Nitrogen form preference of six dipterocarp species Mariko Norisada * , Katsumi Kojima Asian Natural Environmental Science Center, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan Received 20 November 2004; received in revised form 8 April 2005; accepted 10 May 2005 Abstract We investigated the nitrogen form preference of six dipterocarp species: Anisoptera costata Korth., Dipterocarpus obtusifolius Teijsm. ex Miq., Hopea odorata Roxb., Neobalanocarpus heimii (King) P. Ashton, Shorea faguetiana Heim, and Shorea roxburghii G. Don. Seedlings were supplied with nitrogen as nitrate, ammonium, or both in sand culture in a controlled environment. Except for N. heimii, all species showed greater shoot growth when supplied with ammonium than with nitrate. Higher root mass ratios were observed in all species with nitrate, which would be an adaptive response to limited nitrogen uptake. The five species, which preferred ammonium, showed a higher light-saturated photosynthetic rate with ammonium supply. The lower light-saturated photosynthetic rate with nitrate supply was a result of lower photosynthetic capacity, as indicated by a lower CO 2 -saturated photosynthetic rate. The lower leaf nitrogen content in seedlings supplied with nitrate would be the cause of the lower photosynthetic performance. Nitrate reductase activity in leaf and root of D. obtusifolius, N. heimii, and S. roxburghii showed generally low inducibility with nitrate. # 2005 Elsevier B.V. All rights reserved. Keywords: Nitrate; Ammonium; Growth; Photosynthesis; Nitrate reductase; Dipterocarpaceae 1. Introduction The members of the Dipterocarpaceae are pre- dominant tree species of the upper canopy of tropical rain forests in Southeast Asia (Symington, 1974; Whitmore, 1984). They are the most important timber species in the region, and depletion of the stock is now of concern as a result of overexploitation since their entrance on the international market in the 1950s (Richter and Gottwald, 1996). Examination of sustainable use and enrichment of existing resources has increased the need for knowledge of the environmental responses of the species. Light and water are the two main factors covered in studies of the environmental responses of dipterocarp and other tropical tree species (Chazdon et al., 1996; Mulkey and Wright, 1996; Whitmore, 1996). These two factors have crucial roles in species distribution and thus the species richness of tropical forests. Studies of temperate tree species have shown a correspondence of the site preference of a species with its nutritional characteristics, so the nutrient regime www.elsevier.com/locate/foreco Forest Ecology and Management 216 (2005) 175–186 * Corresponding author. Tel.: +81 3 5841 2785; fax: +81 3 5841 2785. E-mail address: [email protected] (M. Norisada). 0378-1127/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.foreco.2005.05.020
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Nitrogen form preference of six dipterocarp species
Mariko Norisada *, Katsumi Kojima
Asian Natural Environmental Science Center, The University of Tokyo, Bunkyo-ku, Tokyo, 113-8657 Japan
Received 20 November 2004; received in revised form 8 April 2005; accepted 10 May 2005
Abstract
We investigated the nitrogen form preference of six dipterocarp species: Anisoptera costata Korth., Dipterocarpus
obtusifolius Teijsm. ex Miq., Hopea odorata Roxb., Neobalanocarpus heimii (King) P. Ashton, Shorea faguetiana Heim,
and Shorea roxburghii G. Don. Seedlings were supplied with nitrogen as nitrate, ammonium, or both in sand culture in a
controlled environment. Except for N. heimii, all species showed greater shoot growth when supplied with ammonium than with
nitrate. Higher root mass ratios were observed in all species with nitrate, which would be an adaptive response to limited nitrogen
uptake. The five species, which preferred ammonium, showed a higher light-saturated photosynthetic rate with ammonium
supply. The lower light-saturated photosynthetic rate with nitrate supply was a result of lower photosynthetic capacity, as
indicated by a lower CO2-saturated photosynthetic rate. The lower leaf nitrogen content in seedlings supplied with nitrate would
be the cause of the lower photosynthetic performance. Nitrate reductase activity in leaf and root ofD. obtusifolius,N. heimii, and
S. roxburghii showed generally low inducibility with nitrate.
city was determined at saturated CO2 concentration
(1000 mmol m�2 s�1) under the same PAR. The leaf
chamber temperature was controlled to 28 8C. Therelative humidity ranged from 70 to 90%.
