Ethnobotanical Leaflets 13: 301-309. 2009.
Studies on the Impact of Altitudinal Gradient on
Ammonium Assimilatory Metabolism in Glycine
max L. (Fabaceae)
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,
Planta Medica, Salem � 636 004, Tamil Nadu, IN
Department of Botany, Government Arts College, Salem � 636 007, Tamil Nadu, IN
author; C. Rajasekaran, #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 15 February 2009
of Glycine max L. were grown, transplanted and acclimatized for 60 days
at different altitudes (250, 400 and 1600 m). Response to shift in altitude
was observed in the plants. Shoot length decreased with the increase in the
altitude, while root length followed a reverse trend. Biomass accumulation in
shoot and roots of G. max was the
maximum at high altitude. Total soluble protein content was significantly
high at low altitude in the shoot and the roots. Free tissue ammonia level in
this species showed positive correlation with increasing altitude. Ammonium
assimilatory enzymes viz., glutamine synthetase (GS), glutamate synthase
(GOGAT) and glutamate dehydrogenase (GDH) were studied. GS/ GOGAT specific
and total activity were altitude sensitive, whereas GDH activity exhibited
inverse trend. Results indicate that there is a positive shift in ammonium
assimilatory pathway in plants growing at high altitude.
Assimilatory Pathway; Glutamine Synthetase (GS); Gluatamate Synthase (GOGAT);
Glutamate Dehydrogenase (GDH); Glycine
Nitrogen 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 the plant 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 is converted to amino
acids in plants (Miflin and Lea, 1980). Enzymes namely Glutamine synthetase
(GS) (EC 220.127.116.11)/ Glutamate
synthase (GOGAT) (EC 18.104.22.168) and
Glutamate dehydrogenase (GDH) (EC
22.214.171.124-4) play a vital role in ammonia assimilation and
detoxification. Off the three
enzymes, GDH occupies a key role in plant metabolism.
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 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 that plants which are least influenced by
altitudinal changes are much resistant to multiple stresses.
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 play an important
role in growth and development and has been extensively studied in various 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 Glycine max
(L.) an annual leguminous food crop, grown at different altitudes in
MATERIALS AND METHODS
Seeds of Glycine max
L. (Fabaceae) 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. Free 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.
RESULTS AND DISCUSSION
Plant growth, biochemical and enzymes of
ammonium assimilation in G. max were estimated. In G. max shoot and root length from
three different altitudes is summarized in Fig. 1. Shoot and root length
decreased with increase in altitude. Similar observation has been reported
for different plant species by Nautiyal and Purohit (1980), Rajasekaran et al., (1998), Rajasekaran (2000).
However, Bhatt and Purohit (1984) and Pankaj Prasad (1997) reported more root
length than shoot at high altitudes 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).
soluble protein (TSP) content decreased with
increasing altitude (Fig 3).
However, free tissue ammonia (FTA) in G. max increased with increasing altitude (Fig 4). 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). 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).
Decrease in GS activity under water stress in Albizzia stipulata and Oeugenia
dalbergioides indicates 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
accumulation 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
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 34% 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). Specific and total activities of GDH-amination in G. max (shoot and root) showed a
positive correlation with increase in altitude (Fig. 9-10). 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).
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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Fig. 1. Changes in shoot and root growth
patterns of G. max acclimatized at������ different altitudes.
Fig. 2. Changes in shoot and root dry
weights of G. max acclimatized at
Fig. 3. Changes in total soluble protein
of shoot and root of G. max acclimatized at different altitudes.
Fig. 4. Changes in shoot and root free
tissue ammonia levels of G. max acclimatized at different altitudes
Fig. 5. Changes in shoot and root GS
(Specific) activity of G. max acclimatized at different altitudes.
Fig. 6. Changes in shoot and root GS
(total) activity of G. max acclimatized at different altitudes.
Fig. 7. Changes in shoot and root GOGAT
(Specific) activity of G. max acclimatized at different altitudes.
Fig. 8. Changes in shoot and root GOGAT
(total) activity of G. max acclimatized at different altitudes.
Fig. 9. Changes in shoot and root
GDH-amination (specific) activity of G. max acclimatized at different
Fig. 10. Changes in shoot and root
GDH-amination activity of G. max acclimatized at different altitudes.