Botanical Studies (2006) 47: 175-184.
3
Current Address: Department of Biochemistry and
Molecular Biology, University of Nevada, Reno, Reno, NV
89557, USA
*
Corresponding author: E-mail: choumasa@mail.npust.edu.
tw
Ecotypic variation of Imperata cylindrica populations in
Taiwan: II. Physiological and biochemical evidence
Ing-Feng CHANG
1,3
and Chang-Hung CHOU
2,*
1
Institute of Botany, Academia Sinica, Taipei, Taiwan
2
Office of President and Department of Tropical Agriculture and International Cooperation, National Pingtung
University of Science and Technology, Pingtung 912, Taiwan
(Received April 21, 2005; Accepted November 10, 2005)
ABSTRACT.
Cogon grass [Imperata cylindrica (I. cylindrica ) L. Beauv. var. major], one of the top ten
weeds in the world, is one genus and one species in Taiwan. In the field, the alcohol dehydrogenase (ADH)
activity, proline, and sodium content in tissues of I. cylindrica showed variation between the wetland
(Chuwei) ecotype and the other two non-wetland ecotypes (Neihu and Sarlun). Moreover, in the greenhouse,
flooding and salt treatment on the vegetative shoot of I. cylindrica showed that the Chuwei ecotype has
higher survivability to flood and salt. A three-month flooding treatment led to a differential increase of ADH
activity in leaf tissues of plantlet of Chuwei and Sarlun ecotypes. In addition, a four-day salt treatment led to
a significant accumulation of proline in leaf tissues of Chuwei ecotype plantlets, yet to a significant amount
of sodium accumulation in root and stem tissues of the Chuwei ecotype following an eight-day (short-term)
and two-month (long-term) treatment. These physiological and biochemical differences revealed the ecotypic
variation among I. cylindrica ecotypes, and the wetland ecotype in Chuwei is physiologically distinct.
Keywords: Alcohol dehydrogenase; Imperata cylindrica; Proline; Sodium.
INTRODUCTION
Imperata cylindrica (L.) Beauv. var. major (Nees)
Hubb., a top-ten weed (Holm et al., 1977), is only one
genus of one species and is widely distributed in Taiwan
(Hsu, 1975). A special population was found in the Chu-
wei Mangrove Forest in the estuary area located at the
river mouth of the Tamshui River in northern Taiwan.
It was also found as a non-endemic species invading
Florida and neighboring states in both dry and wetlands
of the United States (King and Grace, 2000). Cheng and
Chou (1997a) examined the leaves of I. cylindrica in the
Chuwei population with a scanning electron microscope
(SEM). They found the lower stem surfaced with wax
instead of trichomes and the stele empty instead of solid.
The phenotype of the Chuwei plant remained unchanged
after transplanting into the greenhouse. The molecular
polymorphism of Imperata populations was investigated
by use of Random Amplified Polymorphic DNA (RAPD)
(Cheng and Chou, 1997b) and restriction fragment length
polymorphism (RFLP) on the ribosomal DNA (rDNA)
(Chiang et al., 1998; Chou and Tsai, 1999). The Chuwei
population appeared to be a distinct ecotype. However,
little physiological or biochemical evidence of variation
among ecotypes appeared.
