Colloids and Surfaces B: Biointerfaces 59 (2007) 150157

Alterations in osmoregulation, antioxidant enzymes and indole alkaloid

levels in Catharanthus roseus exposed to water deficit
C. Abdul Jaleel, P. Manivannan, A. Kishorekumar, B. Sankar, R. Gopi ,
R. Somasundaram , R. Panneerselvam
Stress Physiology Lab, Department of Botany, Annamalai University, Annamalainagar 608002, Tamilnadu, India
Received 28 March 2007; received in revised form 23 April 2007; accepted 2 May 2007
Available online 6 May 2007

Abstract
Catharanthus roseus (L.) G. Don plants were grown in different water regimes in order to study the drought induced osmotic stress and proline
(PRO) metabolism, antioxidative enzyme activities and indole alkaloid accumulation. The plants under pot culture were subjected to 10, 15 and 20
days interval drought (DID) stress from 30 days after sowing (DAS) and regular irrigation was kept as control. The plants were uprooted on 41 DAS
(10 DID), 46 DAS (15 DID) and 51 DAS (20 DID). The drought stressed plants showed increased aminoacid (AA), glycine betaine (GB) and PRO
contents and decreased proline oxidase (PROX) and increased -glutamyl kinase (-GK) activities when compared to control. The antioxidative
enzymes like peroxidase (POX) and polyphenol oxidase (PPO) increased to a significant level in drought stressed plants when compared to control.
The drought stressed C. roseus plants showed an increase in total indole alkaloid content in shoots and roots when compared to well-watered
control plants. Our results suggest that the cultivation of medicinal plants like C. roseus in water deficit areas would increase its PRO metabolism,
osmoregulation, defense system and the level of active principles.
2007 Elsevier B.V. All rights reserved.
Keywords: Osmolytes; Proline oxidase; -Glutamyl kinase; Water stress; Amino acid; Glycine betaine; Proline; Antioxidants; Alkaloid

1. Introduction
Drought occurs in many parts of the world every year, often
with devastating effects on crop production [1]. Worldwide
losses in crop yields from water deficits probably exceed the
losses from all other causes combined [2]. The environmental stresses such as drought, temperature, salinity, air pollution,
heavy metals, pesticides and soil pH are major limiting factors in
crop production because, they affects almost all plant functions
[3,4]. Water deficit (commonly known as drought) can be defined

Abbreviations: DID, days interval drought; POX, peroxidase; DAS, days

after sowing; FW, fresh weight; AA, aminoacid; GB, glycine betaine; PRO,
proline; PROX, proline oxidase; -GK, -glutamyl kinase; PPO, polyphenol oxidase; QAC, quaternary ammonium compound; -GPR, -glutamyl phosphate
reductase; P5C, pyrroline-5-carboxylate; P5CR,  -pyrroline-5-carboxylate
reductase
Corresponding authors. Tel.: +91 4144 238248x354; fax: +91 4144 222265.
E-mail addresses: abdul79jaleel@rediffmail.com (C.A. Jaleel),
suriyagopi@yahoo.co.in (R. Gopi), kalaisomu 20@rediffmail.com
(R. Somasundaram).
0927-7765/$ see front matter 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.colsurfb.2007.05.001

as the absence of adequate moisture necessary for a plant to grow

normally and complete its life cycle [5]. The lack of adequate
moisture leading to water stress is common occurrence in rainfed areas, brought about by infrequent rains and poor irrigation
[6].
Plant experiences drought stress either when the water supply
to roots becomes difficult or when the transpiration rate becomes
very high and these two conditions often coincide under arid
and semiarid climates. Water stress tolerance is seen in almost
all plant species but its extent varies from species to species
[7]. Although the general effects of drought on plant growth
are fairly well known, the primary effects of water deficit at the
biochemical and molecular levels are not well understood [8].
Accumulation of proline (PRO) has been advocated for use as a
parameter of selection for stress tolerance [9]. PRO accumulation can be met with the stresses such as temperature, drought
and starvation [10]. High levels of PRO enabled the plant to
maintain low water potentials. By lowering water potentials, the
accumulation of compatible osmolytes, involved in osmoregulation allows additional water to be taken up from the environment,
thus buffering the immediate effect of water shortages with in the

