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Growth and nitrogen metabolism of

Catasetum fimbriatum

(orchidaceae) grown with different nitrogen sources

N. Majerowicz

_{*, G.B. Kerbauy}

_{, C.C. Nievola}

_{, R.M. Suzuki}

b a_{Department of Physiological Sciences}_,_{Institute of Biology}_,_{Federal Rural Uni}

6ersity of Rio de Janeiro,

Rod.BR465–Km7 (Km47-Antiga Rio-SP),Serope´dica,RJ.CEP23.890-000,Brazil b_{Department of Botany}_,_Uni

6ersity of Sa˜o Paulo,PO Box11461,Sa˜o Paulo,SP,CEP05422-970,Brazil

Received 19 January 2000; received in revised form 11 May 2000; accepted 30 June 2000

Abstract

Catasetum fimbriatumis an epiphytic orchid from South America that has been used for 15 years as a model plant for metabolic and developmental studies in our laboratory. In this work,C.fimbriatumplants were aseptically grown

with 6 mol m−3 _{of either glutamine or inorganic nitrogen forms (NO}

−_:NH

+ _{ratios). The highest biomass}

accumulation was found in plants supplied with glutamine; no significant difference was observed in plants incubated in the presence of inorganic nitrogen sources. Nitrogen assimilation was limited in the presence NO3− as a sole

nitrogen source.C.fimbriatumdid not accumulate NO3−and very low rates of in vivo nitrate reductase activity were

observed. Most nitrate reductase activity (70%) was detected in the 2 cm apical roots. Nitrate-treated plants exhibited relatively lower amounts of free amino-N, chlorophyll and free NH4+contents and higher soluble sugar contents than

the NH4+-treated plants. While shoot glutamine synthetase activity was only slightly affected by nitrogen sources, root

glutamine synthetase activity was not modified by any nitrogen form. Glutamate dehydrogenase-NADH activity in shoot tissues was not influenced by any nitrogen source. However, the glutamate dehydrogenase-NADH activity in

roots was enhanced when NH4+ tissue contents was augmented by increasing NH4+ in the medium and by the

presence of glutamine. Our results strongly suggest that organic nitrogen and NH4+are probably the most important

Keywords:Ammonium; Glutamine; Nitrate; Nitrogen assimilation; Orchid

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1. Introduction

Catasetum fimbriatum (Morren) Lindl. is an

ornamental epiphytic orchid found in the south-east and central-west of Brazil, Bolivia and Paraguay (Bicalho, 1965). It is an especially inter-esting native orchid for physiological – biochemi-cal studies because of its relatively fast growth

Abbre6iations:Amino N: free aminoacids; GH:d

-glutamyl-hydroxamate; GS: glutamine synthetase; NADH-GDH: NADH-dependent glutamate dehydrogenase; NR: nitrate re-ductase; ptn: protein.

* Corresponding author.

E-mail address:[email protected] (N. Majerowicz).

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habit (Kerbauy, 1984; Kerbauy et al., 1995). The

Catasetum genus is comprised of species with C3

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 NO3− or NH4+, 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 NH4NO3 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 (NH3, NOx), N2 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 associamineraliza-tions 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

mmol 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 cm3 _{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 cm3 _{of Murashige and Skoog (1962)}

media and incubated with a 12 h photoperiod with fluorescent lamps (120 mmol m−2 _s−1_{) at}

2592°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 light/dark periods.

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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 micronutrimacronutri-ents (Murashige and Skoog, 1962). The initial pH, adjusted to 6.0, dropped to 5.5 after 120°C/20 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 NO3

−

, NH4

, NO3

−

plus NH4

(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 NO3

−_{: NH}

+_{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 cm3 _{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°C/48 h). Shoot/plant 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 [(Wt30−Wt0)/Wt0].

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, NO3− and free NH4+.

Fresh samples were extracted in 80% (v/v) 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 NO3− and NH4+ determinations. NO3− was

deter-mined according to Cataldo et al. (1975), and NH4+ 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 cm3 _{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 cm3 _{were then removed from}

the incubating medium and NO2−was determined

by adding 0.3 cm3

1% sulphanilamide and 0.3 cm3

0.2% N-(1-naphthyl) ethylene-diamine. Ab-sorbance was read at 540 nm after 30 min. NR activity values were expressed as nmoles NO2

−

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

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centrifuge tubes, adding 5 cm3

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 15000g. The supernatants were

collected and centrifuged again for 10 min at 15000g, 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 cm3

) con-sisted of 0.1 mol m−3

imidazole buffer (pH 7.5), 0.048 mol m−3 _NH

2OH, 0.040 mol m−3 MgCl2,

0.32 mol m−3 _{glutamate, 0.05 mol m}−3 _ATP.

Adding 0.15 cm3 _{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 cm3_{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 cm3_{assay mixture consisted of 0.1}

mol m−3 _{Tris – HCl buffer (pH 8.2), 0.15 mol}

m−3 _(NH

4)2SO4, 0.02 mol m

−3

a-ketoglutarate, 0.001 mol m−3

CaCl2, and 0.14 mol m

−3

NADH. Adding 0.5 cm3

of the crude enzyme extract started the reaction. NADH consumption was followed spectrophotometrically at 340nm. The blank did not havea-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 difdif-ferent at *_P_B_0.05.

