Appl Biochem Biotechnol (2014) 174:693707

DOI 10.1007/s12010-014-1098-5

Synergistic Effects of Drought Stress and Photoperiods

on Phenology and Secondary Metabolism of Silybum
marianum
Adnan Zahir & Bilal Haider Abbasi & Muhammad Adil &
Sumaira Anjum & Muhammad Zia & Ihsan-ul-haq

Received: 23 March 2014 / Accepted: 22 July 2014 /

Published online: 3 August 2014
# Springer Science+Business Media New York 2014

Abstract Silybum marianum is an important medicinal plant of the family Asteraceae, well
known for its set of bioactive isomeric mixture of secondary metabolites silymarin, primarily
acting as a hepato-protective agent. Abiotic stress augments plant secondary metabolism in
different plant tissues to withstand harsh environmental fluctuations. In the current study, our
aim was to induce drought stress in vitro on S. marianum under the influence of different
photoperiod treatments to study the effects, with respect to variations in secondary metabolic
profile and plant growth and development. S. marianum was extremely vulnerable to different
levels of mannitol-induced drought stress. Water deficiency inhibited root induction completely and retarded plant growth was observed; however, phytochemical analysis revealed enhanced accumulation of total phenolic content (TPC), total flavonoid content (TFC), and total
protein content along with several antioxidative enzymes. Secondary metabolic content was
positively regulated with increasing degree of drought stress. A dependent correlation of seed
germination frequency at mild drought stress and antioxidative activities was established with
2 weeks dark+2 weeks 16/8 h photoperiod treatment, respectively, whereas a positive
correlation existed for TPC and TFC when 4 weeks 16/8 h photoperiod treatment was applied.
The effects of drought stress are discussed in relation to phenology, seed germination
frequency, biomass build up, antioxidative potential, and secondary metabolites accumulation.
Keywords Drought stress . Phenology . Secondary metabolites . Silymarin . Mannitol
Abbreviations
TPC
Total phenolic content
TFC
Total flavonoid content
ROS
Reactive oxygen species
TDZ
Thidiazuron
BA
6-Benzyladenine
A. Zahir : B. H. Abbasi (*) : M. Adil : S. Anjum : M. Zia
Department of Biotechnology, Quaid-i-Azam University, Islamabad 45320, Pakistan
e-mail: bhabbasi@qau.edu.pk
Ihsan-ul-haq
Department of Pharmacy, Quaid-i-Azam University, Islamabad 45320, Pakistan

694

GA3
FW
DW
WC
DPPH
PVP
BSA
OD
POD
SOD
NBT
ANOVA
DMRT
GAE
QUE
FRSA
SDS-PAGE
CAT

Appl Biochem Biotechnol (2014) 174:693707

Gibberellic acid
Fresh weight
Dry weight
Water content
1, 1-Diphenyl-2-picrylhydrazyl
Polyvinyl pyrrolidone
Bovine serum albumen
Optical density
Peroxidase
Superoxide dismutase
Nitro blue tetrazolium
Analyses of variance
Duncan multiple range test
Gallic acid equivalent
Quercetin equivalent
Free radical scavenging activity
Sodium dodecyl sulfate-poly acrylamide gel electrophoresis
Catalase

Introduction
Secondary metabolites are not only central to plant defense mechanisms but also provide a
great source of novel bioactive compounds, exhibiting potential of various pharmacological
activities against many human disorders and diseases [1, 2]. Although produced in plants at
low levels in normal circumstances, their production levels are regulated by fluctuating biotic
and abiotic environmental factors, such as exposure of plants to higher and lower temperatures,
saline conditions, change in pH, humidity, heat shock, UV irradiation, and drought stress [3].
These conditions trigger to bring about changes in plant metabolic activities affecting overall
plant growth and developmental processes. Plants withstand such harsh and offensive environments by switching to enhanced metabolism of secondary metabolites.
Drought stress is one among many types of abiotic stresses that has been related to low crop
productivity in agriculture but its imposition on medicinal plants during in vitro culturing has
rather seen a different prospect. Water deficiency stress is crucial in the behavior of plant
response by causing a shuffle in metabolism [4]. Generally, drought stress reduces water
uptake by plants, depriving plants of essential nutrients thereby affecting the plants ability to
photosynthesize food, which retards growth and development of the plant, characterized by
reduced leaf size, stem elongation, and proliferation of roots [5]. Drought stress forces stomatal
closure to ensure water-use efficiency which causes reduced CO2 assimilation by leaves
rendering plants to divert metabolic flux through photorespiratory process. Such work overload results in the generation of reactive oxygen species (ROS) that causes membrane damage,
disturbed activities of several enzymes, and injury to biomolecules like DNA, lipids, and
amino acids [6]. To alleviate these harmful effects, plants activates its antioxidative defense
system comprising of both enzymatic and non-enzymatic antioxidants. Non-enzymatic antioxidants are the most important because they constitute a class of compounds such as
phenolics and flavonoids which not only defend plants from excessive damage but also show
medicinal characteristics especially against human health problems [7]. Drought stress has
been documented to increase the amount of phenolic acids and flavonoids in some plants.
Flavonoids are important for plants to provide protection against toxic metals [8]. Anthocyanins, an important class of flavonoids which imparts different attractive colors to fruits and