C.A. Jaleel et al. / Colloids and Surfaces B: Biointerfaces 59 (2007) 150157

organism [11]. Occurrence of quaternary ammonium compound

(QAC) glycine betaine (GB) in response to oxidative stress like
salinity is a common phenomenon [12].
Mechanisms of drought tolerance, not yet clear, can be to
some extent explained by stress adaptation effectors that mediate
ion homeostasis, osmolyte biosynthesis, toxic radical scavenging, water transport and long distance response coordination
[13]. The typical first response of all plants to drought stress
is osmotic adjustment. Compatible solutes accumulation in the
cytoplasm is considered as a mechanism to contribute stress tolerance [14]. To counter with abiotic stress, plants increase the
osmotic potential of their cells by synthesizing and accumulating compatible osmolyte such as PRO and GB that participates
in the osmotic adjustment [15]. PRO and GB are thought to
function as osmoprotectants for proteins [16]. Accumulation
of PRO [17] and GB [18] provide an environment compatible with macromolecular structure and function and helps to
adapt the salinity injury [19]. Proline oxidase (PROX) and glutamyl kinase (-GK) play an important role in controlling
the level of PRO, PROX catalyzes the conversion of PRO to glutamate and -GK plays an important role in the synthesis of PRO
[19]. The enzymes -GK and -glutamyl phosphate reductase
(-GPR) are regarded as an enzyme complex called pyrroline-5carboxylate (P5C) synthetase because the resulting product, glutamic -semialdehyde is non-enzymatically converted to P5C.
From there, the P5C is converted into PRO by  -pyrroline-5carboxylate reductase (P5CR). The regulation of PRO synthesis
is probably controlled by the activity of P5C synthase [20].
In addition to this, an important consequence of abiotic stress
in plants is the excessive generation of reactive oxygen species
(ROS) such as superoxide anion (O2 ), H2 O2 and the hydroxyl
radicals particularly in chloroplast and mitochondria [21,22].
Generation of ROS cause rapid cell damage by triggering off a
chain reaction [23]. In order to survive under stress conditions,
plants are equipped with oxygen radical detoxifying enzymes
such as superoxide dismutase (SOD), ascorbate peroxidase
(APX), catalase (CAT) and antioxidant molecules like ascorbic
acid, -tocopherol and reduced glutathione (GSH) [24]. ROS
scavenging depends on the detoxification mechanism provided
by an integrated system of non-enzymatic reduced molecules
like ascorbic acid and glutathione and enzymatic antioxidants
[25]. Antioxidant mechanisms may provide a strategy to enhance
stress tolerance in plants.
With increasing realization of health hazards and toxicity
associated with the indiscriminate use of synthetic drugs and
antibiotics, more and more people are interested in the use of
plants and plant based drugs revived through out the world. So
exploitation of medicinal plants became more and more popular [26]. For the past several years, several scales of physiology
have been applied to study responses to drought stress tolerance
mechanisms and methods to overcome drought stress in field
crops [13]. However little information is gained about the physiological basis in terms of PRO metabolism under drought stress
in medicinal plants. It seems necessary to do research related to
the correlation between medicinal plants and drought stress for
the increasing need of medicinal plants. In order to meet the
ever increasing demand of medicinal plants, for the indigenous