3. Results 3.1. Plant growth

After 30 days of incubation in different nitro-gen sources, the highest plant growth rates were found in glutamine treated-plants (Table 1). Plant dry matter accumulation in the presence of glu-tamine was about 30 to 63% higher than plants grown in inorganic nitrogen. Dry matter parti-tioning to plant parts was slightly affected by glutamine and inorganic nitrogen sources. The shoot/plant ratio indicated a tendency of dry mat-ter accumulation mainly in the shoots, which was enhanced by glutamine (Table 1).

Table 1

Effects of inorganic and organic nitrogen sources (6 mM) on the dry matter accumulation, height, and root length ofC.fimbriatum plants (n=54) after 30 days of incubation (NO3−:NH4+ratio=2:3)a

Length (cm) Dry matter (mg)

Root Plant

Nitrogen sources Shoot Root Shoot Shoot/Plant ratio

10.690.5b _5.6₉_0.2b

NO3− _5.0₉_0.4ab _3.4₉_0.3b _3.1₉_0.2a _0.53₉_0.02b

12.990.8b _0.52₉_0.02b

NO3−:NH4+ 6.790.4b 6.290.5ab 3.590.2b 3.590.2a

0.5790.02ab

3.090.2a

3.590.2b

4.390.2b

NH4+ 10.390.5b 6.090.4b 16.891.1a _10.5₉_0.6a

Glutamine 6.490.6a _4.2₉_0.2a _3.1₉_0.2a _0.63₉_0.01a

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Fig. 1. Relative growth rates (Wt30−Wt0/Wt0) of entire plants, shoot and root ofC. fimbriatumplants (n=54) incu-bated in different NO3−:NH4+ratios. Bars represent the stan-dard error.

NH4

+ _{concentration was raised (Fig. 2C and Fig.}

3). The maximum free ammonium level was ob-served in the root of plants cultivated with glu-tamine (Fig. 2C).

3.3. Nitrate tissue contents

The methodology used for nitrate ion determi-nation in this work (Cataldo et al., 1975) was not accurate enough to detect the presence of this ion in shoot and root tissues of C.fimbriatum plants, even in those plants grown with NO3− as sole

nitrogen source.

3.4. Chlorophyll contents

Higher concentration of NO3

−_{than NH}

+_{in the}

media reduced shoot chlorophyll content (Fig. 4). The levels of root chlorophyll represented about 20 – 27% of the shoot chlorophyll contents. Maxi-mum root chlorophyll amounts were observed in plants incubated with a 1:1 (NO3−: NH4+) ratio.

3.5. Nitrogen assimilating enzymes acti6ity 3.5.1. NR

In vivo NR activity was detected only in plants grown in NO3

−

added media (Table 2). NH4

ion inhibited NR activity when its concentration was higher than the NO3

−_{concentration in the culture}

media (NO3

−_:NH

+ _{2:3 ratio). In the other three}

NO3

−_:NH

+ _{proportions, the activity of NR was}

quantitatively similar and showed the same pat-tern of distribution among plant parts (Table 2). About 70% of NR activity ofC.fimbriatumplants was observed in the apical root region (2 cm).

3.5.2. GS and GDH

The highest rates of GS activity and GS specific activity were found in the shoots ofC.fimbriatum plants (Tables 3 and 4). According to the treat-ment, the activity and specific activity of GS in roots ranged from about 5 to 13% of the values measured in the shoots.

Shoot GS activity was only slightly influenced by nitrogen sources. Increased levels of GS activ-ity in shoot tissues was found in plants grown in media containing NH4+ as the major nitrogen

form (Table 3). Nevertheless, shoot GS activity NH4+ as the only nitrogen source resulted in

relatively low growth rates when compared to plants grown with different NO3

−_:NH

+ _ratios

(Fig. 1). Root growth appeared to be limited by NH4

+ _{supplied alone. Plants fed with NO}

−_:NH

ratio 3:2 showed better vegetative appearance as well as a more vigorous root system than those grown in the other NO3−:NH4+ratios (not shown).

3.2. Free amino-N, soluble sugars and free ammonium contents

Regardless of the nitrogen form used, the levels of free amino-N and soluble sugars in the shoot were higher than those found in roots (Fig. 2A and B). The free amino-N content of the shoot increased with exogenous NH4+ concentration as

well as in the presence of glutamine. A significant high level of free amino-N was observed in the root of glutamine-treated plants. Soluble sugar contents decreased in the shoot and root of plants supplied with NH4

and glutamine. (Fig. 2B). The free NH4

+_{amounts in both shoot and root}

tissues were relatively low in NO3

− _{treated plants}

(Fig. 2C and Fig. 3). The highest free NH4

contents of shoot tissues were found in plants grown in the presence of glutamine (Fig. 2C). However, the concentration of free ammonium in root tissues increased to the extent that external

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B A B

A A

d dc

b a

0 0,4 0,8 1,2 1,6

1:0 3:2 1:1 2:3 0:1

NO3

-:NH4 +

ratio

H4 + g -1 fr

SHOOT ROOT

0 2 4 6 8 10

moles amino N g

-1 fr. wt

SHOOT ROOT

0 10 20 30 40 50

moles soluble sugar g

-1 fr.wt

D a

b b

B B A

0 0,4 0,8 1,2 1,6

NO3- NO3-/NH4+ NH4+ Glutamine

Nitrogen sources

moles free NH

+g -1 fr wt.

