrid of E. deglupta
E. camadulensis (H) to different CO2 and nitrogen
Accepted 5 June 2012
levels. High nitrogen load significantly increased plant growth, leaf N, net photosynthetic rate (Agrowth),
and photosynthetic water use efficiency (WUE). High CO2 significantly increased Agrowth, photosynthetic
Keywords:
nitrogen use efficiency (PNUE) and WUE. Secondary metabolite (SM, i.e. total phenolics and condensed
Eucalypts
Elevated [CO2]
tannin) was specifically altered; as SM of U increased by high N load but not by elevated [CO2], and vice
Nitrogen loading versa for SM of H.
Defense chemical Ó 2012 Elsevier Ltd. All rights reserved.
Resource allocation

1. Introduction eucalypts and acacias (e.g. Alves et al., 2002; Armstrong, 1998;
Laclau et al., 2008; Novriyanti et al., 2012) to be able to moderate
Indonesia’s environment is in a severe state of degradation. environment damages via afforestation and reforestation projects.
Deforestation and industrialization that contribute to the degra- Elevated [CO2] usually stimulates plant growth (e.g. Ghannoum
dation have taken place at an alarming rate. Deforestation in et al., 2010). Since N is one of the main limiting resources for plant
Indonesia is noted as the world’s third largest emitter of green- growth in nature, however, and most leaf N is allocated to photo-
house gases (GHGs) (PEACE, 2007). Therefore, afforestation and synthetic organs (e.g. Feng et al., 2009; Schulze et al., 2005), the
reforestation have been initiated by government and environ- extent of plant development would be fostered by elevated [CO2] in
mentalists to reduce the GHGs emission and restore the environ- the presence of high supply of nutrient (i.e. nitrogen) is then
mental conditions. questioned.
Moreover, rapid industrialization has contributed to pollutions The availability of N and C influences plant leaf chemistry, which
and environment damages, including atmospheric nitrogen depo- in turn would define plant defense status (e.g. Bryant et al., 1983;
sition (Gillett et al., 2000). In fact, the rapid increasing of nitrogen Koike et al., 2003) as well as photosynthetic capacity. Increasing the
(N) deposition due to industrial development and the use of N availability of these resources may induce changes not only in plant
fertilizer not only happens in Indonesia but is taking place in many growth traits but also in plant defense strategies (e.g. Gleadow
parts of Asia (Galloway et al., 2004; Ogawa et al., 2006). Further- et al., 1998). Several studies have reported that elevated [CO2] and
more, the world atmospheric CO2 concentration ([CO2]) continuous N deposition may change the physical and chemical defense traits
to increase (e.g. Grubb, 2003; IPCC, 2007). of leaves (e.g. Gleadow et al., 1998; Koike et al., 2006).
Under these environmental conditions, we expect the high CO2 The certain behavior of plants with specified nutrient and
fixation and storage capacity of fast growing species, such as resources availability can be explained by plant defense theory. The
Growth Differentiation Balance hypothesis (GDB) predicts that
* Corresponding author.
trade-off exist between growth processes and differentiation
E-mail addresses: kee_november09@[Link], keenove@[Link] processes (e.g. the production of SM) (Herms and Mattson, 1992).
(E. Novriyanti), tkoike@[Link] (T. Koike). The trade-off has ecological consequences that affect the resources

0269-7491/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
[Link]
 E. Novriyanti et al. / Environmental Pollution 170 (2012) 124e130 125