2.3. Chlorophyll
The chlorophyll content of the leaves in which
photosynthesis was measured was determined with a
chlorophyll meter (SPAD-502, Minolta) in Experi-
ment I or spectrophotometrically in Experiment II. In
spectrometric measurement, a 1-cm leaf disc was
homogenized in 80% acetone, and the homogenate
was centrifuged for 10 min at 1870 � g. The pellet
was extracted with 80% acetone again, and the
supernatants were bulked. Acetone (80%) was added
to the supernatant to give a final volume of 10 mL.
A645 and A663 of the supernatant was measured, and
M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186178
Table 2
Nitrogen form effects on biomass (root, stem, leaf, shoot, and total) and allocation (root mass ratio, stem mass ratio, and leaf mass ratio) of three
dipterocarp species, A. costata, H. odorata, and S. roxburghii, at DAT 42
Species Parameter Treatment F P
NH4 NH4 + NO3 NO3
A. costata Biomass (g DW)
Root 2.94 (1.72) b 4.55 (2.84) a 4.55 (2.61) a 4.91 0.015Stem 2.45 (1.79) a 2.69 (1.65) a 2.66 (1.80) a 0.30 0.741
Leaf 4.99 (3.50) a 5.17 (2.89) a 2.75 (1.89) b 9.65 <0.001Shoot 7.44 (5.24) ab 7.86 (4.49) a 5.41 (3.66) b 5.92 0.008Total 10.38 (6.93) a 12.41 (7.25) a 9.96 (6.24) a 2.33 0.117
Allocation (% of total biomass)
Root 0.30 (0.05) c 0.37 (0.05) b 0.48 (0.05) a 29.82 <0.001Stem 0.23 (0.04) ab 0.21 (0.02) b 0.26 (0.04) a 4.95 0.015Leaf 0.46 (0.07) a 0.42 (0.06) a 0.26 (0.04) b 29.68 <0.001
H. odorata Biomass (g DW)
Root 0.71 (0.34) a 0.96 (0.42) a 0.79 (0.46) a 0.90 0.421
Stem 0.73 (0.38) a 0.66 (0.32) ab 0.51 (0.39) b 10.97 <0.001Leaf 1.67 (0.74) a 1.42 (0.50) a 0.92 (0.49) b 13.42 <0.001Shoot 2.40 (1.12) a 2.07 (0.82) a 1.43 (0.87) b 13.06 <0.001Total 3.12 (1.43) a 3.03 (1.04) a 2.22 (1.31) b 6.24 0.006
Allocation (% of total biomass)
Root 0.23 (0.04) b 0.32 (0.09) a 0.35 (0.04) a 9.31 <0.001Stem 0.23 (0.03) a 0.21 (0.04) a 0.22 (0.03) a 0.73 0.493
Leaf 0.54 (0.03) a 0.47 (0.06) b 0.43 (0.05) b 14.23 <0.001
S. roxburghii Biomass (g DW)
Root 0.58 (0.17) a 0.60 (0.17) a 0.56 (0.18) a 0.18 0.836
Stem 0.63 (0.24) a 0.66 (0.17) a 0.44 (0.11) b 5.16 0.013Leaf 1.81 (0.63) a 1.83 (0.50) a 0.93 (0.28) b 11.87 <0.001Shoot 2.45 (0.86) a 2.49 (0.66) a 1.37 (0.37) b 10.20 <0.001Total 3.02 (0.97) a 3.09 (0.79) a 1.93 (0.53) b 7.31 0.003
Allocation (% of total biomass)
Root 0.20 (0.05) b 0.20 (0.03) b 0.29 (0.04) a 17.99 <0.001Stem 0.21 (0.03) a 0.22 (0.02) a 0.23 (0.03) a 1.62 0.216
Leaf 0.60 (0.03) a 0.59 (0.03) a 0.48 (0.03) b 52.07 <0.001
One-year-old seedlings were grown for 72 days with supply of nitrogen as ammonium, nitrate, or both. Means are presented, with standard
deviation in parentheses.F andP values of ANCOVA are presented as well. Means with significant treatment effect (P < 0.05) are shown in bold.
