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Acta Physiologiae Plantarum ISSN 0137-5881Volume 33Number 4 Acta Physiol Plant (2011)33:1399-1409DOI 10.1007/s11738-010-0674-8

Anatomical and physiologicalcharacteristics relating to ionic relationsin some salt tolerant grasses from the SaltRange, Pakistan

Mansoor Hameed, Muhammad Ashraf &Nargis Naz

1 23

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ORIGINAL PAPER

Anatomical and physiological characteristics relating to ionicrelations in some salt tolerant grasses from the Salt Range,Pakistan

Mansoor Hameed • Muhammad Ashraf •

Nargis Naz

Received: 11 September 2010 / Revised: 26 October 2010 / Accepted: 7 December 2010 / Published online: 24 December 2010

� Franciszek Gorski Institute of Plant Physiology, Polish Academy of Sciences, Krakow 2010

Abstract Populations of three salt tolerant forage grasses

(Cynodon dactylon, Imperata cylindrica, and Sporobolus

arabicus) were collected from the salt-affected soils of the

Salt Range and normal non-saline soils of the Faisalabad

region to assess their mechanism of adaptation to saline

stress by determining ion relations and some specific ana-

tomical modifications. The population of S. arabicus from

the Salt Range showed increased growth (root and shoot

length, and root and shoot dry weights) under saline con-

ditions. Salt tolerance in this species was related to struc-

tural modifications such as increased area of root, stem,

leaf blade, and leaf sheath for toxic ion accumulation,

increased vesicular hair density in leaves and aerenchyma

formation in leaf sheath for ion exclusion. Uptake of toxic

ions was high in the Salt Range population of C. dactylon

and salt tolerance was related to ion exclusion through

specific leaf structural modifications such as vesicular

hairs. Salt tolerance in the Salt Range population of I. cyl-

indrica was mainly associated with restricted uptake of

toxic Na? and Cl- at root level, and accumulation of toxic

ions via increased succulence in leaf blades and leaf

sheaths in addition to some excretion of toxic ions through

leaf sheath aerenchyma.

Keywords Adaptation � Aerenchyma � Salt tolerance �Succulence � Vesicular hairs

Introduction

Plants growing on naturally salt-affected soils must have

evolved a multitude of morpho-anatomical and physio-

logical adaptive characteristics in view of considerable

length of time they have been exposed to high selection

pressure of the habitat like high salinity and aridity

(Ashraf 1994; Hameed and Ashraf 2008). The Salt

Range in Pakistan is a conspicuous site where soil in

most places is impregnated with high content of sodium

chloride. The full description of the Salt Range has been

given elsewhere (Hameed and Ashraf 2008). However,

the plant species whether dicot or monocot inhabiting

particularly salt-affected soils of the Salt Range are

supposed to be salt tolerant. Of a number of grass species

recorded from the Range, Sporobolus arabicus Boiss,

Cynodon dactylon (L.) Pers, and Imperata cylindrica (L.)

Raeuschel have considerable importance as they provide

valuable forage to sheep and goats being reared in the

Salt Range.

Specific anatomical and physiological modifications in

plants exposed to stressful environments may enable them

to thrive well on such environments. Although plants use a

variety of physiological phenomena to counteract salt

stress, regulation of ion homeostasis is one of the premier

physiological processes operating in plants exposed to salt

stress (Zhu 2003). This includes selective ion uptake

(Flowers and Colmer 2008), accumulation of toxic ions,

partially in terms of increased succulence (Hameed et al.

2009), and excretion of such unwanted toxic ions (Ramadan

and Flowers 2004; Naz et al. 2009).

Communicated by J. Franklin.

M. Hameed (&) � M. Ashraf � N. Naz

Department of Botany, University of Agriculture,

Faisalabad, Pakistan

e-mail: hameedmansoor@yahoo.com

M. Ashraf

Department of Botany and Microbiology,

King Saud University, Riyadh, Saudi Arabia

123

Acta Physiol Plant (2011) 33:1399–1409

DOI 10.1007/s11738-010-0674-8

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In view of all these, it was hypothesized that the eco-

types of the three grass species inhabiting the salt-affected

soils of the Salt Range differ considerably in taking up and

accumulating toxic inorganic ions such as Na? and Cl- and

excretion of such ions through leaf sheath or leaf surface.

