pg_0001

Botanical Studies (2006) 47: 175-184.

Current Address: Department of Biochemistry and

Molecular Biology, University of Nevada, Reno, Reno, NV

89557, USA

Corresponding author: E-mail: choumasa@mail.npust.edu.

Ecotypic variation of Imperata cylindrica populations in

Taiwan: II. Physiological and biochemical evidence

Ing-Feng CHANG

1,3

and Chang-Hung CHOU

2,*

Institute of Botany, Academia Sinica, Taipei, Taiwan

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 plantsˇ 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

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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

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 ⊙ 20 ⊙

20 cm

) in greenhouse for two weeks before being trans-

planted to Kimuraˇs 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C, 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C

for 24 h) was measured in order to calculate soil water

content ([W1-W2] / W1 ⊙ 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

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 ⊙g Centrifuge

(Sigma, Model 2K15, Taiwan) at 4C for 10 min, and

the supernatant was obtained as ADH crude extract. All

extraction steps were performed at 4C, and the extracts

pg_0003

CHANG and CHOU  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C

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

儁

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

儁

per

gram fresh weight. One unit (u) of enzyme activity was

defined as the amount that reduced 1 mole of NAD

儁

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 ⊙g (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C for one h, and samples were then put into

a refrigerator at -20C 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C 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C

for 16 h, then wet-ashed with 7 ml 4.7 M HCl distilled

water and 7 ml 8 M HNO

. The mixture was heated on

a digestion block at 80C 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)

Aug. 3

210.86∮10.5

Aug. 5

209.21∮12.7

Aug. 13

222.11∮15.6

273.70∮25.6

Aug. 21

250.19∮ 4.6

Sep. 11

270.06∮9.6

330.37∮21.3

Sep. 20

290.44∮13.1

Sep. 28

280.80∮17.0

298.63∮11.8

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 (g gfw

-1

)

Neihu

10.27∮0.8

0.18∮0.01

123.41∮6.7

17.64∮1.8

Sarlun

4.21∮0.2

0.10∮0.00

158.57∮7.8

31.68∮1.1

Chuwei

24.7∮4.0

1.01∮0.04

302.97∮7.6

120.55∮8.9

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  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 g gfw

-1

average) was much greater than that of leaves from others.

The proline content was 6.83 times that of Neihu (17.64

g gfw

-1

on average), and 7.4 times that of Sarlun (31.68

g 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 g gfw

-1

, 47.87 times control at 2% salinity, and 3.12

g 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 Duncanˇs 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

∮2.3

32.49

∮3.1

23.01

∮0.9

Sarlun

31.68

∮6.0

39.93

+ 3.8

23.39

∮1.0

Chuwei

215.79

∮9.2

178.23

∮29.5

21.68

∮1.0

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 Duncanˇs multiple

range test. Error bars were S.E. at the 5% level.

pg_0007

CHANG and CHOU  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 Duncanˇs 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 Duncanˇs multiple

range test. Error bars were S.E. at the 5% level.

pg_0008

182

Botanical Studies, Vol. 47, 2006

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Acknowledgments. We were very grateful to Dr. Y.H.

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Taiwan and by a fellowship to I.F. Chang by the Ministry

of Education, Taiwan.

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Imperata cylindrica

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