Physiological aspects of tolerance in Atriplex halimus L. to NaCl and drought

ORIGINAL PAPER

Physiological aspects of tolerance in Atriplex halimus L. to NaCland drought

Mamdouh M. Nemat Alla • Abdel-Hamid A. Khedr •

Mamdouh M. Serag • Amina Z. Abu-Alnaga •

Reham M. Nada

Received: 22 December 2009 / Revised: 29 June 2010 / Accepted: 23 July 2010

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

Abstract Forty-day-old seedlings of Atriplex halimus

were treated either with NaCl (50, 300 and 550 mM) for

the subsequent 30 days or with 15% PEG for the sub-

sequent 10 days. As much as 50 mM of NaCl significantly

increased shoot fresh and dry weight and height; never-

theless, 300 or 550 mM NaCl seemed to have no effect. On

the other hand, these growth parameters were not affected

by drought after 3 or 6 days, but were reduced after

10 days. The gas exchange parameters (photosynthetic

rate, stomatal conductance and transpiration rate) were

increased by 50 mM NaCl, but decreased by 300 and

550 mM. These parameters were decreased in response to

drought only after 10 days of withholding water. In con-

trast to Na?, K? was significantly decreased by NaCl but

not by drought. The time course effect revealed that

phosphoenol pyruvate carboxylase (PEPC) protein was

doubled in response to NaCl after 1 and 5 h and continued

thereafter, higher than control, while drought had no sig-

nificant effect. Rubisco seemed unchanged by NaCl or

drought. It could be concluded that the decrease in fresh

weight might be attributed to the decrease in water content.

Moreover, the decrease in photosynthesis could result from

a decrease in stomatal conductance, a protective mecha-

nism against water loss to improve water use efficiency.

These findings indicate that Atriplex halimus tolerates

NaCl and drought through decreasing growth, reducing gas

exchange parameters to improve water use efficiency,

uptake Na? and saving, if any, the photosynthetic enzyme

particularly PEPC.

Keywords Atriplex halimus � Drought � Gas exchange �Growth � NaCl � PEPC � Tolerance

Introduction

Stresses cause a reduction in plant growth. One cause of

growth rate reduction is inefficient photosynthesis. Salinity

affects adversely seed germination by osmotic effect and/or

ionic toxicity that could result in molecular damage,

growth arrest and even death of plants (Duan et al. 2007).

Three physiological mechanisms are suggested to be

involved in the reduction of plant growth resulting from

osmotic stress caused by salinity as well as drought stress:

(1) reduction in turgor pressure, (2) reduction in photo-

synthetic activity and (3) effects of accumulated salts on

the metabolic activities (Neumann 1997). Stress can inhibit

cell expansion in both leaves and roots (Neumann 1995).

Cell expansion is powered by inward water flow. It

depends on the hydraulic conductivity of water uptake

pathway, the uptake of solutes to maintain osmotic

potential and the volume of the surrounding cells. The

reduction occurs due to nutrient deficiency (Hu et al. 2007)

and ion toxicity.

The gas exchange parameters (photosynthetic rate,

transpiration rate and stomatal conductance) are affected

by salinity and drought stress (Chaves 1991; Zhu and

Meinzer 1999; Munns et al. 2006; Redondo-Gomez et al.

2007). Photosynthetic activity is affected due to malfunc-

tion of Rubisco, the key enzyme in Calvin cycle, and of

PEPC, the key enzyme of C4 plants. PEPC catalyzes the

carboxylation of PEP to form oxaloacetate in mesophyll

Communicated by K. Trebacz.

M. M. Nemat Alla (&) � A.-H. A. Khedr �M. M. Serag � A. Z. Abu-Alnaga � R. M. Nada

Botany Department, Faculty of Science at Damietta, Mansoura

University, Damietta, Egypt

e-mail: [email protected]

123

DOI 10.1007/s11738-010-0578-7

Acta Physiol Plant (2011) 33:547–557

/ Published online: 201013 August

tissue, from which CO2 is released again in bundle sheath

tissue for the decarboxylation of ribulose-1,5-bisphosphate,

the first enzymatic reaction in Calvin cycle that is catalyzed

by Rubisco. Halophytes and xerophytes undertake the same

measures to counter osmotic stress (Kefu et al. 2003). In

this study, the effects of salinity (NaCl) and drought

(polyethyleneglycol, PEG) in the xero-halophyte Atriplex

halimus were studied to relate tolerance to the harsh con-

ditions with some physiological aspects such as gas

exchange parameters and contents of Na?, K?, PEPC and

Rubisco.