C a

C C

c B

b b

A AB

B B

c cb a

b cb

0 50 100 150 200 250 300

1:0 3:2 1:1 2:3 0:1 NO3-:NH4+ RATIO

mg chlorophyll g

-1 fr. wt

Shoot Root

was unaffected in a second group of experiments (Table 4). Shoot GS specific activity was not affected by the nitrogen sources. Nitrogen sources had no influence on GS activities in roots (Tables 3 and 4).

Glutamine and inorganic forms of nitrogen had no effect on GDH activity in the shoots (Tables 3 and 4). However, root GDH activity was enhanced by NH4+ and by glutamine. A positive

Fig. 3. Free NH4+amounts in shoot and root ofC.fimbriatum plants grown for 30 days in different NO3−:NH4+ratios.

Fig. 2. Tissue contents of amino N (1A), soluble sugar (1B) and free NH4+ (1C) in C. fimbriatum plants incubated on glutamine and inorganic nitrogen forms (6 mol m-3_{) for 30} days. The NO3−:NH4+ratio was 2:3.

Fig. 4. Shoot and root chlorophyll contents ofC.fimbriatum plants incubated in different NO3−_:NH4+_{ratios (6 mol m}−3_). N=6.

correlation between NH4+ concentration in the

media and the level of GDH activity in root tissues was observed.

3.6. pH of culture media

C. fimbriatum plants grown for 30 days caused

a substantial decrease in the pH of culture media in all nitrogen sources studied (Table 5). This effect was attenuated by the presence of NO3

−

ions and intensified by NH4+ as isolated nitrogen

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Table 2

Nitrate reductase activity (nmoles NO2−_g−1_{fr. wt h}−1_{) in various parts of}_C_._fimbriatum_{plants (}_n₌_{6) grown for 30 days in media} with different NO3−_:NH4+_{ratios (6 mol m}−3₎a

NO3−:NH4+Ratio 3:2

Plant part 1:0 1:1 2:3 0:1

65.4911.8A _62.9₉_12.3A

Root apice _64.7₉_6.4A 25.093.5B _ndb

Mature root 6.292.5A _9.8₉_3.9A _5.0₉_1.5A _3.8₉_1.6A _nd

12.394.0A _13.8₉_3.4A _9.2₉_1.7A _nd

Bulb 21.097.7A

6.091.3A _7.0₉_1.6A

6.791.7A _4.6₉_0.9A

Leaf nd

Total _98.6₉_17.3A 93.5920.4A _88.7₉_19.0A _42.6₉_6.6B _nd

a_{Bold superscript capital letters compare values among the columns.} b_{nd means non detected NR activity.}

Table 3

Ammonium assimilating enzyme activity in shoot and root tissues ofC.fimbriatumplants (n=6) grown for 30 days in glutamine and inorganic nitrogen forms (6 mol m−3_{). In this experiment the NO}

−_:NH

+_{ratio was 2:3}a Root

Shoot

GS GDH GS GDH

Activityd Activityd _{Specific activity}c

Nitrogen sources _Activityb _{Specific activity}c Activityb

0.2890.04a _0.08₉_0.01c

NO3− _3.3₉_0.1b _1.01₉_0.02a _0.08₉_0.02a _0.39₉_0.03a

0.3690.02a _0.24₉_0.02a

NO3−:NH4+ 4.290.2a 0.9690.02a 0.1090.01a 0.1490.01b 0.2590.03a

0.4690.05a _0.19₉_0.01a

NH4+ 4.090.1a 0.9890.01a 0.1090.02a

0.3690.04a _0.24₉_0.02a

Glutamine 3.890.01ab _1.00₉_0.03a _0.08₉_0.01a _0.18₉_0.01a

a_{Superscript bold letters compare values inside the columns.} b_{GS activity — moles glutamyl hydroxamate min}−1_g−1_{fr. wt.}

c_{GS specific activity — moles glutamyl hydroxamate min}−1_mg−1_protein. d_{GDH activity — moles NADH oxidized min}−1_g−1_{fr. wt.}

Table 4

Ammonium assimilating enzyme activity in shoot and root tissues ofC.fimbriatumplants (n=6) grown for 30 days in five different NO3−:NH4+ratios (6 mol m−3)a

Root Shoot

GS GDH GS GDH

Activityd _Activitya _{Specific activity}c

NO3−:NH4+ratio Activityb Specific activityc Activityd 0.3890.02a _0.29₉_0.01a

1:0 3.190.1a _1.02₉_0.01a _0.06₉_0.02a _0.06₉_0.01d

0.4190.02a _0.31₉_0.02a _0.08₉_0.01d

3:2 _3.7₉_0.2a _1.05₉_0.01a 0.0890.03a

0.3690.01a _0.28₉_0.01a

1:1 3.290.2a _1.00₉_0.04a _0.07₉_0.03a _0.09₉_0.01c

0.1390.02b

0.2190.01a

0.3290.01a

2:3 _3.8₉_0.1a _1.00₉_0.04a 0.0890.01a

0.0790.02a _0.39₉_0.02a _0.23₉_0.02a _0.15₉_0.01a

0:1 3.390.1a _0.94₉_0.01a

a_{Superscript bold letters compare values inside the columns.} b_{GS activity — moles glutamyl hydroxamate min}−1_g−1_{fr wt.}

c_{GS specific activity — moles glutamyl hydroxamate min}−1_mg−1_protein. d_{GDH activity — moles NADH oxidized min}−1_g−1_{fr. wt.}