partitioning and allocation, thus not only soil nutrient condition a photosynthetic photon flux of 1500 mmol m2 s1. The leaf temperature and vapor
(Bryant et al., 1983), but any environmental factor that retards pressure deficit (VPD) were maintained at 25  C  1  C and 1.2 kPa, respectively. We
measured the net photosynthetic rate (Agrowth) (Farquhar et al., 1980; Long and
growth more than photosynthesis can increase the availability of Bernacchi, 2003), stomatal conductance (gs), and transpiration rate (E) at growth
resource pool for allocation to SM. Therefore, the relationship [CO2] (i.e. 380 and 760 mmol mol1 for ambient and elevated treatments, respec-
between growth and SM production is nonlinear and has a peak in tively). The water use efficiency (WUE, mmol mol1) was calculated as Agrowth
defense chemical production (Herms and Mattson, 1992). divided by E. All gas exchange parameters were expressed on the basis of the pro-
jected (one-sided) leaf area covered by the chamber area.
Altered growth and photosynthesis under higher [CO2] is well
documented (e.g. Schulze et al., 2005). However, the magnitude of
2.3. Leaf traits and chemical measurement
plant response to elevated [CO2] among species and conditions
varies with soil nutrient status (Ainsworth and Long, 2005; Zhao Subsequent to gas exchange measurements, leaves were excised from the plants
et al., 2011). for measurement of the leaf mass per area (LMA, g m2) by dividing the leaf mass
The growth and defense traits of eucalypts have been studied in (oven-dried at 70  C for one week) with the leaf area. Leaf N and C content was
determined by using combustion method with NC analyzer (NC-900, Sumica-
relation to their growth condition. Low soil nutrient availability and Shimadzu, Kyoto, Japan). Photosynthetic N use efficiency (PNUE, mmol g1 s1)
elevated CO2 increase C/N ratio that leads to higher level of defense was calculated by dividing Agrowth by Narea. Total phenolics and condensed tannin
chemicals, even though carbon allocation may differ between were measured following the Folin-Ciocalteu method that modified by Julkunen-
growth habits because of different investment in storage and Tiitto (1985) and Matsuki et al. (2004). Lignin content in the leaf was measured
followed Iiyama and Wallis (1990). Chlorophyll content was extracted with dimethyl
structural components (Lawler et al., 1997).
sulfoxide (DMSO) and the optic density of the extracts was measured with spec-
In this present study, Eucalyptus urophylla and hybrid trophotometer (Shinano et al., 1996).
E. deglupta
E. camaldulensis were examined. Their photosynthetic
rate was expected to increase under elevated [CO2] and high N. 2.4. Statistical analysis
Under N-rich environment, C/N ratio should decrease and apparent
trade-off between high growth and low production of secondary Two way analysis of variance (ANOVA) was used to evaluate the difference
among treated seedling by using SPSS 16.0.2 (SPSS Inc., USA).
metabolites may take place as predicted by GDB. In contrast,
elevated [CO2] should increase C/N ratio that would lead to higher
concentration of SM, but the growth is not necessarily to decrease 3. Results
since the enhanced-resource use efficiency may cause the possi-
bility of positive correlation between growth and SM production 3.1. Net photosynthetic rate, growth, and defense chemical
(Herms and Mattson, 1992).
In order to access these predictions, we investigated the In both eucalypts, Agrowth was increased by N load and elevated
response of chemical defense, leaf and photosynthetic traits of two [CO2]; interaction of both factors was also detected. High N load,
eucalypts to high N load and elevated [CO2]. These results will but not elevated [CO2], stimulated the diameter and height of both
provide a plausible understanding of the role of excess N accom- species (Fig. 1).
panied by elevated [CO2] give rise to changes in the defense The hybrid H contained a significantly higher concentration of
chemical of eucalypts (i.e. total phenolics and condensed tannin). condensed tannin (p < 0.05) and marginally lower total phenolics
We hope to contribute our findings to ecosystem rehabilitation in content (p < 0.1) by elevated [CO2] but those were not changed by
Indonesia. high N load. Total phenolics of H showed significant interaction
between N load and elevated [CO2]. The concentration of phenolics
2. Materials and methods in H was reduced by elevated CO2 under N0 condition, however, no
difference was found between ambient and elevated CO2 under N1
2.1. Plant materials and growth condition condition (Table 1).
High N load significantly decreased the total phenolics and
We used seedlings of Eucalyptus urophylla (U) and cuttings of hybrid
E. deglupta
E. camaldulensis (H). The latter was originally developed for materi- although not significant, it reduced the tannin content of U by 50.1%.
alizing high growth performance to supply plant materials for pulpwood planta- However, the effect of elevated [CO2] was insignificant on the
tions, therefore cuttings were used due to mass production of seedlings with similar concentration of total phenolics and condensed tannins of U (Table 1).
traits among them. The seed of U was obtained from Australia Tree Seed Centre of
CSIRO, Kingston, Australia. E. urophylla is species native to Indonesia islands (e.g.
ERDB, 2009), while based on the parent characteristics, H is expected to survive the 3.2. Leaf and photosynthetic traits
tropic/sub tropic environments.
At the initiation of the experiment that was lasted on JanuaryeMay 2010, the For both species, elevated [CO2] did not significantly affect the
average height and diameter of U were 40.5 cm and 4.1 mm, while of H were 57.0 cm
chlorophyll content, LMA, Nmass, Narea, and lignin content (Table 1),
and 4.9 mm. The seedlings and cuttings were 7e8 months old. The plants were
grown in phytotron chambers of the Forest and Forestry Research Product Institute, but significantly increased PNUE and WUE (Table 2). For U, high N
Sapporo, Japan (43 00 N, 141 20 E, 180 m a.s.l.). The chambers were maintained at load significantly increased chlorophyll content, Nmass, Narea, lignin
daily temperatures around 25/20  C under natural light, supplemented by sodium content, and WUE, but decreased the LMA. For H, high N load
halide lamps for adjusting day-length of 14 h. increased the chlorophyll content, Nmass, Narea, and WUE, but its effect
The growth media were pumice soil and clay soil (1:1, v/v) in 7 L pots that are
commonly used in nursery practices. As basal dressing, we supplied 500-fold diluted
was insignificant on LMA, lignin, and PNUE. Phosphorous (P) content
liquid fertilizer (balanced nutrient; N:P:K ¼ 6:10:5, Hyponex Corp. JAPAN, Osaka, in the H foliage was significantly increased by N addition but no
Japan) at a rate of 1 kg N ha1. The pots were watered periodically to sustain the soil effects of elevated [CO2] were found in either H or U foliage (Table 1).
moisture. Under high N condition, lignin content significantly increased in
As a treatment on the seedlings, we Supplied two levels of N (N0 ¼ 0 kg ha1 and
U and tended to increase by 36.6% in H. Elevated [CO2] also slightly
N1 ¼ 50 kg ha1 of (NH4)2SO4 þ balance nutrient) and two levels of [CO2] (ambient
(A) ¼ 380 mmol mol1 and elevated (E) ¼ 760 mmol mol1) into the growth cham- increased foliar lignin content of the eucalypts, by 39.3% and 31.2%
bers. There were three replications for each plant species for the measurement. for U and H, respectively (Table 1).

2.2. Measurement of gas exchange rates 3.3. Interaction effect

The gas exchange rates were measured on mature leaf (counted third or fourth
from the shoot top) by using an open gas exchange system (LI-6400, LI-Cor, Lincoln, In general, lack evidences were found in this study for interac-
Nebraska, USA) in late April 2010. Measurement was carried out under tion effect of N load and elevated [CO2]. For U, interaction between
126 E. Novriyanti et al. / Environmental Pollution 170 (2012) 124e130

