Aquatic Botany 97 (2012) 1723

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Aquatic Botany
journal homepage: www.elsevier.com/locate/aquabot

Salinity increases the triterpenoid content of a salt secretor and a non-salt

secretor mangrove
Mohammad Basyuni a,b , Shigeyuki Baba a , Yuji Kinjo a,c , Hirosuke Oku a,
a
b
c

Tropical Biosphere Research Center, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan
Department of Forestry, Faculty of Agriculture, University of Sumatra Utara, Medan 20155, Indonesia
Graduate School of Agriculture, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan

a r t i c l e

i n f o

Article history:
Received 14 March 2011
Received in revised form 16 October 2011
Accepted 21 October 2011
Available online 29 October 2011
Keywords:
Avicennia marina
Mangrove plant
Phytosterol
Rhizophora stylosa
Salinity tolerance
Triterpenoid

a b s t r a c t
The present study describes the effect of salinity on the triterpenoid content of the salt secretor mangrove Avicennia marina and the non-secretor Rhizophora stylosa. Mangrove seedlings were grown for eight
months in 0%, 0.5%, 1.5%, 2.0% and 3.0% salt concentration. The growth of both species was increased by
salt with maximal stimulation at 1.5%, and this elevation appeared to be attenuated by increasing the salt
concentration above 1.5%. The triterpenoid compositions of three types of chemical structures, lupane
(lupeol, lupenone), oleanane (-amyrin, taraxerol, germanicol), and ursane (-amyrin), were studied.
In addition, the phytosterol components campesterol, stigmasterol and -sitosterol were analyzed. The
total triterpenoid contents in the roots and leaves of A. marina for the 0% group were 87.0 and 66.2 g g1 ,
respectively, and increased signicantly to 173.1 and 142.6 g g1 with 3% salinity. The higher salinity
also signicantly increased the total concentration of phytosterols in the leaves and roots of this species. A
similar increase in the concentration of both triterpenoids and phytosterols was observed in the roots and
leaves of R. stylosa with increasing salt concentration. Thus, the triterpenoid concentration was increased
by salinity in the roots and leaves of both A. marina and R. stylosa irrespective of their differences in
salt management by salt excretion or by a non-excretion mechanism. Comparison of the triterpenoid
concentration in four species of growing mangrove seedlings revealed a correlation between the total
triterpenoid content and the salt tolerance based on the habitat zonation on Iriomote Island. A. marina
thrives closest to sea and had the highest content of triterpenoids (173.1 g g1 in 3% salt group). Therefore, it is likely that the triterpenoid content play an important role in mangrove plants for protection
from salinity in both salt-secretors and non-secretors.
2011 Elsevier B.V. All rights reserved.

1. Introduction
Mangrove plants are halophytes that thrive in the inter-tidal
zone of tropical and subtropical regions. One striking feature
of mangrove plants is their ability to grow in various levels
of salinity, ranging from fresh water to hypersaline environments (Tomlinson, 1986). Mangrove plants can be classied into
two groups, salt-secretors and non-secretors, based on their way
of coping with salinity (Scholander et al., 1962; Scholander,
1968; Tomlinson, 1986). The salt-secretors, including Avicennia marina (Forsk.) Vierh. (Acanthaceae), have either salt glands
or salt hairs to remove excess salt. By contrast, non-secretors,
exemplied by Rhizophora stylosa Griff. (Rhizophoraceae), do not
have such morphological characteristics for excretion of excess
salt. A. marina and R. stylosa are common mangrove species on

Corresponding author. Tel.: +81 98895 8972; fax: +81 98895 8972.
E-mail address: okuhiros@comb.u-ryukyu.ac.jp (H. Oku).
0304-3770/$ see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aquabot.2011.10.005

Okinawa Island, Japan, and are considered to be representative of

each group.
Although mangroves are rich in triterpenoids, the physiological role of these compounds remains obscure (Ghosh et al., 1985;
Basyuni et al., 2007a). Our previous studies have shown that
salinity increased the triterpenoid concentration in the roots and
leaves of non-secretor mangrove species (Oku et al., 2003; Basyuni
et al., 2009). We on the basis of our above studies hypothesized
that triterpenoids may function as a plasma membrane component and fortify the membrane structure to limit the permeation
of salt into the cells. This postulate represents a novel biological strategy of plants to confer salinity tolerance, and appeared
to merit further investigation. Because our previous studies have
only dealt with salt-dependent changes in the triterpenoid concentration in non-secretor mangrove species (Oku et al., 2003;
Basyuni et al., 2009), the present study aimed to describe the effect
of salinity on the triterpenoid content of the salt-secretor mangrove A. marina in comparison to that of the non-secretor species
R. stylosa.