Under low oxygen conditions, plants increase ADH
activity to survive. Hageman and Fisher (1960) first found
that anaerobiosis induced ADH activity in root of maize
seedlings. Freeling (1973) analyzed the protein level of
ADH in seedlings of maize under 5-72 h anaerobiosis
and found that they increased dramatically. Furthermore,
Schwarte (1969) demonstrated that the induction of ADH1
in maize seedlings correlated to the plants¡¦ tolerance to
anaerobiosis. The induction of ADH activity is regulated
on a transcriptional level. In 1984, Dennis and his
coworkers cloned the anaerobiosis response region (ARE),
a promoter of the ADH1 gene. Later, Walker and his
coworkers sequenced the ARE (Walker et al., 1987). ADH
isozymes of maize (Freeling, 1973), tomato (Tanksley and
Johns, 1981), and Echinochloa (Fox et al., 1988) were
induced in anaerobiosis. Smits et al. (1990) reported that
the number of ADH isozymes in hydrophytes correlated
with their alcohol content and that the polymorphism
of isozymes provided natural selection superiority. The
induction of Adh gene expression is also regulated
on a translational level (Bailey-Serres, 1999). Maize
Adh1 mRNA is selectively translated under low oxygen
conditions (Fennoy and Bailey-Serres, 1995; Fennoy et al.,
1998). In Arabidopsis, RopGap4 regulates ADH activity
under low oxygen conditions (Baxter-Burrell et al., 2002).
ECOLOGY
pg_0002
176
Botanical Studies, Vol. 47, 2006
Under high salt conditions, special organic compounds
like proline, glycinebetaine, choline, glycerol, and sor-
bitol accumulate in tobacco (Binzel et al., 1987), barley
(Stewart and Michelle, 1983), spinach (Coughlan and
Wyn Jones, 1980), and eggplant (Jain et al., 1987). Proline
accumulates with increased salinity, and the accumula-
tion is regulated by abscisic acid (Stewart, 1980; Stewart
and Voeberg, 1985). Proline plays many roles in stress
physiology. For example, Stewart and Lee (1974) indi-
cated that the accumulation of proline in plants was cor-
related with salt tolerance. They also pointed out that high
proline levels would protect many N metabolism-related
enzymes from harm. A large amount of proline would
inhibit 1-aminocyclopropane-1-carboxylate (ACC) from
converting to ethylene, thus protecting the plant from eth-
ylene damage (Chrominski et al., 1988; Chrominski et al.,
1989). Furthermore, proline could be an osmo-protectant,
maintaining an osmotic potential balance (Jain et al.,
1987). In addition to accumulating proline, plants also
regulate sodium ion homeostasis in response to salt stress.
A Salt-Overly-Sensitive 1 (SOS1) protein acts as a Na
+
/K
+
antiporter to confer salt tolerance in Arabidopsis (Wu et
al., 1996; Shi et al., 2002; Shi et al., 2003).
We examined the variation of salt and flooding stress
responses of I. cylindrica wetland and non-wetland eco-
types on both a biochemical and physiological level. Both
field and greenhouse studies of three ecotypes showed that
ADH activity, proline content in leaf tissues, and sodium
content in tissues were differentially up-regulated in the
Chuwei ecotype, which is flood and salt tolerant. Here
we provide a new model to study flooding and salt stress
physiology in plants. Our findings support the previous
discovery of the variation among ecotypes on a molecular
level (Cheng and Chou, 1997a), and we found that the
Chuwei ecotype is physiologically distinct.
MATERIAL AND METHODS
Plant material and sampling sites
Imperata cylindrica (L.) Beauv. var. major (Nees)
Hubb, Cogon grass, was sampled from Chuwei mangrove
salt-marsh wetland (wetland site) (Hwang and Chen,
1995). The area had been periodically flooding, and the
grass grown at this site had been designated as a salt-toler-
ant ecotype. The other two representative sites designated
as control sites were Sarlun (sandy beach) and Neihu (in-
land-park), no flooding (non-wetland) sites. Plant leaves
were harvested from each site every two weeks. Plant
samples from the Chuwei sites were collected on both
neap tide days (low tide and no flooding; during August
and September in 1995) and spring tide days (high tide
and flooding; during the same period of time). During the
harvesting, each leaf sample was washed with de-ionized
water, prepared and excised by sterilized scissors, stored
in Ziplock bags in an ice bucket with dry ice to keep it
fresh, and brought back to the lab immediately before use.