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Table 5

pH values of culture media after 30 days of growth of C. fimbriatumplants (n=5) in different nitrogen forms (6 mol m−3_{): the initial pH was 5.5}

Nitrogen forms pH

4.6290.05a

NO3−

3.9890.05b

NO3−:NH4+

NH4+ 3.5590.04c

4.6090.03a

Glutamine

pH Nitrogen forms NO3−:NH4+

4.6190.04a

1:0

3:2 4.0590.04b

1:1 4.0090.04b

2:3 3.9690.03b

0:1 3.7090.03c

than when treated with inorganic nitrogen (Chapin et al., 1993). Seedlings of Hakea (Proteaceae) metabolize glycine, with rapid trans-fer of 15_{N to serine and other amino acids.}

Fur-thermore, plant species from the subtropical heathland of Australia were shown to take up on average three-fold greater quantities of glycine than NO3− (Schmidt and Stewart, 1999).

While free amino-N contents increased in both ammonium and glutamine-treated plants, soluble sugars presented the highest levels in nitrate-fed plants (Fig. 2A and B). In NH4+ cultured plants,

ammonium is converted to amino acids and amides in roots, and transported to the leaves (Barneix and Causin 1996). Absorbed ammonium ions are immediately assimilated by GS-GOGAT (glutamine synthetase and glutamine-2-oxoglu-tarate-amino-transferase) to form glutamine and glutamate (Lea et al., 1990; Sechley et al., 1992). Besides its role in primary NH4+ assimilation, the

GS-GOGAT cycle assimilates NH4+ from the

photorespiratory nitrogen cycle and from catabolism of storage and transport compounds (Lea et al., 1990). Ammonia-grown plants often have higher free amino-N concentration in their tissues than nitrate fed plants (Barneix and Causin, 1996). The availability of carbon skele-tons is necessary to prevent the toxic effects of NH4

+ _{(Magalha˜es et al., 1992) and may be}

re-lated to the depletion of soluble carbohydrates observed in ammonium and glutamine fed plants (Fig. 2B and C). Feng et al. (1998) have shown that the uptake and assimilation of exogenous ammonium by maize roots is followed by an intense endogenous production of ammonium from the catabolism of soluble organic N.

Our results suggest that glutamine-treated plants accumulate amides (glutamine and as-paragine) in their roots (Fig. 2A). Glutamine can be used as a substrate in a number of transamida-tion reactransamida-tions, e.g. glutamate synthase (GOGAT), asparagine synthetase (AS) and carbamoyl-phos-phate synthetase (CPS). Glutamine is also transaminated or used in the synthesis of proteins and in the export of carbon and nitrogen to other parts of the plant (Sechley et al., 1992). The transfer of amino nitrogen from glutamine to keto acids or amino acids requires the availability of

4. Discussion

Asymbiotically grown plants of C. fimbriatum were able to use both glutamine and inorganic nitrogen sources. However, maximum growth rates were found in plants supplied with glu-tamine, suggesting that free amino acids may be an important natural source of nitrogen for this plant species. Benzing (1990), characterizedCata

-setum plants as ‘‘humus based epiphytes’’.

Fre-quently, layers of suspended organic matter accumulate on the abundant upward-growing roots ofCatasetumspecies (Benzing, 1990). It has also been reported that plants of this genus usu-ally grow better in rotting tree stems (Silva and Silva, 1998). Analysis of soil – water extracts indi-cates that organic N is an important N form in plant communities where organic N mineraliza-tion is limited by low temperatures (Chapin et al., 1993), and by deficiency or excess of water in the environment (Schmidt and Stewart, 1999). In ter-restrial habitats, mineralization is subjected to inhibition by anoxia, low pH and possibly by plant-produced defence compounds (Raven et al., 1992). Heng and Goh (1984) suggested that differ-ences in vegetation cover and overlying tree cover play a major role in soil N availability to trees due to differences in the concentration of polyphenols. Eriophurum6aginatum, an arctic sedge, absorbs

amino acids rapidly and accumulates more nitro-gen and biomass when supplied with amino acids

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carbon skeletons and energy. Thus, glutamine-cul-tured plants (Fig. 2B) may require additional consumption of carbohydrates from the soluble pool.

The activities of GS and GDH can vary under distinct N regimes (Oaks, 1994; Magalha˜es et al., 1995), according to the plant tissue (Oaks et al., 1980) and species (Magalha˜es and Huber, 1991). In the present study, shoot GS activity was only slightly affected by the form of nitrogen supplied (Tables 3 and 4). For a large number of species, it has been verified that the N form has little or no influence on the GS activity (Magalha˜es and Huber, 1991; Claussen and Lenz, 1999). However, it has also been shown that GS activity increases in response to increased levels of NH4

+ _{in the}

rhizosphere of various plant species (Magalha˜es and Huber, 1991). Regardless of the N form, GS activity and carbohydrate levels were sufficient to control the free ammonium contents in the C.

fimbriatum shoot. On the other hand, the low

levels of GS activity in root tissues were not effective in regulating the free ammonium con-tents in ammonium and glutamine-fed plants. In these treatments, GDH activity in roots increased in response to increased free ammonium contents (Fig. 2C and Fig. 3; Tables 3 and 4). This increase suggest that high GDH activity may be related to a high incorporation of ammonium into amino acids to prevent toxicity (Cammaerts and Jacobs, 1985). However, GDH could be functioning as a deaminating enzyme to ensure sufficient carbon skeletons for an effective ammonium assimilation (Cammaerts and Jacobs, 1985) and for glutamine metabolism.