et al., 1983; Hamilton et al., 2001; Herms and Mattson, 1992) or

food reserve (e.g. Chapin et al., 1990; Herms and Mattson, 1992).
The lignin content and LMA of the plants were presumably
credited for the insignificant effect of elevated [CO2] on the growth
increments. The LMA of both eucalypts tended to decrease (17.2% in
U and 8.4% in H) which indicated that leaf mass per unit area was
decreased under high [CO2] (Table 1). Meanwhile the lignin content
tended to increase (38.8% in U and 31.1% in H) under elevated [CO2].
Thus, the most likely reason for the insignificant growth under
elevated [CO2] was that the plants which had the stimulated-
Agrowth could not beneficially deploy the newly fixed carbohydrates
into new growth, perhaps allocated to e.g. non-structural carbo-
hydrates or lignin synthesis.
P is a macronutrient that most frequently limits plant growth
next to N (e.g Buenemann et al., 2011; Schachtman et al., 1998). The
foliar P content of the eucalypts varied 4-fold and was less than
0.20% of dry weight (Table 1). It was lower than P-requirement for
the optimal growth of common plants, 0.30e0.50% of dry weight
(Marschner, 1995).
Although eucalypts generally could survive in a relatively lower
P soil (e.g. Beadle, 1962; Dell et al., 1983; Mulligan, 1988); however,
P deficiency likely hampered the growth of the eucalypts under
elevated [CO2] despite of higher rate of Agrowth. This may be
attributed to the fact that shoot growth usually more severely
impaired than photosynthetic rate in P deficiency plants (e.g. Dell
et al., 1987; Plénet et al., 2000). Several studies have reported
that growth reduction of eucalypts seedlings is caused by P-star-
vation (e.g. Godoy and da Silva Rosado, 2011; Gonçalves et al., 2004;
Xu et al., 2005). In fact, nutrient stress may enable the reduction of
growth stimulation under elevated [CO2] (Conroy, 1992; Lynch and
[Link], 2004; Poorter and Pérez-Soba, 2001; Tobita et al., 2010). P-
deficiency also stimulates the root growth despite the shoot growth
declines (e.g. Hawkesford et al., 2012). Further, since in general root
responses to elevated [CO2] are often greater than aboveground
responses, therefore, allocations of assimilates to belowground
(Jackson et al., 2009) is possible explanation.
The higher Agrowth of the eucalypts under high N load sustained
high growth rate despite of P-deficiency because plant under
excessive N condition may enhance the efficiency of P-resorption
(Conroy et al., 1992; Lü and Han, 2010).
Fig. 1. Net photosynthetic rate (Agrowth), D diameter and height of E. urophylla (U) and
hybrid E. degluptax E. camaldulensis (H). A ¼ ambient [CO2] 380 mmol m1, E ¼ elevated
Under high N supply, both eucalypts exhibited enhanced-Agrowth
[CO2] 760 mmol m1, N0 ¼ no N addition, N1 ¼ N supply at rate of 50 kg ha1, and growth increments (height and diameter), but their chemical
N ¼ nitrogen treatment, CO2 ¼ CO2 treatment, N
CO2 ¼ interaction of nitrogen and defense responded differently. In the U both total phenol and total
CO2 treatment. P values are presented in the table next to each graph. Data are average tannin tended to decline. The increased nutrient uptake would
values  SD (n ¼ 3).
decreased the C/N ratio in U, lead the SM of U to decline as growth
received priority for resource allocation (Herms and Mattson,
N and [CO2] strengthened the increased Agrowth, and Narea. Mean- 1992). Further, the LMA of U decreased under high N supply
while for H, the interaction strengthened Agrowth, phenolics content (Table 1), thus the concentration of total phenolics and condensed
and gs but weaken E. tannin were not diluted by biomass of the leaves, suggested that SM
synthesis was reduced more than biomass accumulation (e.g.
4. Discussion Koricheva, 1999; Lavola et al., 1998).
In contrast to U, the defense compound of H did not respond to
4.1. Net photosynthetic rate, growth and defense chemical high N, although C/N ratio also significantly decreased and growth
was increased as it received priority for the available-resource
Growth increments of the two eucalypts were increased under (Fig. 1, Table 1). Therefore, the resource allocation might be
high N load, but were not profoundly influenced by elevated [CO2], species specific. While H showed a trade-off between high growth
despite Agrowth were increased on both conditions. In many cases rate and lower SM concentration, H maintained high growth rate
(e.g. Ghannoum et al., 2010), high [CO2] usually increases the but did not decrease SM concentration. It may imply that H is
photosynthetic rate and thereby the growth rate. However, in the probably more resistant to herbivory than U.
present study and some other experiments (e.g. Arp et al., 1998), Contrary to our prediction, elevated [CO2] did not increase C/N
there were no positive effects of elevated [CO2] on growth of both ratio of the eucalypts. Despite of that, the [CO2] treatment signifi-
eucalypts. Some environmental limitations may retard growth than cantly decreased total phenolics and increased total tannin in H
photosynthesis (Herms and Mattson, 1992). Thus, supposedly, (Table 1). In regard to the increased tannin content, supposedly,
assimilates were not allocated more to growth of those species, but elevated [CO2] might actually increase the N available for tannin
shifted to other allocation, instead, e.g. defense system (e.g. Bryant production because the efficiency of photosynthesis was increased.
 E. Novriyanti et al. / Environmental Pollution 170 (2012) 124e130 127

Table 1
Leaf traits and chemicals of E. urophylla and hybrid E. deglupta
E. camaldulenis.