Materials and methods

Plant material and growth conditions

The seeds of Atriplex halimus used in this study were

collected from the Baltim area in Egypt. The seeds were

collected from one bush, mixed properly and stored in

paper bags at room temperature until use. The seeds were

germinated in 10 9 15 cm trays containing vermiculite

and watered daily with Long Ashton nutrient solution. The

trays were incubated in a growth room at 28/20�C day/

night temperature, 16-h photoperiod, 60% RH and

300 lmol m-2 s-1 light intensity. After 19 days, seedlings

with uniform sizes were selected for treatments. For NaCl

stress treatment, the seedlings were transplanted into

4 9 6 cm pots (one seedling per pot) containing vermic-

ulite and the pots were kept in 50 9 35 cm trays contain-

ing Long Ashton nutrient solution (3 L per tray). For

drought treatment, the seedlings were transplanted into

10 9 10 cm pots (one seedling per pot) containing com-

post (150 g per pot) and watered daily with deionized

water.

The salt stress treatment started when the plants were

40 days old. Four sets of plants were used and each set

included 50 plants. The control set was watered with Long

Ashton nutrient solution, while the other three sets were

treated with 50, 300 or 550 mM NaCl for 10 days. For

drought stress treatment, two sets of 40-day-old seedlings

were used. The control set was watered with deionized

water up to the end of experiment and the other set was

exposed to drought stress by withholding water. Twenty

plants from each set were harvested 10 days after applying

NaCl or 3, 6 or 10 days after withholding water. The

leaves were frozen immediately in liquid nitrogen and

then stored at -80�C until use for further analyses. After

harvest, the fresh weights of shoots were recorded and

dried in an oven at 80�C for 2 days and the dry weights

were then recorded. The water contents were calculated on

fresh weight basis. Five plant replicates were used for

each treatment.

Determination of Na? and K?

Na? and K? were extracted as described by Hansen and

Munns (1988). About 100 mg of frozen leaf tissue was first

homogenized in liquid nitrogen and then 2 ml of boiled

water was added. The mixture was kept in a water bath at

100�C for 1 h. The residue was removed by centrifugation

at 14,0009g for 20 min. The diluted supernatant (1:10 with

H2O) was used to measure Na? and K? by flame pho-

tometry. Three replicates were used for each treatment.

Measurement of photosynthesis, transpiration

and stomatal conductance

Photosynthetic rate, transpiration rate and stomatal con-

ductance of the third leaves were measured by using

LCA-4 portable gas exchange system (Analytical Devel-

opment Company Limited, Hertfordshire, England). Leaf

area was measured by using leaf area meter (Delta-T,

Cambridge, UK). Leaf was fitted into a 625-cm2 PLC4 leaf

chamber. Gas exchange measurements were made in the

growth chamber under saturating light conditions (light

sources were filtered using a small heat absorbing filter)

and adjusted to an intensity of 1,000 lmol m-2 s-1. For

each treatment, three replicates were used.

Time course effect of NaCl and drought on Rubisco

and PEPC contents

Another experiment was conduct in which NaCl (0, 50, 200

and 500 mM) or osmotic stress (by using 15% PEG alone

or combined with 50 mM NaCl) were applied to 40-day-

old seedlings. Third leaf samples from each treatment were

harvested after 1, 5, 24, 48 h and 10 days. The leaves were

frozen immediately in liquid nitrogen and then stored at

-80�C until use for further analyses (the protein levels of

Rubisco and PEPC contents).

Extraction of total proteins for SDS-PAGE

The extraction buffer used in protein dissociation was: 4%

SDS, 2% b-mercaptoethanol and 20% glycerol in 100 mM

Tris–HCl (pH 8.5). About 100 mg of frozen leaves was

ground to a fine powder in a pre-cooled mortar and 4 ll of

extraction buffer was added for each 1 mg of leaf material.

The homogenate was heated up to 80�C for 3 min and the

debris was removed by centrifugation at 14,0009g for

10 min. The proteins were resolved as described by

Laemmlli (1970) by using the Bio-Rad Mini Protean 3

(Bio-Rad laboratories, Hercules, CA, USA). The resolving

gel contained 11% acrylamide and the stacking gel con-

tained 5% acrylamide. Proteins from 3 mg leaf fresh

weight were loaded onto each lane. The proteins were

123


resolved at 100 V for 90 min. The gels were stained with

brilliant blue R-250 (Bio-Rad) and then destained with 20%

methanol. The gels were dried using Bio-Rad gel drier,

scanned and used for Rubisco quantification. Rubisco bands

were quantified using ‘‘Lab Image V 2.7.2’’ program.