18

M. Basyuni et al. / Aquatic Botany 97 (2012) 1723

2. Materials and methods

2.3. RNA isolation and real-time reverse transcription PCR

(RT-PCR)

2.1. Plant material

Mature and healthy propagules of two mangrove plants A.
marina and R. stylosa were collected from Iriomote Island, Okinawa, Japan and planted in Wagner pots with varying salinity in a
glass house. Mature propagules of A. marina were pericarp green in
colour, 1.52.5 cm long and 1.52.0 cm wide, while mature propagules of R. stylosa were yellowish green in colour, 2030 cm long
and 1.52.0 cm diameter. The germinated seedlings were grown
for eight months with exposure to natural temperature and sunlight in an uncontrolled glass house. The maximum irradiance in
the glass house was 950 mol m2 s1 , and the average temperature was 24.1 C. A seawater solution was prepared by dissolving
commercial salt powder (red sea salt, Houston, TX, USA) to make
samples with 0%, 0.5%, 1.5%, 2.0% and 3.0% (equal to sea water level)
salinities according to the manufacturers protocol. In this study,
salinity is dened as the mass of salt powder/mass of the solution
(Fofonoff and Lewis, 1979). In each treatment, the salt concentration was checked once weekly by an S/Mill-E salinity refractometer
(Atago Co., Ltd., Tokyo, Japan). The salinity was adjusted by adding
tap water to the control (0%) and pure water (salt treatments) to
compensate for evapotranspiration. Five to six plants in separate
pots i.e., ve to six pots per species per salinity treatment were
grown for eight months. After eight months of cultivation, the two
species of mangrove plants were harvested and washed. The leaves
and roots were frozen in liquid nitrogen and stored at 80 C for
isoprenoid analysis and RNA extraction (in the case of R. stylosa).

2.2. Analysis and identication of isoprenoids

The leaves and roots (35 g in wet weight) of R. stylosa and A.
marina were powdered in liquid nitrogen along with cholesterol
as an internal standard for isoprenoid analysis and then extracted
with 25 times volume of chloroformmethanol (2/1, v/v) (CM21).
The cell wall debris insoluble to CM21 was removed by ltration
through No. 2 lter paper (Advantec, Tokyo, Japan). The method
used for isoprenoid analysis was described previously (Basyuni
et al., 2007a). The absolute content (concentration) of isoprenoids
was calculated from the ratio of the peak area of the respective compound to that of the internal standard cholesterol. Isoprenoids in
the lipid extracts were analyzed by gas chromatography (GC 2010,
Shimadzu, Kyoto, Japan) equipped with ame ionization detection
(GC-FID).
The chemical structures of isoprenoids were mainly identied
by comparison of their retention time on the GC column with those
of authentic standards. Authentic standards of -amyrin, -amyrin,
lupeol, lupenone and cycloartenol were purchased from Extrasynthese (Genay, France). -Sitosterol, stigmasterol and campesterol
were purchased from Tama Biochemical (Tokyo, Japan), cholesterol
from Wako Pure Chemical Industries (Osaka, Japan) and lanosterol
from SigmaAldrich (USA). The chemical structures for germanicol
and taraxerol were identied by comparing their retention times
and MS spectra with those in our previous report (Basyuni et al.,
2007b). A CBPI-M50-025 (0.25 mm ID 50 m, Shimadzu) column
was used. The column temperature program was 50 C for 1 min,
then increased to 300 C at a rate of 10 C min1 , and held at 300 C
for 26 min. The carrier gas was helium, with a ow rate of 20 cm s1 ,
and the temperatures for the injector and detector were 250 C
and 300 C, respectively. A GCMS QP-2010 (Shimadzu) mass spectrometer was used. The column and GC conditions were described
above. The sample was ionized by electron impact (EI) at 70 eV to
determine the chemical structure or by chemical ionization with
methane as a reaction gas to determine the molecular weight.