A E P-value

N0 N1 N0 N1 N CO2 N
CO2
E. urophylla
Total phenolics (mg g1) 131.75  26.50 109.55  12.33 134.83  18.78 76.85  9.46 0.005 0.192 0.124
Condensed tannins (mg g1) 5.63  3.64 4.23  1.97 9.34  7.49 3.23  2.32 0.180 0.610 0.384
Lignin (%) 4.53  1.09 6.24  3.57 4.97  1.42 9.98  2.11 0.033 0.145 0.245
LMA (g m2) 111.85  5.53 76.27  24.55 93.90  20.05 66.58  24.38 0.027 0.270 0.732
Chlorophyll (mg mg1) 1.92  0.67 5.91  1.19 1.51  0.35 5.23  0.18 0.000 0.339 0.804
Nmass (%) 0.58  0.08 3.04  0.36 0.76  0.16 3.38  0.55 0.000 0.225 0.688
Narea (g m2) 0.53  0.24 2.94  0.34 0.67  0.26 2.36  0.39 0.000 0.255 0.084
C/N 57.88  39.18 33.54  31.48 68.82  19.76 14.95  2.39 0.037 0.812 0.372
P (%) 0.05  0.02 0.13  0.07 0.11  0.04 0.15  0.12 0.167 0.361 0.636
E. deglupta
E. camaldulensis
Total phenolics (mg g1) 108.39  20.63 73.55  13.81 53.15  11.29 98.92  6.01 0.506 0.097 0.001
Condensed tannins (mg g1) 5.99  1.79 3.36  1.33 8.77  4.00 8.51  1.91 0.391 0.040 0.476
Lignin (%) 7.07  3.69 8.53  3.87 8.17  3.40 12.28  1.01 0.171 0.227 0.495
LMA (g m2) 92.19  3.41 80.80  20.94 83.00  12.21 76.65  15.06 0.316 0.445 0.769
Chlorophyll (mg mg1) 1.75  0.02 4.60  0.15 2.16  0.32 4.12  0.58 0.001 0.437 0.673
Nmass (%) 0.92  0.09 2.23  0.27 0.88  0.68 2.68  0.38 0.000 0.176 0.115
Narea (g m2) 0.85  0.55 1.77  0.37 0.81  0.65 1.83  0.29 0.000 0.954 0.723
C/N 51.27  5.53 21.55  2.19 51.90  4.28 18.14  2.13 0.000 0.547 0.388
P (%) 0.06  0.02 0.20  0.08 0.07  0.03 0.19  0.06 0.010 0.918 0.712

LMA ¼ leaf mass per area, Nmass ¼ leaf N content per unit dry mass, Narea ¼ leaf N content per unit area, C/N ratio ¼ carbon to nitrogen ratio, P ¼ phosphorous, N is nitrogen
treatment (N0 ¼ 0 kg ha1, N1 ¼ 50 kg ha1 of (NH4)2SO4 þ balance nutrient), CO2 is CO2 treatment (A ¼ ambient: 380 mmol m1, E ¼ elevated: 760 mmol m1). Data are mean
values  SD (n ¼ 3).

In other words, elevated [CO2] may allow the plant to reallocate N when N was limited. However, high N diminished the effect of
from photosynthesis to secondary metabolites, specifically tannin elevated [CO2] (Table 1). Supposedly, when N is not a limiting factor
(Hamilton et al., 2001; Jones and Hartley, 1999). in the elevated [CO2] environment, the plant could enhanced the
Another possible explanation for the increased condensed Agrowth, thus there were enough assimilates to be allocated to
tannins is a shifting resource allocation from retarded-growth phenolics synthesis.
under elevated [CO2]. Environmental constrain can mitigate the Unlike H, total phenolics and condensed tannins of U did not
cost of defense as when the growth is retarded more than the respond significantly to the elevated [CO2]. This fact further
photosynthesis then it will increase the pool of resource availability strengthens our consideration that both eucalypts respond differ-
for SM production with little or no trade-off with growth (Herms ently to elevated [CO2] and high N with regard to the resource
and Mattson, 1992). P deficiency likely limited the growth under allocation to secondary metabolites. Productions of secondary
elevated [CO2] (Table 1), however, a trade-off was only apparent metabolite are deeply related to evolutionally processes, and
between the impaired-growth and condensed tannins but not total therefore, the responses to environmental change may be varied
phenolics. widely even among the same genus.
The high [CO2] tended to decrease total phenolics in H. The
decreased total phenolics may be partly attributed to the tendency 4.2. Characteristic of leaf and photosynthetic responses
of increased leaf N content and of decreased C/N ratio (by 3.81% in
H) (Table 1). Elevated [CO2] usually decreases leaf N content A higher supply of C and N in elevated [CO2] and N load could
because it inhibits the assimilation of nitrate into organic nitrogen promote a higher photosynthetic rate, and hence a higher growth
compound in leaves (e.g. Bloom et al., 2010). Consequently, total rate (e.g. Poorter and Pérez-Soba, 2001). In this study, however, no
phenolics seemed to decrease under elevated [CO2] as C/N ratio significant increase was found in the growth of either eucalyptus,
tended to decreased (Herms and Mattson, 1992). despite the higher values of Agrowth under elevated [CO2]. We found
The increased total phenolics content in H was influenced that lignin tend to increase with N load and elevated [CO2] (Table 1).
significantly by high N load. Elevated [CO2] decreased the SM of H Lignin is another end-product of the available-resources in plants

Table 2
Photosynthetic traits of E. urophylla and hybrid E. deglupta
E. camaldulenis.

A E P-value

N0 N1 N0 N1 N CO2 N
CO2
E. urophylla
PNUE (mmol mol1 s1) 103.86  94.53 98.12  34.94 142.01  29.53 194.62  40.81 0.492 0.071 0.392
WUE (mmol mol1) 4.04  0.82 6.79  0.99 5.10  2.57 9.84  0.74 0.002 0.044 0.282
gs (mol m2 s1) 0.04  0.01 0.25  0.15 0.12  0.08 0.25  0.02 0.012 0.480 0.480
E (mol m2 s1) 0.70  0.23 3.06  1.12 1.64  1.08 3.26  0.31 0.003 0.253 0.450
E. deglupta
E. camaldulensis
PNUE (mmol mol1 s1) 157.71  44.57 140.75  62.11 198.83  47.28 256.23  42.59 0.501 0.026 0.232
WUE (mmol mol1) 2.35  0.50 4.75  0.93 6.30  2.68 9.10  1.22 0.021 0.002 0.829
gs (mol m2 s1) 0.37  0.17 0.26  0.07 0.143  0.08 0.37  0.15 0.451 0.451 0.055
E (mol m2 s1) 4.12  1.06 3.54  0.45 2.00  0.71 3.68  0.68 0.249 0.055 0.033