Appl Biochem Biotechnol (2014) 174:693707

695

protect cells from high light damage acting as sunscreen in photosynthetic tissues, have
neuroprotective effects, also accumulate when plants are exposed to drought stress [9].
S. marianum is an important traditional medicinal plant of the family Asteraceae. This plant
grows wild in nature and is also being cultivated on large areas in some parts of the world for
commercial production of its novel secondary metabolites. The major component of secondary
metabolites includes a set of isoflavonoids, collectively known as silymarin along with other
several bioactive compounds [10]. The major traditional medicinal application of silymarin
being in practice is its hepato-protective activity and is also a part of modern day medication
for the treatment of liver diseases [11]. Current study was carried to examine if mannitolinduced drought stress would increase the secondary metabolic content of S. marianum and to
evaluate how seed germination and growth of S. marianum counter in such a stressed
condition under the influence of different photoperiod treatments.

Material and Methods

Seed Germination
Prior to inoculation, seeds of S. marianum were surface-sterilized by immersing in 0.1 %
HgCl2 solution for 2 min, 70 % ethyl alcohol for 3 min, and washed three times with distilled
water. These sterilized seeds were inoculated on Murashige and Skoog (MS) [12] medium
containing 30 g l1sucrose, 8 g l1 agar as solidifying agent, and a single combination of
different plant growth regulators (0.5 mg l1 BA+1 mg l1 TDZ+1.5 mg l1 GA3). For
induction of drought stress, various concentrations of mannitol (0.25, 0.5, 1.0, 2.0, 4.0, 6.0,
8.0, and 10.0 g l1, respectively) to each treatment were added.
Plant Growth Conditions
Culture flasks each containing 46 seeds were kept in growth room under the influence of
three different photoperiod treatments: (a) complete darkness for 4 weeks, (b) 2 weeks
complete darkness+2 weeks 16/8 h dark and light photoperiod using cool white fluorescent
tubes at an irradiance level of 4050 mol/m2/s, and (c) 4 weeks 16/8 h dark and light
photoperiod. MS basal medium (MS0) was used as a control. Plants were allowed to grow at a
maintained temperature of 252 C till the end of 4 weeks. For dark treatment, culture flasks
were kept in closed paper boxes.
Plant Phenology
After 4 weeks of growth, plants were collected for determination of various growth parameters.
Plants were weighed for determination of fresh weight (FW) and then oven dried at 60 C for
48 h for dry weight (DW) determination. Water content (WC) was measured by the given
formula WC=FWDW/DW. Length of radical and shoot, leaf length and width were also
measured.
Estimation of Total Phenolic Content and Total Flavonoid Content
For estimation of total phenolic and flavonoid content, 300 mg powdered plant material,
1,500 l methanol (100 % pure) was added and vortexed followed by sonication for 30 min.
The mixture was vortexed again for proper shaking and mixing. Samples were centrifuged at

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Appl Biochem Biotechnol (2014) 174:693707