Total RNA from the leaves and roots of R. stylosa were extracted
by using a modied CTAB method as described previously (Basyuni
et al., 2007b). Total RNA (approximately 25 g L1 ) was dissolved into 20 L of diethylpyrocarbonate treated water. The
rst-strand cDNA was synthesized from 2 to 5 g of total RNA
by the Superscript III rst-strand synthesis system (Invitrogen,
Carlsbad, CA, USA) in a total volume of 20 L for 5 min at 65 C,
1 h at 50 C, and 5 min at 85 C, according to the manufacturers
instructions. The resultant cDNA mixture was diluted with 50 L
of Tris/EDTA (10 mM Tris/HCl, 1 mM EDTA, pH 8.0) and was then
used as a template for real-time RT-PCR analysis. The real-time
RT-PCR was used to quantitate the mRNA level. The PCR amplication was carried out in a 20 L total volume containing 1.2 L
of template DNA, 0.2 M each primer, 2.5 mM dNTP mixture,
0.1 L of 100 times diluted SYBR Green I (Takara Bio Inc., Otsu,
Shiga, Japan) and 5 units of Takara Ex TaqTM HS (Takara Bio Inc.).
The real-time RT-PCR was performed using an iCycler (Bio-Rad,
Tokyo, Japan) and cycled 35 times as follows: 30 s denaturation
at 94 C, 30 s annealing at 5862 C, and 30 s polymerization at
72 C. The PCR primers were designed based on our previous
studies (Basyuni et al., 2007b,c, 2011): R. stylosa RsM1 (F: 5 CGCTTGGAGGACTAGCAGCTG-3 ; R: 5 -CATCTGCCCAGCATGAACAAGAG-3 ), R. stylosa RsM2 (F: 5 -GAAAACACAGTGTCAAGATGG3 ; R: 5 -AGGGTCTCTCTCCGCCTGCCCCG-3 ), R. stylosa RsCAS (F:
5 -ATAATTGCTCTAGCATTCG-3 ; R: 5 -TGGGATCTGTTGCCTTCAAG3 ), R. stylosa RsAct1 (F: 5 -TTCGACCATGTTCCCGGGCA-3 ; R:
5 -TGCACAATTGATGGTCCAGA-3 ). The real time RT-PCR product
was separated with 3% agarose gel electrophoresis and showed a
discrete single band of the predicted size. Each data point represents the average of six independent measurements and the error
bars indicate the standard errors of individual experiments. To
quantitate the expression level, a standard curve was constructed
by performing PCR with known concentrations of serially diluted
template DNA for genes of interest (RsM1, RsM2 and RsCAS). An
actin gene RsAct1 (Basyuni et al., 2011) was used as an internal
control to normalize the PCR efciency as described previously
(Larionov et al., 2005). The amplication specicity was checked by
a melting curve analysis. The actin gene is stably expressed under
salinity conditions in a number of plant species (Gu et al., 2004;
Jang et al., 2004; Basyuni et al., 2011).
2.4. Growth measurement and chlorophyll content (SPAD value)
The growth of A. marina and R. stylosa seedlings was determined
under varying salt concentrations. The stem heights of A. marina
(614 seedlings) and R. stylosa (912 seedlings) after eight months
of cultivation were the indices of growth used in this study.
The chlorophyll content of the leaves was measured by an SPAD502 chlorophyll meter (Konica Minolta, Osaka, Japan). The SPAD
meter is a simple portable instrument that measures the greenness or relative chlorophyll content of leaves (Kariya et al., 1982;
Inada, 1985). The units of the measurements are given in SPAD (Soil
Plant Analysis Development), which are values dened by Minolta
Company (Inada, 1985). Five successive readings across the whole
surface area were taken, and the mean value was used as an index
of the chlorophyll content in this study.
2.5. Statistical analysis
Data were analyzed by one-way analysis of variance (ANOVA)
followed by Dunnetts test for comparisons of all treatments against
the control. The statistical signicance of the correlation coefcient
was evaluated by Students t-test. The value of P < 0.05 was selected

5.0 1.4

142.6 6.1a

20.9 1.8a
101.1 6.4a
20.6 1.6a

88.8 6.3a

3.0% (n = 5)

as a limit of statistical signicance. All statistical analyses were performed using the SAS 9.1 statistical software program (SAS Institute
Inc., Cary, NC, USA).