Water content of plant leaves harvested from each site was
assayed. Plants rhizomes collected from the field were
washed by sterilized water and cultured in pots (60 ¡Ñ 20 ¡Ñ
20 cm
3
) in greenhouse for two weeks before being trans-
planted to Kimura¡¦s culture solution (Ma et al., 2001) to
grow plantlets and vegetative shoots. The culture solution
was aerated with an air pump (NS, Model 8200, Taiwan)
for 24 h. The culture solution was replaced every week.
After two weeks, 14-day-old plantlets of the Chuwei eco-
type (designated PC14), Sarlun ecotype (PS14), and Neihu
ecotype (PN14) were used as the study material. After two
months, 90-day-old plantlet, Chuwei ecotype (designated
PC90), Sarlun ecotype (PS90), and Neihu ecotype (PN90),
were obtained. Greenhouse temperature was controlled at
25 to 30¢XC, and these plantlets were harvested after flood-
ing and salt treatment.
Soil water content and salinity assay
Soils from three different habitats were harvested from
each site every month in 1995. Fresh weight (W1) and
dry weight (W2) of soil (after drying in oven at 100¢XC
for 24 h) was measured in order to calculate soil water
content ([W1-W2] / W1 ¡Ñ 100 %). Electrical conductivity
(MS/CM) of soil was determined by use of an electrical
conductivity meter (Jenco, Model 1010, Taiwan) (Kalra
and Maynard, 1991). Soil salinity (M) was calculated by
converting soil electrical conductivity into soil salinity
in consideration of soil water content with NaCl as the
standard.
Flooding and salt treatment
A flooding experiment was conducted in water culture
in a greenhouse of the Institute of Botany, Academia
Sinica, Taipei, Taiwan. Flooding was achieved by growing
plantlets in culture solution without air pumping. A
culture solution aerated with an air pump was used as
the control. For the flooding treatment, PN14, PS14 and
PC14 were flooded for three months. Dissolved oxygen
(DO) concentration was measured by use of an O
2
meter (Consort, Model Z521, Taiwan). A salt treatment
experiment was also conducted in water culture. In the
salt treatment group, NaCl was added to water to make
salt water with salinities of 1%, 2%, and 3% (w/v). PN90,
PS90 and PC90 were cultured for four days, eight days
(short term) and two months (long term) in the salt water.
For the control group, NaCl was not added, and the culture
solution was sodium-free.
ADH activity assay
Half a gram of fresh leaves frozen by liquid nitrogen
were ground and homogenized in 5 mL extraction buffer
[0.1 M Tris-HCl, pH 8.0, 0.01 M beta-mercaptoethanol, 1
mM dithiothreitol, 0.02 mM phenylmethylsulfonyl fluo-
ride (PMSF)(Sigma)] with 5 grams sea sand added. The
homogenates were centrifuged at 5510 ¡Ñg Centrifuge
(Sigma, Model 2K15, Taiwan) at 4¢XC for 10 min, and
the supernatant was obtained as ADH crude extract. All
extraction steps were performed at 4¢XC, and the extracts
pg_0003
CHANG and CHOU ¡X Ecotypic variation of
Imperata cylindrica
177
were kept on ice. ADH enzyme activity was assayed spec-
trophotometrically at a wavelength of 340 nm by use of
a spectrometer (Hitachi, Model U-2000, Taiwan) at 30¢XC
by the method previously described (Irish and Schwartz,
1987). The reaction mixture contained 0.1 M Tris-HCl,
pH 8.5, 0.06 M ethanol, 0.002 M semicarbazide-Hcl, and
0.003 M beta-nicotinamide adenine dinucleotide (£]-NAD
ƒy
).
Data were the average of four replicates. Enzyme activity
was calculated based on a standard curve of pure NADH
and expressed as the initial rate of reduction of NAD
ƒy
per
gram fresh weight. One unit (u) of enzyme activity was
defined as the amount that reduced 1 £gmole of NAD
ƒy
per
min per gram of fresh weight. The optical density (absor-
bance expressed as A value) of the base line at wavelength
340 nm was measured with ten replicates for each data set.