151

systems of medicine as well as for the pharmaceutical industry,

some medicinal plants need to be cultivated commercially, but
the soil salinity and other forms of pollutions pose serious threats
to plant production [27]. So it seems valuable, to test the important medicinal plants for their abiotic stress tolerance capacity.
Catharanthus roseus (L.) G. Don (family: Apocynaceae) is
one of the highly exploited and studied medicinal plants. This
plant contains alkaloids which are valuable source of antitumour
agents like vinblastine and vincristine used in chemotherapy of
leukemia and in the treatment of Hodgkins disease, and also a
popular ornamental plant [28]. Despite the relative great number of reports on the medicinal aspects of C. roseus plants [29],
there are only a few attempts to explain the physiological basis
of drought effects and osmoregulation. To the best of our knowledge, no information on the physiological response interms of
PRO metabolisms of C. roseus to water deficit stress is available.
The objectives of this study were to provide additional information on the osmolyte concentration (AA, GB and PRO contents),
PRO metabolizing enzymes (-GK and PROX), antioxidant
enzyme activities (POX and PPO) and total indole alkaloid
accumulation in C. roseus under different water regimes.
2. Materials and methods
2.1. Plant materials and drought stress induction
The seeds of C. roseus (L.) G. Don (family: Apocynaceae)
were collected from the Department of Horticulture, Faculty
of Agriculture, Annamalai University, Tamilnadu, India. Seeds
were surface sterilized with 0.2% HgCl2 solution for 5 min with
frequent shaking and thoroughly washed many times with deionized water to remove HgCl2 . Six seeds were sown in each pot of
30 cm 30 cm containing 3 kg of soil mixture composed of red
soil, sand and the farmyard manure at 1:1:1 ratio. All the pots
were watered to the field capacity with ground water up to 30
days after sowing (DAS). The seedlings were thinned to 2 pot1
on 20 DAS. Pots were irrigated with ground water regularly as a
control and other treatment is 10, 15 and 20 days interval drought
(DID) from 30 DAS. The pots were covered with a rain out shelter, made up of plastic sheets, whenever rainfall was anticipated
and immediately after rain, rain out shelter was pulled back so
that, pots received maximum sunlight. Further, the pots were
regularly covered with rain out shelter during night time. Using
this system the pots were protected from rainfall and any external moisture entry. Plants were uprooted randomly on 41, 46
and 51 DAS, washed carefully and separated into root, stem and
leaf for analyses.
2.2. Osmolyte concentration
2.2.1. Estimation of AA content
Extraction and estimation of the AA content was followed by
the method of Moore and Stein [30]. 0.5 g of plant material taken
in a pestle and mortar and homogenized with 10 ml of 80% boiling ethanol. The extract was centrifuged at 800 g for 15 min
and the supernatant was made up to 10 ml with 80% ethanol and
used for the estimation of free AAs. 1 ml of ethanol extract was

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C.A. Jaleel et al. / Colloids and Surfaces B: Biointerfaces 59 (2007) 150157

taken in a 25 ml test tube and neutralized with with 0.1 N sodium

hydroxide using methyl red indicator. To which, 1 ml ninhydrin
reagent was added. The contents were boiled in a boiling water
bath for 20 min then 5 ml of diluting reagent was added, cooled
and diluted to 25 ml with distilled water. The absorbance was
read at 570 nm in a spectrophotometer. The standard graph was
prepared by using glycine. The AA content was calculated using
the standard graph.

5 mM MgCl2 , 0.1 ml of 0.5 mM NADP, 0.1 ml of 1 mM KCN,

0.1 ml of 1 mM phenazine methosulphate (PMS), 0.1 ml of
0.06 mM 2,6-dichlorophenol indophenol (DCPIP) and 0.1 ml
distilled water instead of PRO. The reaction was monitored
at 600 nm at 25 C using PRO to initiate reaction, the OD
value increased was noted at 0, 1, 2, 3, 4 and 5 min. PROX
activity was expressed in U mg1 protein (one U = mM DCPIP
reduced min1 mg1 protein).

2.2.2. Estimation of PRO content

The PRO content was estimated by the method of Bates
et al. [31]. The plant material was homogenized in 3% aqueous sulfosalicylic acid and the homogenate was centrifuged at
10000 rpm. Supernatant was used for the estimation of PRO content. The reaction mixture consisted of 2 ml acid ninhydrin and
2 ml of glacial acetic acid, which was boiled at 100 C for 1 h.
After termination of reaction in ice bath, the reaction mixture
was extracted with 4 ml of toluene and absorbance was read at
520 nm.