Western blotting and immunochemical detection

of PEPC

Proteins were resolved on 11% acrylamide as described

before by loading 3 mg of fresh weight in a volume of

15 ll onto each lane and then transferred to PVDF mem-

branes (Bio-Rad) using Bio-Rad mini protean 3 transblot-

ter. The transfer buffer consisted of 5.8 g l-1 Tris base and

2.9 g l-1 glycine. The proteins were transferred for 1 h at

100 V and 4�C. The membranes were then blocked in 2.5%

dry non-fat dried milk in TBS (Tris-buffered saline:

150 mM NaCl in 20 mM Tris–HCl, pH 7.4). The blots

were used for immunochemical detection of PEPC. PEPC

was detected by using a primary antibody developed in

rabbit. The blots were incubated in 1:1,000 dilution of the

primary antibody in 2.5% milk TBS for 2 h. After five

washes (3 min each) in TBS, the membranes were incu-

bated in 1:1,000 dilution of the secondary antibody (goat

anti-rabbit IgG-horseradish peroxidase conjugate) (Sigma)

for 90 min and washed as before. The signals were

detected by ECL kit (Pharmacia Amersham) and visualized

according to the manufacturer’s instructions.

The full data were statistically analyzed using SPSS V

12.0.1 for ANOVA test.

Results

Figure 1 shows that the soil water content remained at

81%. Withholding water after the addition of PEG resulted

in significant decreases in soil water content. This decrease

was augmented as the time of withholding water elapsed.

Soil water content decreased from 81% in the control to 70,

49 and 22% after 3, 6 and 10 days of withholding water,

respectively.

Shoot fresh weight of Atriplex halimus seedlings was

maximum at 50 mM NaCl (Fig. 2). However, the other

concentrations showed significant reduction in weight

compared to 50 mM, but had no effects relative to control.

Similarly, the shoot dry weight was significantly greater in

the presence of 50 mM NaCl than that of the control.

However, shoot dry weights remained unchanged with

other concentrations. On the other hand, water content at

all concentrations of NaCl was comparable to control.

Nevertheless, water content at 300 and 550 mM was sig-

nificantly lower than that at 50 mM NaCl. On the other

hand, drought stress had no significant effect on fresh and

dry weight 3 and 6 days after withholding water. On the

contrary, shoot fresh weight significantly decreased by

about 45%, 10 days after withholding water; however,

shoot dry weight was not significantly changed. On the

other hand, shoot water content did not show significant

reduction due to drought 3 or 6 days after withholding

water. Nevertheless, shoot water content significantly

decreased 10 days after withholding water to reach 81%

compared to that of the well-watered seedlings.

In Fig. 3, NaCl at 300 and 550 mM significantly

decreased the shoot height by about 15 and 27%, respec-

tively; nevertheless, 50 mM had no effect. Also, there was

no significant change in leaf numbers of seedlings under 50

or 300 mM NaCl, but 550 mM resulted in a significant

decrease up to 31% compared to the control. Also in Fig. 3,

there was no significant effect of drought on the shoot

height 3 or 6 days after withholding water. However, shoot

height was significantly reduced by 15% of the control

after 10 days. The same trend was observed for the number

of leaves; there was a significant decrease in leaf number

by about 37% compared to the well-watered seedlings only

after 10 days.

The photosynthetic rate increased significantly in the

presence of 50 mM NaCl compared to the control (Fig. 4).

However, it was significantly reduced by about three and

fourfolds at 300 and 550 mM NaCl, respectively. The same

trend was observed for stomatal conductance where the

highest value was shown at 50 mM NaCl, but decreased at

300 and 550 mM of NaCl by 45 and 62% of the control,

respectively. The transpiration rate remained similar to the

control at 50 mM NaCl, but decreased at 300 and 550 mM

of NaCl, by about three and fourfolds as compared to the

control, respectively. At 3 and 6 days after withholding

water, there was no significant change in photosynthetic

Days after withholding water3 6 10

Soil

wat

er c

onte

nt (

%)

0

20

40

60

80

100

120ControlDrought

a

b

c

d

aa

Fig. 1 Soil water content 3, 6 and 10 days after withholding water

from 40-day-old Atriplex halimus. Values are mean (±SE) of at least

five replications. Data labeled with different letters are significantly

different at P \ 0.05

123

Acta Physiol Plant (2011) 33:547–557 549

rate, stomatal conductance or transpiration rate; neverthe-

less, there was a significant decrease after 10 days by about

48, 63 and 57%, respectively.

