habit Kerbauy, 1984; Kerbauy et al., 1995. The Catasetum genus is comprised of species with C
3
photosynthetic metabolism and annual life cycles controlled by wet and dry periods Benzing,
1990. Nitrogen is one of the most limiting factors for
plant growth and plants have various mechanisms for maximizing N metabolism efficiency. Complex
systems of uptake, assimilation and mobilization usually avoid the waste of N and energy
Fernandes and Rossiello, 1995. Most vascular plants acquire nitrogen as NO
3 −
or NH
4 +
, the main available forms of this element in the soils
Marschner, 1995. However, preference for ni- trate or ammonium varies according to the plant
species, which is generally related to the physio- logical adaptations of plants to natural ecosys-
tems Adams and Attiwill, 1982.
Soluble organic nitrogen, including protein and free amino acids, is often of great importance for
plant N nutrition in diverse environments, from arctic to tropical communities Schmidt and
Stewart, 1999. The concentration of free amino acids can exceed that of inorganic nitrogen in
communities with low rates of mineralization. Plant species adapted to these environments grow
better in the presence of organic nitrogen Chapin et al., 1993; Schmidt and Stewart, 1999. Plants of
three species of epiphytic bromeliads efficiently used glutamine and NH
4
NO
3
as nitrogen sources Mercier et al., 1997.
Epiphytic plants are nutritionally diverse and usually found in acid organic substrate Benzing,
1990. The potential sources of nitrogen for these plants are the dry and wet atmospheric deposi-
tions NH
3
, NO
x
, N
2
fixation by associations with microorganisms Raven, 1988; Stewart et al.,
1995 and stemflow leachates Awasthi et al., 1995. The process of organic matter mineraliza-
tion and the associations between plant and ani- mals or plant and mycorrhizas also provide
nitrogen to the epiphytes Benzing, 1990; Stewart et al., 1995. However, scientific studies of mineral
nutrition and nitrogen metabolism of orchids are rather scarce Hew et al., 1993; Majerowicz,
1997. As C. fimbriatum is a fast growing epi- phytic orchid and develops very well in rotting
tree stems, we tested the hypothesis that it had a preference for organic nitrogen forms, such as
glutamine, over inorganic ones. The study of the activity of key nitrogen assimilating enzymes may
give valuable information on the ability of plant species
in using
different nitrogen
sources Stewart et al., 1988, 1992; Claussen and Lenz,
1999. This study examined the effects of inorganic
nitrogen and glutamine on: i the accumulation and partitioning of dry matter in shoots and roots,
ii concentrations of free amino-N, soluble carbo- hydrates, free ammonium, nitrate and chlorophyll
in plant tissues; and iii the activity of nitrate reductase NR, glutamine synthetase GS and
glutamate
dehydrogenase NADH-GDH
in shoots and roots of C. fimbriatum plants.
2. Material and methods
2
.
1
. Obtention of micropropagated plants Seedlings of C. fimbriatum Morren Lindl.
were obtained asymbiotically, as previously de- scribed by Colli and Kerbauy 1993. Both germi-
nation and seedling growth were conducted with a 16 h photoperiod under fluorescent lamps 40
m
mol m
− 2
s
− 1
and 25 9 2°C temperature. After about 3 months, a vigorous seedling was selected
and transferred to a culture flask containing 80 cm
3
of Murashige and Skoog culture media 1962, 0.1 mg dm
− 3
of 6-benzyladenine added, gelled with 1.6 g dm
− 3
of Phytagel and incubated in the dark. In this condition, C. fimbriatum
seedlings maintained the shoot apical meristem activity, giving rise to a whitish, slender and
lengthy stem structure. Hundreds of etiolated and leafless stem nodes are formed during dark incu-
bation Kerbauy et al., 1995. After 4 – 6 months, the etiolated stem structures were sectioned and
the node segments were transferred to flasks con- taining 80 cm
3
of Murashige and Skoog 1962 media and incubated with a 12 h photoperiod
with fluorescent lamps 120 mmol m
− 2
s
− 1
at 25 9 2°C. Light induces the development of lat-
eral bud of the stem node segments, originating normal plantlets after about 5 weeks of incuba-
tion under lightdark periods.
2
.
2
. Experimental treatments The basal culture media consisted of 20 g cm
− 3
of sucrose, 1.6 g cm
− 3
of Phytagel, macronutri- ents Vacin and Went, 1949 and micronutrients
Murashige and Skoog, 1962. The initial pH, adjusted to 6.0, dropped to 5.5 after 120°C20 min
autoclaving. The original nitrogen sources of Vacin and Went 1949 were modified, with the
final concentration being maintained at 6 mol m
− 3
. Two sets of experiments were conducted. In the
first group, the nitrogen sources used were NO
3 −
, NH
4 +
, NO
3 −
plus NH
4 +
2:3 ratio and glutamine. The solution of glutamine was filter sterilized
using a 0.45 mm pore membrane. In the second group, plants were grown in the presence of five
different NO
3 −
: NH
4 +
ratios: 1:0, 3:2, 1:1, 2:3 and 0:1. Each treatment consisted of three experimen-
tal flasks, each one containing nine plants in 80 cm
3
of culture media. Plants were maintained under a 12 h photope-
riod with daylight fluorescent lamps 120 mmol m
− 2
s
− 1
and 25 9 2°C temperature. After 30 days of incubation, plants were carefully rinsed
with distilled water, separated into shoot and root, and the fresh and dry weights were deter-
mined 70°C48 h. Shootplant ratio was calcu- lated as the ratio of shoot dry weight over total
plant dry weight. The relative growth rate was calculated as the ratio of dry weight difference
between dry weight at the end of 30 days of incubation and dry weight at the beginning of the
experiment over dry weight at the beginning of the experiment [Wt
30
− Wt
Wt ].