2.4. Assay of Antioxidative enzymes

2.2.3. Estimation of GB content

The amount of GB was estimated according to the method of
Grieve and Grattan [32]. The plant tissue was finely ground,
mechanically shaken with 20 ml deionized water for 24 h at
25 C. The samples were then filtered and filtrates were diluted
to 1:1 with 2 N H2 SO4 . Aliquots were kept in centrifuge tubes
and cooled in ice water for 1 h. Cold KI-I2 reagent was added and
the reactants were gently stirred with a vortex mixture. The tubes
were stored at 4 C for 16 h and then centrifuged at 10000 rpm
for 15 min at 0 C. The supernatant was carefully aspirated with
a fine glass tube. The periodide crystals were dissolved in 9 ml
of 1,2-dichloroethane. After 2 h, the absorbance was measured
at 365 nm using GB as standard.
2.3. Assay of PRO metabolizing enzymes
2.3.1. -Glutamyl kinase [ATP: l-glutamate
5-phosphotransferases (EC 2.7.2.11)] activity
-GK activity was assayed by the method of Hayzer and
Leisinger [33]. Plant samples (1 g) were extracted with 50 mM
Tris-HCl buffer and centrifuged at 40,000 rpm for 30 min at 4 C.
0.1 ml reaction buffer was prepared by adding 0.1 ml 10 ATP
and 1.8 ml of extract and incubated at 37 C for 30 min, 2 ml of
stop buffer was added. -GK activity was measured at 535 nm
and expressed in units (U mg1 protein). One U of enzyme activity is defined as g of -glutamylhydroxamate formed min1
mg1 protein.
2.3.2. Proline oxidase [L. proline: O2 oxidoreductase (EC
1.4.3.1)] activity
PROX activity was determined according to the method
outlined by Huang and Cavalieri [34]. Plant samples (1 g)
were extracted with 5 ml of TrisHCl buffer (pH 8.5) grinding medium and centrifuged at 10,000 rpm for 10 min at 4 C.
The supernatant was again centrifuged at 25,000 rpm at 20 min
at 4 C. 3 ml assay mixture was prepared by taking 0.1 ml of
extract, 1.2 ml of 50 mM Tris HCl buffer (pH 8.5), 1.2 ml of

2.4.1. Peroxidase (POX, EC 1.11.1.7) activity

POX was assayed by the method of Kumar and Khan [35].
Assay mixture of POX contained 2 ml of 0.1 M phosphate
buffer (pH 6.8), 1 ml of 0.01 M pyrogallol, 1 ml of 0.005 M
H2 O2 and 0.5 ml of enzyme extract. The solution was incubated for 5 min at 25 C after which the reaction was terminated
by adding 1 ml of 2.5 N H2 SO4 . The amount of purpurogallin
formed was determined by measuring the absorbance at 420 nm
against a blank prepared by adding the extract after the addition of 2.5 N H2 SO4 at zero time. The activity was expressed
in U mg1 protein. One unit is defined as the change in the
absorbance by 0.1 min1 mg1 protein.
2.4.2. Polyphenol oxidase (PPO, EC 1.10.3.1) activity
The assay of PPO was carried out by the method of Kumar
and Khan [35]. Assay mixture for PPO contained 2 ml of 0.1 M
phosphate buffer (pH 6.0), 1 ml of 0.1 M catechol and 0.5 ml
of enzyme extract. This was incubated for 5 min at 25 C, after
which the reaction was stopped by adding 1 ml of 2.5 N H2 SO4 .
The absorbancy of the purpurogallin formed was read at 495 nm.
To the blank 2.5 N H2 SO4 was added of the zero time of the
same assay mixture. PPO activity is expressed in U mg1 protein
(U = change in 0.1 absorbance min1 mg1 protein). For all the
enzymatic calculations protein was determined by the method
of Bradford [36], using bovine serum albumin (BSA, Sigma,
USA) as the standard.
2.5. Indole alkaloid accumulation
Total alkaloid extraction and determination was carried out
by following the standard extraction method of Endo et al. [37]
with modification of Misra and Gupta [38].
2.6. Statistical analysis
Statistical analysis was performed using one way analysis of
variance (ANOVA) followed by Duncans Multiple Range Test
(DMRT). The values are mean S.D. for six samples in each
group. P values 0.05 were considered as significant.
3. Results
3.1. Effects of drought stress on AA content
Drought stress caused an increase in AA content in leaves
and root of C. roseus plants when compared with well-watered

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153

Fig. 2. Effect of drought on Glutamyl kinase (a) and proline oxidase (b) activities
in leaves and roots of C. roseus plants. Values are given as mean S.D. of six
samples in each group. Values are not sharing a common superscript (a and b)
differ significantly at P 0.05 (DMRT).