As shown in Fig. 5, Na? accumulated to significantly

higher levels in the leaves of Atriplex halimus under NaCl

stress. The magnitude of accumulation increased with

increasing NaCl concentration. At 50 mM NaCl, Na?

content was 79% higher than that of control. Moreover, at

300 and 550 mM NaCl, the Na? contents of leaves were

about 6 and 6.5 times higher than that of the control,

respectively. In contrast to Na?, K? was significantly

decreased with the increase of NaCl treatment. K? at 50,

300 and 550 mM NaCl was decreased to about 42, 31 and

23% of that accumulated in control leaves, respectively.

Under drought treatment, there was no significant change

in the Na? contents of leaves 3 or 6 days after withholding

water. On the other hand, Na? content was significantly

increased in leaves of the drought-treated seedlings

10 days after treatment compared to the control. However,

drought had no significant effect on K? accumulation in

Atriplex halimus leaves.

In Fig. 6, the amount of PEPC protein was doubled in

response to all concentrations of NaCl (50, 200 and

500 mM) 1 and 5 h after stress onset. From 24 h up to the

end of the treatment at 10 days, the magnitude of increase

of PEPC protein level of the stressed leaves decreased

slightly, but still remained higher than that of control

leaves. Although drought increased PEPC protein level,

these increased levels did not reach double value. In fact,

the effect of PEG was small, with no significance, either

alone or combined with NaCl on PEPC protein level. In

contrast to PEPC, there was no significant change in

Rubisco contents of leaves following treatment with PEG

(Fig. 7). In spite of the slight fluctuations detected in

Rubisco contents, NaCl and drought did not result in sig-

nificant changes of Rubisco at any time interval.

Discussion

The present results showed that 50 mM NaCl significantly

improved the growth of Atriplex halimus. Moreover, 300

and 550 mM NaCl did not reduce the growth. All these

results prove the halophytic properties of this species. Khan

et al. (2000) found that halophytes such as Atriplex spp.

showed improved growth at moderate concentrations of

NaCl (mM)0 50 300 550

Shoo

t fre

sh w

eigh

t (g)

0

2

4

6 a

b b b

Shoo

t dry

wei

ght (

g)

NaCl (mM)0 50 300 550

Shoo

t wat

er c

onte

nt (

%)

0

20

80

85

90

a

b

ab

b

1A 1C

Shoo

t dry

wei

ght (

g)

NaCl (mM)0 50 300 550

0.0

0.2

0.4

0.6

0.8

1.0 a

bab

ab

1B


0.0

0.2

0.4

0.6

0.8

1.0

1.2ControlDrought


0

20

80

100

ControlDrought

*

C2

Shoo

t wat

er c

onte

nt (

%)

B2


Shoo

t fre

sh w

eigh

t (g)

0

2

4

6

8

10 ControlDrought

***

A2

Fig. 2 Effect of NaCl treatments (1) and drought (2) for 10 days on

shoot fresh weight (A), shoot dry weight (B) and water content (C) of

40-day-old Atriplex halimus. Values are mean (±SE) of at least five

replications. Data labeled with different letters are significantly

different at P \ 0.05. (*) and (***) are significantly different from the

respective control at P \ 0.05 and P \ 0.001, respectively

123


NaCl, which seem to be inhibitory for the growth of non-

halophyte plants. They concluded that Atriplex griffithii

showed little inhibition in seedling growth in a medium

containing up to 180 mM NaCl. They, moreover, con-

firmed that deleterious effects of salt stress on plants are

thought to result from low water potentials, ion toxicities,

nutrient deficiencies or a combination of all of these

factors.

Salinity imposed by 50 mM NaCl increased shoot fresh

and dry weights of Atriplex halimus, but shoot height and

the number of leaves were unchanged. The higher con-

centrations seemed to have no effect on fresh and dry

weights, but decreased the shoot height and leaf number.

