Nitrogen Form Preference of Six Dipterocarp Species ada 2004)

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.

# 2005 Elsevier B.V. All rights reserved.

Keywords: Nitrate; Ammonium; Growth; Photosynthesis; Nitrate reductase; Dipterocarpaceae

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Forest Ecology and Management 216 (2005) 175–186

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

* 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

(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

.

M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186176

likely will also be important for seedling establish-

ment (Kronzucker et al., 1997). The nutritional

characteristics of trees are of great importance in

silviculture as well. Nevertheless, our knowledge of

the influence of nutrient conditions on tropical tree

species is less than that of light and water (Whitmore,

1996). Nutritional studies in dipterocarp species are

mostly limited to some fertilizer experiments (e.g.,

Fetcher et al., 1996; Gunatilleke et al., 1997).

However, a study by Bungard et al. (2000) showed

the effects of nitrogen availability on the photosyn-

thetic characteristics of four dipterocarp species under

different light regimes and on responses of these

characteristics to sudden changes of light regime. The

results suggested the importance of variations in

nitrogen availability in regeneration dynamics and in

the distribution of canopy-dominating dipterocarp

species.

Both the amount and the form of nitrogen affect

tree growth by affecting nitrogen uptake. Nitrate and

ammonium are the major inorganic forms of nitrogen

taken up by plant roots (Marschner, 1995). Ammo-

nium can be readily assimilated into amino acids, but

nitrate has to be first reduced to ammonium via

nitrate reductase followed by nitrite reductase. In

studies of closed- and open-forest communities of

Australian rainforests, Stewart et al. (1988, 1990)

reported low levels of nitrate reductase in the roots

and shoots of most of the closed-forest species

examined but high levels in the leaves of pioneer

species.

In the field, the soil nitrogen regime depends on

climate, soil type, vegetation, and microenvironment

(Vitousek and Matson, 1988; Maggs, 1991; Smith

et al., 1998; Silver et al., 2000). The composition of

nitrogen can vary over time (Maithani et al., 1998) and

change in response to disturbances (Vitousek et al.,

1989). Investigations of nitrogen composition changes

with succession have found ammonium to dominate as

a result of low nitrification at late successional stages

(Attiwill and Adams, 1993). Studies of changes of

nitrogen composition after burning or clear-cutting

have reported an increase in nitrogen mineralization

and nitrification, after such disturbances (Matson

et al., 1987; Attiwill and Adams, 1993). Most of the

limited studies of tropical tree species have examined

the effects of quantity (e.g., Lawrence, 2001) but not

quality of nitrogen. Consequently, responses of

tropical tree species to qualitative changes in soil

nitrogen remain to be shown.

Understanding of nitrogen characteristics of dip-

terocarps is essential for clarifying mineral cycling

and species establishment in tropical forests in

Southeast Asia and for development of silvicultural

techniques. The effects of nitrogen on dipterocarps,

however, have been reported only quantitatively (e.g.,

Mirmanto et al., 1999; Bungard et al., 2000, 2002), not

qualitatively. Given that dipterocarps are climax-

forest species, it would seem that they should prefer

ammonium to nitrate, as in the case of Australian

rainforest species. However, considering that the

Dipterocarpaceae consist of nearly 500 species with a

broad range of light demand (Symington, 1974),

variation in nitrogen characteristics cannot be ruled

out. Here, we report responses of growth and

photosynthesis to ammonium, nitrate, or a mixture

in six dipterocarp species: Anisoptera costata Korth.,

Dipterocarpus obtusifolius Teijsm. ex Miq., Hopea

odorata Roxb., Neobalanocarpus heimii (King) P.

Ashton, Shorea faguetiana Heim, and Shorea roxbur-

ghii G. Don. All the six species are distributed in

Thailand and chosen because of the availability of

seeds. The objective of the study was to clarify

whether each species prefers ammonium to nitrate for

shoot growth. Owing to the irregular fruiting habit of

dipterocarps, we carried out two independent experi-

ments with three species each. In Experiment II, we

assessed in vivo nitrate reductase activities (NRA),

growth, and photosynthesis.