13,000 rpm for 5 min. Supernatant was taken and used for further analysis. Aluminum chloride
colorimetric method [13] was used for total flavonoid content (TFC) determination. TFC was
quantified with respect to standard curve of quercitin. For total phenolic content (TPC)
determination, Folin-Ciocalteu method was used [14]. TPC was quantified with respect to
standard curve of gallic acid. Absorption of extracts for determination of TFC and TPC were
taken at 415 and 630 nm, respectively, using UVVis spectrophotometer (Halo DR-20, UV
Vis spectrophotometer, Dynamica Ltd., Victoria, Australia). Values of the phytochemical
content were expressed in milligram equivalents per gram dry weight of plantlets.
Free Radical Scavenging Assay
The method of Lee et al. [15] was followed to study the free radical scavenging potential of
samples spectrophotometrically at 517 nm using 1,1-diphenyl-2-picrylhydrazyl (DPPH). The
capacity of samples to scavenge DPPH free radical was calculated by the following equation:
% scavenging Absorbance of control Absorbance of sample=Absorbance of control
 100
Protein Content and Enzymatic Assays
For estimation of total protein content and enzymatic assays, extraction from fresh samples
was done by the method of Nayyar and Gupta [16] with some modifications. Briefly, 100 mg
whole fresh plant sample was homogenized with 1 ml of 50 mM potassium phosphate buffer
containing 1 % PVP (buffer pH 7). The homogenate obtained was centrifuged at 15,000g for
30 min and the resultant supernatant was isolated and utilized for protein estimation and
different enzymatic assays.
Estimation of Total Protein Content
The method of Lowry et al. [17] was followed for the assessment of total protein content.
Bovine serum albumen (BSA) was used as a standard, and calibration curve was made to
quantify total protein content.
Protease Activity Assay
This assay was performed following the method of Ullah et al. [18]. Enzyme activity was
described as increase in absorbance of reaction mixture per unit time under given conditions of
assay. Activity of proteases (EC; 3.4) was calculated by the given formula:
Protease Activity OD=0:1 U=g FW; U=g FW units= gram fresh weight:
Peroxidase and Superoxide Dismutase Activity Assay
The modified method of Lagrimini [19] was used for the assessment of peroxidase activity
(POD; EC 1.11.1.7). POD is involved in catalytic conversion of guaiacol into tetra guaiacol in
the presence of H2O2 which is the principle of this assay. Superoxide dismutase (SOD; EC
1.15.1.1) assay was performed with accordance to the protocol adapted by Giannopolitis and

Appl Biochem Biotechnol (2014) 174:693707

697

Ries [20]. SOD inhibits photochemical reduction of nitro blue tetrazolium (NBT) which is the
principle of this assay. Activity of enzymes was calculated according to the given formula: A=
ELC, where A=absorbance, E=extinction coefficient (6.39 mM-1 cm-1), L=length of each
wall (0.25 cm), C=concentration of enzyme (value of C measured in nM/min/mgFW), FW=
fresh weight of the sample.
Statistical Analysis
Six culture flasks were used in each treatment, and data was recorded as mean values from
triplicates. ANOVA (analyses of variance) and DMRT (Duncan multiple range test) were used
for comparisons of means of different treatments. Significance was tested among data of the
same photoperiod treatments using Statistix 8.1 at p<0.05.

Results and Discussion

Effect of Drought Stress on Seed Germination
Mild drought stress levels enhanced the seed germination frequency of S. marianum up to
80 % at concentration of 1.0 g l1 mannitol (Table 1). Several reports suggest that mild level of
drought stress might regulate the critical moisture content for the metabolism of nutrients
required for seed germination [21]. Studies conducted on carrot supported the supposition that
early stages of seed activation could occur at osmotic potentials lesser than those that allowed
radicle projection from the seed [22]. Drought stress delays and reduces seed germination
frequency or may even cause seed germination failure [23]. Increasing drought stress negatively impacted seed germination frequency, lowest germination frequency was recorded at
concentration of 10.0 g l1 mannitol. The role of 2 weeks dark+2 weeks 16/8 h photoperiod
treatment was prominent in highest seed germination frequencies obtained, which affirmed the
results of the previous study conducted to evaluate the effect of different light regimes on seed
germination of S. marianum [24].
Effect of Drought Stress on Plant Phenology
Drought stress significantly affected the development of roots and growth of shoots. A
comparatively reduced plantlet height was observed in all the treatments. Four weeks dark
photoperiod treatment showed a little enhancing influence on shoots growth but none of the
different photoperiod treatments had an effect on root induction (Table 1). Only the radicle
protruded and elongated in some plantlets, lateral branching off or any other similar root
growth was completely inhibited. Roots play a key role during drought stress to retain water,
and plant resistance to drought stress is dependent on root performance. Even low concentrations of mannitol have been reported to block protein degradation and utilization in the storage
vacuoles of root cells leading to retarded growth [25]. Drought stress significantly affected the
growth of shoots of S. marianum. These results were similar with the effects of salinity and
drought stress on Helianthus annus L. (sunflower), a plant from the same family [26].
Drought stress lowered water content in all the treatments applied by a great margin
compared to control with some variations. Highest water content was obtained at mild stress
and lowest value was recorded for intense stress level. Different photoperiod treatments
applied appeared to have caused negligible variation in effect on water content levels of
S. marianum (Fig. 1).

