plastic and cytoplasmic compartments of leaves contain these enzymes. The two important antioxi-
dant enzymes in chloroplasts are SOD and APX, which allow the efficient detoxification of ROS
generated from photooxidation [7]. Though many studies have focused on the antioxidant responses
of plants to environmental stresses [4], not much work has been devoted to study such changes in
relation to leaf development.
In the present study, the photosynthetic perfor- mance and antioxidant responses of phyllodes ex-
panded petioles that form simple, photosynthezing laminae [8] of 220 day-old A. mangium Willd.
seedlings were investigated. The aims of this study were to understand the distribution of phyllode
photosynthetic capacity in seedlings and its rela- tionship with the in vivo PSII photosystem II
function and antioxidant response. A. mangium is a fast growing tree, with an increasingly important
role in reforestation programs. This study should enable an understanding of the physiological fac-
tors that contribute to the rapid growth and estab- lishment of the seedlings.
2. Material and methods
2
.
1
. Growth of seedlings and site description Seeds of A. mangium were collected from
Hainan Province, P.R. China. Seedlings were grown under natural conditions in the Department
of Biological Sciences, National University of Sin- gapore. The temperature was 29 9 6°C. Daily at-
mospheric relative humidity ranged from 70 to 100. The daily photosynthetic photon flux den-
sity PPFD ranged from a minimum of 80 mmol m
− 2
s
− 1
at 08:00 and 18:00 h to 1650 mmol m
− 2
s
− 1
at 12:00 h. Seeds were immersed in boiling water for 30 s
and then soaked in cold water for 24 h to facilitate uniform germination. Pretreated seeds were sown
in vermiculite in plastic pots diameter 32.5 cm, height 26.0 cm. Three to four seeds were sown in
each pot. Plants were kept well watered and were fertilized twice a week with Hoagland’s solution.
Experiments were conducted when the plants were 220-days-old and 65 cm tall, on average. All
experiments were repeated three times. A total of six seedlings were used for each experiment.
2
.
2
. Position of phyllodes Seedlings were selected for experiments based
on their uniform appearance, in terms of the aver- age height of the plants, the total number of
phyllodes present on each plant and the size of emerging phyllodes. The emerging phyllode at the
shoot apex, with a length of 5 – 6 cm, was consid- ered as the phyllode at position 1 phyllode 1.
Phyllodes 2, 4, 6, 8, 10 and 12 were labeled in all seedlings, counting down from the top of the
plant. Seedlings used for experiments had a total of 13 – 14 phyllodes.
2
.
3
. Determination of photosynthetic pigments Six discs diameter 55 mm were randomly re-
moved from each phyllode and were ground with 100 vv acetone. The concentrations of chloro-
phylls and carotenoids were determined by the methods of Arnon [9] and Embry and Nothnagel
[10], respectively.
2
.
4
. Gas exchange measurements Changes in photosynthetic CO
2
exchange of phyllodes at different positions still attached to
plants, in response to changes in PPFD levels, were determined with a CIRAS-1 portable differ-
ential CO
2
H
2
O infrared gas analyzer connected to a Parkinson broad leaf cuvette equipped with an
automatic light unit PP Systems, Hitchin, UK. A phyllode area of 2.5 cm
2
was enclosed in the leaf cuvette. Gas exchange rates were determined at an
airflow rate of 200 cm
3
min
− 1
with ca. 365 mmol mol
− 1
CO
2
in the cuvette and a constant leaf temperature of 29°C. After the determination of
dark respiration rate, the rates of CO
2
exchange over a range of PPFD from 25 to 1400 mmol m
− 2
s
− 1
were determined.
2
.
5
. Chlorophyll fluorescence measurements Chlorophyll fluorescence was determined with a
pulse amplitude modulation fluorometer PAM 101 – 103, Waltz, Effeltrich, Germany; experi-
ments were conducted at 25°C. Following a 30- min dark adaptation, F
o
was determined at a light level lower than 0.01 mmol m
− 2
s
− 1
. After F
o
was recorded, a 600-ms saturating light pulse 5000
mmol m
− 2
s
− 1
was given and F
m
was determined.
