Plant Science 252 (2016) 358366

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Plant Science
journal homepage: www.elsevier.com/locate/plantsci

The coordinated regulation of Na+ and K+ in Hordeum brevisubulatum

responding to time of salt stress
Chun-Mei Wang a,1 , Zeng-Run Xia b,1 , Guo-Qiang Wu c , Hui-Jun Yuan c , Xin-Rui Wang d ,
Jin-hua Li a , Fu-Ping Tian a , Qian Zhang a , Xin-Qiang Zhu a , Jiong-Jie He a , Tanweer Kumar b ,
Xiao-Li Wang a, , Jin-Lin Zhang b,
a

Lanzhou Institute of Husbandry and Pharmaceutical Science, Chinese Academy of Agricultural Sciences, Lanzhou 730050, Peoples Republic of China
State Key Laboratory of Grassland Agro-ecosystem, College of Pastoral Agriculture Science and Technology, Lanzhou University, Lanzhou 730020, Peoples
Republic of China
c
School of Life Science and Engineering, Lanzhou University of Technology, Lanzhou 730050, Peoples Republic of China
d
College of Animal Science, South China Agricultural University, Guangzhou 510642, Peoples Republic of China
b

a r t i c l e

i n f o

Article history:
Received 15 June 2016
Received in revised form 10 August 2016
Accepted 13 August 2016
Available online 16 August 2016
Keywords:
Rapid Na+ accumulation
Na+ efux
K+ inux
Coordinated ion regulation
Na+ secretion

a b s t r a c t
Hordeum brevisubulatum, called as wild barley, is a useful monocotyledonous halophyte for soil improvement in northern China. Although previously studied, its main salt tolerance mechanism remained
controversial. The current work showed that shoot Na+ concentration was increased rapidly with stress
time and signicantly higher than in wheat during 0168 h of 100 mM NaCl treatment. Similar results
were also found under 25 and 50 mM NaCl treatments. Even K+ was increased from 0.01 to 50 mM in
the cultural solution, no signicant effect was found on tissue Na+ concentrations. Interestingly, shoot
growth was improved, and stronger root activity was maintained in H. brevisubulatum compared with
wheat after 7 days treatment of 100 mM NaCl. To investigate the long-term stress impact on tissue Na+ ,
100 mM NaCl was prolonged to 60 days. The maximum values of Na+ concentrations were observed at
7th in shoot and 14th day in roots, respectively, and then decreased gradually. Micro-electrode ion ux
estimation was used and it was found that increasing Na+ efux while maintaining K+ inux were the
major strategies to reduce the Na+ concentration during long-term salt stress. Moreover, leaf Na+ secretions showed little contribution to the tissue Na+ decrease. Thereby, the physiological mechanism for H.
brevisubulatum to survive from long-term salt stress was proposed that rapid Na+ accumulation occurred
in the shoot to respond the initial salt shock, then Na+ efux was triggered and K+ inux was activated
to maintain a stable K+ /Na+ ratio in tissues.
2016 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Salinity is a serious threat to crop yield as well as the environment protection [1]. Fortunately, halophytes have evolved
various mechanisms to cope with soil salinity in long-term natural
selection processes [2,3]. Most of the halophytes are dicotyledonous. However, the majority of the economically important
crops are monocotyledonous [4,5]. Therefore, understanding of

This research was supported by the National Natural Science Foundation of China
(31201841 and 31222053), and the Agricultural Science and Technology Innovation
Program of Chinese Academy of Agricultural Sciences (CAAS-ASTIP-2014-LIHPS-08).
Corresponding authors.
E-mail addresses: [email protected] (X.-L. Wang), [email protected]
(J.-L. Zhang).
1
These authors have contributed equally to this work.
http://dx.doi.org/10.1016/j.plantsci.2016.08.009
0168-9452/ 2016 Elsevier Ireland Ltd. All rights reserved.

the salt-tolerance mechanism(s) of monocotyledonous halophytes,

especially the wild relatives of cultivated cereals, will aid effective
improvement of the salt tolerance of cereal crops [46].
Hordeum brevisubulatum (Trin.) Link, also known as wild barley,
is a close relative of barley and wheat. It is well-known monocotyledonous halophytes and could be used as saline grass for soil
improvement in north China [7]. The prime researches regarding the salt tolerance of H. brevisubulatum mainly focused on the
description of the biological characteristics [8], along with the preliminary analysis of the physiological indexes during 19802000s
[9]. Researches regarding the salt tolerance of H. brevisubulatum
have gradually increased in recent years, but few have focused on its
key physiological salt tolerance mechanism. Li et al. proposed that
Na+ compartmentation in leaves and Na+ secretion from the leaf
surface were the key adaptive mechanisms, but no obvious morphological evidence of secretory structures was found [10] except

