Ethnobotanical Leaflets 13: 413-21 , 2009.
Altitudinal Gradient on Ammonium Assimilatory Enzymes in Rauvolfia teraphylla L. (Apocyanaceae) � A Perennial Medicinal
C. Rajasekaran1#*, T. Kalaivani2#, R. Jayakumararaj3,
Abhijeet Singh4, V.R. Pusalkar5 and R. Marimuthu6
1Periyar University, Salem � 636 011, Tamil Nadu, IN
2Department of Biochemistry, A.V.S. College, Salem �
636015, Tamil Nadu,
3Department of Botany, Government Arts College,
Dharmapuri � 636705,
Tamil Nadu, IN
4M.N. Institute of Applied Sciences, Bikaner � 334 001,
Medica, Salem � 636 004, Tamil Nadu, IN
Department of Botany, Government Arts College, Salem � 636 007, Tamil Nadu, IN
#Present address: School of Biotechnology & Chemical Engineering, Vellore
Institute of Technology University, Vellore -636 014, TN, India, Phone No:
9414903209; Email: firstname.lastname@example.org
Issued 01 March 2009
Seedlings of Ravuolfia tetraphylla L. were grown,
transplanted and acclimatized for 60 days at different altitudes gradient
(250, 400 and 1600 m) in Yercaud, Salem, Tamil Nadu, India. Response to shift
in altitude was observed in the test plants. Shoot length decreased with the
increase in the altitude, while increase in the root length was directly
proportional to the increase in the altitudinal gradient. Biomass
accumulation in roots of R. tetraphylla
recorded the maximum at high altitude at the same time shoot biomass was
maximum at an intermediate height (400m), thereafter reduction in biomass was
observed with the increase in the altitude. Total soluble protein content was
significantly high at low altitude in the shoot while it followed a reverse
trend in the roots. Likewise, free tissue ammonia level in this species
showed positive correlation with increase in the altitude. Ammonium
assimilatory enzymes viz., glutamine synthetase (GS), glutamate synthase
(GOGAT) and glutamate dehydrogenase (GDH) were analyzed. GS/ GOGAT activity
and specific activity were altitude sensitive, whereas GDH activity exhibited
inverse trend. A positive shift in ammonium assimilatory pathway in test
plants growing at high altitude was observed in R. tetraphylla.
Key� Words: Altitudinal gradient, Ravuolfia tetraphylla, Ammonium Assimilatory Pathway;� Glutamate Dehydrogenase (GDH).
metabolism in plants is a complex process, and is regulated by the form of
nitrogen that is available to the plant (Magalhaes and Huber, 1989). Major
source of nitrogen to plants is ammonium, and is largely assimilated by the
roots (Yoneyama and Kumazawa, 1974). Generally, plants prefer ammonium (NH4+)
compared to nitrate (NO3-) and nitrite (NO2-)
as ammonia is the starting point for nitrogen assimilation in higher plants.
Internal source of ammonia is photorespiration and amino acid catabolism
(Srivastava and Singh, 1987). However, high level of ammonia is toxic,
therefore it has to be converted to amino acids in plants to maintain its low
level (Miflin and Lea, 1980). Enzymes namely Glutamine synthetase (GS) (EC 184.108.40.206)/ Glutamate synthase
(GOGAT) (EC 220.127.116.11) and
Glutamate dehydrogenase (GDH) (EC
18.104.22.168-4) play a vital role in ammonia assimilation, detoxification
and regulation of nitrogen metabolism in plants. Among three enzymes, GDH occupies a key role in plant
����� The ability of
plant to acquaint to new environmental conditions depends upon its
morphological adaptation and physiological response. In mountainous
environment, variation in altitude offers wide variety of environmental
conditions. In general, with increase in elevation, stressors such as
temperature, pressure, light intensity, rainfall, partial pressure of
metabolic gases are known to influence plant metabolism (Woodcock, 1976;
Purohit, 1977). Like any other metabolic process, nitrogen metabolism in
plants is significantly influenced by variation in the altitude. Increased
nitrogen content has been reported in plants with the increase in the
altitude (Korner, 1989). This implies that plants which are least influenced
by altitudinal changes are much resistant to multiple stressors.
����� Several workers
have compared morpho-physiological response of plants to the change in the
altitude (Bhadula et al., 1985;
Rajasekaran et al., 1998;
Rajasekaran, 2000). Ammonium assimilation in plants plays an important role
in growth and development. Therefore it has been extensively studied in
various plant species. However, studies on ammonium assimilation in plants from
the various climatic zones have been limited and no studies have been
undertaken in Shervaroyan hills, part of Eastern Ghats, Tamil Nadu,
India.� The present work is an attempt
to study growth behavior and analyze the activity of ammonium assimilation
enzymes in R. tetraphylla L. a
perennial medicinal plant, grown at different altitudes in Shervaroyan hills.