PNUE ¼ photosynthetic nitrogen use efficiency, WUE ¼ photosynthetic water use efficiency, gs ¼ stomatal conductance, E ¼ leaf transpiration rate, N is nitrogen treatment
(N0 ¼ 0 kg ha1, N1 ¼ 50 kg ha1 of (NH4)2SO4 þ balance nutrient), CO2 is CO2 treatment (A ¼ ambient: 380 mmol m2 s1, E ¼ elevated: 760 mmol m2 s1). Data are mean
values  SD (n ¼ 3).
128 E. Novriyanti et al. / Environmental Pollution 170 (2012) 124e130

(e.g. Herms and Mattson, 1992). Contrary to our result, the present study found only few interaction effects of N load and
decreased C/N ratio by N load usually leads to the lower lignin enhanced [CO2].
content (e.g. Henry et al., 2005). Growth and lignin content usually When N was not a limiting factor, the performance of Agrowth
show negative correlation (Novaes et al., 2010), yet both were under elevated [CO2] increased. Given a high N supply, elevated
increased in this study. GDB explains that the enhanced-Agrowth [CO2] increased Agrowth of the U by 58.93% and of the H by 95.20%.
under condition of increased-resource availability may create the For the H, high Agrowth was associated with lower phenolics content
possibility for a positive correlation between the aforementioned when the N supply was high. The presence of high N would prob-
variables (i.e. growth and lignin synthesis) despite competition for ably allow the H to sustain high Agrowth, thus it ameliorated the
a common resource base (Herms and Mattson, 1992). Similar effect of elevated [CO2] in which adequate assimilates could be
tendency was reported that lignin content was increased by allocated to SM synthesis (e.g. Bryant et al., 1983; Mattson et al.,
elevated [CO2] and N load (Blaschke et al., 2002; Cotrufo, 1994). 2005; Simon et al., 2010).
Some studies found increased chlorophyll content with elevated
[CO2], with or without N supply (e.g. Li and Gupta, 1993) or affected 5. Conclusion
by only elevated [CO2] or only N deposition (Zhao et al., 2011).
However, this study found no difference in chlorophyll content Resources allocation in the two eucalypts, especially to defen-
with CO2 treatment, but high chlorophyll content were detected sive chemicals, was affected differently by N load and elevated
with high N supply. Leaf chlorophyll concentration is sensitive to N [CO2], suggesting that induced defense vary widely in eucalyptus
supply, and it decreases markedly in condition of low N (Burns species. The different response of the two eucalypts to the envi-
et al., 2002). We therefore expect the opposite effect of increasing ronmental change might imply their competitiveness in the
chlorophyll under high N. High N supply significantly increased leaf growing environment. While at the same time it could maintain
N, and therefore increased the total chlorophyll content (Table 1). rapid growth, unaltered defensive chemicals of H may favorable to
Higher N content in leaves (Nmass and Narea) was found in the overcome the frequent herbivory attack in tropical/subtropical
high N environment (Table 1). The increased growth rate of both environment.
species by high N load (Fig. 1) did not offset N tissue content by Moreover, another environmental constraints could hamper the
a dilution effect. Both species could therefore maintain their high N fertilizer effect of elevated [CO2] on growth of the eucalypts. The
status despite the greater biomass. The N tissue content usually retarded-growth is then could further define the concentration of
reduces at high [CO2], because the high growth rate would dilutes defense chemicals as resources allocation is shifted to defense
the N tissue concentration and necessitates further N uptake in system.
plant (Coleman et al., 1993; Johnson, 2006), thus under high N and Despite the photosynthetic rate of the two eucalypts performed
high [CO2] plant may have lower N tissue content. Although tended well under elevated N and [CO2], the strategy of the eucalypts in
to slightly increased, there was no significant effect of elevated allocating the available resource to growth and defense under
[CO2] on Nmass and Narea in either species, or in their growth altered-environment should define their prosperity as afforestation
increments (height and diameter). The seedlings could presumably plants. Considering the advantageous response to high N load and
maintain a high N content due to the balance nutrients we supplied its interaction with elevated [CO2], H is therefore the more prom-
together with N. ising material for afforestation and reforestation to repair degraded
PNUE of both species increased significantly with elevated [CO2]. areas in Indonesia.
In general, high PNUE would be attained by species which have
lower leaf N content. However, both species sustained high PNUE Acknowledgemnts
despite the foliar N content was insignificant under elevated [CO2].
This higher PNUE was probably due to the lower LMA of the euca- The authors are grateful for partly financial support from
lypts in this environment (e.g. Harrison et al., 2009) as shown in a grant-in-aid from the Japan Society for the Promotion of Science
Table 2. We assumed that the balance-nutrient supplied together through the Grant-in-Aid for Scientific Research on Innovative
with (NH4)2SO4 also allowed those seedlings to maintain high PNUE. Areas program (21114008) to T.K, and Research Fellowships for
The increased Nmass and Narea under high N load to some extent Young Scientists Program (20.1143) to M. W. We are indebted to
decreased the PNUE of both species (5.5% and 10.8% for U and H, Prof. Amane Makino and Dr. Toshihiro Watanabe for invaluable
respectively), although the effects were insignificant (Table 2). It comments on plants nutrient physiology. We thank Dr. Anthony
consistent with many studies, high N load usually increases leaf N Garrett of the Scitext Cambridge, U.K. for the guidance in improving
content and thereby decreases PNUE (e.g. Poorter and Evans, 1998). our English.
However, elevated [CO2] were likely sustained the increased-PNUE
of both eucalypts (by 27.0% in U and 22.4% in H) despite high N was References
supplied (Table 1). Presumably, elevated [CO2] may increase the
PNUE independent of N content by increase in Agrowth (Davey et al., Ainsworth, E.A., Long, S.P., 2005. What have we learned from 15 years of free-air
1999). CO2 enrichment (FACE)? A meta-analytic review of the responses of photo-
synthesis, canopy properties and plant production to rising CO2. New Phytol-
N load and elevated [CO2] also significantly increased the WUE ogist 165, 351e372.
of both species. This finding consent with other observations (e.g. Alves, P., Magalhães, A., Barja, P., 2002. The phenomenon of photoinhibition of
Cao et al., 2007) that elevated [CO2] enhances the overall rate of photosynthesis and its importance in reforestation. The Botanical Review 68,
193e208.
Agrowth and greatly increased the WUE, with regard to the Armstrong, W.P., 1998. The Unforgettable Acacias, a Large Genus of Trees and
decreasing of E and gs in all N treatment. However, decreased E and Shrubs. Zoonooz, pp. 28e31.
gs are observed only in the H under elevated [CO2]. Under N load, Arp, W.J., Van Mierlo, J.E.M., Berendse, F., Snijders, W., 1998. Interactions between
elevated CO2 concentration, nitrogen and water: effects on growth and water
the values of E and gs of U in fact increased (Table 2).
use of six perennial plant species. Plant, Cell & Environment 21, 1e11.
Some studies state that enhanced [CO2] alters plant photosyn- Beadle, N.C.W., 1962. Soil phosphate and the delimitation on plant communities in
thetic traits (e.g. Ghannoum et al., 2010) and growth or defense Eastern Australia II. Ecology 43, 281e288.
traits (Mattson et al., 2005); other report that the effect of [CO2] is Blaschke, L., Forstreuter, M., Sheppard, L.J., Leith, I.K., Murray, M.B., Polle, A., 2002.
Lignification in beech (Fagus sylvatica) grown at elevated CO2 concentrations:
coordinated with the presence of high nutrient supply (i.e. interaction with nutrient availability and leaf maturation. Tree Physiology 22,
nitrogen) (e.g. Crous et al., 2008; Finzi et al., 2006). However, this 469e477.
 E. Novriyanti et al. / Environmental Pollution 170 (2012) 124e130 129