Different letters indicate significant difference between N forms (Tukey HSD, P < 0.05).
chlorophyll-a and -b contents were determined
according to Arnon (1949).
2.4. Leaf nitrogen
In Experiment II, the nitrogen content of the leaves
in which photosynthesis was measured was deter-
mined with an NC analyzer (NA1500, Carlo Erba).
Leaf discs (diameter, 4 mm) were taken from the
leaves and dried at 80 8C for further analysis.
Measurement was duplicated for each sample.
2.5. Nitrate reductase activity
The in vivo NRA of leaf and root samples was
determined according to Gebauer et al. (1998) with
some modifications. Ten 7-mm leaf discs were taken
from each of the leaves in which photosynthesis was
measured and immersed immediately in 5 mL of
incubation buffer in a 15-mL tube. Leaf discs were
incubated for 2 h at 28 8C in the dark in a N2
atmosphere. Incubation was terminated by boiling at
100 8C for 1 min. Then, 0.6 mL of 5% (w/v)
M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186 179
sulfanilamide in 3N HCl, 0.6 mL of 0.1% (w/v) N-
(1-naphthyl) ethylene-diamine-dihydrochloride, and
0.8 mL of Milli-Q water were added to 2 mL of
incubated solution, and the mixture was kept at
room temperature for 30 min. A540 was measured,
and the generated nitrite was determined against a
series of standards. About 200–300 mg of fresh fine
roots were chopped and similarly treated for NRA
measurement.
2.6. Statistical analysis
Generally, differences among treatments were
evaluated with ANOVA or Kruskal–Wallis test and
Scheffe’s test for each species. Analysis of covar-
iance was used for data obtained repeatedly from the
same seedlings over time. Differences in height
and diameter increment and leaf production among
N forms in Experiment II were tested with the
Tukey–Welsch method. Differences in biomass
were tested by ANCOVA with initial plant size
(d2h for leaf, stem, and total biomass and d2 for root
biomass) as the covariate. Data were log- or arcsine-
transformed as necessary to ensure homogeneity of
variance.
Fig. 1. Nitrogen formeffects on height increment of three dipterocarp
species:A. costata (a),H. odorata (b), and S. roxburghii (c). One-year-
old seedlings were grown with supply of nitrogen as ammonium,
nitrate, or both.Heights are expressed relative to initial size. Error bars
denote standard deviations. Different letters indicate significant dif-
ference between nitrogen forms at the end of the experiment (Scheffe,
3. Results
3.1. Experiment I
3.1.1. Growth
All the three dipterocarp species showed less
height growth when supplied with nitrate as a sole
nitrogen source (Fig. 1). Similar nitrogen form effects
were also observed in diameter growth (data not
shown). Aboveground biomass, especially leaf
biomass, was less when supplied with nitrate as a
sole nitrogen source (Table 2). Root biomass, on the
other hand, was less affected in H. odorata and S.
roxburghii seedlings or increased in A. costata
seedlings, resulting in higher root mass ratio for all
the three species when supplied with nitrate (Table 2).
Less growth ofH. odorata and S. roxburghii seedlings
supplied with nitrate was observed on total plant
biomass as well, while A. costata seedlings showed
similar total plant biomass between the two nitrogen
forms (Table 2).
P < 0.05).