2. Materials and methods

2.1. Plant materials and treatments

2.1.1. Experiment I

Seeds of three dipterocarp species—A. costata, H.

odorata, and S. roxburghii—were collected in south-

ern Thailand and sown in a greenhouse (28/25 8C,natural light) in Tokyo, Japan. One-year-old seedlings

were transplanted into a 1/10000-a Wagner pot

(100 cm2 surface area and 18.5 cm depth) in sand.

Two to three weeks after transplanting, 10 pots of each

species were set in each of six watering systems.

Seedlings were watered with a nutrient solution at the

level of sand surface of pots, differing in nitrogen

M. Norisada, K. Kojima / Forest Ecology and Management 216 (2005) 175–186 177

Table 1

Nutrient solution composition of the three nitrogen form treatments

Treatment

NH4 NH4 + NO3 NO3

NaNO3 0 0 0.3

KNO3 0 1.5 3

Ca(NO3)2�4H2O 0 0.25 0.35

(NH4)2SO4 2 1 0

Na2SO4�10H2O 0.15 0.15 0

K2SO4 1.5 0.75 0

CaCl2�2H2O 0.35 0.1 0

MgSO4�7H2O 0.25 0.25 0.25

NaH2PO4�2H2O 0.6 0.6 0.6

FeSO4�7H2O 0.01 0.01 0.01

H3BO3 0.02 0.02 0.02

MnCl2�4H2O 0.002 0.002 0.002

ZnSO4�7H2O 0.002 0.002 0.002

CuSO4�5H2O 0.002 0.002 0.002

Na2MoO4�2H2O 0.0005 0.0005 0.0005

CoCl2�6H2O 0.0005 0.0005 0.0005

Concentrations are in mM.

form, three times a day for 15 min. Three treatments of

different forms of 4 mM nitrogen were provided:

ammonium, nitrate, and both. The nutrient solution

composition is shown in Table 1. All nutrient contents

other than nitrogen, sulfur, and chloride were the same

among the treatments. The pH of nutrient solutions

was adjusted to 5.8 with HCl or NaOH. Nutrient

solutions were replaced once a week. Two watering

systems were provided for each treatment, and

watering systems were exchanged within each of

the treatments 3 days after solution refreshment and

exchanged across the treatments every week to

eliminate effects of watering system. Pots were

rotated within each watering system every 3–4 days

to eliminate the effects of location effects. Treatment

was continued for 72 days. Height and diameter of

stem were measured once a week. At 41 and 42 days

after treatment (DAT), photosynthesis at ambient CO2

concentration under saturated light was measured on

fully developed leaves of 10–15 seedlings per

treatment, then all seedlings in one watering system

in each treatment were harvested for dry weight

measurement. The harvested seedlings were separated

into leaf, stem and root, then dried to a constant weight

at 80 8C for biomass determination. The remaining

seedlings were divided among the two watering

systems per treatment for further growth. Photosynth-

esis of fully developed leaves under saturated light was

measured at ambient CO2 concentration and at

saturated CO2 concentration at 64 and 65 DAT and

at 71 and 72 DAT, respectively.

2.1.2. Experiment II

Seeds of three dipterocarp species—D. obtusifo-

lius, N. heimii, and S. faguetiana—were collected in

southern Thailand and sown in a greenhouse as above.

Each 2.5-month-old seedling of D. obtusifolius and N.

heimii and each 9-month-old seedling of S. faguetiana

was transplanted into a 1/10000-a Wagner pot in sand.

The same three nitrogen treatments as in Experiment I

were applied for 127 days. Height and diameter of

stem were measured once a week. The number of

newly developed leaves was counted weekly as well.