Regardless of nitrogen source, C. fimbriatum plants caused a drop in the pH of the media.

Dendrobium plants, epiphytic orchids, dropped

the pH of media during the period in which the rate of ammonium uptake exceeded that of nitrate uptake; the pH of the medium raised when the ammonium had already been depleted (Hew et al., 1988). Assimilation of NH4+ and NO3− involves,

theoretically, the generation of an excess of about 1.22 H+_{and 0.78 OH}− _{per molecule assimilated,}

respectively (Raven, 1988). However, the presence of NO3−as a sole nitrogen, source did not prevent

a pH decline byCatasetumcultured plants. Other factors may cause acidification of culture media such as the accumulation of CO2from root

respi-ration (Williams, 1993), and root exudation of organic acids and other organic compounds (Minocha, 1987).

Ammonium, as a unique nitrogen source, slightly limited C. fimbriatum growth, despite the low pH values of culture media (Table 5) and the high levels of free NH4

+ _{detected in root tissues}

(Fig. 2C and Fig. 3). Nevertheless, no visual symptom of ammonium toxicity was observed. Contrarily, NH4

+_{-grown plants showed higher}

chlorophyll contents in shoots when compared to NO3−-treated ones (Fig. 4). C. fimbriatum plants

seem to be tolerant of low pH and relatively high ammonium concentration in their rhizospheres and this tolerance may explain why epiphytes normally grow in acid habitats (Benzing 1990) and suggests that NH4

+ _{ions may represent an}

important natural nitrogen source for these plants.

Plants from acidic soils may be less sensitive to ammonium toxicity and have a low capacity for nitrate assimilation because of low nitrate reduc-tase activity (Smirnoff, 1995; Stewart et al., 1992). C.fimbriatumplants had low levels of NR activity when compared to other native and crop species (Gebauer et al., 1988; Magalha˜es and Huber, 1991). The NR values found in C. fimbriatum plants were similar to those observed in leaves of species from climax communities of the Brazilian southeast tropical rainforest (Stewart et al., 1992). Claussen and Lenz (1999) reported that blueberry plants, supplied with nitrate nitrogen, contained low NR activity only in roots. This species is known to be well adapted to raw humus, low soil pH, and ammonium nitrogen (Claussen and Lenz, 1999).

The activity of NR was 2.0 – 4.0 fold higher in roots than in shoots, with about 70% of the total NR activity performed in the root apices (Table 2). The same pattern of distribution of NR activ-ity was observed in seedlings ofCeratonia siliqua, a traditional cultivated tree in Mediterranean ar-eas.C.siliquaroots exhibited a declining gradient of nitrate uptake rates, nitrate concentration, and

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NR activity from the root tip to the insertion point of cotyledons. The combination of low capacity of loading nitrate into the xylem and low transport of nitrate from the root to the shoot would explain this pattern of nitrate assimilation in C. siliqua (Cruz et al., 1993).C.fimbriatum plants showed a limited ability to assimilate NO3−ions. The failure

in the detection of NO3− in plant tissues indicates

that this species possesses a limited capacity to absorb this ion. However, once absorbed, NO3−

should be immediately reduced by NR. Hew et al. (1993) observed low rates of NO3− and NH4+

uptake by roots of three orchid species, which were about 10-fold lower than that of barley.

Nitrate ions supported C. fimbriatum plant growth but caused a drop in both amino acid and chlorophyll tissue contents (Fig. 2A and Fig. 3). In addition, the high contents of soluble sugars and the low free NH4+ contents found in both shoots

and roots of nitrate-fed plants could reflect a quantitative limitation on nitrogen assimilation. It is well-known that carbon and nitrogen metabolism share organic carbon and energy sup-plied from both photosynthesis and respiration (Huppe and Turpin, 1994). Restriction on absorp-tion and assimilaabsorp-tion of nitrogen into organic molecules could be associated with the high levels of sugars in the metabolic pool of the nitrate-fed C. fimbriatum plants. Transgenic plants of Nico -tiana plumbaginifolia, with low levels of NR ex-pression, showed lower levels of chlorophyll, protein and amino acids and higher contents of carbohydrate (starch and sucrose) than wild type or high NR expressors (Foyer et al. 1994).

Our main hypothesis was accepted as C.

fimbriatum plants developed best when supplied

with an amino acid. It has been suggested that plant species showing preferential use of NH4+

(Cruz et al., 1993), organic nitrogen sources or dependent on biological N2 fixation (Oaks 1992),

tend to reduce NO3

−

mainly in roots. Our results suggest that organic N and NH4

+_{ions are the main}

sources of nitrogen toC.fimbriatum. Some of the physiological characteristics detected in C.

fimbriatum plants are typical of plant species

adapted to tropical and subtropical rainforest cli-max communities (Stewart et al., 1992) and of plants species adapted to extreme soil conditions

(Claussen and Lenz, 1999; Schmidt and Stewart 1999).