Bloom, A.J., Burger, M., Asensio, J.S.R., Cousins, A.B., 2010. Carbon dioxide enrich- Iiyama, K., Wallis, A.F.A., 1990. Dissolution of wood with acetyl bromide solutions e
ment inhibits nitrate assimilation in wheat and Arabidopsis. Science 328, reactions of lignin model compounds. Journal of Wood Chemistry and Tech-
899e903. nology 10, 39e58.
Bryant, J.P., Chapin III, F.S., Klein, D.R., 1983. Carbon/nutrient balance of boreal plants IPCC, 2007. Summary for policy makers. In: Metz, B., Davidson, O.R.,
in relation to vertebrate herbivory. Oikos 40, 357e368. Bosch, P.R., Dave, R., Meyer, L.A. (Eds.), Climate Change 2007: Mitigation.
Buenemann, E.K., Oberson, A., Frossard, E., 2011. Phosphorous in Action. Springer, Contribution of Working Group III to the Fourth Assessment Report of the
Berlin, Heidelberg. Intergovernmental Panel on Climate Change. Cambridge University Press,
Burns, A., Gleadow, R., Woodrow, I., 2002. Light alters the allocation of nitrogen to New York, USA.
cyanogenic glycosides in Eucalyptus cladocalyx. Oecologia 133, 288e294. Jackson, R.B., Cook, C.W., Pippen, J.S., Palmer, S.M., 2009. Increased belowground
Cao, B., Dang, Q.-L., Zhang, S., 2007. Relationship between photosynthesis and leaf biomass and soil CO2 fluxes after a decade of carbon dioxide enrichment in
nitrogen concentration in ambient and elevated [CO2] in white birch seedlings. a warm-temperate forest. Ecology 90, 3352e3366.
Tree Physiology 27, 891e899. Johnson, D.W., 2006. Progressive N limitation in forests: review and implications for
Chapin, F.S., Schulze, E.-D., Mooney, H.A., 1990. The ecology and economics of long-term responses to elevated CO2. Ecology 87, 64e75.
storage in plants. Annual Review of Ecology and Systematics 21, 423e447. Jones, C.G., Hartley, S.E., 1999. A protein competition model of phenolic allocation.
Coleman, J.S., McConnaughay, K.D.M., Bazzaz, F.A., 1993. Elevated CO2 and plant Oikos 86, 27e44.
nitrogen-use: is reduced tissue nitrogen concentration size-dependent? Oeco- Julkunen-Tiitto, R., 1985. Phenolic constituents in the leaves of northern willows:
logia 93, 195e200. methods for the analysis of certain phenolics. Journal of Agricultural and Food
Conroy, J.P., 1992. Influence of elevated atmospheric CO2 concentrations on plant Chemistry 33, 213e217.
nutrition. Australian Journal of Botany 40, 445e456. Koike, T., Matsuki, S., Matsumoto, T., Yamaji, K., Tobita, H., Kitao, M., Maruyama, Y.,
Conroy, J.P., Milham, P.J., Barlow, E.W.R., 1992. Effect of nitrogen and phosphorus 2003. Bottom-up regulation for protection and conservation of forest ecosys-
availability on the growth response of Eucalyptus grandis to high CO2. Plant, Cell tems in Northern Japan under changing environments. Eurasian Journal of
& Environment 15, 843e847. Forest Research 6, 177e189.
Cotrufo, M.F., 1994. Decomposition of tree leaf litters grown under elevated CO2: Koike, T., Tobita, H., Shibata, T., Matsuki, S., Konno, K., Kitao, M., Yamashita, N.,
effect of litter quality. Plant and Soil 163, 121e130. Maruyama, Y., 2006. Defense characteristics of seral deciduous broad-leaved
Crous, K.Y., Walters, M.B., Ellsworth, D.S., 2008. Elevated CO2 concentration affects tree seedlings grown under differing levels of CO2 and nitrogen. Population
leaf photosynthesisenitrogen relationships in Pinus taeda over nine years in Ecology 48, 23e29.
FACE. Tree Physiology 28, 607e614. Koricheva, J., 1999. Interpreting phenotypic variation in plant allelochemistry:
Davey, P.A., Parsons, A.J., Atkinson, L., Wadge, K., Long, S.P., 1999. Does photosyn- problems with the use of concentrations. Oecologia 119, 467e473.
thetic acclimation to elevated CO2 increase photosynthetic nitrogen-use effi- Laclau, J.P., Bouillet, J.P., Gonçalves, J.L.M., Silva, E.V., Jourdan, C., Cunha, M.C.S.,
ciency? A study of three native UK grassland species in open-top chambers. Moreira, M.R., Saint-André, L., Maquère, V., Nouvellon, Y., Ranger, J., 2008.
Functional Ecology 13, 21e28. Mixed-species plantations of Acacia mangium and Eucalyptus grandis in Brazil:
Dell, B., Jones, S., Wilson, S., 1987. Phosphorus nutrition of jarrah (Eucalyptus mar- 1. Growth dynamics and above ground net primary production. Forest Ecology
ginata ) seedlings. Plant and Soil 97, 369e379. and Management 255, 3905e3917.
Dell, B., Lorenagan, J.F., Plaskett, D., 1983. The phosphorous responses of Eucalyptus Lavola, A., Julkunen-Tiitto, R., Roininen, H., Aphalo, P., 1998. Host-plant preference of
seedlings grown in a Pallid zone clay treated with three levels of lime. an insect herbivore mediated by UV-B and CO2 in relation to plant secondary
Australian Journal of Botany 31, 231e238. metabolites. Biochemical Systematics and Ecology 26, 1e12.
ERDB, 2009. Eucalyptus urophylla and Acacia mangium. In: Baltazar, E.M., Asis, L.E., Lawler, I.R., Foley, W.J., Woodrow, I.E., Cork, S.J., 1997. The effects of elevated
Cambay, M.V. (Eds.), Research Information Series on Ecosystems. Ecosystem CO 2 atmospheres on the nutritional quality of Eucalyptus foliage and its
Research and Development Bureau, Laguna. interaction with soil nutrient and light availability. Oecologia 109,
Farquhar, G.D., von Caemmerer, S., Berry, J.A., 1980. A biochemical model of 59e68.
photosynthetic CO2 assimilation in leaves of C3 species. Planta 149, 78e90. Li, Y., Gupta, G., 1993. Photosynthetic changes in soybean with and without nitrogen
Feng, Y.-L., Lei, Y.-B., Wang, R.-F., Callaway, R.M., Valiente-Banuet, A., Inderjit, Li, Y.- and increased carbon dioxide. Plant Science 89, 1e4.
P., Zheng, Y.-L., 2009. Evolutionary tradeoffs for nitrogen allocation to photo- Long, S.P., Bernacchi, C.J., 2003. Gas exchange measurements, what can they tell us