Materials and Methods
of R. tetraphylla L. (Apocyanaceae)
were sown in farmyard manure and garden soil in a ratio of 1:2 at Salem (250m)
to raise seedlings. 40 day-old seedlings were transferred to experimental
sites: Salem (250m), Kurumbapatti (400m) and Yercaud (1600m) in Shervaroyan
hills of Salem, Tamil Nadu. Experimental plants acclimatized for 60 days at
respective sites and growth performance was analyzed (shoot and root length
and dry weight). For biochemical analysis, plant samples were frozen in
liquid nitrogen after collection.�
Samples were homogenized in 0.1 M, Tris-HCl pH 7.5 containing 2 mM
EDTA and 0.1% -mercaptoethanol, PMSF was added to prevent proteolysis. All
the studies were conducted at 4�C. Total soluble protein was estimated using
Bradford (1976) method.
tissue ammonia level was estimated using method of Chaney and Marbach (1983). GS
activity was determined by the method of Truax et al. (1994). One hundred l of supernatant was incubated
with 900 l of assay buffer (0.1 M - imidazole-HCl, 65 mM - L-
Glutamate, 4 mM - MnCl2, 0.75 ADP, 33 mM sodium
arsenate and 17mM hydroxylamine, pH 6.8) at 30�C for one hour. The reaction
was terminated by adding 1ml of stop solution (0.37 M FeCl3, 0.2 M
TCA in 0.67 N HCl). After centrifugation,
absorbance was determined at 540nm. Glutamylhydroxamate (Sigma Chemical Co,
USA) was used to develop standard curve.
����� A modified method of Dougall (1974) for
the assay of NADH-GOGAT and NADH-GDH by following the rate of reduction of
NADH at 340nm for 5 min. GOGAT assay mixture contain 20 mM Tricine, pH 7.5,
12.5 mM, - ketoglutarate, 12.5 mM L-Glutamine and 0.15 mM NADH. Assay
mixture for GDH contained 20 mM Tricine, pH 7.5, 1 mM -ketoglutarate, 1
mM CaCl2, 100 mM NH4Cl and 0.15 mM NADH.
����� All the estimations were carried out in
triplicate (n = 3) following standard methods.
���� Plant growth, biochemical and enzymes of
ammonium assimilation in R. tetraphylla
were estimated. In R. tetraphylla shoot and root length
from three different altitudes is summarized in Fig. 1. Shoot length
decreased with increase in altitude, while the root length increased with
increase in the altitude. Similar observation with regards to the shoot has
been reported for different plant species by Nautiyal and Purohit (1980),
Rajasekaran et al., (1998),
Rajasekaran (2000). Likewise, increase in the root length with increase in
the altitude has previously been reported by Bhatt and Purohit (1984) and
Pankaj Prasad (1997). This could be due to non-availability of water in
higher altitude and other factors such as soil, temperature. Sharma, (1980)
reported dwarfism as an environment stress at high altitudes. Decrease in
cumulative height and growth rate was steep in low land species than high
land species (Todaria, 1980). Whereas
shoot and root dry weight increased with the increase in the altitude (Fig
2). This could be due to higher concentration of carbon dioxide in the
atmosphere (Purohit, 1998).
Fig. 1. Changes in
shoot-root growth patterns of R. tetraphylla acclimatized at different altitudes.
Fig. 2. Changes in
shoot-root dry weight of R. tetraphylla acclimatized
at different altitudes
����� Total soluble
protein (TSP) content in the shoot decreased with
increasing altitude while reverse trend was observed in the root system (Fig
3). However, free tissue
ammonia (FTA) in the shoot and the root system of R. tetraphylla increased with increasing altitude (Fig 4).
Interestingly, the FTA level in the shoot was comparatively high than the
root system. In roots maximum FTA was recorded at high altitude and minimum
at low altitude. The present
observations are in agreement with previous studies on Selinum
vaginatum (Rajasekaran, 2000;
Rajasekaran et al., 2009).
Fig. 3. Changes in TSP of shoot
and root of R. tetraphylla acclimatized at different
������ Specific and
total activities of GS are presented in Fig 5-6. GS specific and total
activities of both the parts showed an inverse relation with the increasing
altitude. Maximum activity was observed at the low and minimum at high
altitudes. Variation in activity of GS under certain environmental conditions
has been attributed to reassimilation
of ammonia released during photorespiration (McNally et al., 1983; Wallsgrove et
al., 1983). However, seasonal variation in GS of temperate deciduous tree
leaves strongly indicated that decline in light intensity and temperature in
late season accounted for drop in GS activity (Pearson and Ji, 1994), similar
has already been reported in G. max and S. vaginatum (Rajasekaran
et al., 1998; Rajasekaran, 2000; Rajasekaran et al., 2009).