Beyond representing an interesting model plant for physiological studies, the genus Catasetum comprises several species of economic value. Our results pointed out the advantageous use of glutamine for a more efficient development of Catasetum plants. It was also evident that NO3−

alone is a poor nitrogen source for Catasetum growth.

Acknowledgements

N. Majerowicz is grateful for Coordenadoria de Aperfeic¸oamento do Ensino Superior (CAPES) and PICDT/UFRRJ for the doctoral fellowship. The authors thank Dr Ricardo L. Berbara for the helpful English revision of the text.

References

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Table 2

Nitrate reductase activity (nmoles NO2−g−1fr. wt h−1) in various parts ofC.fimbriatumplants (n=6) grown for 30 days in media

with different NO3−:NH4+ratios (6 mol m−3)a

NO3−:NH4+Ratio

3:2

Plant part 1:0 1:1 2:3 0:1

65.4911.8A _62.9₉_12.3A

Root apice _64.7₉_6.4A 25.093.5B _ndb

Mature root 6.292.5A _9.8₉_3.9A _5.0₉_1.5A _3.8₉_1.6A _nd

12.394.0A _13.8₉_3.4A _9.2₉_1.7A _nd

Bulb 21.097.7A

6.091.3A _7.0₉_1.6A

6.791.7A _4.6₉_0.9A

Leaf nd

Total _98.6₉_17.3A 93.5920.4A _88.7₉_19.0A _42.6₉_6.6B _nd

a_{Bold superscript capital letters compare values among the columns.} b_{nd means non detected NR activity.}

Table 3

Ammonium assimilating enzyme activity in shoot and root tissues ofC.fimbriatumplants (n=6) grown for 30 days in glutamine and inorganic nitrogen forms (6 mol m−3_{). In this experiment the NO}

−_:NH

+_{ratio was 2:3}a

Root Shoot

GS GDH GS GDH

Activityd

Activityd _{Specific activity}c

Nitrogen sources _Activityb _{Specific activity}c Activityb

0.2890.04a _0.08₉_0.01c NO3− 3.390.1b 1.0190.02a 0.0890.02a 0.3990.03a

0.3690.02a _0.24₉_0.02a

NO3−:NH4+ 4.290.2a 0.9690.02a 0.1090.01a 0.1490.01b

0.2590.03a

0.4690.05a _0.19₉_0.01a NH4+ 4.090.1a 0.9890.01a 0.1090.02a

0.3690.04a _0.24₉_0.02a

Glutamine 3.890.01ab _1.00₉_0.03a _0.08₉_0.01a _0.18₉_0.01a

a_{Superscript bold letters compare values inside the columns.} b_{GS activity — moles glutamyl hydroxamate min}−1_g−1_{fr. wt.}

c_{GS specific activity — moles glutamyl hydroxamate min}−1_mg−1_protein. d_{GDH activity — moles NADH oxidized min}−1_g−1_{fr. wt.}

Table 4

Ammonium assimilating enzyme activity in shoot and root tissues ofC.fimbriatumplants (n=6) grown for 30 days in five different NO3−:NH4+ratios (6 mol m−3)a

Root Shoot

GS GDH GS GDH

Activityd _Activitya _{Specific activity}c

NO3−:NH4+ratio Activityb Specific activityc Activityd

0.3890.02a _0.29₉_0.01a

1:0 3.190.1a _1.02₉_0.01a _0.06₉_0.02a _0.06₉_0.01d

0.4190.02a _0.31₉_0.02a _0.08₉_0.01d 3:2 _3.7₉_0.2a _1.05₉_0.01a 0.0890.03a

0.3690.01a _0.28₉_0.01a

1:1 3.290.2a _1.00₉_0.04a _0.07₉_0.03a _0.09₉_0.01c

0.1390.02b 0.2190.01a

0.3290.01a 2:3 _3.8₉_0.1a _1.00₉_0.04a 0.0890.01a

0.0790.02a _0.39₉_0.02a _0.23₉_0.02a _0.15₉_0.01a 0:1 3.390.1a _0.94₉_0.01a

a_{Superscript bold letters compare values inside the columns.} b_{GS activity — moles glutamyl hydroxamate min}−1_g−1_{fr wt.}

c_{GS specific activity — moles glutamyl hydroxamate min}−1_mg−1_protein. d_{GDH activity — moles NADH oxidized min}−1_g−1_{fr. wt.}

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Table 5

pH values of culture media after 30 days of growth of C.

fimbriatumplants (n=5) in different nitrogen forms (6 mol m−3_{): the initial pH was 5.5}

Nitrogen forms pH

4.6290.05a NO3−

3.9890.05b NO3−:NH4+

NH4+ 3.5590.04c

4.6090.03a Glutamine

pH Nitrogen forms NO3−:NH4+

4.6190.04a 1:0

3:2 4.0590.04b

1:1 4.0090.04b

2:3 3.9690.03b

0:1 3.7090.03c

than

when

treated

with

inorganic

nitrogen

(Chapin

et

al.,

1993).

Seedlings

of

Hakea

(Proteaceae) metabolize glycine, with rapid

trans-fer of

_{N to serine and other amino acids.}

Fur-thermore, plant species from the subtropical

heathland of Australia were shown to take up on

average three-fold greater quantities of glycine

than NO

3−

(Schmidt and Stewart, 1999).