Proline content assay
L-proline content was measured by the method previ-
ously described (Bates et al., 1973). Half a gram of fresh
leaf tissues were chopped into pieces, and frozen in liquid
nitrogen. Five ml extraction buffer [3% (w/v) 5-sulfosali-
cylic acid] was added to 5 grams of sea sand for grinding
and homogenization. The homogenate was centrifuged at
5380 ¡Ñg (Sigma, Model 2K15) for 10 min. The superna-
tant was obtained as proline crude extract. The reaction
mixture contained 2 ml acid-ninhydrin solution containing
0.14 M ninhydrin, 60% (v/v) acetic acid, 2.4 M phosphatic
acid, and 2 ml proline crude extract. The reaction was per-
formed at 100¢XC for one h, and samples were then put into
a refrigerator at -20¢XC to stop the reaction immediately.
Four ml methanol was added into the samples and vor-
texed. The solution was then fractionated into two layers,
the upper methanol layer and the lower water layer. Three
ml of the upper methanol layer solution was transferred
into a cuvete. Proline content was analyzed spectrophoto-
metrically at wavelength 520 nm using a spectrophotom-
eter (Beckman, Model DU-50) with twenty replicates for
each proline content assay and quantified based on a stan-
dard curve of pure proline.
Sodium content assay
Plant material was dried in an oven at 110¢XC for 24 h
and chopped into pieces. Half a gram of leaf, 0.1 gram
root and 0.1 gram stem, were dry-ashed individually by
a furnace (NEY Box Furnace, Model 6-1350A) at 470¢XC
for 16 h, then wet-ashed with 7 ml 4.7 M HCl distilled
water and 7 ml 8 M HNO
3
. The mixture was heated on
a digestion block at 80¢XC for 2 h in the hood until the
solution turned colorless. The final remains were diluted
to 50 ml with distilled water prior to analysis. Afterwards,
20 ml of 3 M HCl were added. Each sample was filtered
by a filter paper (Whatman no. 42). Sodium content was
measured frame-photometrically using a frame photometer
(Siba, Model 410) by the method decribed by Kalra and
Maynard (1991) and quantified based on a standard curve
of pure Na
+
.
Statistical analysis
Data were analyzed by Duncan
¡¦
s multiple range test
in an SAS statistical package (Academia Sinica, Taiwan)
and by a Student
¡¦
s Pairwise T-test in an SPSS statistical
package (Microsoft, Taiwan).
RESULTS AND DISCUSSION
Chuwei soil has much higher water content and
salinity than other dry habitats
Since a soil physical property reflects the environmen-
tal conditions of each sampling site, soil chemistry was
taken into consideration for comparison. To determine
the water content in the soil of three habitats, soil samples
were collected at monthly intervals from August to Octo-
ber in 1995 (Supplemental Table I). Our results showed
that the water content of soil from Chuwei mangrove for-
est sampling sites (24.7 % on average, ranged between
17.3 and 33.3 %) was much greater than that of soil from
other sites (Table 1). The soil water content was 2.4 times
that of Neihu (10.27% on average, ranged between 2.6
and 17.4 %), and 5.87 times that of Sarlun (4.21% on
average, ranged between 1.9 and 6.4%). We concluded
that soil from the Chuwei mangrove forest sampling sites
is much wetter than that from other sites. On the other
hand, soil oxidation-reduction potential from the topsoil
(5-10 cm from ground zero) was measured by use of an
oxidation-reduction potential meter (Jenco. Model 62,
Taiwan). Chuwei soil showed low oxidation-reduction po-
tential (between -88 and 175 mV) compared to other sites
(between 19 and 117 mV), suggesting a low oxygen status
(Hseu and Chen, 2000). In addition, chemical properties
Supplemental Table I. Average ADH activity in leaves of I.
cylindrica from Chuwei before and after flooding. Sampling
was perform ed from August to Septem ber in 1995. ADH
activity on neap-tide (low tide) days was shown in regular
font. ADH activity on spring-tide (high tide) days was shown
in bold. ND, not determined.