C.-M. Wang et al. / Plant Science 252 (2016) 358366

for some called salt hairs [11], and no succulence was observed
in the genus of Hordeum. Some other researchers suggested that
restricting Na+ inux along with enhancing the osmotic adjustment
by accumulating organic compounds can contribute to an overall
enhancement of the salt tolerance in H. brevisubulatum [12,13]. On
the other hand, construction of a cDNA library, the screening of
expressed gene fragments induced by salt stress [1417], analysis
of the known salt tolerance related genes, such as HbNHX1 [18],
DREB1 [19], DREB2 [20], rbcS [21], HbCDPK [22] and HbCIPK2 [7],
and the subcellular localization of the protein for salt stress signal
transduction gene, such as CIPK [23], HbCBL1 and HbCBL2 [24], were
conducted by other researchers in order to nd the main genes that
control the salt tolerance of H. brevisubulatum. However, the main
genes and physiological mechanisms for salt tolerance still remain
unclear.
In current work, in order to nd the key physiological salt tolerance mechanisms of H. brevisubulatum, rstly, the growth response
indexes of shoot and root were determined after 7 days salt treatment of 100 mM NaCl. Interestingly, signicantly higher shoot Na+
was accumulated rapidly in H. brevisubulatum than that in wheat.
We also veried this under low concentrations (25, 50 mM). Meanwhile, treatment time was prolonged to 60 days to nd the time
point of peak values for Na+ and K+ concentration in tissues and
their trends with the stress time prolonging. Surprisingly, Na+
was declined obviously during long-term stress. So, to assess the
contribution of Na+ and K+ uxes to the decline of tissue Na+ concentration, a micro-electrode ion ux estimation technique (MIFE)
for net Na+ and K+ uxes was used. Moreover, K+ concentration
were expanded from 0.01 to 50 mM in medium to conrm whether
high Na+ accumulation in shoot was caused by K+ deciency in
medium. Na+ content on the leaves surface was also measured
to clarify the contribution of salt secretion to the salt tolerance
of the plant species. This work would be also helpful to answer
whether various mechanisms found by various researches to date
are adopted during different growth stages in H. brevisubulatum.

2. Materials and methods

2.1. Plant materials and growth conditions
H. brevisubulatum seeds were collected from saline-alkaline
wetlands (E100 06 , N 39 11 ) in northwestern China. Then, H.
brevisubulatum and wheat (Triticum aestivum L. cv. Longchun
26) seeds were germinated on bibulous paper saturated with
sterile water in rectangular dishes (15 cm 8 cm 5 cm) in the
dark at 25 C. The germination period lasted 7 days for the
H. brevisubulatum and 3 days for the wheat. After the leaf
emergence occurred, seedlings were cultured with a modied
Hoaglands nutrient solution (5 mM KNO3 , 1 mM NH4 H2 PO4 ,
0.5 mM Ca(NO3 )2 , 0.5 mM MgSO4 , 60 M Fe-Citrate, 92 M H3 BO3 ,
18 M MnCl2 4H2 O, 1.6 M ZnSO4 7H2 O, 0.6 M CuSO4 5H2 O and
0.7 M (NH4 )6 Mo7 O24 4H2 O) and the pH values were regulated
as 6.0. Once the second leaf appeared, seedlings were transferred
into black-painted containers with the same solution. Seedlings
were grown in an environment controlled chamber at 25 C in
the day and 18 C at night. There was a photon ux density of
2300 mol m2 s1 in the day with a photo period of 16/8 h for the
day/night cycle and a relative humidity from 6575%. The solution
was renewed every three days to avoid ion concentration imbalance.
For the pre-experiment, the plasma membrane permeability
of the roots was determined under 0300 mM NaCl with two,
three and four-leaf-old plants in order to use the appropriate
NaCl concentrations and growth stages (Supplementary Fig. S1).
Then, four-leaf-old plants and 0100 mM NaCl concentrations of