19

3.9 0.8
19.6 1.3a
65.3 5.2a

M. Basyuni et al. / Aquatic Botany 97 (2012) 1723

4.4 1.1
4.8 2.0
1.2 0.3
3.8 0.9
9.6 1.1a
9.1 1.1a
11.6 4.3a
10.8 2.8a
33.0 3.1
Lanosterol

Data are represented as the mean SE.

a
Signicantly different from 0% at P < 0.05 using Dunnetts test.

126.7 6.9a
99.5 11.5a
83.5 6.1
66.2 4.7
173.1 6.7a
151.9 9.3a
102.5 9.4
87.0 5.4
Total

132.0 6.4a

19.3 0.7a
91.9 6.8a
15.5 1.1a
10.4 2.7a
82.7 9.3
6.4 1.3
3.9 0.4
74.6 6.3
5.0 0.4
2.6 0.1
59.1 4.6
4.5 0.5
17.7 2.5a
116.7 6.4a
38.7 2.6a
15.0 2.4a
106.3 4.1a
30.6 4.4a
9.7 0.6
96.2 7.5a
26.1 3.1
8.7 0.9
71.0 7.5
22.8 3.8
3.7 0.6
66.1 6.7
17.2 3.2
Triterpenoid
-Amyrin
Lupeol
-Amyrin

76.8 9.3a
57.4 3.9
51.3 3.9
35.7 3.1
150.0 11.5a
122.5 6.9a
102.3 12.8
Total

80.0 6.3

108.3 8.7

3.0 0.2
16.5 2.3a
57.3 6.7a
3.4 0.8
13.5 2.5
40.5 5.7
1.5 0.2
12.4 0.6
37.4 4.1
3.5 0.9
6.8 0.7
25.4 1.9
12.4 0.8a
35.5 2.0a
102.1 5.6a
11.9 1.7a
32.0 4.0a
78.6 1.1
8.0 1.0a
28.0 2.7
72.3 6.8
9.8 1.0a
23.3 4.5
69.2 9.6

2.0% (n = 5)
1.5% (n = 5)
0.5% (n = 5)

2.8 0.5
16.7 3.0
60.5 3.8

0.5% (n = 4)
0% (n = 4)
0% (n = 5)

3.0% (n = 4)

Leaf
Root

Content (g g1 )
Isoprenoids

Table 1
Effect of salt stress on the isoprenoid content of A. marina roots and leaves.

The isoprenoid contents in the mangrove roots and leaves from

A. marina and R. stylosa are listed in Tables 1 and 2. The triterpenoids
largely fall into three types of carbon skeletons: lupane (lupeol,
lupenone), oleanane (-amyrin, taraxerol, germanicol), and ursane
(-amyrin). Campesterol, stigmasterol, -sitosterol, lanosterol and
cycloartenol were the phytosterol components identied in the
mangrove roots and leaves. Thus, six triterpenoids and ve phytosterols were quantied in this study.
Table 1 lists the isoprenoid concentration in the roots of A.
marina grown for eight months with various salinities. The sum
of triterpenoid content for the 0% salt treatment was 87.0 g g1 ,
and this value increased almost 2-fold to 173.1 g g1 for the 3% salt
treatment, with lupeol as the major component. However, the total
of phytosterol content in the roots of the 0% group (80.0 g g1 ) was
almost comparable to the triterpenoid content, and also increased
to 150.0 g g1 in the 3% salt group. -Sitosterol was the major
phytosterol component throughout the salt treatment conditions.
The relative proportion of triterpenoids in the leaves was much
higher than that for root (Table 1). The concentrations of triterpenoid and phytosterol increased by approximately 2-fold and
2.5-fold, respectively, from 0% salt to 3% salt treatment. The major
triterpenoid and phytosterol were lupeol and -sitosterol, respectively, in accordance with the results from the roots.
Table 2 summarizes the isoprenoid concentration in the roots
and leaves of R. stylosa grown for eight months with various salinities. More triterpenoids were observed in the roots of R. stylosa
than in A. marina. Taraxerol, germanicol and lupenone, which were
absent in A. marina, occurred at appreciable levels in R. stylosa. The
concentration of each triterpenoid was comparable with that in A.
marina. Lupeol, the largest component, comprised approximately
76% of the total triterpenoids and predominated over the other
components. The total triterpenoid concentration was 57.5 g g1
in freshwater and increased to 126.4 g g1 in the 3% salt group. The
concentration of phytosterol in the roots was increased by almost
2-fold in the 3% salinity group. Likewise, salinity increased the concentrations of both triterpenoids and phytosterols in the leaves, as
shown in Table 2.
Table 3 lists the correlation coefcients between the salinity and
isoprenoid concentration. The concentrations of -amyrin, lupeol
and -amyrin were positively correlated with the salt concentration in the roots and leaves of A. marina. In addition, -sitosterol
and stigmasterol showed a positive and statistically signicant correlation with salinity in the leaves and roots of A. marina (Table 3).
Similarly, there were signicantly high correlations between the
triterpenoid content (-amyrin, lupeol, taraxerol and -amyrin)
and salt concentration in the leaves and roots of R. stylosa. Among
the phytosterols, we found positive correlations between the major
component of -sitosterol and salt concentration in both the roots
and leaves of R. stylosa. By contrast, the concentrations of campesterol and stigmasterol were correlated positively with salinity only
in the root tissue of R. stylosa.
The relative proportion of triterpenoids and phytosterols in the
total content of isoprenoids was calculated. Thus, the triterpenoid
and phytosterol values represent the sum proportion of the individual component for each group. In the roots of A. marina, the
relative proportion of the triterpenoids was higher than that of the
phytosterols and increased with increasing salt concentration. The