These results indicate that high concentrations of NaCl

seemed to have no dramatic effect on growth of Atriplex

halimus. Moreover, the optimal growth of Atriplex halimus

appeared to require low concentration of NaCl. Atriplex

halimus, like other halophytes such as Suaeda salsa (Kefu

et al. 2003), could adapt to conditions of low salinity

without reduction in dry matter by using Na? in shoot

growth, accumulating it inside the vacuole to maintain a

lower water potential (Nobel 1988). Kefu et al. (2003)

found that Suaeda salsa adapts very well to saline soil,

concluding that it could absorb quite a lot of Na and Cl

from soil and compartmentalize them in the vacuoles to

lower the plant water potential, hence it can absorb water to

maintain life. They related this adaptation to the relatively

small contribution of organic osmotica to osmotic adjust-

ment and also to the high activity of H?-ATPase and H?-

PPase in the tonoplast, which endow the plant with more

energy to translocate Na into vacuoles through Na?/H?

antiports. Moreover, the water content of Atriplex halimus

decreased significantly at 300 and 550 mM NaCl. There-

fore, the decrease in shoot fresh weight might be attributed

to the decrease in water content rather than to decrease in

the dry weight. The decrease in water content during salt

NaCl (mM)0 50 300 550

Shoo

t hei

ght (

cm)

0

10

20

30 a

bc

a

NaCl (mM)0 50 300 550

Num

ber

of le

aves

0

20

40

60

80

100

a

bab

a


Shoo

t hei

ght (

cm)

0

5

10

15

20

25 Control

Drought

*


Num

ber

of le

aves

0

20

40

60

80

100 Control

Drought

***

1B1A

2A 2B

Fig. 3 Effect of NaCl

treatments (1) and drought (2)

for 10 days on shoot height

(A) and number of leaves (B) of

Atriplex halimus. Values are

mean (±SE) of at least five

replications. Data labeled with

different letters are significantly

different at P \ 0.05. (*) and

(***) are significantly different

from the respective control at

P \ 0.05 and P \ 0.001,

respectively

123


stress seems to be an adaptive mechanism for many plants

including the tolerant ones (Munns 2002).

On the other hand, Atriplex halimus seedlings were

able to tolerate 10 days of withholding of water. None-

theless, there were no differences in fresh and dry

weights, shoot height, number of leaves or water content

between control and stressed seedlings after 3 or 6 days.

The plants reached the highest level of dehydration con-

dition 10 days after withholding water as soil water con-

tent was least. Martinez et al. (2005) found a significant

decrease in the water content of Atriplex halimus leaves

after 15 days of withholding water under soil water

potential less than -3 MPa.

Similarly, the growth of Atriplex halimus was signifi-

cantly reduced by withholding water for 10 days as shown

by 45% loss in the fresh weight, 16% reduction in the shoot

height and 37% reduction in the number of leaves. These

reductions of leaf growth and shoot elongation seemed to

be important mechanisms to avoid water stress as reported

by Munns and Tester (2008). Therefore, slower growth of

NaCl (mM)

Tra

nspi

ratio

n ra

te (

mm

ol m

-2 s

-1 )

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

a

b

a

b

Phot

osyn

thet

ic r

ate

(µm

ol m

-2 s

-1)

0

2

4

6

8

10

12

14

16

18

20

a

b

c

c

Stom

atal

con

duct

ance

(m

ol m

-2 s

-1 )

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

a

b

cc

1A

1B

1C

0

5

10

15

20

25

30Control

Drought

**

0.0

0.1

0.2

0.3

**

Days after withholding water

0 50 300 550 3 6 100

2

4

6

**

2B

2C

2AFig. 4 Effect of NaCl


for 10 days on photosynthetic

rate (A), stomatal conductance

(B) and transpiration rate (C) of

Atriplex halimus. Values are

means (±SE) of at least five

replications. Data labeled with

different letters are significantly

different from control at

P \ 0.05. (**) is significantly

different from the respective

control at P \ 0.01

123


Atriplex halimus under salt or drought stress could be an

adaptive feature that enables plants to rely on other

resources as building blocks (e.g. producing compounds

that can be used as compatible solutes and/or osmolytes to

maintain its normal status) and save energy to affect

adaptation under these harsh conditions (Zhu 2001).