17.53a

27.54a

12.32a

24.1d

LL

LW

SF

705a

RL

SL

28.6g

1128a

RL

6.11a

41.4d

SF

SF

12.42a

LW

LW

28.23a

LL

808a
21.34a

40.65a

SL

SL
LL

617a

RLa

25d

7.51.3b

13.72b

10.12b

4.41.2c

62a

4.51cde

20.35b
8.22e

82cd

78.6a

5.21.2bc

10.32c

10.53f

8.452bc

0.25

28.1c

7.41.4b

13.82b

92.8bc

4.51.3bc

50c

4.71bcd

19.254bc
8.71.3d

9.51.8bc

76.7b

5.81.6bc

12.13.7b

14.74bc

92.5b

0.5

24d

5.61d

11.41.8d

10.81b

4.651.1d

55.2b

4.91b

196bc
9.62c

103b

80.7a

5.61.2bc

11.62.5b

15.53b

8.62bc

1.0

51.5a

6.252cd

12.23d

9.51.4bc

4.851.5b

50c

4.451de

20.35bc
8.652.5d

7.81.4cd

32.1f

5.21.3bc

10.21.2cd

12.43.6def

7.61.8bc

2.0

53.6a

6.82.2cd

10.62.2d

8.72.1bc

3.81de

35.7f

4.81.2bc

14.63e
91.7d

5.91.9ef

50c

5.51bc

9.71.6cd

134cd

4.81d

4.0

40b

6.21.6bc

13.81.6b

9.81.5bc

3.61.4e

40.6e

5.21.5b

17.52.3cd
10.12c

6.92.5de

34.4e

6.42b

10.152c

112ef

5.41.6d

6.0

37.1c

6.852bc

1221d

6.31c

2.71f

44.8d

4.41.3e

14.12.9e
8.12.1e

5.51.2ef

32.3f

4.51.3c

9.351d

12.74.5cde

72c

8.0

9e

61.8d

7.52.5b

82bc

2.51.2f

8.8h

52b

163de
121.5b

4.71.2f

21.9g

6.21bc

11.71.6b

11.21.2def

3.851e

10.0

Root length in case of MS0

X 2 weeks dark+2 weeks 16/8 h, Y 4 weeks dark, Z 4 weeks 16/8 h, RL radicle length, SL shoot length, LL leaf length, LW leaf width (all values expressed in millimeter), SF percent
seed germination frequency

Similar letters are significantly not different

Values are meanstandard error of triplicates and significance was tested among data of the same photoperiod treatments

Photoperiod treatments

MS0

Mannitol concentration (gl1)

Table 1 Effects of different drought stress levels on various growth parameters of S. marianum

698
Appl Biochem Biotechnol (2014) 174:693707

Appl Biochem Biotechnol (2014) 174:693707

699

55

2 weeks dark + 2 weeks 16/8 h photoperiod

4 weeks dark
4 weeks 16/8 h photoperiod

50

40

a
c

bc
d

de

15

c
e

ef

20

bc

bb
b

cd
de

25

de

30

de

35

cd
cd

water content (g/g)

45

10
5
0
-1

mannitol concentration (gl )

Fig. 1 Effect of drought stress on water content, water content reduced with increasing concentration of
mannitol in the medium. Values are meanstandard error of triplicates and significance was tested among data
of the same photoperiod treatments

We observed reduced growth rates of leaves and other photosynthetic parts of S. marianum,
judged against control, especially in dark treatments where photosynthesis was affected by
absence of a key factor light, which is critical in the processes of photosystems. Leaves
surface area measured in terms of length and width was found to be reduced and only two
cotyledonous leaves were formed in all the plantlets except control where first true leaves were
also formed. Four weeks 16/8 h photoperiod treatment supported the leaf growth (Table 1).
These results were in agreement with experimental studies conducted to test plants
800

a
a
d

400

b
300

fe
f

c
c

de

500

ef

600

2 weeks dark + 2 weeks 16/8 h phtoperiod

4 weeks dark photoperiod
4 weeks 16/8 h photoperiod

cd

mean fresh weight (mg)

700

c
e

g
200

100
0
-1

mannitol concentration (gl )