Likewise, decrease in GS activity under water stress in Albizzia stipulata and Oeugenia
dalbergioides indicate that
GS in both the plants is sensitive to water stress (Pankaj Prasad, 1997;
Purohit, 1998), although in some
plant species GS has been insensitive to water stress (Becana et al., 1984). FTA accumulation
reflected in decreased GS activity in shoot and root (Figs. 4-6). Kamachi et
al., (1992) reported that environmental conditions may induce FTA
Fig. 4. Changes in shoot-
root FTA levels of R. tetraphylla
acclimatized at different altitudes
Fig. 5. Changes in
shoot -root GS specific activity of R. tetraphylla at
that may affect GS activity at high
altitude. This is in accordance with previous reports (Cren and Hiral, 1999;
Rajasekaran, 2000). This could be due to the fact that GS is less effective
under high ammonia accumulation
Fig. 6. Changes in
shoot-root GS activity of R. tetraphylla acclimatized
at different altitudes.
����� Both, specific and total activity of
NADH-GOGAT at three different altitudes is shown in Fig 7-8. NADH-GOGAT
specific and total activity in shoot and root followed similar trend as in
GS. NADH-GOGAT activity in shoots showed similar trend in low and middle
altitudes. A decrease of 31% was observed at high altitude. Likewise, with
the increase in the altitude decrease in shoot and root GOGAT specific and
total activity was observed (Fig. 7-8). This indicates that FTA content in
the tissues has complex regulatory effects on GOGAT activity as several
stresses have been reported to be operative along different altitudes
(Bhadula and Purohit, 1994; Pankaj Prasad, 1997; Purohit, 1998; Rajasekaran,
2000; Rajasekaran et al., 2009).
Specific and total activities of GDH-amination in R. tetraphylla (shoot and root) showed a positive correlation
with increase in altitude (Fig. 9-10).
Fig. 7. Changes in
shoot-root GOGAT specific activity of R. tetraphylla at different
Fig. 8. Changes in
shoot-root GOGAT activity of R. tetraphylla
acclimatized at different altitudes.
����� Increased NADH-GDH activity has been
reported along an altitudinal gradient (Pankaj Prasad, 1997; Rajasekaran et
al., 1998; Rajasekaran, 2000). However, increased GDH activity is an
indicator of detoxification of ammonia released during the breakdown of
proteins and amino acids (Becana et
al., 1984). In the present investigation, GDH-amination activity
increased with the increase in the altitude (Fig. 9-10). Srivastava and
Singh, (1987) reported that GDH pathway is active under certain nutritional
and environmental conditions. Although, GS/GOGAT pathway is predominant in
ammonia assimilation in higher plants, plants do switch over to GDH pathway
under certain conditions of low energy and high ammonia (Yamaya, 1999;
Rajasekaran et al., 1998; Rajasekaran, 2000; Rajasekaran et al., 2009).
Fig. 9. Changes in
shoot-root GDH specific activity of R. tetraphylla at
Fig. 10. Changes in
shoot-root GDH activity of R. tetraphylla
acclimatized at different altitudes.
����� Specific activity
of GS decreased at high altitude (Fig. 5), whereas at these altitudes GDH
specific activity increased (Fig. 9). GDH plays a complementary role to GS/GOGAT
cycle in synthesis of glutamate (Srivastava and Singh, 1987; Rajasekaran et
al., 1998; Rajasekaran, 2000) or could catalyze the oxidation of
glutamate to provide carbon-skeletons to the TCA cycle. At high altitude,
FTA levels also were found to increase (Fig. 4). Increased levels of ammonia
may have resulted in increase in de
novo synthesis of GDH (Srivastava and Singh, 1987; Loulakakis and
Angelakis 1990a; Watanebe et al., 1992).
This may be one of the reasons for enhancement of GDH activities at high
altitudes. However, investigations on
isoforms, isoform patterns of
ammonium assimilatiory enzymes, in-vitro studies using inhibitors of
ammonium assimilatory enzymes along an altitudinal gradient is
required to elucidate mechanism for this behavior.
����� Council of Scientific and Industrial
Research, Ministry of Human Resource Development, Government of India, New
Delhi, is gratefully acknowledged for financial support (SRF- Extended
fellowship, file no. 9/810(1)/2001-EMR- I) to CR. Authors
are grateful to Dr. RSR Vice Chancellor, Periyar University, Prof. KVK for
consistent encouragement to carryout this work.
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