Sampling date
Before flooding After flooding
ADH (u)
ADH (u)
Aug. 3
210.86¡Ó10.5
ND
Aug. 5
209.21¡Ó12.7
ND
Aug. 13
222.11¡Ó15.6
273.70¡Ó25.6
1
Aug. 21
250.19¡Ó 4.6
ND
Sep. 11
270.06¡Ó9.6
330.37¡Ó21.3
1
Sep. 20
290.44¡Ó13.1
ND
Sep. 28
280.80¡Ó17.0
298.63¡Ó11.8
1
Values showed significant difference in Student
¡¦
s t-test at the
5% level. S.E. was at the 5% level.
pg_0004
178
Botanical Studies, Vol. 47, 2006
of soil from three different sampling sites were compared.
Soil samples from three habitats were collected at monthly
intervals from August to October in 1995. Data showed
that the salinity of soil from Chuwei sampling sites (1.01
M on average, ranging between 0.5 and 1.4 M) was much
greater than that from others (Table 1). The soil salinity
was 5.6 times that of Neihu (0.18 M on average, ranging
between 0.1 and 0.4 M), and 10.1 times that of Sarlun
(0.10 M on average, ranging between 0.05 and 0.2 M). It
is concluded that the soil from Chuwei wetland sampling
sites is much saltier than that from other sites. Therefore,
Chuwei soil is under both low oxygen and high salt condi-
tions.
The Chuwei ecotype showed higher
survivability to flood and salt
To test the variation in growth of three ecotypes, PN14,
PS14 and PC14 were grown under flooding conditions
for three months. Leaf length of PN14, PS14 and PC14
were measured at weekly intervals, and growth of plantlet
was expressed as the increase in shoot (leaf) length.
Results showed that plantlets from the Chuwei sampling
site could survive anaerobic conditions and were flood
tolerant (Figure 1C). PN14 did not grow throughout
the whole culturing (Figure 1A) while PS14 grew well
but slower than control (Figure 1B). PC14 grew well
and with an enhanced growth rate. After three months,
the height (shoot length) of PN14, PS14 and PC14 was
measured. PN14 showed a 100% growth inhibition while
PS14 showed 43%. However, PC14 showed no growth
inhibition at all. The result showed that the Chuwei
ecotype had a higher survivability to flood.
To test the variation in growth of three ecotypes, PN14,
PS14 and PC14 were grown under salt conditions for
two months. Leaf lengths of PN14, PS14 and PC14 were
measured at weekly intervals, and growth of plantlet was
indexed by the increase of leaf length. Results showed
that the plantlets of Chuwei wetland ecotypes could
survive in 1% salt water and were salt tolerant. PN14
stopped growing after 41 days at 1% salinity. After 31
days, it stopped growing at 2% salinity (Figure 2A).
PS14 stopped growing at 1% salinity after 45 days, but
did not grow at 2% salinity (Figure 2B). Nevertheless,
PC14 grew well, kept growing at 1% salinity even after
two months, and stopped growing at 2% salinity after 50
days (Figure 2C). Our result showed that Chuwei ecotype
showed higher survivability to salt. The salt tolerance of
the Chuwei ecotype (1%) was even stronger than Kandelia
candel (tolerant to 0.8% salt), a dominant mangrove forest
species in the Chuwei salt-marsh wetland (Huang and
Chen, 1995).
Table 1. Variation of soil water content, salinity, and average ADH activity and proline content in leaves of three I. cylindrica
ecotypes in the field. Values were means of the samples collected between July and October in 1995. Values followed by the same
letter showed no significant difference in Duncan
¡¦
s multiple-range test at the 5% level. S.E. was at the 5% level.