1.5% (n = 4)

3.1. Effect of salinity on the isoprenoids composition

Phytosterols
Campesterol
Stigmasterol
-Sitosterol

2.0% (n = 5)

3. Results

9.3 1.1a
0.6 0.0

11.7 1.3a
2.5 1.5

Table 3
Correlation coefcient between salt concentration and isoprenoid component in the
leaves and roots of mangrove trees.
Isoprenoids

Campesterol
Stigmasterol
-Sitosterol
Lanosterol
Taraxerol
Germanicol
-Amyrin
Cycloarenol
Lupenone
Lupeol
-Amyrin

R. stylosa
Leaf

Root

Leaf

0.82
0.90*
0.91*
0.71
nd
nd
0.96*
nd
nd
0.97*
0.91*

0.55
0.89*
0.92*
0.57
nd
nd
0.93*
nd
nd
0.90*
0.88*

0.86
0.42
0.94*
0.64
0.98*
0.94*
0.99*
0.59
0.54
0.95*
0.96*

0.89*
0.89*
0.94*
0.83
0.97*
nd
0.95*
0.53
nd
0.97*
0.97*

opposite trend was observed for the relative proportion of phytosterols. The relative proportions of these two components were not
inuenced by the salinity (data not shown).
The proportion of triterpenoids was lower than that of the phytosterols in the roots of R. stylosa, which is different from the
prole for A. marina. However, similar to A. marina, the triterpenoid
concentration in the roots of R. stylosa increased with increasing
salinity. No changes in the relative proportion of triterpenoids and
phytosterols were observed in the leaves of R. stylosa with salt treatment, similar to the results obtained for the A. marina leaves (data
not shown).
The R. stylosa cDNA of triterpenoid synthases (RsM1 and
RsM2) and cycloartenol synthase (RsCAS), which are involved
in triterpenoid and phytosterol synthesis, respectively, were
cloned in our laboratory as described previously (Basyuni et al.,
2007b,c). Changes in the mRNA levels of triterpenoid synthase and
cycloartenol synthase were studied to gain insight into the saltdependent response of this species.
The mRNA levels of RsM1 and RsM2, two triterpenoid synthase
genes were found to increase with salinity in the roots and leaves
of R. stylosa (Fig. 1A and B). By contrast, the mRNA level of RsCAS, a
phytosterol synthase gene, increased with salt concentration only
in the roots (Fig. 1A) but not in the leaves (Fig. 1B).
No measurement was made for the mRNA levels of triterpenoid
synthase and cycloartenol synthase in A. marina because no information on their cDNA is available.

4.5 0.6
1.1 0.3
Data are represented as the mean SE. nd, not detected.
a
Signicantly different from 0% at P < 0.05 using Dunnetts test.