synthesis versus cell walls in an invasive plant. Proceedings of the National about the underlying limitations to photosynthesis? Procedures and sources of
Academy of Sciences 106, 1853e1856. error. Journal of Experimental Botany 54, 2393e2401.
Finzi, A.C., Moore, D.J.P., DeLucia, E.H., Lichter, J., Hofmockel, K.S., Jackson, R.B., Lynch, J.P., [Link], S.B., 2004. Mineral stress: the missing link in understanding how
Kim, H.-S., Matamala, R., McCarthy, H.R., Oren, R., Pippen, J.S., Schlesinger, W.H., global climate change will affect plants in real world soils. Field Crops Research
2006. Progressive nitrogen limitation of ecosystem processes under elevated 90, 101e115.
CO2 in a warm-temperate forest. Ecology 87, 15e25. Lü, X.-T., Han, X.-G., 2010. Nutrient resorption responses to water and nitrogen
Galloway, J.N., Dentener, F.J., Capone, D.G., Boyer, E.W., Howarth, R.W., amendment in semi-arid grassland of Inner Mongolia, China. Plant and Soil 327,
Seitzinger, S.P., Asner, G.P., Cleveland, C.C., Green, P.A., Holland, E.A., Karl, D.M., 481e491.
Michaels, A.F., Porter, J.H., Townsend, A.R., Vöosmarty, C.J., 2004. Nitrogen Marschner, H., 1995. Mineral Nutrition of Higher Plants. Academic Press Limited,
cycles: past, present, and future. Biogeochemistry 70, 153e226. London.
Ghannoum, O., Phillips, N.G., Conroy, J.P., Smith, R.A., Attard, R.D., Woodfield, R., Matsuki, S., Sano, Y., Koike, T., 2004. Chemical and physical defence in early and late
Logan, B.A., Lewis, J.D., Tissue, D.T., 2010. Exposure to preindustrial, current and leaves in three heterophyllous birch species native to Northern Japan. Annals of
future atmospheric CO2 and temperature differentially affects growth and Botany 93, 141e147.
photosynthesis in Eucalyptus. Global Change Biology 16, 303e319. Mattson, J.W., Julkunen-Tiitto, R., Herms, D.A., 2005. CO2 enrichment and carbon
Gillett, R.W., Ayers, G.P., Selleck, P.W., Tuti, M.H.W., Harjanto, H., 2000. Concentra- partitioning to phenolics: do plant responses accord better with the protein
tions of nitrogen and sulfur species in gas and rainwater from six sites in competition or the growth differentiation balance models? Oikos 111,
Indonesia. Water, Air, & Soil Pollution 120, 205e215. 337e347.
Gleadow, R.M., Foley, W.J., Woodrow, I.E., 1998. Enhanced CO2 alters the relationship Mulligan, D.R., 1988. Phosphorus concentrations and chemical fractions in Euca-
between photosynthesis and defence in cyanogenic Eucalyptus cladocalyx F. lyptus seedlings grown for a prolonged period under nutrient-deficient condi-
Muell. Plant, Cell & Environment 21, 12e22. tions. New Phytologist 110, 479e486.
Godoy, T.G., da Silva Rosado, S.C., 2011. Efficiency of phosphorous use in young Novaes, E., Kirst, M., Chiang, V., Winter-Sederoff, H., Sederoff, R., 2010. Lignin and
plants of Eucalyptus urophylla S.T. Blake. Cerne 17, 303e308. biomass: a negative correlation for wood formation and lignin content in trees.
Gonçalves, J.L.d.M., Stape, J.L., Laclau, J.-P., Smethurst, P., Gava, J.L., 2004. Silvicultural Plant Physiology 154, 555e561.
effects on the productivity and wood quality of eucalypt plantations. Forest Novriyanti, E., Watanabe, M., Makoto, K., Takeda, T., Hashidoko, Y., Koike, T., 2012.
Ecology and Management 193, 45e61. Photosynthetic nitrogen and water use efficiency of acacia and eucalypt seed-
Grubb, M., 2003. The economics of the Kyoto Protocol. World Economics 4, lings as afforestation species. Photosynthetica 50, 273e281.
143e189. Ogawa, A., Shibata, H., Suzuki, K., Mitchell, M.J., Ikegami, Y., 2006. Relationship of
Hamilton, J.G., Zangerl, A.R., DeLucia, E.H., Berenbaum, M.R., 2001. The car- topography to surface water chemistry with particular focus on nitrogen and
bonenutrient balance hypothesis: its rise and fall. Ecology Letters 4, 86e95. organic carbon solutes within a forested watershed in Hokkaido, Japan.
Harrison, M.T., Edwards, E.J., Farquhar, G.D., Nicotra, A.B., Evans, J.R., 2009. Hydrological Processes 20, 251e265.
Nitrogen in cell walls of sclerophyllous leaves accounts for little of the PEACE, 2007. In: Sari, A.P., Maulidya, M., Butarbutar, R.N., Sari, R.E., Rusmantoro, W.
variation in photosynthetic nitrogen-use efficiency. Plant, Cell & Environment (Eds.), Indonesia and Climate Change: Current Status and Policy. World Bank,
32, 259e270. Jakarta.
Hawkesford, M., Horst, W., Kichey, T., Lambers, H., Schjoerring, J., Moller, s.I., Plénet, D., Mollier, A., Pellerin, S., 2000. Growth analysis of maize field crops under
White, P., 2012. Nutritional physiology: functions of macronutrient. In: phosphorus deficiency. II. Radiation-use efficiency, biomass accumulation and
Marschner, P. (Ed.), Marschner’s Mineral Nutrition of Higher Plants. Elsevier yield components. Plant and Soil 224, 259e272.
Ltd., San Diego, USA, pp. 135e178. Poorter, H., Evans, J.R., 1998. Photosynthetic nitrogen-use efficiency of species that
Henry, H.A.L., Cleland, E.E., Field, C.B., Vitousek, P.M., 2005. Interactive effects of differ inherently in specific leaf area. Oecologia 116, 26e37.
elevated CO2, N deposition and climate change on plant litter quality in a Cal- Poorter, H., Pérez-Soba, M., 2001. The growth response of plants to elevated CO2
ifornia annual grassland. Oecologia 142, 465e473. under non-optimal environmental conditions. Oecologia 129, 1e20.
Herms, D.A., Mattson, W.J., 1992. The dilemma of plants: to grow or defend. The Schachtman, D.P., Reid, R.J., Ayling, S.M., 1998. Phosphorus uptake by plants: from
Quarterly Review of Biology 67, 283e335. soil to cell. Plant Physiology 116, 447e453.
130 E. Novriyanti et al. / Environmental Pollution 170 (2012) 124e130