Lea

f N

a+ co

nten

t (µm

ol g

-1 F

Wt)

0

50

100

150

200

250

Control

Drought**


Lea

f K

+co

nten

t (µm

ol g

-1 F

Wt)

0

50

100

150

200

Control

Drought

NaCl (mM)

Lea

f N

a+co

nten

t (µm

ol g

-1 F

Wt)

0

200

400

600

800

1000

1200

1400

1600

1800

a

b

c

a

NaCl (mM)0 50 300 550 0 50 300 550

Lea

f K

+ c

onte

nt (

µmol

g-1

FW

t)

0

20

40

60

80

100

120

140

160

180

200

a

b

bc

c

1B1A

2B2A

Fig. 5 Effect of NaCl


for 10 days on Na? (A) and K?

(B) content in Atriplex halimusleaves. Values are mean (±SE)

of at least five replications. Data

labeled with different letters are

significantly different at

P \ 0.05. (**) is significantly

different from the respective

control at P \ 0.01

Fig. 6 Time course effect of NaCl treatments (1) and drought (2) on PEPC protein level of Atriplex halimus. A Western blot and B the protein

level (±SE) of at least four replications from two independent experiments

123


Gas exchange parameters were affected by both salt and

drought stresses. There was a significant increase in pho-

tosynthetic rate and stomatal conductance in plants treated

with 50 mM NaCl and a significant decrease by 300 and

550 mM of NaCl. The transpiration rate was reduced only

under higher concentrations. This is consistent with pre-

vious findings. Zhu and Meinzer (1999) found that 4 weeks

after imposing stress, photosynthetic rate and stomatal

conductance were moderately enhanced at 50 and 100 mM

NaCl, but significantly decreased at higher levels (400, 500

and 600 mM NaCl). Na? has a positive impact on C4

photosynthesis, where it is found to increase the conversion

of pyruvate into phosphoenolpyruvate in the light condition

(Murata et al. 1992). Conversely, Redondo-Gomez et al.

(2007) found significant decreases in net photosynthetic

rate and stomatal conductance of Atriplex portulacoides

with increasing NaCl from 20 to 700 mM.

Photosynthesis is among the primary processes affected

by drought (Chaves 1991) and salinity (Munns et al. 2006).

The effect can be direct through decrease in CO2 assimi-

lation and diffusion from the stomata to mesophyll cells

(Flexas et al. 2007) or through the alteration in the pho-

tosynthetic mechanism (Lawlor and Cornic 2002) and

indirect through oxidative stress that impairs the photo-

synthetic machinery (Chaves et al. 2009). The decrease in

photosynthetic rate in Atriplex halimus under salt and

drought treatment might be attributed to the decrease in the

availability of CO2 resulting from reduction of stomatal

conductance, but not due to injuries in the photosynthetic

apparatus (Demmig and Bjorkman 1987).

The gas exchange parameters of Atriplex halimus did

not show significant changes 3 and 6 days after withhold-

ing water, but decreased after 10 days. Brown and Pezeshki

(2007) reported that the stomatal conductance and the net

photosynthetic rate of dehydrated Spartina alterniflora

significantly declined after 8 days of drought stress. Simi-

larly, the stomatal conductance of Carrizo citrange was

negatively affected either after a dehydration period of

8–18 days or after exposure to 100 mM NaCl (Perez-Perez

et al. 2007).

Plants rely on reducing their stomatal conductance as a

protective mechanism against excessive water loss (Chaves

et al. 2009). The present study indicates that the decline in

stomatal conductance under high concentrations of NaCl

was not accompanied with change in water content.

Redondo-Gomez et al. (2007) observed that the decrease in

stomatal conductance was not accompanied with a decline

in the relative water content in the salt-stressed Atriplex

portulacoides. They attributed the decline in stomatal

conductance to the signaling process rather than to the loss

of turgor. Therefore, Atriplex portulacoides was able to

increase water use efficiency under high salinities. There-

fore, the decline in the transpiration rate of Atriplex hali-

mus subjected to 300 and 550 mM NaCl as well as to

drought for 10 days could be considered as one of the

tolerance mechanisms, as supported by Veselov et al.

(2008).

Chen et al. (2003) reported that lowering the transpira-

tion rates under salt stress contributes to improving their

ability to control Na? influx into roots and subsequently to

the shoots. However, Atriplex halimus actively accumu-

lates Na? to high concentrations in its shoots at high

concentrations; therefore, the decrease in the transpiration

rate does not seem to be essentially for the purpose of

avoidance of excessive Na? influx. The reduction in the

transpiration rate in Atriplex halimus could be an avoidance

mechanism to prevent water loss under salt and drought

stress, i.e. an improvement in the water use efficiency.