Fig. 1. Effect of drought on aminoacid (a) and proline (b) glycine betaine
(c)contents in leaves and roots of Catharanthus roseus plants. Values are given
as mean S.D. of six samples in each group. Values are not sharing a common
superscript (a and b) differ significantly at P 0.05 (DMRT).

control (Fig. 1a). The AA content was more in roots when compared to leaves. In leaf samples, extend of increase in AA content
at 10 and 15 DID was not significant. But there was significant enhancement in 20 DID (P 0.05). In the case of root
also, there was negligible increase at 10 DID, but significantly
increased (P 0.05) under 15 and 20 DID, when compared to
well-watered control plants.
3.2. Effects of drought stress on PRO content
A compatible solute, which accumulates under abiotic stress
in plants, is PRO. In the present study, an increase in PRO
accumulation (Fig. 1b) in C. roseus under drought stress with a
concomitant increase in -GK (PRO synthesizing enzyme) and
a decrease in PROX (PRO degrading enzyme) activities. In both
leaf and root samples at all DID, there was significant (P 0.05)
enhancement in PRO content when compared to control plants.
3.3. Effects of drought stress on GB content
One of the most important mechanisms exerted by higher
plants under stress conditions is the accumulation of compatible solutes such as GB. In the present study, the amount of
GB content increased with water deficit stress in C. roseus
plants (Fig. 1c). The effect of water deficit was not so profound

(P 0.05) in root samples in the case of GB accumulation when

compared to leaf samples, where the increase was significant
(P 0.05).
3.4. Effects of drought stress on -GK activity
The -GK activity has been increased in shoot and root to
a large extent in the drought stressed C. roseus plants when
compared with well-watered control (Fig. 2a). In both leaf and
root samples, the effect of water deficit stress was significant
(P 0.05) when compared to control plants. All the treatments
(10, 15 and 20 DID) significantly increased the -GK activity in
leaf and root samples.
3.5. Effects of drought stress PROX activity
PROX activity has been inhibited by drought stress to a large
extent (P 0.05) in all parts of C. roseus when compared with
control (Fig. 2b). Both leaf and root samples, showed decreased
PROX activity, but it was more profound in the case of leaf
samples.
3.6. Effects of drought stress on POX activity
The POX activity has been increased in leaf and root to a large
extent in the drought stressed C. roseus plants when compared
with well-watered control (Fig. 3a). In leaf samples, the effect
of water deficit stress were not significant when compared to
control plants in 10 and 15 DID. All the treatments (10, 15 and
20 DID) significantly increased (P 0.05) the POX activity in
root samples when compared to control.

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C.A. Jaleel et al. / Colloids and Surfaces B: Biointerfaces 59 (2007) 150157

Fig. 4. Effect of drought on total indole alkaloid accumulation in leaves and

roots of C. roseus plants. Values are given as mean S.D. of six samples in each
group. Values are not sharing a common superscript (a and b) differ significantly
at P 0.05 (DMRT).

Fig. 3. Effect of drought on peroxidase (a) and polyphenol oxidase (b) activities
in leaves and roots of C. roseus plants. Values are given as mean S.D. of six
samples in each group. Values are not sharing a common superscript (a and b)
differ significantly at P 0.05 (DMRT).

3.7. Effects of drought stress on PPO activity

The PPO activity has been increased in leaf and root to a large
extent in the drought stressed C. roseus plants when compared
with well-watered control (Fig. 3b). In leaf samples, the effect
of water deficit stress were not significant when compared to
control plants in 10 DID but it significantly increased (P 0.05)
under 15 and 20 DID. All the treatments (10, 15 and 20 DID) significantly increased the PPO activity in root samples (P 0.05)
when compared to control.
3.8. Effects of drought stress on indole alkaloid
accumulation
The indole alkaloid content increased significantly (P 0.05)
in leaves and roots of drought stressed C. roseus plants when
compared to well-watered control plants (Fig. 4). The highest
alkaloid accumulation was recorded in roots than leaves.
4. Discussion
Drought stress caused increased AA content in leaves and root
of C. roseus plants when compared with well-watered control.
The AA content has been shown to increase under drought condition in sorghum [39], pepper [40] and Arachis hypogaea [41].
The accumulation of AA may be due to the hydrolysis of protein
and also may be occurring in response to the change in osmotic
adjustment of their cellular contents [42]. Free AA accumulation
is more important account for most of the changes in osmotic
potential. The accumulation of free AA under stress indicates the possibility of their involvement in osmotic adjustment
[39]. Osmotic adjustment is one of the important mechanisms,