At the end of the experiment, photosynthesis was

measured at ambient and saturated CO2 concentration

in a fully developed leaf in each of 10 seedlings per

treatment (123–126 DAT), and NRAwas measured in

leaves and roots (127 DAT). Plants were then

harvested and separated into leaf, stem, and root as

above for dry mass determination.

2.2. Photosynthesis

Photosynthesis of fully developed leaves was

measured with a portable photosynthesis system

(LI6400, Li-Cor). Light-saturated photosynthetic rates

were measured at 350 mmol mol�1 CO2 concentration

and 1000 mmol m�2 s�1 PAR. Photosynthetic capa-

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


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


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


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)


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).


Fig. 2. Nitogen form effects on light-saturated photosynthetic rate

(Pn) (a), stomatal conductance (gs) (b), internalCO2 concentration (Ci)

(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


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


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).


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


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


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


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


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


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

canopy-dominating dipterocarp species. Considering

that nutrient regime varies across and within site,

nutritional traits would also play a role in ecological

patterns of distribution, which have beenmostly related

to physiological traits in terms of light and water

acquisition. Since nutrient availability is determined by

both amount and form, the nitrogen form regime seems

also to play an important role in these ecological

aspects. Information about natural habitat relating to

soil nitrogen regime is lacking for the examined species

so far, but the outcome of this studywill shed a new light


on their distribution and regeneration in future research.

An increase in the ratio of ammonium to nitrate with

succession progress has been reported in tropical

(Vitousek and Matson, 1988) and temperate forests

(e.g., Robertson and Vitousek, 1981). Stewart et al.

(1988, 1990) reported a low level of nitrate assimilation

in closed forest species in Australia. A preference for

ammonium would be an advantage at sites where

ammonium dominates. On the other hand, given that

soil nitrate content increases after disturbance (e.g.,

Vitousek et al., 1982; Denslow et al., 1998), a low

ability to use nitrate would be a disadvantage in

competition for nitrogen.

Nitrate caused a higher root mass ratio in seedlings

of all the examined species (Tables 2 and 3). A higher

root mass ratio under nitrogen-limiting conditions has

been well documented (e.g., Andrews et al., 1999; Cruz

et al., 2003a; de Groot et al., 2003; Nguyen et al., 2003;

for review, see Andrews et al., 2001). More photo-

assimilate distribution to roots could have compensated

for the lower nitrogen uptake when ammonium-

preferring species were supplied with nitrate, while it

reduced carbon gain at the same time. Interestingly, N.

heimii, which showed no apparent preference between

ammonium and nitrate, also showed higher root mass

ratio when supplied with nitrate as a sole nitrogen

source (Table 3), while the reason is unclear.

4.2. Photosynthesis of six dipterocarp species

with different nitrogen forms

The five dipterocarp species which showed pre-

ference for ammonium had a higher light-saturated

photosynthetic rate with ammonium supply (Figs. 2a

and 5a). This is likely the cause of the better growth

of these species when nitrogen was supplied as

ammonium. Lower photosynthetic rate in leaves of the

five species supplied with nitrate was not accompanied

by lower stomatal conductance (Figs. 2b and 5b),

indicating reduction in photosynthetic capacity as the

cause of the lower photosynthetic rate. An impairment

in photosynthetic capacity was suggested by the

higher internal CO2 concentration (Figs. 2c and 5c)

and apparently revealed by the lower CO2-saturated

photosynthetic rate (Figs. 2d and 5d) with nitrate

supply. A higher internal CO2 concentration suggests a

lower carboxylation efficiency resulting from lower

rubisco activity. A lower CO2-saturated photosyn-

thetic rate indicates lower RuBP regeneration rate and/

or lower rubisco activity. Lower rubisco activity in

seedlings supplied with nitrate likely resulted from

lower rubisco content, since the rubisco activation

state increases as leaf nitrogen decreases (Cheng and

Fuchigami, 2000).