359

were used for the following experiments. Once the plants achieved
four leaves, they were used for the salt treatment experiments.
NaCl concentrations were increased by 25 mM/12 h in advance to
achieve the nal concentrations for treatments. The solutions were
aerated and renewed every day to avoid oxygen and ion alterations.
2.2. Plant growth measurements
The plants of H. brevisubulatum and wheat were cultivated with
a modied Hoaglands nutrient solution containing 25, 50, and
100 mM NaCl for 7 days. Then, the plants were harvested, gently
blotted, separated into shoots and roots, weighed for fresh weights
immediately and oven-dried at 80 C for 3 d to obtain constant dry
weights. Then, the water content and root/shoot ratio was calculated.
At the same time, root morphological parameters were determined using a root automatism scan apparatus (Perfection V700
Photo, Seiko Epson Corp, Japan) equipped with WinRHIZO software
(Regent Instruments Co). The specic procedures were described in
the references [25]. In each replicate, roots were placed in a transparent plastic tray lled with distilled water, and placed on the
scan apparatus. Image recordings were performed at a resolution of
800 dpi, and saved as a tagged image le (TIF) format. The root phenotype traits, including the total root length, volume, surface area
and number of root tips, were assayed using WinRHIZO software.
The root-absorbing area was determined by a methylene-blue colorimetric method as described by Zou et al. [26]. The percentages
of active absorption area and specic surface area were calculated
according to Wang et al. [25].
2.3. Various terms of salt stress treatments
The plants of H. brevisubulatum and wheat were treated with
a modied Hoagland nutrient solution supplemented with 25, 50
and 100 mM NaCl. Then, they were harvested for Na+ and K+ ion
concentration measurements at 0, 6, 12, 24, 48, 96, and 168 h after
salt stress for the short-term experiment and 7, 14, 28, and 60 days
for the long-term experiment, respectively. The measurements of
Na+ and K+ contents were carried out as described by Wang et al.
[4]. Proline content in the tissues was tested according to Zhao [13].
2.4. Net Na+ and K+ uxes measurements by MIFE
The net Na+ and K+ uxes of H. brevisubulatum plants subjected
to 100 mM NaCl were measured using a MIFE technique (BIO-IM,
Younger USA LLC, Amherst, MA 01002, USA) as previously described
[2729] at Xuyue Science and Technology Co., Ltd., Beijing, China.
In principal, prior to the ux measurement, the microelectrode was
calibrated with different concentrations of the Na+ (0.3 mM, 0.9 mM
and 3 mM), and K+ (0.1 mM, 0.5 mM and 5 mM) buffers. Only electrodes with a Nernstian slope >50 mV/decade were used in this
study. The roots (with shoots retained) of the H. brevisubulatum
were then washed gently with measuring solution and incubated
in a petri dish containing a 10 ml of measuring solution (0.1 mM
KCl, 0.1 mM CaC12 , 0.1 mM MgC12 , 0.5 mM NaCl, 0.2 mM Na2 SO4 ,
0.3 mM MES, pH 6.0, adjusted with Tris) to equilibrate for 15 min.
The net Na+ ux measurements commenced from the root tip and
were repeated along the root at various positions from the root tip
in order to determine the optimal position (the zone that corresponded to the site where the Caparian bands developed, and the
zone of the lateral roots initial development). The steady-state of
ion uxes were then recorded until the values variation amplitude
was relatively stable (approximately 400 s). The micro-electrode
oscillated with an excursion of 30 m, and completed an entire
cycle in 5.36 s. The net ion uxes (pmol cm2 s1 ) were calculated
using Mage Flux software developed by Xuyue (http://xuyue.net/