While free amino-N contents increased in both

ammonium and glutamine-treated plants, soluble

sugars presented the highest levels in nitrate-fed

plants (Fig. 2A and B). In NH

cultured plants,

ammonium is converted to amino acids and

amides in roots, and transported to the leaves

(Barneix and Causin 1996). Absorbed ammonium

ions are immediately assimilated by GS-GOGAT

(glutamine synthetase and

glutamine-2-oxoglu-tarate-amino-transferase) to form glutamine and

glutamate (Lea et al., 1990; Sechley et al., 1992).

Besides its role in primary NH

assimilation, the

GS-GOGAT cycle assimilates NH

from the

photorespiratory

nitrogen

cycle

and

from

catabolism of storage and transport compounds

(Lea et al., 1990). Ammonia-grown plants often

have higher free amino-N concentration in their

tissues than nitrate fed plants (Barneix and

Causin, 1996). The availability of carbon

skele-tons is necessary to prevent the toxic effects of

NH

_{(Magalha˜es et al., 1992) and may be}

re-lated to the depletion of soluble carbohydrates

observed in ammonium and glutamine fed plants

(Fig. 2B and C). Feng et al. (1998) have shown

that the uptake and assimilation of exogenous

ammonium by maize roots is followed by an

intense endogenous production of ammonium

from the catabolism of soluble organic N.

Our

results

suggest

that

glutamine-treated

plants accumulate amides (glutamine and

as-paragine) in their roots (Fig. 2A). Glutamine can

be used as a substrate in a number of

transamida-tion reactransamida-tions, e.g. glutamate synthase (GOGAT),

asparagine synthetase (AS) and

carbamoyl-phos-phate

synthetase

(CPS).

Glutamine

is

also

transaminated or used in the synthesis of proteins

and in the export of carbon and nitrogen to other

parts of the plant (Sechley et al., 1992). The

transfer of amino nitrogen from glutamine to keto

acids or amino acids requires the availability of

4. Discussion

Asymbiotically grown plants of

C. fimbriatum

were able to use both glutamine and inorganic

nitrogen sources. However, maximum growth

rates were found in plants supplied with

glu-tamine, suggesting that free amino acids may be

an important natural source of nitrogen for this

plant species. Benzing (1990), characterized

Cata-setum

plants as ‘‘humus based epiphytes’’.

Fre-quently, layers of suspended organic matter

accumulate on the abundant upward-growing

roots of

Catasetum

species (Benzing, 1990). It has

also been reported that plants of this genus

usu-ally grow better in rotting tree stems (Silva and

Silva, 1998). Analysis of soil – water extracts

indi-cates that organic N is an important N form in

plant communities where organic N

mineraliza-tion is limited by low temperatures (Chapin et al.,

1993), and by deficiency or excess of water in the

environment (Schmidt and Stewart, 1999). In

ter-restrial habitats, mineralization is subjected to

inhibition by anoxia, low pH and possibly by

plant-produced defence compounds (Raven et al.,

1992). Heng and Goh (1984) suggested that

differ-ences in vegetation cover and overlying tree cover

play a major role in soil N availability to trees due

to differences in the concentration of polyphenols.

Eriophurum

6 aginatum, an arctic sedge, absorbs

amino acids rapidly and accumulates more

nitro-gen and biomass when supplied with amino acids

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carbon skeletons and energy. Thus,

glutamine-cul-tured plants (Fig. 2B) may require additional

consumption of carbohydrates from the soluble

pool.

The activities of GS and GDH can vary under

distinct N regimes (Oaks, 1994; Magalha˜es et al.,

1995), according to the plant tissue (Oaks et al.,

1980) and species (Magalha˜es and Huber, 1991).

In the present study, shoot GS activity was only

slightly affected by the form of nitrogen supplied

(Tables 3 and 4). For a large number of species, it

has been verified that the N form has little or no

influence on the GS activity (Magalha˜es and

Huber, 1991; Claussen and Lenz, 1999). However,

it has also been shown that GS activity increases

in response to increased levels of NH

_{in the}

rhizosphere of various plant species (Magalha˜es

and Huber, 1991). Regardless of the N form, GS

activity and carbohydrate levels were sufficient to

control the free ammonium contents in the

C. fimbriatum

shoot. On the other hand, the low

levels of GS activity in root tissues were not

effective in regulating the free ammonium

con-tents in ammonium and glutamine-fed plants. In

these treatments, GDH activity in roots increased

in response to increased free ammonium contents

(Fig. 2C and Fig. 3; Tables 3 and 4). This increase

suggest that high GDH activity may be related to

a high incorporation of ammonium into amino

acids to prevent toxicity (Cammaerts and Jacobs,

1985). However, GDH could be functioning as a

deaminating enzyme to ensure sufficient carbon

skeletons for an effective ammonium assimilation

(Cammaerts and Jacobs, 1985) and for glutamine

metabolism.

Regardless of nitrogen source,

C. fimbriatum

plants caused a drop in the pH of the media.