The differences in their salinity tolerance may not be only

due to specific physiological adaptations, but also due to

their structural modifications in root, stem, leaf blade and

leaf sheath. This is perhaps due to some of the specific

anatomical features they have developed so far during

the length of time they have been growing there. Thus, the

major objective of the present study was to elucidate the

mechanism of ion regulation in salt tolerant ecotypes of

three potential grasses and relate these physiological dis-

tinctions with their anatomical adaptations particularly in

their leaves and roots.

Materials and methods

The present investigation was carried out to asses the adapt-

ability in relation to ionic balance of three potential salt tol-

erant grasses from the Salt Range, Pakistan. A population of

C. dactylon (L.) Pers. was collected from the edges of a

natural salt lake, the Uchhali Lake, in the Salt Range, Punjab

(pH 6.62, ECe 19.92 dS m-1). This species was in direct

contact of highly saline water of the lake. A population of

I. cylindrica (L.) Raeuschel was collected from some distance

from the edges of the salt lake, which is seasonally inundated

by diluted less saline waters during the rainy season (pH 7.38,

ECe 15.40 dS m-1). However, that of S. arabicus Boiss was

collected from the valley near the foothills, which was greatly

affected by the salt deposition as a result of dissolved salts

from the exposed hills (pH 8.12, ECe 34.36 dS m-1). The

ecotypes of all three grasses were also collected as checks

(control population) from normal non-saline habitats within

the Faisalabad region (pH 9.20, ECe 1.82 dS m-1 for

C. dactylon, pH 7.70, ECe 2.29 dS m-1 for I. cylindrica, and

pH 8.86, ECe 1.52 dS m-1 for S. arabicus).

The soil taken from the rhizosphere of each grass popu-

lation from each habitat was analyzed for physico-chemical

characteristics. Saturation percentage, pH and electrical

conductivity were determined by preparing saturated soil

paste of each sample. The soil pH and ECe were determined

using a combined pH/electrical conductivity meter. Inor-

ganic ions such as Na?, K? and Ca2? were determined with

a flame photometer and Cl- with a chloride meter.

Populations of both ecotypes of all three grasses were

grown in a mixture of sand, peat and clay mixed in equal

quantities for a period of 6 months under the climatic

conditions of Faisalabad (average day and night tempera-

tures 37 ± 3 and 24 ± 3�C, respectively, photoperiod

11–12 h, relative humidity 45.9–58.6%). Ramets of equal

size with three mature tillers were detached from each

plant and grown in half-strength Hoagland’s nutrient

solution (Hoagland and Arnon 1950) in containers of fiber

glass (25 L capacity) till their establishment in hydropon-

ics. The containers were aerated 12 h daily by air pumps.

Two salt levels, 0 (Hoagland’s nutrient solution with no

salt added) and 150 mM of NaCl in Hoagland’s nutrient

solution, were established in hydroponics. The experiment

was arranged in a completely randomized design (CRD)

with three factors (grasses, ecotypes and salinity levels)

and 12 replicates. After a growth of 60 days, plants were

carefully collected from the hydroponics for determining

physiological and anatomical characteristics.

For determining tissue ionic content of plants, dried

ground plant material (leaves and roots) was digested in

concentrated H2SO4 following Wolf (1982). For the

determination of excreted ions from leaf sheath and leaf

blade surface, 12 samples of the topmost fully expanded

leaf along with leaf sheath from the main tiller of all three

grass species were carefully separated, each sample

immediately washed in 20 ml of deionized water, and leaf

wash determined for the excreted ions. A flame photometer

was used to determine cations (Na?, K? and Ca2?) and a

chloride meter used to determine Cl-.

For studying stem and leaf sheath anatomy, a 2 cm piece

from the base of 3rd internode, for leaf blade anatomy a

2 cm piece from the base of top fully developed leaf, and

for root anatomy a 2 cm piece of the thickest adventitious

root from the root-culm junction were selected. All plant

samples so selected were first fixed in formalin acetic

alcohol fixative (formaldehyde 10%, acetic acid 5%, eth-

anol 50% and distilled water 35%) for 48 h and subse-

quently transferred to acetic alcohol solution (acetic acid

25% and ethanol 75%) for long-term storage. Free-hand

sectioning slides were prepared by a series of dehydrations

in ethanol using a standard double-stained technique of

safranine and fast green stains following Ruzin (1999).