Fig. 2 Mean fresh weight of S. marianum plantlets, a sudden decline in fresh mass occurred upon induction of
drought stress and fresh mass values were relative to water content of the plantlets as nutrient availability was
dependent on water content. Values are meanstandard error of triplicates and significance was tested among
data of the same photoperiod treatments

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Appl Biochem Biotechnol (2014) 174:693707

photosynthetic performance under drought stress whereby it was concluded that during water
deficiency, plant growth is first limited by a decline in the growth rate of assimilatory surface
(absorption and utilization of nutrients and required converting factors) and then suppression
of leaf photosynthesis [27].
Drought stress badly affected fresh weight and dry weight of S. marianum compared to
control. The enhancement of fresh weight was positively correlated with water content of the
plantlets irrespective of the photoperiod treatments applied (Fig. 2). An increasing trend in dry
weight was also observed along the scale of all levels of stress applied (Fig. 3). The low values
of fresh weight and dry matter accumulation obtained in the current study could be attributed
to the affected metabolism of carbon and nitrogen [28]. Drought stress causes reduced uptake
of water along with dissolved nutrients and has been indicated to decrease nitrogen fixation
ability of soybean plants. A correlation between soil moisture content and nitrogen-fixing
activity existed with the progressive increase in drying. It was suggested that water stress
affected nodule activity directly [29]. Study conducted to evaluate the effect of drought stress
on photosynthetic carbon metabolism in spinach revealed that even low levels of water deficit
caused suppression of starch synthesis [30]. Water stress also causes a reduction in biosynthesis of lignin, a biopolymer which constitutes a quarter to a third of dry mass [31].
Effect of Drought Stress on Secondary Metabolic Content
In the current study, we obtained higher concentrations of total phenolics and total flavonoids
and maximum DPPH free radical scavenging activity in drought-affected S. marianum plantlets compared to control. Total phenolic and total flavonoid accumulation was enhanced with
the increasing levels of stress. Maximum phenolic accumulation of 55.2 mg GAE/g DW was
recorded at 10.0 g l1 mannitol (Fig. 4). A similar study also showed that water deficit
enhanced artemisinin content in leaves and whole plants [of Artemisia annua L. [32].
26
2 weeks dark + 2 weeks 16/8 h photoperiod
4 weeks dark
4 weeks 16/8 h photoperiod

24
22

bcd
de

ab
bc

bc
cd

c
cd

12

cd

14

de

16

aa

cd
ab

mean dry weight (mg)

18

ab

20

10
8
6
4
2
0
-1

mannitol concentration (gl )

Fig. 3 Dry weight of S. marianum plantlets, reduced dry weight depicted the affected primary metabolism as
photosynthetic process was arrested by drought stress. Values are meanstandard error of triplicates and
significance was tested among data of the same photoperiod treatments

Appl Biochem Biotechnol (2014) 174:693707

701

2 weeks dark + 2 weeks 16/8 h phtoperiod

4 weeks dark photoperiod
4 weeks 16/8 h photoperiod

70

60

50
40

f
e

cd

total phenolic content (mg GAE/g DW)

80

d
c a

b
a

b
b

b
b

30
20
10
0
-1

mannitol concentration (gl )

Fig. 4 Total Phenolic Content (TPC) obtained at different levels of drought stress, TPC accumulation was
correlative to dry weight at moderate and higher levels of drought stress, which clearly showed the role of
phenolics in alleviating stress. Values are meanstandard error of triplicates and significance was tested among
data of the same photoperiod treatments

Optimum phenolic accumulation occurred when 4 weeks 16/8 h photoperiod treatment was
applied. A dependent correlation of accumulation of total phenolic content existed with dry
weight of plantlets of S. marianum. Similar effects were also observed for total flavonoid
content but total flavonoid accumulation was relative to total phenolic accumulation. Maximum accumulation of flavonoid content, 4.8 mg of QUE/g DW occurred at concentration of
10.0 g l1 mannitol used (Fig. 5). Sucrose-induced osmotic stress has great effect on biomass,
metabolic content, and antioxidant capacity in root suspension cultures of Hypericum
perforatum L. Higher sucrose concentrations resulted in improved build up of phenols,
flavonoids, and chlorogenic acid. It was suggested that higher sucrose concentration causes
osmotic stress which induced the amassing of secondary metabolites [33]. Induction of
drought stress markedly increased the FRSA of S. marianum. Comparatively, maximum
values were obtained for all the stress levels applied. Higher FRSA of 96 % was obtained at
concentration of 10.0 g l1 mannitol. Two weeks dark+2 weeks 16/8 h photoperiod treatment
proved to be the most effective, increasing free radical scavenging potential of S. marianum
(Fig. 6). In a study performed on Salvia officinalis L., moderate water deficit increased
individual and total polyphenols and a severe deficit resulted in the action of enzymes
responsible for the biosynthesis of phenolic compounds. Results also showed increase in
reducing power and DPPH activity with increase in stress severity [34].
Effect of Drought Stress on Total Protein Content and Enzymatic Activities
A significant increase in total protein content of S. marianum occurred upon the induction of
drought stress. Maximum protein content of 610 and 599 g BSAE/mg FW were recorded at
concentration of 0.25 and 10 g l1 mannitol, respectively. Four weeks dark and 2 weeks dark+
2 weeks 16/8 h photoperiod treatment proved to be the most influential for enhancement of
total protein content (Fig. 7). Enhanced protein content obtained was in contradiction to a