M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186180
Fig. 2. Nitogen form effects on light-saturated photosynthetic rate
(c), andCO2-saturated light-saturated photosynthetic rate (Pnsat) (d) of
fully developed leaves in threedipterocarp species:Ac,A. costata;Ho,
H. odorata; Sr: S. roxburghii. One-year-old seedlingsweregrownwith
supply of nitrogen as ammonium, nitrate, or both and gas exchange
wasmeasured at 64 and 65DATunder ambientCO2 concentration and
at 71 and 72 DAT under saturated CO2 condition. Error bars denote
standard deviations. Different letters indicate significant difference
between nitrogen forms for each species (Scheffe, P < 0.05). NS: not
significant (ANOVA, P < 0.05).
Fig. 3. Nitrogen form effects on chlorophyll content of fully
developed leaves in three dipterocarp species: Ac, A. costata; Ho,
H. odorata; Sr, S. roxburghii. One-year-old seedlings were grown
with supply of nitrogen as ammonium, nitrate, or both for 72 days
and chlorophyll content was measured at the end of the experiment.
Chlorophyll content is shown as value from SPAD meter. Error bars
denote standard deviations. Different letters indicate significant
difference between nitrogen forms for each species (Scheffe,
P < 0.05).
3.1.2. Photosynthesis
All the three dipteorcarp species showed lower Pn
when supplied with nitrate as a sole nitrogen source,
which was accompanied with higher internal CO2
concentration (Ci) and lower CO2-saturated photo-
synthetic rate under saturated light (Pnsat; Fig. 2).
Stomatal conductance (gs) was not affected by the
nitrogen forms (Fig. 2b).
3.1.3. Chlorophyll
The chlorophyll content of fully developed leaves
was lower in the seedlings supplied with nitrate for all
the three dipterocarp species (Fig. 3).
3.2. Experiment II
3.2.1. Growth
D. obtusifolius and S. faguetiana seedlings showed
less height growth when supplied with nitrate as a sole
nitrogen source (Fig. 4). Diameter growth in the two
species was also less when supplied with nitrate (data
not shown). Aboveground biomass was less in the two
species when nitrate was supplied as a sole nitrogen
source (Table 3). In contrast, root biomass was
increased in D. obtusifolius or less affected in S.
faguetiana when supplied with nitrate as a sole
nitrogen source, resulting in higher root mass ratio
(Table 3). In contrast to the two species, N. heimii
seedlings showed similar growth between the two
nitrogen forms and more growth when supplied with
ammonium plus nitrate (Fig. 4; Table 3). Root mass
ratio of N. heimii seedlings was higher when nitrate
was supplied as a sole nitrogen source (Table 3).
3.2.2. Photosynthesis
D. obtusifolius and S. faguetiana seedlings showed
lower Pn when supplied with nitrate as a sole nitrogen
source (Fig. 5a). Both species showed lower Pnsat
when supplied with nitrate as a sole nitrogen source
(Fig. 5d). D. obtusifolius seedlings showed lower gs
M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186 181
Fig. 4. Nitrogen formeffects on height increment of three dipterocarp
species: D. obtusifolius (a), S. faguetiana (b), and N. heimii (c). 2.5-
month-old seedlings ofD. obtusifolius andN. heimii, and 9-month-old
S. faguetiana seedlings were grown with supply of nitrogen as
ammonium, nitrate, or both. Heights are expressed as relative to
initial size. Error bars denote standard deviations. Different letters
indicate significant difference between nitrogen forms at the end of the
experiment (Tukey–Welsch, P < 0.05). NS: not significant.
Fig. 5. Nitrogen form effects on light-saturated photosynthetic rate
(Pn) (a), stomatal conductance (gs) (b), internal CO2 concentration
(Ci) (c), and CO2-saturated photosynthetic rate (Pnsat) (d) of fully
developed leaves in three dipterocarp species: Do, D. obtusifolius;
Sf, S. faguetiana; Nh, N. heimii. 2.5-month-old seedlings of D.
obtusifolius and N. heimii and 9-month-old S. faguetiana seedlings
were grown with supply of nitrogen as ammonium, nitrate, or both
and gas exchange was measured at 123–126 DAT. Error bars denote
standard deviations. Different letters indicate significant difference
between nitrogen forms for each species (Scheffe, P < 0.05). NS:
not significant (P < 0.05).