Measurements were taken with a light microscope, using

an ocular micrometer, which was calibrated with a stage

micrometer. Micrographs of stained sections were taken

with a digital camera fitted on a stereo-microscope.

Data for anatomical characteristics were recorded using

all the 12 replicates. The analysis of variance (ANOVA)

for each attribute was computed using the MSTAT Com-

puter Program (MSTAT Development Team, 1989). LSD

at 5% level of probability was calculated to test the dif-

ferences among mean values (Steel et al. 1997).

Results

Ecotypes of all three grass species, C. dactylon, I. cylind-

rica and S. arabicus, responded differently in relation to

1400 Acta Physiol Plant (2011) 33:1399–1409

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both tissue ionic content and their anatomical characteris-

tics to saline stress.

Soil analysis

The Salt Range populations of all three grasses were col-

lected from highly salt-affected soils. However, S. arabicus

was collected from the salt-affected soils with highest

salinity (ECe 34.36 dS m-1), and C. dactylon and I. cyl-

indrica from relatively less saline habitats.

Growth attributes

Shoot length decreased significantly (P B 0.01) at 150 mM

NaCl in Faisalabad ecotypes of all three grass species

(Fig. 1), while this growth attribute increased in the Salt

Range ecotypes. In contrast, root length increased in the

ecotypes of C. dactylon and S. arabicus, but decreased in

the ecotypes of I. cylindrica from both Faisalabad and the

Salt Range (Fig. 1).

Shoot dry weight decreased with increase in salt level in

all cases except the Salt Range ecotype of S. arabicus,

where a slight increase in shoot dry weight was recorded

(Fig. 1). Root dry weight increased considerably in the

ecotypes of C. dactylon and S. arabicus from the Salt

Range. However, the Salt Range ecotypes of C. dactylon

produced the maximum root dry weight under 150 mM

NaCl. Although both ecotypes of I. cylindrica were little

affected by increasing salt level in terms of growth attri-

butes, they produced the minimum root and shoot dry

weights as compared to the other grasses.

Tissue ionic content

Leaf Na? increased at 150 mM NaCl as compared to

control in all ecotypes of the three grasses (Fig. 2). In

general, the Faisalabad ecotypes accumulated more Na? in

the leaves than that recorded in the Salt Range ecotypes of

all three grass species. The Salt Range ecotype of C. dact-

ylon was the lowest in accumulating Na? in its leaves at

150 mM NaCl among all the grasses.

The ecotypes of all three grasses showed a marked

increase in root Na? at higher salt level (Fig. 2), but the

Salt Range ecotypes of all three grasses accumulated less

Na? in their roots than that recorded in the Faisalabad

ecotypes except S. arabicus, in which both ecotypes did not

differ significantly in root Na?. The Salt Range ecotype of

I. cylindrica was the lowest in accumulating Na? in roots

of all ecotypes.

Increasing salt levels resulted in a significant (P B 0.01)

increase in root and leaf Cl- of both ecotypes of all three

grasses (Fig. 2), but the Salt Range ecotypes in all cases

accumulated much less Cl- in their leaves under 150 mM

NaCl level than that in their counterparts from the Faisal-

abad region. However, the ecotypes of S. arabicus did not

differ significantly in root Cl- under saline or non-saline

conditions.

Leaf K? decreased in both ecotypes of each grass spe-

cies at 150 mM NaCl, but the Salt Range ecotype of

S. arabicus showed a little decrease in leaf K?. Both

ecotypes of C. dactylon were the lowest in leaf K? content

of all ecotypes under saline or non-saline conditions

(Fig. 2). Root K? decreased with increasing salt level in all

0

30

60

90

Sh

oo

t le

ng

th(c

m)

0

30

60

90

Ro

ot

len

gth

(cm

)

0

1

2

3

4

5

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

Fsd SR Fsd SR Fsd SR

Cda Icy Sar

Sh

oo

rt d

ry w

eig

ht

(g p

lan

t-1)

0.0

0.5

1.0

1.5

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

Fsd SR Fsd SR Fsd SR

Cda Icy Sar

Ro

ot

dry

wei

gh

t(g

pla

nt-1

)