Fig. 7 Time course effect of NaCl treatments (1) and drought (2) on Rubisco protein level of Atriplex halimus. A SDS-Page and B the protein

level (±SE) of at least four replications from two independent experiments

123


Similar responses were reported in Argyranthemum cor-

onopifolium (De Herralde et al. 1998).

Vacuoles represent about 90% of mature cell volume, so

ions can act as ‘‘cheap’’ osmolytes, if they are sequestered

properly in the vacuole. Ions, particularly Na?, require less

energy compared to other organic compounds such as

proline and glycine betaine used by plants for osmotic

adjustment (Raven 1985). Growth and survival of halo-

phytes is attributed to their capacity to accumulate high

concentrations of Na? in their vacuoles and use it in the

maintenance of turgor and osmotic adjustment (Flowers

1972).

The stimulation of Atriplex halimus growth at 50 mM

NaCl was associated with Na? accumulation, indicating its

ability to resist salt stress by accumulating Na? and not to

restrict its uptake as in glycophytes (Lutts et al. 1996).

Similar results were obtained by Silveira et al. (2009) who

found that Na? concentrations gradually increased inside

Atriplex nummularia leaves with increasing NaCl concen-

trations from 0 to 600 mM.

In some Atriplex species, the increased sequestration of

Na? appears to be mainly into leaf vacuoles and special-

ized leaf structures such as trichomes and vesicles, where

toxic concentrations of Na? and Cl- are kept away from

the other biochemically active cell compartments (Bradley

and Morris 1991). The accumulation of Na? inside the

vacuole requires an active transport against a concentration

gradient (Silveira et al. 2009). The accumulation of high

concentrations of Na?, in the present study, was accom-

panied by a slight decline in water content. Conversely, the

leaf succulence of Cakile maritima was significantly

enhanced by NaCl concentrations up to 500 mM (Debez

et al. 2006). Also, the accumulation of Na? inside the

leaves of Atriplex halimus improved the water status of the

leaves and had positive consequences on the metabolic and

osmotic processes (Debez et al. 2003). Therefore, leaf

succulence and/or the ability to conserve hypertony are

considered to be one of the important features that char-

acterize the halophytes.

Under water deficit conditions, Na? was increased only

10 days after starting dehydration. As in salt stress, Na?

might be involved in osmotic adjustment; nevertheless, the

insignificant change in Na? content after 3 and 6 days might

be attributed to the absence of stress at these points as

revealed by unchanged growth and soil water content. Under

water deficit, the plant is exposed to a combination of water

stress and ionic stress as the soil matrix potential decreases

simultaneously with decreasing soil moisture. It was found

that the dehydrated Atriplex halimus accumulated more Na?

than the control plants even without the addition of NaCl to

the stressed plants (Martinez et al. 2003).

According to these findings, specific Na? uptake could

be concluded as an integral part of the response of Atriplex

halimus to salt stress as well as to drought stress. Despite

accumulating high concentrations of Na? in leaves of

Atriplex halimus, its growth did not show a consequent

dramatic decrease as reported in glycophytes in which

growth was considerably inhibited by exposure to 200 mM

NaCl (Zhang et al. 2001). Glenn and Brown (1998) con-

cluded that tolerance of Atriplex canescens to water and

salt stress was linked through a common mechanism of

accumulating Na? for osmotic adjustment.

In contrast to Na? accumulation inside Atriplex halimus

leaves, K? concentrations were significantly decreased by

NaCl concentrations. Bajji et al. (1998) found that K?

decreased with the increasing NaCl concentration in the

external medium from 0 to 600 mM. Similar results were

detected in Atriplex nummularia (Silveira et al. 2009). This

decrease in K? content in the salt-stressed Atriplex halimus

may be attributed to the competitive uptake of Na? and K?

(Pitman 1981), the depletion of K? in leaves in which Na?

replaces K? (Redondo-Gomez et al. 2007), depolarization

of the plasma membrane with a consequent K? loss

(Shabala et al. 2005) and down-regulation of the genes

involved in K? transport (Zhu 2003).

On the other hand, drought had no effect on the K?

content. These findings are consistent with the unchanged

K? content found in Atriplex halimus exposed to water

stress for 22 days (Martinez et al. 2003). Such unchanged

K? content in dehydrated Atriplex halimus leaves in the

present study might be attributed to the limited Na?

already found in the soil that compete with the K? uptake

by the plant. This conclusion could be supported by the

data from salt experiment, in which K? content decreased

with increasing application of external Na?.