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Appl Biochem Biotechnol (2014) 174:693707

cd

ab

ab

ab

ab

bcd
bc
ab
bc

bcd
bc

b
3

bcd

ab
bcd

ab

ab

ab

2 weeks dark + 2 weeks 16/8 h phtoperiod

4 weeks dark photoperiod
4 weeks 16/8 h photoperiod

cd
cd

total flavonoid content (mg QUE/g DW)

0
-1

mannitol concentration (gl )

Fig. 5 Total Flavonoid Content (TFC) accumulated at different levels of drought stress, TFC accumulation was
found to be related with TPC accumulation. Values are meanstandard error of triplicates and significance was
tested among data of the same photoperiod treatments

range of studies conducted on drought stress. Water stress is said to inhibit incorporation of
amino acids into proteins and cause a decrease in protein content of different plant tissues [35].
In a study conducted on Bermuda grass, results showed severalfold increase in the accumulation of free proline and free asparagines during drought stress but protein synthesis was
affected and decrease in protein levels was recorded [36]. The effect of water stress on protein
synthesis in Brassica napus was assessed by SDS-PAGE and largely decline in protein
DPPH free radical scavanging activity (%)

130
120
110
100
90
80
70

60
50

2 weeks dark + 2 weeks 16/8 h phtoperiod

4 weeks dark photoperiod
4 weeks 16/8 h photoperiod
a
a
b
b
c
d
a
a
d
a aa
d
b
b b
b
b
b
d
c
d
d
d

40
30
20
10
0

mannitol concentration (g/l)

Fig. 6 Effects of drought stress on FRSA of S. marianum, induction of drought stress effectively increased
FRSA activity of the plantlets. Values are meanstandard error of triplicates and significance was tested among
data of the same photoperiod treatments

Appl Biochem Biotechnol (2014) 174:693707

703

total protein content (g BSAE/mg FW)

750
2 weeks dark + 2 weeks 16/8 h photoperiod
4 weeks dark
4 weeks 16/8 h photoperiod

a
a

600

a cd

d
d

450

b a aab

c
e

f
g

300

150

0
-1

mannitol concentration (gl )

Fig. 7 Effects of drought stress on total protein content of S. marianum, despite of plantlets facing a stressful
condition, protein synthesis increased enough to cope with the detrimental circumstances. Values are mean
standard error of triplicates and significance was tested among data of the same photoperiod treatments

synthesis with the introduction of drought stress was found, protein synthesis was resumed by
rehydration [37]. In the current study, maximum protein buildup can be attributed to the lower
activity of proteases observed. Despite some major variations, protease activity was decreased
comparatively to control. Enhanced protease activity was observed in plantlets kept under the
influence of 4 weeks 16/8 h photoperiod treatment (Fig. 8). The role of protease could be
2.5
2 weeks dark + 2 weeks 16/8 h photoperiod
4 weeks dark
4 weeks 16/8 h photoperiod

bc
ab

ab

cd
abc

bc cd

ab cd

cd
1.0

abc

ab cd

bc

1.5

bc

ab

ab
ab

bc

protease activity (U/gFW)

2.0

0.5

0.0
-1

mannitol concentration (gl )

Fig. 8 Protease activity at different drought stress levels, lowered protease activity was considered helpful for
proteins carrying out their specific functions against stress. Values are meanstandard error of triplicates and
significance was tested among data of the same photoperiod treatments