Cynodon dactylon

Cynodon dactylon

Imperata cylindrica

Imperata cylindrica

Sporobolus arabicus

Sporobolus arabicus

71.6=DSL23.7=DSL

870.0=DSL39.0=DSL

Fig. 1 Shoot and root length

and dry weights of 60-day-old

plants of three grass species

from Faisalabad (Fsd) and Salt

Range (SR) grown

hydroponically under 0 and

150 mM NaCl in greenhouse

conditions (mean ± SE;

n = 12; LSD least significant

difference)

Acta Physiol Plant (2011) 33:1399–1409 1401

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the grasses, but this decrease in root K? was much less in

the both ecotypes of C. dactylon as compared to that in the

other two grasses. Although the Salt Range ecotypes

showed slightly more accumulation of K? in their roots, it

was otherwise in S. arabicus (Fig. 2).

The pattern of Ca2? accumulation in the three grass

species was different. For example, leaf Ca2? in the Salt

Range ecotype of C. dactylon increased considerably

whereas that in the Faisalabad ecotype it decreased sig-

nificantly. The Salt Range ecotype of I. cylindrica also

showed similar trend but in leaf Ca2? content was signif-

icantly (P B 0.01) lower than that in the Salt Range eco-

types of C. dactylon (Fig. 2). The ecotypes of S. arabicus

from both Faisalabad and the Salt Range showed increased

accumulation of Ca2? in their shoots at 150 mM NaCl.

Root Ca2? also showed variable behavior in all grasses

as the salt concentration increases in external growth

medium (Fig. 2). Both I. cylindrical and S. arabicus

showed a decrease in Ca2? under salt stress, but the eco-

types of I. cylindrica were much less affected than those of

S. arabicus (Fig. 2).

Excreted ionic content

Ecotypes of both S. arabicus and C. dactylon excreted

substantial amounts of Na? and Cl- from leaf blade sur-

face, particularly the Salt Range ecotypes (Fig. 3). In

contrast, S. arabicus excreted both Na? and Cl- in a sig-

nificant amount through leaf sheath under 150 mM NaCl.

I. cylindrica also excreted Na? and Cl- through leaf sheath

LSD 5% = 5.23

0

20

40

60

Ro

ot

Na

+

(mg

g-1 d

.w.)

LSD 5% = 5.81

0

20

40

60

Lea

f N

a+

(mg

g-1 d

.w.)

LSD 5% = 5.12

0

20

40

60

Ro

ot

Cl-

(mg

g-1 d

.w.)

LSD 5% = 2.76

0

7

14

21

28

35

Lea

f C

l-

(mg

g-1 d

.w.)

LSD 5% = 2.73

0

10

20

30

Ro

ot

K+

(mg

g-1 d

.w.)

LSD 5% = 2.56

0

10

20

30

Lea

f K

+

(mg

g-1 d

.w.)

LSD 5% = 1.56

0

5

10

15

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

Fsd SR Fsd SR Fsd SR

Cynodondactylon

Imperatacylindrica

Sporobolusarabicus

Ro

ot

Ca

2+

(mg

g-1 d

.w.)

LSD 5% =2.36

0

5

10

15

20

25

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

Fsd SR Fsd SR Fsd SR

Cynodondactylon

Imperatacylindrica

Sporobolusarabicus

Lea

f C

a2+

(mg

g-1 d

.w.)

Cynodon dactylon

Imperata cylindrica

Sporobolus arabicus

Cynodon dactylon

Imperata cylindrica

Sporobolus arabicus

Fig. 2 Leaf and root ionic

content of 60-day-old plants of

three grass species from

Faisalabad (Fsd) and Salt Range

(SR) grown hydroponically

under 0 and 150 mM NaCl in

greenhouse conditions

(mean ± SE; n = 12; LSD least

significant difference)

1402 Acta Physiol Plant (2011) 33:1399–1409

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but to a lesser extent as compared to that in S. arabicus.

The Salt Range ecotypes of both these grasses excreted

relatively more amount of Na? and Cl- than their coun-

terparts from the Faisalabad region (Fig. 3). C. dactylon,

on the other hand, showed negligible excretion through leaf

sheath.