20.7 1.2a
0.6 0.2
18.5 1.0
0.3 0.2
17.5 4.3
2.1 0.8
16.0 2.4
1.0 0.3
14.8 2.8
2.3 1.0
Lanosterol
Cyloartenol

A. marina
Root

Correlation coefcient was calculated using Excel 2000 for Windows. nd, not
detected.
*
Statistically signicant at P < 0.05 using Students t-test.

5.0 0.6
0.9 0.3

7.0 0.3
0.5 0.1

155.5 3.8a
138.8 2.3a
124.1 2.4a
101.7 2.2a
76.1 5.1a
Total

57.5 4.1

97.7 2.4a

126.4 5.6a

76.6 1.6

98.9 4.3a

52.5 3.1a
nd
90.9 4.5a
nd
5.2 1.3
6.9 1.5a
42.2 2.0
nd
88.0 0.7a
nd
4.1 0.4
4.5 0.5
41.3 0.6
nd
75.2 2.3a
nd
3.6 0.3
4.0 0.2
1.3
0.8
1.4a
0.3
1.1a
0.7a

Triterpenoid
Taraxerol
Germanicol
-Amyrin
Lupenone
Lupeol
-Amyrin

11.5
12.6
14.4
3.5
4.1
11.4

2.6
0.6
1.4
1.3
0.8
2.8

12.1
14.9
20.8
3.2
6.7
18.4

1.8
1.0
2.3
1.1
0.7
1.0a

14.0
15.1
36.8
3.1
9.0
19.7

1.7
1.3
1.1a
1.3
2.6a
0.4a

14.4
15.8
38.8
0.6
9.9
22.2

18.9
17.6
49.3
2.6
10.6
27.4

0.8a
1.2a
1.8a
1.5
0.5a
1.8a

34.4 1.0
nd
36.7 1.7
nd
3.0 0.4
2.5 0.2

36.3 3.5
nd
56.2 3.6a
nd
3.2 0.5
3.2 0.6

42.5 5.2a
32.0 4.6
24.3 3.2
25.9 6.0
123.9 8.4a
127.1 6.9a
Total

79.0 3.6

131.4 6.8a

164.7 5.0a

16.3 0.9

2.3 0.5
3.8 0.5
25.9 3.8a
1.9 0.2
2.9 0.4
19.5 2.3
19.1 1.6a
41.3 3.6
63.5 3.4a
17.6 0.8a
56.5 3.0a
53.0 3.8
5.7 2.3a
27.6 2.0
45.7 2.6

18.9 0.4a
49.4 3.2a
63.1 4.2a

25.6 1.3a
51.4 1.9a
87.7 2.3a

1.5 0.1
2.0 0.5
12.8 0.5

1.8 0.5
3.4 1.0
20.7 4.3

2.0% (n = 5)
0% (n = 3)

0.5% (n = 5)

1.5% (n = 5)

2.0% (n = 5)

3.0% (n = 5)

0% (n = 5)

0.5% (n = 5)

1.5% (n = 4)

Phytosterols
Campesterol
Stigmasterol
-Sitosterol

Leaf
Root

Content (g g1 )
Isoprenoids

Table 2
Effect of salt stress on the isoprenoid content of R. stylosa roots and leaves.

4.2 1.0a
5.8 1.7a
32.5 3.1a

M. Basyuni et al. / Aquatic Botany 97 (2012) 1723

3.0% (n = 5)

20

3.2. Effect of salinity on the growth and chlorophyll content of A.

marina and R. stylosa
Plant growth was measured by the height of the plants, as
described in the Section 2. The growth of both species increased
in the presence of salt with maximal stimulation at 1.5%. This elevation appeared to be attenuated when the salt concentration was
increased more than 1.5% (Fig. 2A). The shapes of the growth curves
for the two species were mostly similar.
The SPAD values of A. marina depicted a similar shape as the
growth curve. With increasing salt concentration, the SPAD values
increased to 1.5% and then decreased at concentrations above 1.5%
(Fig. 2B). On the other hand, the SPAD values for R. stylosa remained
constant up to a 1.5% salt concentration, and signicantly increased
at 3%.
4. Discussion
Our previous studies implied that an increase of triterpenoids in salt-stressed mangroves may be associated with the