Saturating light was supplied by a Schott flash lamp KL 1500, Schott, Mainz, Germany. After
the measurement of F
o
and F
m
, the actinic light 300 mmol m
− 2
s
− 1
was switched on. To deter- mine maximum fluorescence F
m
under light-satu- rating conditions, a 600-ms saturation light pulse
of 5000 mmol m
− 2
s
− 1
was switched on at 60 s intervals. After 30 min, the actinic light was
switched off and a far-red light was turned on for the accurate measurement of F
o
. Values of various chlorophyll fluorescence parameters were calcu-
lated according to Van Kooten and Snel [11] and Genty et al. [12].
2
.
6
. Phyllode area and dry weight After the measurement of chlorophyll fluores-
cence, the phyllode was used for the determination of phyllode area and dry weight. For the determi-
nation of phyllode area, the outline of each lamina was traced on graph paper and weighed. Phyllode
area was calculated according to an area-weight standard curve. The phyllodes were dried in an
oven at 80°C for 7 days until dry weight was constant.
2
.
7
. Determinations of enzyme acti6ities and total soluble protein content
For the determination of total SOD activity, fresh phyllode tissues were removed from the mid-
dle portion of each phyllode and ground in a chilled mortar in 50 mM phosphate buffer pH
7.8, containing 0.1 mM EDTA, for the extraction of total SOD enzyme. Similarly, for the extraction
of total APX, phyllode tissues were ground in 50 mM phosphate buffer pH 7.0 containing 0.1 mM
EDTA, 5 mM ascorbate, 0.5 wv PVP molecu- lar weight 10 000, 0.1 vv Triton X-100 and
0.05 vv b-mercaptoethanol. After centrifuging at 11 700 × g at 4°C for 15 min, the supernatant
of each extract was used as the crude enzyme extract for determinations of soluble protein con-
centration and activities of SOD and APX.
The SOD activities of phyllodes 1, 4, 8 and 12 were assayed by determining the ability of the
extracted enzymes to inhibit the photochemical reduction of nitro-blue tetrazolium NBT using
the method of Beauchamp and Fridovich [13]. The 3-ml reaction mixture contained 50 mM phosphate
buffer pH 7.8, 0.1 mM EDTA, 56 mM NBT, 13 mM methionine, 0.02 mM NaCN, 1.2 mM ri-
boflavin and 15 ml enzyme extract. The reaction was started by illuminating the reaction mixture
PPFD = 120 mmol m
− 2
s
− 1
at 28°C for 15 min. The absorbance of the reaction mixture at 560 nm
was then recorded. One unit of SOD U was defined as the amount of SOD that caused 50
inhibition of the photo-reduction of NBT [13].
The APX activities of phyllodes 1, 4, 8 and 12 were determined according to Nakano and Asada
[14] by monitoring the rate of ascorbate oxidation at 290 nm extinction coefficient = 2.8 mM
− 1
cm
− 1
. The 3-ml reaction mixture contained 50 mM potassium phosphate buffer pH 7.0, 0.1 mM
EDTA, 0.5 mM ascorbate, 0.3 mM H
2
O
2
and 40 ml enzyme extract. One unit of APX U was
defined as the amount of enzyme that oxidized 1 mmol of ascorbate per min at room temperature
[14].
The soluble protein concentration was measured by the protein – Coomassie dye binding method
using bovine serum albumin as a standard [15].
2
.
8
. Statistical analysis Each result is presented as the mean 9 S.E.
Multiple analysis of variance Tukey test, P 5 0.05, n = 6 was conducted to analyze the effect of
leaf position
on the
different physiological
parameters determined.
3. Results and discussion