Both ecotypes of S. arabicus excreted K? and Ca2?

through leaf sheath and leaf blade surface in relatively

larger amounts than those by the other grasses, but the Salt

Range ecotype was relatively more efficient in excretion of

these ions (Fig. 3). C. dactylon also excreted considerable

amount of both K? and Ca2?, but only through leaf blade

surface. In contrast, I. cylindrica excreted a significant

amount of Ca2?, but only through leaf sheath.

Anatomical studies

In C. dactylon, root area increased with increased salt level

in both the Faisalabad and the Salt Range ecotypes (Fig. 4).

The Faisalabad ecotype showed thicker stems than that

recorded in the Salt Range ecotype. In I. cylindrica, higher

salt level results a reduction in root area, but the Salt Range

ecotype showed more or less stability in this characteristic

under both normal and saline conditions. Root area in

S. arabicus decreased at 150 mM NaCl in the Faisalabad

ecotype, but a considerable increase was recorded in this

attribute in the Salt Range ecotype.

Stem area was decreased in the C. dactylon ecotype

from the Faisalabad region at 150 mM NaCl salt level.

LSD 5% = 61.31

0

200

400

600

Lea

f b

lad

e N

a+

(mg

L-1)

LSD 5% = 63.4

0

200

400

600

Lea

f sh

eath

Na

+

(mg

L-1)

LSD 5% = 12.91

0

30

60

90

120

Lea

f b

lad

e C

l-

(mg

L-1)

LSD 5% = 13.97

0

50

100

150

Lea

f sh

eath

Cl-

(mg

L-1)

LSD 5% = 2.42

0

5

10

15

20

25

Lea

f b

lad

e K

+

(mg

L-1)

LSD 5% = 0.78

0

2

4

6

8

Lea

f sh

eath

K+

(mg

L-1)

LSD 5% = 2.88

0

10

20

30

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

Cynodondactylon

Im peratacylindrica

Sporobolusarabicus

Lea

f b

lad

e C

a2+

(mg

L-1)

LSD 5% = 2.21

0

5

10

15

20

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

0mM

150m

M

Cynodondactylon

Im peratacylindrica

Sporobolusarabicus

Lea

f sh

eath

Ca

2+

(mg

L-1)

Cynodon dactylon

Cynodon dactylon

Imperata cylindrica

Imperata cylindrica

Sporobolus arabicus

Sporobolus arabicus

Fig. 3 Ionic content in leaf

blade and leaf sheath washings

of 60-day-old plants of three

grass species from Faisalabad

(Fsd) and Salt Range (SR)

grown hydroponically under 0

and 150 mM NaCl in

greenhouse conditions

(mean ± SE; n = 12; LSD least

significant difference)

Acta Physiol Plant (2011) 33:1399–1409 1403

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In contrast, in the Salt Range ecotype, stem area was

slightly increased in salt treatment (Fig. 4). Stem area in

both ecotypes of S. arabicus increased under salt, but this

increase in the Salt Range ecotype was very much pro-

nounced as compared to that in its counterpart from the

Faisalabad region.

Midrib thickness in the Faisalabad ecotype of C. dact-

ylon remained constant under both non-saline and saline

treatments (Fig. 4). This characteristic was slightly affec-

ted under salt treatment in the Salt Range ecotype of

C. dactylon. Midrib thickness increased considerably in

the Faisalabad ecotype of I. cylindrica, whereas the Salt

Range ecotype showed a decrease in this character under

salt treatment, but it had much thicker leaf midrib than

that recorded in the Faisalabad ecotype (Figs. 4, 5). The

shape of midrib in the Faisalabad ecotype is conical and

that in the Salt Range ecotype is rounded (Fig. 5). In

S. arabicus, the midrib thickness increased in the Faisal-

abad ecotype due to salt stress but in the Salt Range

ecotype, there was a slight decrease in this anatomical

characteristic (Fig. 4).

Lamina thickness showed a variable response to salt

treatment in the ecotypes of all three grasses (Fig. 4). In

C. dactylon, lamina thickness was least affected under salt

stress, because it was little increased in the Faisalabad

ecotype and slightly decreased in the Salt Range ecotype.

In S. arabicus, a similar pattern of lamina thickness was

recorded as in the case of C. dactylon, but the impact of salt

stress on lamina thickness was relatively more pronounced

(Fig. 4). In I. cylindrica, salt caused a decrease in lamina

thickness in the Faisalabad ecotype and an increase in the

Salt Range ecotype.