(Fig. 5b) and higher Ci (Fig. 5c) when supplied with
nitrate, but S. faguetiana seedlings showed no
difference in the two parameters by nitrogen form
treatments (Fig. 5b and c). N. heimii seedlings showed
similar Pn and Pnsat between the two nitrogen forms
and highest Pn when supplied with ammonium plus
nitrate (Fig. 5a and d).
M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186182
Table 3
Nitrogen form effects on biomass (root, stem, leaf, shoot, and total) and allocation (root mass ratio, stem mass ratio, and leaf mass ratio) of three
dipterocarp species, D. obtusifolius, S. faguetiana, and N. heimii, at the end of the experiment
Species Parameter Treatment F P
NH4 NH4 + NO3 NO3
D. obtusifolius Biomass (g DW)
Root 1.53 (0.55) b 1.35 (0.54) b 2.06 (0.75) a 4.71 0.013Stem 0.95 (0.37) a 0.75 (0.29) b 0.58 (0.23) b 15.08 <0.001Leaf 3.46 (1.34) a 2.72 (1.09) a 1.31 (0.43) b 25.77 <0.001Shoot 4.41 (1.69) a 3.48 (1.37) a 1.89 (0.64) b 23.27 <0.001Total 5.94 (2.21) a 4.82 (1.86) ab 3.95 (1.32) b 9.69 <0.001
Allocation (% of total biomass)
Root 0.26 (0.03) b 0.29 (0.05) b 0.52 (0.06) a 161.44 <0.001Stem 0.16 (0.03) a 0.16 (0.02) a 0.15 (0.03) a 1.70 0.192
Leaf 0.58 (0.04) a 0.56 (0.05) a 0.33 (0.04) b 178.76 <0.001
S. faguetiana Biomass (g DW)
Root 1.01 (0.62) a 1.42 (1.44) a 0.84 (0.47) a 0.94 0.396
Stem 1.88 (1.33) a 2.53 (2.32) a 1.33 (0.76) a 2.65 0.080
Leaf 2.39 (1.88) a 3.40 (2.82) a 1.51 (1.18) a 2.45 0.096
Shoot 4.28 (3.15) ab 5.94 (5.08) a 2.84 (1.92) b 2.88 0.065
Total 5.29 (3.75) a 7.36 (6.48) a 3.68 (2.38) a 2.53 0.089
Allocation (% of total biomass)
Root 0.21 (0.05) ab 0.20 (0.04) b 0.24 (0.04) a 5.50 <0.001Stem 0.38 (0.08) a 0.35 (0.05) a 0.38 (0.06) a 0.87 0.424
Leaf 0.41 (0.12) a 0.45 (0.09) a 0.38 (0.08) a 2.68 0.078
N. heimii Biomass (g DW)
Root 0.47 (0.25) b 0.61 (0.27) ab 0.66 (0.14) a 3.46 0.039Stem 0.75 (0.30) b 0.97 (0.44) a 0.98 (0.29) a 4.47 0.016Leaf 1.11 (0.61) b 1.95 (0.97) a 1.22 (0.44) ab 6.96 0.002Shoot 1.86 (0.87) b 2.92 (1.38) a 2.20 (0.68) ab 7.11 0.002Total 2.33 (1.10) b 3.53 (1.62) a 2.85 (0.77) ab 4.36 0.018
Allocation (% of total biomass)
Root 0.20 (0.03) b 0.18 (0.03) b 0.24 (0.05) a 12.67 <0.001Stem 0.34 (0.07) ab 0.29 (0.07) b 0.34 (0.05) a 4.84 0.012Leaf 0.46 (0.08) b 0.54 (0.07) a 0.42 (0.07) b 13.50 <0.001
2.5-month-old seedlings of D. obtusifolius and N. heimii and 9-month-old S. faguetiana seedlings were grown for 127 days with supply of
nitrogen as ammonium, nitrate, or both. Means are presented, with standard deviation in parentheses. F and P values of ANCOVA are presented
as well. Means with significant treatment effect (P < 0.05) are shown in bold. Different letters indicate significant difference between N forms
(Tukey HSD, P < 0.05).