360

C.-M. Wang et al. / Plant Science 252 (2016) 358366

mageux). In order to eliminate the error of free diffusion during

the testing caused by the differences of ion concentrations between
the plants and test solutions during MIFE measuring processes, ux
values of the control were considered as the reference values.
2.5. Various K+ level treatments
KNO3 (5 mM) in Hoaglands nutrient solution was replaced by
0.01 mM KNO3 , 5 mM KNO3 , 5 mM KNO3 + 15 mM KCl, and 5 mM
KNO3 + 45 mM KCl to achieved the nal K+ concentrations of 0.01,
5, 20 and 50 mM, respectively, while the NO3 concentration was
retained, under 100 mM NaCl for 14 days. 0.01 mM replaced 0 mM
there to avoid nutrition deciency. Then, the plants were harvested
for Na+ and K+ ion concentration measurements. The selective
absorption (SA) and selective transport (ST) values were calculated
according to Wang et al. [30].
2.6. Salt secretion analysis
The salt secretion from the leaves was analyzed according to
Wang et al. [4]. Briey, four-leaf-old plants of H. brevisubulatum
were cultured in hydroponic tanks and treated with 100 mM NaCl
for 60 days; while control was irrigated in the same modied
Hoagland solution without NaCl. Then, the leaves were carefully
separated from the shoot bases, and rinsed in 30 ml of deionized
water for 1.5 min, holding the incision above the water to avoid any
Na+ loss from the cut end. At this point, the leaves were removed
from the washing water, blotted and weighted. The roots with the
remaining shoots were harvested, and after washing off any surface
soil with tap water, they were immediately blotted and weighed.
The Na+ and K+ contents in the deionized water and the entire
plants were measured as described below.
2.7. Statistical analysis
The results of the growth, ion concentration and net Na+ and
K+ uxes of the plants were presented as a means with standard
deviations (n = 6). Statistical analyses, one-way analysis of variance
(ANOVA), and Duncans multiple range tests were performed using
statistical software at P < 0.05 (Ver.16.0, SPSS Inc., Chicago, IL, USA).
3. Results
3.1. Shoot growth was improved and stronger root activity was
maintained in H. brevisubulatum compared with wheat during
short-term salt stress
The growth responses in both plant species were investigated.
Interestingly, it was found that fresh weight increased signicantly
in the shoots of H. brevisubulatum in 50 and 100 mM NaCl. However,
no signicant increases were observed in wheat (Fig. 1). The dry
weights showed the similar results (Supplementary Fig. S2).
Total root length, root volume, root surface area, the numbers
of root tip and percentage of active absorption area of wheat were
signicantly reduced by 7 day treatment of 100 mM NaCl compared
to the control (Table 1). However, no signicant negative effect was
found for H. brevisubulatum, furthermore, the numbers of root tips,
the percentage of active absorption and specic surface areas were
even enhanced by 100 mM NaCl (Table. 1).
3.2. Rapid Na+ uptake and higher Na+ levels were maintained in
the shoots of H. brevisubulatum during short-term (within 168 h)
salt stress
Shoot Na+ concentration of H. brevisubulatum was increased
gradually with prolonged time and over 53-fold after 168 h treat-

ment of 100 mM NaCl, and it was signicantly higher (2.66.4 folds)

than that of wheat during 6168 h (Fig. 2a). Na+ concentrations in
roots were relatively stable in both species (Fig. 2d). However, H.
brevisubulatum showed an obviously lower root Na+ concentration
than in shoot. K+ concentrations were relatively stable in shoot
of both species during 0168 h. Root K+ concentrations decreased
gradually from 48 h, but more stable and 2940% higher K+ concentrations were maintained in H. brevisubulatum than in wheat
during 96168 h of treatments (Fig. 2b and e). These results led to
a lower K+ /Na+ ratio in shoot, but a higher K+ /Na+ ratio in root of H.
brevisubulatum compared with wheat (Fig. 2c and f). Interestingly,
even in the absence of NaCl, both shoot and root Na+ concentrations
were signicantly higher (approximately 3 times) in H. brevisubulatum than those in wheat (Fig. 2a and d), leading to a signicantly
lower K+ /Na+ ratio in H. brevisubulatum (Fig. 2c and f).
To clarify whether the high Na+ concentration accumulation
occurred under mild salt treatment in H. brevisubulatum, 25 and
50 mM NaCl were used. Similar result was obtained that higher Na+
concentration was accumulated in shoot of H. brevisubulatum than
wheat (Supplementary Figs. S3 and S4).

3.3. Na+ concentration was reduced while K+ was retained in H.

brevisubulatum during long-term (760 days) NaCl treatments
To further assess whether the high Na+ accumulation in H.
brevisubulatum persisted over long-term treatments, time-courses
(from 0 to 60 days) of the tissue K+ and Na+ concentrations were
analyzed (Fig. 3a,b,c). The results indicated that shoot Na+ concentration increased rapidly when subjected to 100 mM NaCl and
reached the maximum value (2.15 mmol gDW1 ) at 7th days, and
then gradually reduced with the prolonged NaCl treatments (1460
days). Although root Na+ concentration increased not so dramatically compared with shoot during 07 days, it increased sharply
from 7th day and reached the maximum value (2.43 mmol gDW1 )
at 14th days, then decreased gradually till 60th day. It was noticeable that, although shoot Na+ concentration increased rapidly and
higher than in root during initial salt shock (07 days), relatively
lower Na+ levels was maintained in shoot during the following
long-term stress (1460 days) (Fig. 3a).
Although K+ concentration had a slight decline during the shortterm (07 day) stress, a relatively stable level of K+ was maintained
during the following stress. Consequently, K+ /Na+ ratio had a slight
decrease during 0168 h (Fig. 2c and f), and then increased gradually (Fig. 3c). Moreover, K+ concentrations were higher in shoots
than those in roots during all time-courses (Fig. 3b).