LSD 5% = 0.26

0

1

2

3

4

Ro

ot

area

(m

m2 )

LSD 5% = 1.36

0

4

8

12

16

Ste

m a

rea

(mm

2 )

LSD 5% = 49.68

0

100

200

300

400

500

Lam

ina

thic

knes

s (µ

m)

LSD 5% = 151.34

0

300

600

900

1200

1500

Mid

rib

th

ickn

ess

(µm

)

LSD 5% = 2.21

0

5

10

15

20

Lea

f tr

ich

om

e d

ensi

ty

LSD 5% = 3.22

0

10

20

30

Lea

f ve

sicu

lar

hai

r d

ensi

ty

LSD 5% = 41.31

0

150

300

450

0mM

150m

M

0mM

150m

M0m

M

150m

M

0mM

150m

M0m

M

150m

M

0mM

150m

M

Fsd SR Fsd SR Fsd SR

Cynodondactylon

Imperatacylindrica

Sporobolusarabicus

Lea

f sh

eath

th

ickn

ess

(µm

)

LSD 5% = 1502.16

0

5000

10000

15000

0mM

150m

M

0mM

150m

M

0mM

150m

M0m

M

150m

M

0mM

150m

M

0mM

150m

M

Fsd SR Fsd SR Fsd SR

Cynodondactylon

Imperatacylindrica

Sporobolusarabicus

Lea

f sh

eath

aer

ench

yma

area

(µm

2 )

Cynodon dactylon

Cynodon dactylon

Imperata cylindrica

Imperata cylindrica

Sporobolus arabicus

Sporobolus arabicus

Fig. 4 Root, stem and leaf

anatomical characteristics of

60-day-old plants of three grass

species from Faisalabad (Fsd)

and Salt Range (SR) grown

hydroponically under 0 and

150 mM NaCl in greenhouse

conditions (mean ± SE;

n = 12; LSD least significant

difference)

1404 Acta Physiol Plant (2011) 33:1399–1409

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Number of trichomes per unit area increased in both

ecotypes of C. dactylon under salt level (Fig. 4). In the Salt

Range ecotype, salt stress caused a twofold increase in

trichome density as compared to that in non-stress treat-

ment. In S. arabicus, the Faisalabad ecotype showed no

visible change in trichome density under salt stress, but the

Salt Range ecotype showed a marked increase in this

parameter (Figs. 6, 7).

Vesicular (bladder or bicellular microhairs) hairs

increased markedly in both C. dactylon and S. arabicus

under salt stress (Fig. 4). In both ecotypes of C. dactylon,

vesicular hairs were not recorded under non-saline treat-

ment, but their density was increased by about fourfold in

the Salt Range ecotype under salt level as compared to that

in the Faisalabad ecotype (Figs. 4, 8). In I. cylindrica, no

such excretory structures were recorded. In S. arabicus,

vesicular hair density increased greatly in both ecotypes,

but in the Salt Range ecotype, this increase was about

fivefold under salt treatment (Figs. 4, 6).

Leaf sheath thickness decreased in the ecotypes of

C. dactylon from both Faisalabad and the Salt range under

salt stress (Fig. 4). Sheath thickness remained unaffected in

the Faisalabad ecotype of I. cylindrica, but in the Salt

Range ecotype it increased to some extent. However, this

parameter increased greatly in both ecotypes of S. arabicus

due to salt level.

A well-developed aerenchyma was recorded in the leaf

sheaths of both ecotypes of I. cylindrica (Figs. 4, 5). The

Faisalabad ecotype showed slight increase in aerenchy-

matous area under salt stress, but the Salt Range ecotype

showed a twofold increase (Fig. 4), i.e., from 6,172 lm2 at

0 mM to 12,427 lm2 at 150 mM NaCl. In S. arabicus,

aerenchyma formation was recorded in the ecotype from

the Salt Range only under salt stress (Figs. 4, 7). However,

Fig. 5 Anatomical

modifications in Imperatacylindrica to salinity stress:

a thick round and succulent leaf

midrib (0 mM NaCl) and

b increased sclerification in

midrib (150 mM NaCl) in the

Salt Range ecotype; c conical

shape leaf midrib (0 mM NaCl)

and d somewhat round midrib

(150 mM NaCl) in the

Faisalabad ecotype; e leaf

sheath with intact parenchyma

(0 mM NaCl), and f well-

developed aerenchyma

(150 mM NaCl) in leaf sheath

in the Salt Range ecotype

(Ae aerenchyma, P parenchyma,

VB vascular bundles, Scsclerenchyma)

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in contrast, the aerenchymatous region was not recorded in

both ecotypes of C. dactylon.