Sampling site
Soil water (%)
Soil salinity (M)
Leaf metabolites
ADH (u)
Proline (£gg gfw
-1
)
Neihu
10.27¡Ó0.8
b
0.18¡Ó0.01
b
123.41¡Ó6.7
c
17.64¡Ó1.8
b
Sarlun
4.21¡Ó0.2
b
0.10¡Ó0.00
c
158.57¡Ó7.8
b
31.68¡Ó1.1
b
Chuwei
24.7¡Ó4.0
a
1.01¡Ó0.04
a
302.97¡Ó7.6
a
120.55¡Ó8.9
a
Figu re 1. Variation of flood s urvivability of plantlet under
flooding conditions for three months. Growth rate was expressed
as shoot length of plantlet PN14 (A), PS14 (B), and PC14 (C)
versus time duration. Error bars were S.E. at the 5% level.
pg_0005
CHANG and CHOU ¡X Ecotypic variation of
Imperata cylindrica
179
In the United States, Imperata cylindrical was found
in both dryland and wetland in Florida (King and Grace,
2000), and was found with flood-tolerant potential
(King and Grace, 2000). It appears that the flood-
tolerance potential of Imperata cylindrica was noted
worldwide. Since we found that flooding led to the
increase of the growth rate of the I. cylindrica Chuwei
ecotype (Figure 1C) and also found that during spring-
tide day the seedlings emerged from the soil, flooding
stress appears to be an important limiting factor for I.
cylindrica Chuwei ecotype to survive. This induced-
shoot-growth phenomenon has also been characterized
in other species (i.e. rice and Rumex palustris) (Vriezen
et al., 2003; Voesenek et al., 2003). In addition to maize,
rice and Echinochloa, our discovery revealed Imperata as
another monocot model for the study of low oxygen stress
physiology in plants.
ADH activity in leaves of Chuwei ecotype was
much higher in the field and up regulated after
a three-month flooding treatment
To compare the ADH activity in leaves of I. cylindrica
in the field, leaf samples were collected every two weeks
from August to October in 1995 and January to June in
1998 (Table 1, Supplemental Table I). Overall, ADH
activity in leaf of Chuwei ecotype was 302.97 u on
average, 2.45 times that of Neihu (123.41 u on average)
and 1.91 times that of Sarlun (158.57 u on average).
Results showed that the average level of ADH activity in
leaves of I. cylindrica Chuwei ecotype was much greater
than that of leaves from other sites.
In order to test if increase of ADH activity in leaves of
the Chuwei ecotype was due to flooding, a three-month
flooding treatment on plants was performed. Leaves of
PN14, PS14 and PC14 were harvested for activity study
after three months flooding treatment. Data were means
of four individuals. Results showed that ADH activity in
leaves of Chuwei plantlets was differentially up regulated
with decreased dissolved oxygen (D.O.) in comparison
with Sarlun plantlets (Figure 3). Because PN14 did not
grow, there were not enough leave tissues to be analyzed.
Evidence of ADH activity assay from both the field
and the greenhouse suggested that the I. cylindrica
Chuwei ecotype had undergone alcoholic fermentation
in anaerobiosis. It is commonly observed that ADH
activity increases under anaerobiosis in plants, i.e., in
maize (Andrews et al., 1993; Wignarajan and Greenway,
1976), soybean (Sachs et al., 1990), Echinochloa (Cobb
and Kennedy, 1987), and rice (John and Greenway, 1976).
Most of the plants expressed ADH in roots (Andrews et
al., 1993). However, it was reported that rice ADH activity
was detected in leaves (Cobb and Kennedy, 1987). In our
study, ADH was also expressed in leaves of I. cylindrical,
which is consistent with the study of rice. However, we did
not test the ADH activity in other tissues. It may be that
tissue specific expression of ADH activity exists among
different kinds of plants. Differences in the mechanism of
hypoxia tolerance between roots and shoots in Arabidopsis
is possible (Ellis et al., 1999).