Photosynthetic rate could be decreased as a result of

stomatal closures (Garcia-Legaz et al. 1993) as well as

stress-induced damage to Rubisco and other enzymes in the

Calvin cycle (Rivelli et al. 2002). In this study, there was

no significant change in the Rubisco content (not the

activity) under all concentrations of NaCl, whereas the

content of PEPC was doubled by 50, 200 and 500 mM

NaCl within 1 and 5 h after treatment and remained

thereafter higher than control. Bouraima et al. (1987) stated

that the response of PEPC in pearl millet to mild treatment

with NaCl depends on the tolerance of the seedlings to the

salt. PEPC was enhanced in the salt-tolerant genotype and

decreased in the most sensitive one. They concluded that

PEPC may be effective markers of salt tolerance in the C4

plant. Moreover, Li and Chollet (1994) found that treat-

ment of the common ice plant (Mesembryanthemum crys-

tallinum) with high salinity caused an increase in PEPC

protein. Contrarily, Debez et al. (2006) reported that

Rubisco activity of Cakile maritime significantly increased

at salinity levels of 100–200 mM NaCl but declined at

higher levels, while PEPC activity did not show any

123


difference from that of control plants. Also, Zhu and

Meinzer (1999) found a decrease in Rubisco and PEPC

activities in response to salt stress in Atriplex lentiformis.

On the other hand, the contents (not the activities) of

both enzymes were not significantly affected by PEG either

alone or combined with NaCl. In this regard, Majumdar

et al. (1991) stated that exposing Glycine max to drought

for 8 days sharply inhibited the activity of Rubisco within

2 days. They, moreover, found that although PEPC activity

did not show any significant change after 2 days, it

declined to almost zero value within the 5th day. In

dehydrated sugarcane (Saccharum officinarum), Rubisco

activity remained the same as that of a well-watered plant,

but PEPC activity increased with decreasing availability of

water (Saliendra et al. 1996).

The present results indicate that the decrease in stomatal

conductance at high concentration was not accompanied by

a decrease in Rubisco, suggesting that the reduction in

photosynthetic rate was due to the limitation of CO2 rather

than photosynthetic enzyme activity. Moreover, the

unchanged Rubisco content in Atriplex halimus under salt

and/or drought stress is a proof of the ability of this plant to

save the most important enzyme in the photosynthetic

machinery to resume its growth under this harsh environ-

ment. PEPC could be involved in increasing the efficiency

of photosynthesis. Moreover, PEPC content increased

during severe conditions to refix CO2 that passively leaked

from the bundle sheath under salt conditions (Hatch and

Osmond 1976).

In conclusion, the present findings showed that 50 mM

NaCl improved the growth parameters of Atriplex halimus.

On the other hand, growth was reduced by drought treat-

ment after withholding water for only 10 days. The gas

exchange parameters were increased by 50 mM NaCl, but

decreased by drought after 10 days. Despite increasing

Na?, all treatments decreased K?. The insignificant change

in Na? content after 3 and 6 days might be attributed to the

absence of stress. However, the accumulated Na? could be

considered as an integral part of the response of Atriplex

halimus to stress. PEPC protein was increased by NaCl but

was unaffected by drought, while Rubisco seemed

unchanged by all treatments. These results revealed that the

decrease in fresh weight might be attributed to the decrease

in water content. Moreover, the decrease in stomatal con-

ductance was not accompanied by a decrease in Rubisco

suggesting that the reduced photosynthesis was due to

limitation of CO2 rather than inhibited photosynthetic

enzyme activity. However, the decrease in photosynthesis

might be attributed to the decrease in the availability of

CO2 resulting from reduction of stomatal conductance. So,

plants rely on reducing their stomatal conductance as a

protective mechanism against water loss. Such reduction

could improve their water use efficiency. These findings

indicate that Atriplex halimus can tolerate low NaCl con-

centration and also short-term drought. Such tolerance

might be regulated by reducing growth, to save energy, and

gas exchange parameters, to improve water use efficiency,

as well as increasing Na? uptake and PEPC protein to

efficiently refix CO2 that passively leaked from the bundle

sheath under stress conditions.

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Physiological aspects of tolerance in Atriplex halimus L. to NaCl and drought

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