Discussion

The ecotypes of all three grasses, C. dactylon, I. cylindrica,

and S. arabicus, from the Salt Range can be regarded as

more tolerant to salt stress than their counterparts from the

Faisalabad region in terms of growth attributes, particularly

shoot dry weight. On the bases of biomass production

under saline conditions, S. arabicus from the Salt Range

can be rated as the most tolerant as it showed highest shoot

biomass production under salt stress. It was followed by the

Salt Range ecotype of C. dactylon, in which shoot dry

weight was slightly affected by salt stress. The ecotype of

I. cylindrica was the least tolerant among all ecotypes from

the Salt Range, because its growth was significantly

affected by salt stress. The differential tolerance of the

ecotypes of three grass species from the Salt Range relates

well to the soil physico-chemical properties (particularly

ECe of the soil) of the habitats of the Salt Range from

which these ecotypes were collected. Such differences in

the natural populations have also been reported by Ashraf

and Ahmad (1995) and Ashraf (1997) in grasses.

Maintenance of ion balance in plants subjected to saline

conditions is vital for sustaining growth and productivity

under such environmental adversities (Munns and Tester

2008). While examining the ion content in different plant

parts of all ecotypes of the three grass species exposed to

high salt concentration, it is evident that salt application

resulted in an increased Na? content of leaves and roots in

all three grasses from both Faisalabad and the Salt Range.

A consistent increase in Na? in leaves or roots with an

increase in salt stress of growth medium has earlier been

reported in Aeluropus lagopoides (Gulzar et al. 2003),

Panicum turgidum (Mahmood and Athar 2003), Sorghum

bicolor (de Lacerda et al. 2005), and C. dactylon (Hameed

and Ashraf 2008).

The highly salt tolerant ecotype of S. arabicus from the

Salt Range accumulated more toxic ions like Na? and Cl-

in roots than did the ecotypes of C. dactylon and I. cyl-

indrica indicating no selective uptake of toxic ions. Higher

accumulation of Na? and Cl- in tissues by S. arabicus can

be related to its structural modifications such as increased

area of root, stem, leaf blade, and leaf sheath, which may

provide more space for dumping off a large amount of

these ions (Flowers and Colmer 2008). In addition, accu-

mulation of ions like Na? plays a vital role in osmotic

adjustment, which in turn facilitates water uptake along a

soil–plant gradient in highly salt tolerant plants and halo-

phytes (Khan et al. 2000). Furthermore, a substantial

amount of excretory toxic ions depicted its high efficiency

in salt secretions either through leaf sheath or leaf blade

surface. This was again supported by considerably

increased vesicular hair density in this ecotype under high

salt level, which is probably the main mechanism to cope

with high salinities. Such hairs are reported to be involved

Fig. 6 Anatomical

modifications in Sporobolusarabicus to salinity stress: a leaf

lamina (0 mM NaCl) with a few

vesicular hairs and trichomes

and b significantly increased

vesicular hair and trichome

density (150 mM NaCl) in the

Faisalabad ecotype; c thicker

leaves with prominent

sclerenchyma region on adaxial

surface (0 mM NaCl), and

d markedly increased vesicular

hair density, well-developed

bulliform cells and increased

sclerification on both leaf

surfaces and in vascular tissue

(150 mM NaCl) in the Salt

Range ecotype (Bf bulliform

cells, VH vesicular hairs, Scsclerenchyma, Tr trichomes)

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in salt excretion by some earlier findings in different spe-

cies, e.g., Cynodon and Distichlis (Oross and Thomson

1982), in salt tolerant species (Hagemeyer 1997), sub-

family Chloridoideae (Marcum 1999), and Odyssea pau-

cinervis (Somaru et al. 2002). Formation of aerenchyma in

the Salt Range ecotype of S. arabicus and the deposition of

salt crystals on the edges of leaf sheath as was confirmed

through ionic washings reflect that this ecotype also pos-

sesses the mechanism of ion exclusion through aerenchyma

in addition to excretion via bicellular microhairs or secre-

tory trichomes through leaf surface.