which alleviates some of the detrimental effects of water stress

[43].
In most plants, there is an increased accumulation of AA and
amines (eg: PRO, B-alanine, GB) in their tissues in response
to stress. The way these compounds are accumulated differs
between species and ranges form only one to several different
compounds being accumulated. Generally, plant species that
accumulate PRO usually have low amounts of this AA when
grown in well-watered and non-saline soils, increasing its contents upon imposition of drought or salt stresses [44]. Increased
PRO accumulation was reported in water stressed sorghum [39]
and bell pepper [40]. Increased PRO in the stressed plants may be
an adaptation to overcome the stress conditions. PRO accumulated under stressed conditions supplies energy for growth and
survival and thereby helps the plant to tolerate stress [45]. Under
abiotic stress like ultra violet light the PRO content showed an
increase in wheat [46]. NaCl stress showed increased PRO content in rice [47] and peanut [19]. PRO accumulation in plants
might be a scavenger and acting as an osmolyte. The reduced
PROX may be the reason for increasing PRO accumulation.
The GB content increased under drought stress in barley
[48] and in higher plants [49]. The GB content increased under
light stress in Arabidopsis [50]. Aliphatic QAC such as GB,
stachydrine, homostachydrine, trigonelline have been found
to accumulate in a large number of plants exposed to salt
and water stresses. Glycophytes like tomato, peas and beans
showed an increase in GB with increasing salinity [51,19]. GB
is synthesized in chloroplasts as two enzymes, namely choline
monoxygenase (CMO) and betaine aldehydehydrogenase, are
responsible for GB synthesis chloroplastically. The accumulation of GB might serve as an intercellular osmoticum of GB and
could be closely correlated with elevation of osmotic pressure
[52]. GB may maintain the osmoticum, provided that the basal
metabolism of the plant can sustain a high rate of synthesis of
these compounds to facilitate osmotic adjustment for tolerance
to water stress [15].
The -GK activity has been increased in shoot and root to a
large extent in the drought stressed C. roseus plants when compared with well-watered control. Similar results were reported

C.A. Jaleel et al. / Colloids and Surfaces B: Biointerfaces 59 (2007) 150157

in abiotic stressed plants. The NaCl stress induced PRO accumulation in tobacco cells lies at increased -GK level [53]. The
induction of PRO accumulation may be due to an activation of
PRO synthesis through glutamate pathway involving -GK, glutamyl phosphate reductase and P5CR activities [54]. The PRO
accumulation in drought stressed C. roseus can be attributed to
the increased level of -GK activity [55]. The -GK activity can
be inversely correlated with PROX activity and protein content
in salt treated plants [19].
Under water deficit condition the PRO metabolizing enzyme
PROX decreased in C. roseus plants. This report coincides with
earlier reports under water stress in Arabidopsis [56] and in
tomato [54]. The decrease in PROX activity with increasing GK activity might be the reason for higher PRO accumulation
in drought stressed C. roseus plants. This enzyme converts free
PRO into glutamate. Reduction in PROX activity and simultaneous increase in PRO level were reported in low temperature
stressed wheat [57]. PROX, oxidize the PRO and convert it back
to glutamate. This enzyme also influences the level of free PRO.
PRO accumulation in salt stressed cell may also be explained
by a decreased oxidation of PRO during salt stress. The activity
of PRO degrading enzymes, PROX and proline dehydrogenase
(PDH) were significantly inhibited in the salt stressed green
gram seedlings [51]. PRO may act as a non-toxic osmotic solute
preferentially located in the cytoplasm or as an enzyme protectant, stabilizing the structure of macromolecules and organelles.
Accumulated PRO may supply energy to increase salinity tolerance [17,58]. PRO as an osmoprotectant compound, plays a
major role in osomoregulation and osmotolerance [46]. However its definite role in exerting stress resistance continues to be
a debate [59].
Several plants, including halophytes accumulate high PRO
levels in response to osmotic stress as a tolerance mechanism to
high salinity and water deficit [60,61]. In plants, PRO is synthesised from either glutamate or ornithine. However, the glutamate
pathway is primary route used under osmotic stress or nitrogen
limitation conditions whereas the ornithine pathway is prominent under high nitrogen input [15,62]. However, recent data
suggest that glutamate is the major AA involved in PRO synthesis, since transgenic tobacco plants with reduced expression of
cytosolic glutamine synthetase accumulated less PRO than nontransformed plants in response to salt stress [63]. Accumulation
of PRO in plants under stress is a result of the reciprocal regulation of two pathways: [(1 -pyrroline-5-carboxylate synthetase
(P5CS) and P5CR] and repressed activity of PRO degradation
[61]. PRO catabolism is catalyzed by pyrroline-5-carboxylate
dehydrogenase and PDH, a mitochondrial enzyme, whose activity had been shown to reduce during salt stress [64]. The first
two steps of PRO biosynthesis are catalysed by P5CS by means
of its -GK and glutamic--semialdehyde dehydrogenase activities. Subsequently, the P5C formed is reduced by P5CR to PRO
[65].
Although the precise role of PRO accumulation is still
debated, PRO is often considered as a compatible solute involved
in osmotic adjustment [66]. The accumulation of PRO may
be through an increase in its synthesis constantly with inhibition of its catabolism [67] and may be a mechanism for