M. Basyuni et al. / Aquatic Botany 97 (2012) 1723

*
RsM1

0.0

0.0

RsCAS

RsM2

0.5

1.5

2.0

3.0

mRNA (Ratio to internal standard)

*
*
*

0.4

R. Stylosa leaf

1.0

*
*

0.8

1.2

1.2

0.8

1.6

R. Stylosa root

2.0

mRNA (Ratio to internal standard)

21

0.6
0.4
0.2
0.0

RsM1

0.0

RsCAS

RsM2

0.5

1.5

2.0

3.0

Salinity (%)

Salinity (%)

Fig. 1. Effect of salinity on mRNA levels of RsM1 multifunctional triterpenoid synthase (), RsM2 multifunctional triterpenoid synthase () and RsCAS cycloartenol synthase
() in the roots (A) and leaves (B) of R. stylosa. RsAct1 from R. stylosa was used an internal control to normalize the PCR results. Measurements for individual sample were in
quadruple, and the values are the mean SE (n = 56). The asterisk indicates a statistically signicant difference from 0% at P < 0.05 using Dunnetts test.

20.0
A. marina

4.0

8.0

12.0

0.0

SPAD value

16.0

Plant height (cm)

R. stylosa

80.0

60.0

prediction, salinity increased the triterpenoid concentration of this

species, similar to the response of the non-secretor species (Oku
et al., 2003; Basyuni et al., 2009).
The salt concentration-dependent increase in triterpenoids suggests that these components function as cellular constituents to
counteract the osmotic pressure or cytotoxicity caused by salt
(Wahid and Ghazanfar, 2006; Kim et al., 2008). This response
appeared to be true for all cases of mangrove trees despite large
variations in the basal level of these components. Notably, the
total concentration of triterpenoids is well correlated with the
salt-tolerance of mangrove plants growing on Iriomote Island. The
mangrove habitat on Iriomote Island shows zonation from the coast
to inland in the order of A. marina, R. stylosa, K. candel and B.
gymnorrhiza (Wakushima et al., 1994; Baba, 2001; Basyuni et al.,
2007b; Enoki et al., 2009). The salt tolerance of four mangrove
seedlings studied in our laboratory increases in the order of B. gymnorrhiza < K. candel < R. stylosa < A. marina, which is in accordance
with their above-described habitat zonation. A. marina grows closest to sea and had the highest content of triterpenoids. Therefore,
the triterpenoids play an important role in mangrove plants for
protection from salinity in both salt-secretors and non-secretors.
This particularly holds true for root tissues that directly encounter
salt, and are the primary sites of perception and injury for salinity.
The above scenario for triterpenoid function also agrees with our
previous observation that triterpenoids are distributed in greater
proportion in the outer part of the root tissue than in the inner part
(Oku et al., 2003; Basyuni et al., 2007a). The present and previous

salt-tolerance mechanism in this species (Oku et al., 2003; Basyuni

et al., 2007b, 2009). Mangrove plant species can be divided into
two groups based on their salinity management system: saltsecretors and non-secretors (Scholander et al., 1962; Scholander,
1968; Tomlinson, 1986). Our studies hitherto have exclusively dealt
with observing salt-dependent changes in the lipid components of
non-secretor mangrove species (Oku et al., 2003; Basyuni et al.,
2009). The salt-tolerant mechanisms are complex and exhibit variation between mangrove plant species (Takemura et al., 2000; Liang
et al., 2008; Basyuni et al., 2009; Ezawa and Tada, 2009; Parida and
Jha, 2010). We hypothesized that the salt response prole of the
triterpenoid concentration in salt-secretor mangroves may show
divergence from that of non-secretor species.
The triterpenoid concentration of A. marina was the highest
among the mangrove seedlings studied in our laboratory, as shown
in Fig. 3 (for comparison, the data for B. gymnorrhiza and K. candel
were included from our previous paper (Basyuni et al., 2009)). The
higher basal level of the triterpenoid concentration in A. marina
was further increased with salinity in a concentration-dependent
manner. Thus, the data in this study appeared to support our
hypothesis that triterpenoids play a role in conferring salinity tolerance. A. marina excrete salt via the salt glands located within the
abaxial indumentum (Tomlinson, 1986), which may be the principal regulatory mechanism to dispose of absorbed salt (Drennan
and Pammenter, 1982; Clough, 1984; Mallery and Teas, 1984). For
this reason, we expected that the salinity would not inuence the
triterpenoid concentration of this species. However, against our