The Salt Range ecotype of C. dactylon which was next

to S. arabicus in terms of biomass production under salt

stress also showed higher uptake of both Na? and Cl- in

spite of bearing thin roots. However, relatively lower

concentrations of these ions in the leaves indicate that the

ecotype used the exclusion mechanism through some spe-

cific structural modifications, such as vesicular hairs

(Cheng and Chou 1997). Exclusion of toxic ions is a

common phenomenon occurring in most mesophytes

(Munns et al. 1983; Ashraf 1994). However, exclusion of

salts from leaves, roots or other plant parts is regulated by a

multitude of physiological and anatomical characteristics.

In the present study, the Salt Range ecotype of C. dactylon

generally tended to exclude toxic ions through leaves and

this salt exclusion mechanism could have been due to its

greatly increased density of vesicular hairs on both leaf

surfaces (Marcum 1999).

The Salt Range ecotype of I. cylindrica showed specific

structural adaptations to tolerate salt stress. For example, it

showed reduced root area so as to absorb less quantity of

toxic Na? and Cl- at root level, i.e., avoidance by selective

uptake of toxic ions. In view of Munns (2002), selective

transport of toxic ions to leaves is an important survival

mechanism to high salinities. Increased succulence (paren-

chymatous tissue) in leaf blade and leaf sheath as observed

Fig. 7 Anatomical

modifications in Sporobolusarabicus to salinity stress: a leaf

lamina (150 mM NaCl) with

excretory trichomes in the

Faisalabad ecotype, b vesicular

hairs or bicelled microhairs

(150 mM NaCl) in the Salt

Range ecotype, c adaxial leaf

surface in the Faisalabad

ecotype with dense cover of

trichomes (150 mM NaCl), and

d adaxial leaf surface in the Salt

Range ecotype (150 mM NaCl)

with dense cover of vesicular

hairs, e leaf sheath (0 mM

NaCl) with sclerenchyma above

vascular tissue, and f leaf sheath

(150 mM NaCl) with distinct

and thick sclerenchyma layer on

outer surface and aerenchyma

formation in the Salt Range

ecotype (Ae aerenchyma, Scsclerenchyma, Tr trichomes,

VB vascular bundle,

VH vesicular hairs)

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in this ecotype might have enhanced the vacuolar volume so

as to provide greater area for dumping off toxic ions such as

Na? and Cl-, as reported by Hameed et al. (2009). Leaf or

stem succulence is an adaptive feature, which contributes to

the regulation of internal ion concentrations in many halo-

phytes (Short and Colmer 1999), although leaf succulence is

a rare phenomenon in monocots (Hameed and Ashraf 2008).

In addition, the ecotype showed an increased aerenchyma in

leaf sheath under saline stress. This could be an important

feature to excrete toxic ions, as well as in overcoming gas-

eous exchange problems during physiological drought

caused by salinity stress.

In conclusion, anatomical modifications to overcome

high salinities were very specific not only in the grass

species but also in the ecotypes. The Salt Range ecotype of

S. arabicus had increased area of root, stem, leaf blade, and

leaf sheath. These characteristics may enhance the ability

of the ecotype to accumulate salts. In addition, increased

vesicular hair density and aerenchyma formation in leaf

sheath of this ecotype under salt stress seem useful in salt

excretion. The Salt Range ecotype of C. dactylon totally

depended on ion exclusion through specific structural

modifications such as vesicular hairs. The Salt Range

ecotype of I. cylindrica showed restricted uptake of toxic

Na? and Cl- at root level in addition to increased succu-

lence in leaf blade and leaf sheath for dumping off the toxic

ions. The most efficient way for maintaining growth and

survival in adverse environmental conditions seems to be

excessive excretion of toxic ions from leaf sheath and leaf

surface, i.e., via microhairs and leaf sheath aerenchyma.

Perhaps, this requires low amount of energy for its survival

and this might be utilized in the growth. On the other hand,

substantial amount of energy is required in selective uptake

of toxic ions and also for ion sequestering in non-meta-

bolically active areas such as vacuoles.

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