Schulze, E.-D., Beck, E., Müller-Hohenstein, K., 2005. Plant Ecology. Springer- Tobita, H., Uemura, A., Kitao, M., Kitaoka, S., Utsugi, H., 2010. Interactive effects of
Heielberg, Berlin. elevated CO2, phosphorus deficiency, and soil drought on nodulation and nitro-
Shinano, T., Lei, T.T., Kawamukai, T., Inoue, M.T., Koike, T., Tadano, T., 1996. genase activity in Alnus hirsuta and Alnus maximowiczii. Symbiosis 50, 59e69.
Dimethylsulfoxide method for the extraction of chlorophyll a and b from the Xu, D., Dell, B., Yang, Z., Malajczuk, N., Gong, M., 2005. Effect of phosphorous
leaves of wheat, field bean, dwarf bamboo, and oak. Photosynthetica 32, application on productivity and nutrient accumulation of a Eucalyptus urophylla
409e415. plantation. Journal of Tropical Forest Science 17, 447e461.
Simon, J., Gleadow, R.M., Woodrow, I.E., 2010. Allocation of nitrogen to chemical Zhao, H., Xu, X., Zhang, Y., Korpelainen, H., Li, C., 2011. Nitrogen deposition limits
defence and plant functional traits is constrained by soil N. Tree Physiology 30, photosynthetic response to elevated CO2 differentially in a dioecious species.
1111e1117. Oecologia 165, 41e54.