The effects of nitrogen form on leaf chlorophyll

content correspond well with those on photosynthetic

rate (Figs. 2(a and d), 3, 5(a and d), 6a). It is not clear

whether lower chlorophyll content is a cause or a

result of the lower photosynthetic activity. In any case,

although it was examined in only three of the six

species, the lower leaf nitrogen content with nitrate

supply (Fig. 6c) suggests that insufficient nitrogen

uptake caused by inability to assimilate nitrate would

be a cause of the lower chlorophyll content with nitrate

supply. The lower chlorophyll-a/b ratio in leaves ofD.

obtusifolius and N. heimii seedlings supplied with

nitrate resulted from a greater decrease in chlorophyll-

a than in chlorophyll-b. The response of the

chlorophyll-a/b ratio to nitrogen limitation is not

well documented, and contradictory results have been

obtained so far. A decrease in the chlorophyll-a/b ratio

under nitrogen deficiency was reported in cassava

(Manihot esculentaCrantz) plants (Cruz et al., 2003b),

but a common response of increased chlorophyll-a/b

ratio under nitrogen deficiency was reported in four

tropical tree species (Kitajima and Hogan, 2003).

4.3. Nitrate assimilation ability of three

dipterocarp species

NRAwas detected in leaves and roots, with similar

or somewhat higher levels in leaves than in roots of all

three species examined (Fig. 7). The levels of NRAs in

roots and leaves are comparable to those of closed-

forest species in Australia (Stewart et al., 1988, 1990).

The ratio of leaf NRA to root NRA differs among

species and may change depending on nitrogen

availability (Downs et al., 1993). Ratios of leaf to

root nitrate reductase decreased in D. obtusifolius and

increased in S. faguetiana seedlings with nitrate

supply (data not shown). Leaf nitrate reduction uses a

photosynthetic derivative reductant, whereas root

nitrate reduction uses a reductant produced through

carbohydrate breakdown. The increase in the leaf to

root nitrate reductase ratio in S. faguetiana may have

some advantage in this context.


Although nitrate reductase is a substrate-inducible

enzyme (Sivasankar and Oaks, 1996), such induction

was not found except in leaves of S. faguetiana and

roots of D. obtusifolius. Stewart et al. (1988) also

reported low inducibility of nitrate reductase in

closed-forest species, whereas the pioneer species

they tested showed substrate induction of the enzyme.

Soil nitrate content increases after disturbance

(Vitousek et al., 1982; Brouwer and Riezebos,

1998). Even though S. faguetiana was revealed to

prefer ammonium, the increase in leaf nitrate

reductase in response to nitrate may have some

advantage by consuming excess light energy when

leaves are exposed to excess light by gap formation.

Lavoie et al. (1992) concluded that nitrate uptake and

not nitrate assimilation via nitrate reductase limited

the growth of jack pine (Pinus banksiana Lamb.) with

nitrate as the sole N source. We cannot tell from our

results whether assimilation via nitrate reductase

limited the growth of the five dipterocarp species when

nitrate was supplied as the sole N source. Compre-

hensive investigation of the use of nitrogen in different

forms is needed in order to address this question.

In conclusion, we showed that all of the dipterocarp

species examined except for N. heimii prefer ammo-

nium to nitrate as a nitrogen source. The greater growth

with supply of ammonium was due to greater

photosynthesis, whichwas the result of greater nitrogen

absorption. Futurework should examine the response of

the species to different forms of nitrogen at lower

concentrations. Physiological aspects of nitrogen

uptake and assimilation have recently been well

documented in temperate tree species, especially in

coniferous species (e.g., Kronzucker et al., 1996).

Further research on nitrogen characteristics of dipter-

ocarp species with the help of knowledge and tools

derived from studies of temperate tree species would be

important for a better understanding of tropical forests

and for reforestation in Southeast Asia.

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Nitrogen Form Preference of Six Dipterocarp Species ada 2004)