Based on a current model, plants can be categorized
into either carbohydrate-conserving or carbohydrate-
consuming type based on their responses to hypoxia
(Fukao and Bailey-Serres, 2004). In the former type, low
ADH activity and restricted growth were observed. In
the latter type, high ADH activity and rapid shoot growth
were observed. The Chuwei ecotype appeared to be of the
carbohydrate-consuming type.
Proline accumulation in leaves of the Chuwei
ecotype was much higher and up regulated
significantly after a four-day salt treatment
To compare proline content in leaves of I. cylindrica
in the field, samples were collected at monthly intervals
from August to December in 1995. For proline content
Figure 2. Variation of salt survivability of plantlet under salt
conditions for two months. Growth rate was expressed as shoot
length of plantlet PN14(A), PS14(B), and PC14(C) versus time
duration. Error bars were S.E. at the 5% level.
pg_0006
180
Botanical Studies, Vol. 47, 2006
analysis, leaves of 20 individuals were sampled within a
day. Results showed that the amount of proline in leaves
of the I. cylindrica Chuwei ecotype (120.55 £gg gfw
-1
on
average) was much greater than that of leaves from others.
The proline content was 6.83 times that of Neihu (17.64
£gg gfw
-1
on average), and 7.4 times that of Sarlun (31.68
£gg gfw
-1
on average) (Table 1). In order to test if proline
content in leaves of plantlets increased differentially in
response to salt, a salt treatment was performed for 4
days to see the short-term effect on proline accumulation.
Leaves of PN90, PS90 and PC90 were harvested for study
after an eight-day salt treatment. Samples were collected at
four-day intervals. Data were average of nine individuals.
After four days, the proline content in leaves of PC90 was
2.06 £gg gfw
-1
, 47.87 times control at 2% salinity, and 3.12
£gg gfw
-1
, 72.43 times control at 3% salinity (Figure 4).
Our results showed proline accumulated significantly in
Chuwei plantlet (PC90) (Figure 4).
Sodium accumulation in roots and stems of
Chuwei ecotype was much higher in the field
and up regulated significantly after a short and
long-term salt treatment
To compare sodium content of I. cylindrica in the field,
samples were collected at monthly intervals from August
to December in 1995. For sodium content analysis,
sampling was performed with 10 replicates within a day.
Results showed that the sodium content in roots was
215.79 mg gdw
-1
on average, 11.64 times that of Neihu
(18.54 mg gdw
-1
), and 6.81 times that of Sarlun (31.68 mg
gdw
-1
). The sodium content in stems was 178.23 mg gdw
-1
on average, 5.49 times that of Neihu (32.49 mg gdw
-1
),
and 4.46 times that of Sarlun (39.93 mg gdw
-1
). However,
sodium content in leaves of I. cylindrica, showed no
difference (Table 2). Therefore, sodium content in roots
and stems of the Chuwei ecotype was much higher.
Short-term (eight-day) salt treatment was performed
to see its effect on sodium accumulation. To determine
sodium content of plantlet in response to short term salt
treatment, root, stem, and leaves of PN90, PS90 and
PC90 were harvested for study after an eight-day salt
treatment, and data were means of seven individuals.
Results showed that sodium accumulated in roots and
stems of plantlets. Sodium accumulated with increased
Figure 4. Variation of accumulation of proline in leaves of
plantlet of three ecotypes after a four-day salt treatment. Bars
having different letters are s ignificantly different, p=0.05,
ANOVA, with Duncan¡¦s multiple range test. Error bars were S.E.
at the 5% level.
Table 2. Variation of sodium content in roots, stems and leaves of three I. cylindrica ecotypes in the field. Values were means of
the samples collected between July and October in 1995. Values followed by the same letter showed no significant difference in
Duncan
¡¦
s multiple-range test at the 5% level. S.E. was at the 5% level.