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Appl Biochem Biotechnol (2014) 174:693707

restricted to developmental regulation and photosynthetic process [38, 39]; both of these were
significantly affected by drought stress, hence the lower protease activities observed which
ultimately allowed enhanced protein accumulation.
On the contrary, concentrations of POD and SOD increased upon induction of stress.
Plantlets grown under the influence of 2 weeks dark+2 weeks 16/8 h photoperiod treatment
contained higher concentrations of POD and SOD (Fig. 9a, b). We determined activities of

0.50

2 weeks dark + 2 weeks 16/8 h phtoperiod

4 weeks dark photoperiod
4 weeks 16/8 h photoperiod

0.40
0.35

a
ab

a
b
bb

ab

ab

0.15

ab

0.20

a
a
ab

aa

ab

0.25

ab

0.30

ab

POD concentration (nM/min/mg/FW)

0.45

ab

0.10
0.05
0.00
-1

mannitol concentration (gl )

0.30

ab

ab

cd
abc

abc

bc
ab

cd

ad

bc

ab

ab

0.15

ab

0.20

ab

ab

0.25

ab
ab

2 weeks dark + 2 weeks 16/8 h phtoperiod

4 weeks dark photoperiod
4 weeks 16/8 h photoperiod

ab

SOD concentration (nM/min/mg FW)

c
0.10

0.05

0.00
-1

mannitol concentration (gl )

Fig. 9 a Effect of drought stress on POD activity, POD concentration was significantly increased in 2 weeks
dark+2 weeks 16/8 h photoperiod treatment. b Effect of drought stress on SOD activity, SOD concentration was
also raised with induction of drought stress but found to be lower as compared to POD. Values are mean
standard error of triplicates and significance was tested among data of the same photoperiod treatments

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Fig. 10 (1) Seed-derived plantlets of S. marianum on MS0 (control), (2) on MS medium containing 2 g/l
mannitol, (3) 4 g/l mannitol, (4) 6 g/l mannitol, (5) 8 g/l mannitol, (6) 10 g/l mannitol. (Comparison of growth of
seed-derived S. marianum plantlets on MS0 medium with those derived on medium containing different
concentrations of mannitol)

SOD and POD to signify their role in a stressed situation. Drought-tolerant bean
Phaseolus acutifolius showed a better protection mechanism against oxidative damage
by maintaining higher constitutive and induced activities of antioxidant enzymes than
the sensitive common bean Phaseolus vulgaris [40]. POD activity was found higher
than SOD in our study, particularly when plants were subsequently transferred from
dark to light treatment in combination with drought stress. A similar report suggested
that both tall fescue and Kentucky bluegrass were capable of surviving surface soil
drying and this capability could be related to increase in antioxidant activities,
particularly SOD and CAT [41]. The correlation of activities of SOD and POD with
drought stress well implies their role as antioxidants (Fig. 10).

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Appl Biochem Biotechnol (2014) 174:693707

Conclusions
From the current study, it can be concluded that drought stress is a complex phenomenon.
Several physiological and biochemical processes are involved at different developmental
stages of plants to counteract drought stress. These processes include reduction in water loss
by increasing stomatal resistance and production of enzymatic and non-enzymatic free radical
scavengers and various osmolytes to retain cellular water potential. Although plant morphogenesis and phenotypic flexibility of S. marianum was badly affected by drought stress, its
induction seemed to be a useful strategy to obtain high outputs of secondary metabolites, i.e.,
total phenolic and flavonoid content which cannot be achieved in natural environment. The
strategy is being extended to the molecular basis in the contemporary flourishing era of in vitro
secondary metabolites production; whereby, it would be possible to study and mimic the
metabolic pathways resulting in high yield of novel secondary metabolites and up scaling to
production level through bioprocess engineering.
Acknowledgments Financial support of Higher Education Commission (HEC) of Pakistan is gratefully
acknowledged.

References
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.

Stamp, N. (2003). The Quarterly Review of Biology, 78, 2355.