Dendrobium

plants, epiphytic orchids, dropped

the pH of media during the period in which the

rate of ammonium uptake exceeded that of nitrate

uptake; the pH of the medium raised when the

ammonium had already been depleted (Hew et al.,

1988). Assimilation of NH

and NO

3−

involves,

theoretically, the generation of an excess of about

1.22 H

_{and 0.78 OH}

−

_{per molecule assimilated,}

respectively (Raven, 1988). However, the presence

of NO

3−

as a sole nitrogen, source did not prevent

a pH decline by

Catasetum

cultured plants. Other

factors may cause acidification of culture media

such as the accumulation of CO

from root

respi-ration (Williams, 1993), and root exudation

of organic acids and other organic compounds

(Minocha, 1987).

Ammonium, as a unique nitrogen source,

slightly limited

C. fimbriatum

growth, despite the

low pH values of culture media (Table 5) and the

high levels of free NH

_{detected in root tissues}

(Fig. 2C and Fig. 3). Nevertheless, no visual

symptom of ammonium toxicity was observed.

Contrarily, NH

_{-grown plants showed higher}

chlorophyll contents in shoots when compared to

NO

3−

-treated ones (Fig. 4).

C. fimbriatum

plants

seem to be tolerant of low pH and relatively high

ammonium concentration in their rhizospheres

and this tolerance may explain why epiphytes

normally grow in acid habitats (Benzing 1990)

and suggests that NH

_{ions may represent an}

important natural nitrogen source for these

plants.

Plants from acidic soils may be less sensitive to

ammonium toxicity and have a low capacity for

nitrate assimilation because of low nitrate

reduc-tase activity (Smirnoff, 1995; Stewart et al., 1992).

C. fimbriatum

plants had low levels of NR activity

when compared to other native and crop species

(Gebauer et al., 1988; Magalha˜es and Huber,

1991). The NR values found in

C. fimbriatum

plants were similar to those observed in leaves of

species from climax communities of the Brazilian

southeast tropical rainforest (Stewart et al., 1992).

Claussen and Lenz (1999) reported that blueberry

plants, supplied with nitrate nitrogen, contained

low NR activity only in roots. This species is

known to be well adapted to raw humus, low soil

pH, and ammonium nitrogen (Claussen and Lenz,

1999).

The activity of NR was 2.0 – 4.0 fold higher in

roots than in shoots, with about 70% of the total

NR activity performed in the root apices (Table

2). The same pattern of distribution of NR

activ-ity was observed in seedlings of

Ceratonia siliqua,

a traditional cultivated tree in Mediterranean

ar-eas.

C. siliqua

roots exhibited a declining gradient

of nitrate uptake rates, nitrate concentration, and

(4)

NR activity from the root tip to the insertion point

of cotyledons. The combination of low capacity of

loading nitrate into the xylem and low transport of

nitrate from the root to the shoot would explain

this pattern of nitrate assimilation in

C. siliqua

(Cruz et al., 1993).

C. fimbriatum

plants showed a

limited ability to assimilate NO

3−

ions. The failure

in the detection of NO

3−

in plant tissues indicates

that this species possesses a limited capacity to

absorb this ion. However, once absorbed, NO

3−

should be immediately reduced by NR. Hew et al.

(1993) observed low rates of NO

3−

and NH

uptake by roots of three orchid species, which

were about 10-fold lower than that of barley.

Nitrate ions supported

C. fimbriatum

plant

growth but caused a drop in both amino acid and

chlorophyll tissue contents (Fig. 2A and Fig. 3). In

addition, the high contents of soluble sugars and

the low free NH

contents found in both shoots

and roots of nitrate-fed plants could reflect a

quantitative limitation on nitrogen assimilation. It

is

well-known

that

carbon

and

nitrogen

metabolism share organic carbon and energy

sup-plied from both photosynthesis and respiration

(Huppe and Turpin, 1994). Restriction on

absorp-tion and assimilaabsorp-tion of nitrogen into organic

molecules could be associated with the high levels

of sugars in the metabolic pool of the nitrate-fed

C. fimbriatum

plants. Transgenic plants of

Nico-tiana plumbaginifolia, with low levels of NR

ex-pression, showed lower levels of chlorophyll,

protein and amino acids and higher contents of

carbohydrate (starch and sucrose) than wild type

or high NR expressors (Foyer et al. 1994).

Our main hypothesis was accepted as

C. fimbriatum

plants developed best when supplied

with an amino acid. It has been suggested that

plant species showing preferential use of NH

(Cruz et al., 1993), organic nitrogen sources or

dependent on biological N

fixation (Oaks 1992),

tend to reduce NO

3 −

mainly in roots. Our results

suggest that organic N and NH

_{ions are the main}

sources of nitrogen to

C. fimbriatum. Some of the

physiological

characteristics

detected

in

C. fimbriatum

plants are typical of plant species

adapted to tropical and subtropical rainforest

cli-max communities (Stewart et al., 1992) and of

plants species adapted to extreme soil conditions

(Claussen and Lenz, 1999; Schmidt and Stewart

1999).

Beyond representing an interesting model plant

for physiological studies, the genus

Catasetum

comprises several species of economic value.

Our results pointed out the advantageous use of

glutamine for a more efficient development of

Catasetum

plants. It was also evident that NO

3−

alone is a poor nitrogen source for

Catasetum

growth.

Acknowledgements

N. Majerowicz is grateful for Coordenadoria de

Aperfeic¸oamento do Ensino Superior (CAPES)

and PICDT

/

UFRRJ for the doctoral fellowship.

The authors thank Dr Ricardo L. Berbara for the

helpful English revision of the text.

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