2
.
3
. Amino acids, chlorophyll, NO
3
−
and free NH
4
+
contents Plants from each treatment were rinsed in dis-
tilled water, blotted on filter paper and separated into shoot and root fractions. Each organ was cut
into small pieces and mixed in a Petri dish con- taining humid filter paper. Three 0.5 g fresh sam-
ples of shoot and root fragments were collected for determinations of free amino-N, soluble sug-
ars, chlorophyll, NO
3 −
and free NH
4 +
. Fresh samples were extracted in 80 vv etha-
nol for free amino-N analysis Yemm and Cock- ing, 1955 and soluble sugar determinations
Yemm and Willis, 1954. Chlorophyll tissue con- tents were measured spectrophotometrically after
extraction with pure acetone and calculated fol- lowing Lichtenthaler 1987. Aqueous extracts
from centrifuged homogenates were used for NO
3 −
and NH
4 +
determinations. NO
3 −
was deter- mined according to Cataldo et al. 1975, and
NH
4 +
was assayed by the phenol – hypochlorite reaction following Magalha˜es et al. 1992.
2
.
4
. Nitrogen assimilating enzymes After 30 days of culture in different nitrogen
sources, C. fimbriatum plants were harvested 2 – 3 h after the beginning of the light period, rinsed in
distilled water and blotted on filter paper. Plants were separated into shoot and root fractions and
fresh samples were collected for the determination of in vivo NR, GS and NADH-GDH activities
and soluble protein contents.
2
.
4
.
1
. NR acti6ity Leaves, bulbs, mature roots and the 2 cm apical
roots were cut out in small pieces, mixed thor- oughly, separated into three 0.3 – 0.5 g samples
which were transferred to test tubes containing 5 cm
3
of the incubation medium. NR activity was estimated by means of an in
vivo assay Jaworski, 1971. Incubating medium consisted of 0.1 mol m
− 3
potassium phosphate buffer pH 7.5, 0.05 mol m
− 3
KNO
3
and 3 n-propanol. Tissues were vacuum infiltrated three
times for 1 min and dark incubated for 1 h at 30°C. Aliquots of 1 cm
3
were then removed from the incubating medium and NO
2 −
was determined by adding 0.3 cm
3
1 sulphanilamide and 0.3 cm
3
0.2 N-1-naphthyl
ethylene-diamine. Ab-
sorbance was read at 540 nm after 30 min. NR activity values were expressed as nmoles NO
2 −
g
− 1
fresh weight h
− 1
.
2
.
4
.
2
. GS and NADH-GDH acti6ities Triplicate fresh tissue samples 1 g from shoot
and root were homogenized in liquid nitrogen. The fine powder obtained was transferred to cold
centrifuge tubes, adding 5 cm
3
of 0.05 mol m
− 3
imidazole pH 7.9 buffer containing 0.005 mol m
− 3
ditiothreitol. The homogenates were cen-
trifuged 30 min at 15000 g. The supernatants were collected and centrifuged again for 10 min at
15000 g, kept in ice and used for GS and NADH- GDH assays as well as for the determination of
soluble protein contents according to Bradford 1976.
GS was assayed following Pe`rez-Soba et al. 1994. The standard assay mixture 0.5 cm
3
con- sisted of 0.1 mol m
− 3
imidazole buffer pH 7.5, 0.048 mol m
− 3
NH
2
OH, 0.040 mol m
− 3
MgCl
2
, 0.32 mol m
− 3
glutamate, 0.05 mol m
− 3
ATP. Adding 0.15 cm
3
of crude enzyme extract started the reaction. After incubation for 1 h at 30°C, the
d -glutamyl-hydroxamate GH formed was deter-
mined by adding 1.0 cm
3
of ferric chloride reagent 0.37 mol m
− 3
FeCl
2
, 0.67 mol m
− 3
HCl, and 0.2 mol m
− 3
trichloroacetic acid and measuring ab- sorbance at 540 nm with a spectrophotometer.
Activity of GS was expressed as mmoles of GH formed per g
− 1
fresh weight fr. wt. h
− 1
and specific activity of GS as mmoles GH formed
mg
− 1
protein h
− 1
. GDH was assayed by means NADH oxidation
at 30°C according to Cammaerts and Jacobs 1985. The 3.0 cm
3
assay mixture consisted of 0.1 mol m
− 3
Tris – HCl buffer pH 8.2, 0.15 mol m
− 3
NH
4 2
SO
4
, 0.02 mol m
− 3
a -ketoglutarate,
0.001 mol m
− 3
CaCl
2
, and 0.14 mol m
− 3
NADH. Adding 0.5 cm
3
of the crude enzyme extract started the reaction. NADH consumption was
followed spectrophotometrically at 340nm. The blank did not have a-ketoglutarate. GDH activity
was expressed as mmoles NADH g
− 1
fr. wt. min
− 1
.
2
.
5
. Statistical analysis Each experimental treatment consisted of three
replicates, each one containing nine plants. The experiments described in Section 2.2 were carried
out twice and the results represent the mean of two experiments. Therefore, there were 54 values
per growth parameter per treatment n = 54 and six replicates per biochemical analysis per treat-
ment n = 6 Data were statistically analyzed us- ing the one-way analysis of variance. Tukey’s
multiple range test was used to compare the means of all nitrogen treatments. In the tables and
graphic representations, values marked with dif- ferent letters are different at
P B 0.05.
3. Results