Verpoorte, R. (1998). Drug Discovery Today, 3, 232238.
Ramakrishna, A., & Ravishankar, G. A. (2011). Plant Signaling and Behavior, 6, 17201731.
Reddy, A. R., Chaitanya, K. V., & Vivekanandan, M. (2004). Journal of Plant Physiology, 161, 11891202.
Gonzalez-Dugo, V., Durand, J. L., Gastal, F., Bariac, T., & Poincheval, J. (2012). Environmental and
Experimental Botany, 75, 258267.
Maurino, V. G., & Flugge, U. I. (2008). Plant signaling and Behavior, 3, 923.
Bozin, B., Mimica-Dukic, N., Samojlik, I., Goran, A., & Igic, R. (2008). Food Chemistry, 111, 925929.
Chalker-Scott, L. (1999). Photochemistry and Photobiology, 70, 19.
Larson, R. (1988). Phytochemistry, 27, 969978.
Kren, V., & Walterova, D. (2005). Biomedical Papers, 149, 2941.
Braithwaite, M. C., Tyagi, C., Tomar, L. K., Kumar, P., Choonara, Y. E., Pillay, V. (2013). Journal of
Functional Foods doi:10.1016/j.jff.2013.09.022 doi:10.1016/j.jff.2013.09.022#doilink.
Murashige, T., & Skoog, F. (1962). Physiologia Plantarum, 15, 473497.
Haq, I. U., Ullah, N., Bibi, G., Kanwal, S., Ahmad, M. S., & Mirza, B. (2011). Iranian Journal of
Pharmaceutical Research, 11, 241249.
Singleton, Vernon, L., Orthofer, R., Rosa, M., Lamuela-Raventos. (1999). Methods in Enzymology, 299,
152178.
Lee, S. K., Zakaria, H. M., Cheng, H. S., Luyengi, L., Gamez, E. J. C., Mehta, R., Kinghorn, A. D., &
Pezzuto, J. M. (1998). Combinatorial Chemistry and High Throughput Screening, 1, 3546.
Nayyar, H., & Gupta, D. (2006). Environmental and Experimental Botany, 58, 106113.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., & Randall, R. J. (1951). The Journal of Biological Chemistry,
193, 265275.
Ullah, N., Haq, I. U., Safdar, N., & Mirza, B. (2013). Toxicology and Industrial Health, 5, 17.
Lagrimini, L. M. (1991). Plant Physiology, 96, 577583.
Giannopolitis, C. N., & Ries, S. K. (1977). Plant Physiology, 59, 309314.
Sankhla, R., & Chawan, D. D. (1980). Biologia Plantarum, 22, 388391.
Hegarty, T. W. (1977). New Phytologist, 78, 349359.
Almansouri, M., Kinet, J. M., & Lutts, S. (2001). Plant and Soil, 231, 243254.
Khan, M. A., Abbasi, B. H., Ahmed, N., & Ali, H. (2013). Industrial Crops and Products, 46, 105110.

Appl Biochem Biotechnol (2014) 174:693707

707

25. Baranova, E. N., Gulevich, A. A., Kalinina-Turner, E. B., & Koslov, N. N. (2011). Russian Agricultural
Sciences, 37, 1119.
26. Moghanibashi, M., Karimmojeni, H., Nikneshan, P., & Delavar, B. (2012). Plant Knowledge Journal, 1, 10
15.
27. Boyer, J. S. (1970). Plant Physiology, 46, 233235.
28. Martin, T., Oswald, O., & Graham, I. A. (2002). Plant Physiology, 128, 472481.
29. Sprent, J. I. (1972). New Phytologist, 71, 603611.
30. Zrenner, R., & Stitt, M. (1991). Plant, Cell and Environment, 14, 939946.
31. Alvarez, S., Marsh, E. L., Schroeder, S. G., & Schachtman, D. P. (2008). Plant, Cell and Environment, 31,
325340.
32. Yadav, R. K., Sangwan, R. S., Sabir, F., & Sangwan, N. S. (2013). Plant Physiology and Biochemistry, 74,
7083.
33. Cui, X. H., Murthy, H., Wu, C. H., & Paek, K. Y. (2010). Plant Cell, Tissue and Organ Culture, 103, 714.
34. Bettaieb, I., Hamrouni-Sellami, I., Bourgou, S., Limam, F., & Marzouk, B. (2011). Acta Physiologiae
Plantarum, 33, 11031111.
35. Nir, I., Poljaxoff-Mayber, A., & Klein, S. (1970). Israel Journal of Botany, 19, 451462.
36. Barnett, N. M., & Naylor, A. W. (1966). Plant Physiology, 41, 12221230.
37. Good, A. G., & Zaplachinski, S. T. (1994). Physiologia Plantarum, 90, 914.
38. Van der Hoorn, R. A. (2008). Annual Review of Plant Biology, 59, 191223.
39. elisko, A., & Jackowski, G. (2004). Journal of Plant Physiology, 161, 11571170.
40. Turkan, I., Bor, M., Ozdemir, F., & Koca, H. (2005). Plant Science, 168, 223231.
41. Fu, J., & Huang, B. (2001). Environmental and Experimental Botany, 45, 105114.