Sampling site
Plant sodium content (mg gdw
-1
)
Root
Stem
Leaf
Neihu
18.54
b
¡Ó2.3
32.49
b
¡Ó3.1
23.01
a
¡Ó0.9
Sarlun
31.68
b
¡Ó6.0
39.93
b
+ 3.8
23.39
a
¡Ó1.0
Chuwei
215.79
a
¡Ó9.2
178.23
a
¡Ó29.5
21.68
a
¡Ó1.0
Figure 3. Variation of increase of ADH activity in leaf of plantlet
of Chuwei and S arlun ecotypes after a long-te rm fl ooding
trea tment. ADH ac tivity was me asured after three months
treatment. Becaus e PN14 did not grow, there were no leaves
to be analyzed. The dissolved oxygen (D.O.) concentration of
control (aeration) was 0.25 mM. Bars having different letters are
significantly different, p=0.05, ANOVA, with Duncan¡¦s multiple
range test. Error bars were S.E. at the 5% level.
pg_0007
CHANG and CHOU ¡X Ecotypic variation of
Imperata cylindrica
181
salinity with the Chuwei plantlet (PC90) appearing to be
the highest (Figure 5A, B). As the salt concentration was
2%, the sodium content in the stem of PC90 (229.74 mg
gdw
-1
) increased 22.46 fold. On the other hand, as the salt
concentration was 3%, the sodium content increased 45.57
fold in the root of PC90 (350.47 mg gdw
-1
) and 31.37 fold
in the stem of PC90 (213.16 mg gdw
-1
). The accumulation
of sodium was also found in leaves with increased salinity,
but PC90, with extremely low sodium content (Figure
5C), was an exception.
To determine the sodium content of plantlets in
response to long-term (two months) salt treatment, root,
stem and leaves of PN14, PS14 and PC14 were harvested
for study after a two-month salt treatment, and data
were means of seven individuals. Results showed that
sodium accumulated in roots and stems of plantlets. It
increased with increased salinity, and plantlets of the
Chuwei ecotype (PC14) appeared to undergo the highest
increases (Figure 6A; Figure 6B). With salt concentration
at 1%, the sodium content increased 28.87 fold in roots
of PC14 (224.77 mg gdw
-1
), and 32.33 fold in stems of
PC14 (221.94 mg gdw
-1
). With salt concentration at 2%,
the sodium content increased 30.14 fold in roots of PC14
(234.32 mg gdw
-1
), and 34.08 fold in stems of PC14
(234.31 mg gdw
-1
). The accumulation of sodium was also
found in leaves with increased salinity, but PC14 was an
exception. Here, the sodium content was extremely low
(Figure 6C). Therefore, we found sodium accumulated
significantly in roots and stems, but not in leaves, of the
Chuwei ecotype after a short-term and a long-term salt
treatment.
Halophytes were categorized into two types according
to their responses to salt: the regulation type and the ac-
cumulation type (Hellebust, 1976). The salt levels in the
regulation type plants were often low. This type of plant
secretes salt. The regulation type usually consists of man-
grove plants and can be classified into two classes. The
first class is salt-exclusion species. These species allow
Figure 5. Variation of accumulation of sodium in roots (A),
stems (B) and leaves (C) of plantlet of three ecotypes after
an eight-day salt treatment. Bars having different letters are
significantly different, p=0.05, ANOVA, with Duncan¡¦s multiple
range test. Error bars were S.E. at the 5% level.
Figu re 6. Variation of accumulation of sodium in roots (A),
stems (B) and leaves (C) of plantlet of three ecotypes after a
two-month salt treatment. Bars having different letters are
significantly different, p=0.05, ANOVA, with Duncan¡¦s multiple
range test. Error bars were S.E. at the 5% level.
pg_0008
182
Botanical Studies, Vol. 47, 2006
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In conclusion, this study detected variation among Im-
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Acknowledgments. We were very grateful to Dr. Y.H.
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Riverside. This study was supported by grants to Dr.
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Taiwan and by a fellowship to I.F. Chang by the Ministry
of Education, Taiwan.
pg_0009
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Imperata cylindrica
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