3.2.3. Chlorophyll and nitrogen
D. obtusifolius and S. faguetiana seedlings showed
lower chlorophyll content in leaves when nitrate was
supplied as a sole nitrogen source (Fig. 6a). N. heimii
seedlings showed similar leaf chlorophyll contents
between the two nitrogen forms (Fig. 6a). Chlorophyll-
a/b ratio was lower in D. obtusifolius and N. heimii
seedlingswhen suppliedwith nitrate, but not affected in
S. faguetiana seedlings (Fig. 6b). For all the three
species, leaf nitrogen content was lower when supplied
with nitrate as a sole nitrogen source (Fig. 6c).
3.2.4. In vivo nitrate reductase activity in leaves
and roots
In vivo NRA was detected in leaves of all three
species (Fig. 7a). S. faguetiana seedlings showed
higher leaf NRA when supplied with nitrate as a sole
nitrogen source, while N. heimii seedlings showed
lower leaf NRA under the condition (Fig. 7a). D.
obtusifolius seedlings showed no response in leaf
NRA to nitrogen form treatments (Fig. 7a). Similar or
lower levels of NRA were detected in roots than in
leaves of all three species (Fig. 7b). D. obtusifolius
M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186 183
Fig. 6. Nitrogen form effects on chlorophyll content (a), chloro-
phyll-a/b ratio (b), and nitrogen content (c) of fully developed leaves
in three dipterocarp species: Do, D. obtusifolius; Sf, S. faguetiana;
Nh, N. heimii. 2.5-month-old seedlings of D. obtusifolius and N.
heimii and 9-month-old S. faguetiana seedlings were grown for 127
days with supply of nitrogen as ammonium, nitrate, or both. Error
bars denote standard deviations. Different letters indicate significant
difference between nitrogen forms for each species (Scheffe,
P < 0.05). NS: not significant (ANOVA, P < 0.05).
Fig. 7. Nitrogen form effects on in vivo nitrate reductase activity
(NRA) in leaves (a) and roots (b) of three dipterocarp species: Do,D.
obtusifolius; Sf, S. faguetiana; Nh, N. heimii. 2.5-month-old seed-
lings of D. obtusifolius and N. heimii and 9-month-old S. faguetiana
seedlings were grown for 127 days with supply of nitrogen as
ammonium, nitrate, or both. Error bars denote standard deviations.
Different letters indicate significant difference between nitrogen
forms for each species (Scheffe, P < 0.05). NS: not significant
(ANOVA for leaf, Kruskal–Wallis for root, P < 0.05).
seedlings showed higher root NRA when nitrate was
supplied as a sole nitrogen source, while the other two
species showed no response in root NRA to nitrogen
forms (Fig. 7b).
4. Discussion
4.1. Growth of the six dipterocarp species with
different nitrogen forms
Except for N. heimii, all of the dipterocarp species
we examined showed growth reduction in shoots,
especially in leaves, when N was supplied as nitrate,
which indicates a preference of these species for
ammonium in shoot growth (Tables 2 and 3; Figs. 1 and
4). N. heimii seedlings did not show a clear preference
between the two nitrogen forms (Tables 2 and 3;
Figs. 1 and 4). N. heimii grows comparatively slowly
(Soerianegara and Lemmens, 1994). Its relatively low
demand for nitrogen might have hidden its preference
of nitrogen form. Bungard et al. (2000) reported the
involvement of nitrogen availability in the growth
response of dipterocarp species to gap formation, and
these investigators pointed out that nutrient conditions
affect regeneration dynamics and the distribution of