40.0

20.0

R. stylosa

A. marina

0.0
0.0

0.5

1.5

Salinity (%)

2.0

3.0

0.0

0.5

1.5

2.0

3.0

Salinity (%)

Fig. 2. Effect of salinity on growth (A) and SPAD value (B) in the leaves of A. marina () and R. stylosa () seedlings. Data presented are the mean SE (n = 614) for growth
measurements and the mean SE (n = 5) for SPAD values. The asterisk indicates a statistically signicant difference from 0% at P < 0.05 using Dunnetts test.

22

M. Basyuni et al. / Aquatic Botany 97 (2012) 1723

Fig. 3. Effect of salinity on the total content of triterpenoid (g g1 ) in the roots of B. gymnorrhiza, K. candel, R. stylosa and A. marina. Data are represented as the mean SE
(n = 35).

results both show the signicance of triterpenoids as protectants

from salinity in mangrove trees.
Consistent with our previous study (Basyuni et al., 2009),
signicant correlations were observed between the salinity and
triterpenoid concentration in the roots and leaves of A. marina and
R. stylosa. This study also found signicant correlations between
salinity and phytosterol content in both species. -Sitosterol,
showed a highly positive correlation with salinity in the roots and
leaves of both species (Table 3). These results suggest that phytosterols as well as triterpenoids play a role in the salinity tolerance
of salt-tolerant mangrove species such as A. marina and R. stylosa.
Given that triterpenoids function as a membrane component, the
molar ratio between phytosterols and triterpenoids may be another
factor contributing to salt tolerance in triterpenoid-rich species
compared to B. gymnorihiza or K. candel as shown in Fig. 3.
An increase in the triterpenoid concentration was also supported by the increased mRNA level of the triterpenoid synthase
gene with increasing salinity, as shown in Fig. 1. The physiological
response of mangroves to salinity appears to vary depending on
the duration of the salinity treatment (Basyuni et al., 2009; Ezawa
and Tada, 2009). Recent studies on transcriptome proling of B.
gymnorrhiza revealed enhanced gene expression during the early
stages of salt treatment, returning to the basal level after 12 days of
treatment (Ezawa and Tada, 2009; Yamanaka et al., 2009). These
changes indicate immediate physiological responses that occur
with the onset of exposure to salinity. By contrast, changes shown
herein and in our previous study are based on observations during
eight months of salinity treatment. Therefore, it is likely that the
increase in triterpenoids bears a constitutive role in the salt tolerance mechanism of mangrove plants and may be involved in a
more fundamental regulatory system to cope with salinity.
The root tissue plays a pivotal role in controlling the content
of ions such as Na+ and Cl in the whole plant (Munns, 2005).
Scholander (1968) demonstrated that the salt separation process
must occur at or near the root surface, and is mediated by the
physical process alone. Salt separation should be involved in the
ltration of sea water at the interface of the cell surface. Phytosterol is generally accepted to be a lipid component of the plasma
membrane in plant cells (Grunwald, 1974; Mansour et al., 1994;
Hartmann, 1998). Several studies have demonstrated that phytosterols contribute to regulating the ion permeability of the plant cell
membrane despite showing variations in efcacy depending on the
chemical structure of the sterol carbon skeleton (Grunwald, 1974;
Mansour et al., 1994; Hartmann, 1998). Triterpenoids have been
distinguished from phytosterols because they are not considered
to be a lipid membrane constituent despite having similar chemical
structure and biosynthetic pathway (Oku et al., 2003; Basyuni et al.,
2009). However, there has been no scientic rationale to exclude

these compounds from their constitutive role in the lipid membrane. Our forthcoming study will shed light on the physiological
role of triterpenoids and will focus on their localization in the cell.

Acknowledgements
This work was supported by a Grant-in-Aid for a Postdoctoral
Fellow (20.08104 to MB) from the Japan Society for the Promotion
of Science and a Competence Grant (to MB) from the Directorate for
Research and Community Service, Ministry of National Education,
Republic of Indonesia.

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