155

stress tolerance. However, its role in imparting stress resistance

under saline conditions is controversial. Anyway, understanding
the biosynthesis, degradation, transport and role of PRO during stress and the signaling events that regulate stress-induced
accumulation is vital in developing plants for stress tolerance
[15].
ROS scavenging is important in imparting tolerance against
oxidative stress. It may be presumed that enhancement of the
antioxidative system favours water stress resistance [68]. PPO
is believed to be ubiquitous in the plant kingdom and they
are primarily associated with the enzymatic browning and offflavour generation. Increasing evidences suggests that drought
induces oxidative stress in various plants, in which ROS, such
as superoxide radical (O2 ), hydroxy radical ( OH), H2 O2 and
alkoxy radical (RO ) are produced [69]. The toxic superoxide
radical has a half-life of less than one second and is usually
rapidly dismutated by SOD to H2 O2 , a product that is relatively
stable and can be detoxified by CAT and POX. These metalloenzymes constitute an important primary defense of cells against
superoxide free radicals generated under stress conditions and
thereby increased activity is known to confer oxidative stress tolerance [70]. Although the general effects of drought on plants
are fairly well known, the primary effects of water deficit at
the biochemical and molecular levels are not well understood
[7]. Oxidative damage in the plant tissue is alleviated by a
concerted action of both enzymatic and non-enzymatic antioxidant metabolisms. There are many reports in the literature that
underline the intimate relationship between enhanced antioxidant enzyme activities and increased resistance to environmental
stresses [71].
The total alkaloid accumulation in both shoot and root of
C. roseus was found increased significantly under the oxidative
stress resulted form drought stress. It is reported previously that
the abiotic stresses can increase the alkaloid content in plants
[72]. The content of alkaloids in C. roseus have been found
influenced by individual factor, such as stage of plant growth
[73], moisture stress and nitrogen fertilization [28] and triadimefon, a plant growth regulator, treatment [29]. There are reports
on improvement of indole alkaloid production in cell cultures
of C. roseus treated by various chemicals, elicitors, earth elements and bioregulators [74]. Recently it has also been reported
that indole alkaloid production in C. roseus can be increased by
combined treatment of salinity and different nitrogen sources
[38].
5. Conclusion
Thus it is clear that, plants under drought stress are highly
regulated by components of osmoregulative and antioxidative
systems and secondary metabolite contents. Our results indicated that the cultivation of medicinal plants like C. roseus
in water deficit areas would increase its PRO metabolism,
osmoregulation, defense system and the level of active principles. However, the data presented here reflect the importance of
a physiological analysis of plant response, which must accompany field experiments and evaluation. Further investigations are
required to ascertain this conclusion.

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