3.4. Na+ and K+ uxes contribution to Na+ decline during

long-term (760 days) salt stress in H. brevisubulatum
In order to clarify the contribution of net Na+ and K+ uxes to
Na+ decline during long-term stress, a MIFE technique at anatomically distinct zones were used. The results showed that a rapid Na+
efux (140.5 pmol cm2 s1 ) was recorded after 7 days of 100 mM
NaCl treatment compared with control, and then increased to
295.4 pmol cm2 s1 after 60 days of treatment (Fig. 4). Although,
K+ efux (267.2 pmol cm2 s1 ) was observed after 7 days of NaCl
treatment, an obvious K+ inux (-190.4 pmol cm2 s1 ) was record
after 60 days of treatment (Fig. 5). This was consistent with the
results of tissue ion concentrations (Fig. 3) where Na+ concentration
decreased obviously from 7th day and K+ concentration decrease
at initial salt stock (07 days) and increased gradually in the following stages, consequently a higher K+ /Na+ was maintained for H.
brevisubulatum to adapt to salt stress.

C.-M. Wang et al. / Plant Science 252 (2016) 358366

H. brevisubulatum

(mgplant-1)

60

Fresh weight

(a)

20

Soot

(b)

Root

400

300

ab

30

ab

ab

200
c

10

Wheat

500

50
40

361

100

25

50

100

0
0

25

50

100

Treatments NaCl (mM)

Treatments NaCl (mM)

Fig. 1. Fresh weight of shoot (white columns) and root (black columns) in H. brevisubulatum (a) and wheat (b) under 0, 25, 50 and 100 mM NaCl (increased stepwise with
25 mM per 12 h) in modied Hoagland solution for 7 days. Ten plants for H. brevisubulatum and two plants for wheat were pooled in each replicate (n = 6). Values are
means SD and bars indicate SD.
Table 1
Root morphology and physiology indexes of H. brevisubulatum and wheat under control (0) and 100 mM NaCl (increased stepwise with 25 mM per 12 h) in modied Hoagland
solution for 168 h. Ten plants for H. brevisubulatum and two plants for wheat were pooled in each replicate (n = 6). Values are means SD and bars indicate SD.
Treatments
NaCl (mM)

Total root length

(cm plant1 )

Root volume
(mm3 plant1 )

Root surface area

(cm2 plant1 )

Numbers of root tip Percent of active

(plant1 )
absorption area (%)

Specic surface area

(m2 cm3 )

H. brevisubulatum

0
100
0
100

31.30 2.54 a
26.64 3.09 a
159.68 9.74 a
95.29 12.67 b

9.98 1.11 a
8.85 0.70 a
74.58 5.15 a
63.25 4.92 b

1.98 0.22 a
1.71 0.11 a
12.19 1.84 a
8.67 0.72 b

140.68 9.28 a
89.28 2.79 b
525.30 35.24 a
286.80 19.40 b

1.52 0.01b
1.56 0.01 a
1.49 0.01 b
1.54 0.02a

Shoot Na+ concentraon

(mmolg DW-1)

(a)

Shoot K+ concentraon
(mmolg DW-1)

(b)

3.0
Wheat

2.5

H. brevisubulatum

2.0

1.5
c

1.0
e

0.5
0.0
3.0

j ij
BT

hi

ef

gh

hi

hig

d
fg

12 me
24 (hour)
48 96
Stress

168

(d)

3.0
2.5

(e)

2.0
ab
1.5

bc
def

a
bcd
ef cde

bcd def
ef
efef ef

1.0
0.5
0.0

(c) 100

BT

12
24
48
Stress time (hour)

96

168

(f)
Root K+/Na+ rao

Shoot K+/Na+ rao

2.0
1.5
1.0
0.5
g
0.0
3.0

BT

cd
6

abc

ad

ab

aa

96

168

ef
12
24
48
Stress time (hour)

2.0
1.5
1.0
a
0.5
0.0
10
8

80
60
40

46.83 0.26 b
47.97 0.23 a
48.54 0.32 a
47.12 0.20 b

2.5

2.5

Root K+ concentraon
(mmolg DW -1)

Wheat

Root Na+ concentraon

(mmolg DW -1)

Species

20

c
g

12

0
0

gh f hi fg i

24

48

96

ii
168

Stress me (hour)

BT

cc

ab d

abc

de ef fg de

12
24
48
Stress time (hour)

96

de

168

6
4
2

bb
ded

def de ghefg hefgh hfgh

0
0

12

24

48

96

168

Stress me (hour)

Fig. 2. Na+ (a), K+ (b), K+ /Na+ ratio (c) in shoot and Na+ (d), K+ (e), K+ /Na+ ratio (f) in root of H. brevisubulatum (black columns) and wheat (white columns) during 0168 h
treatment of 100 mM NaCl (increased stepwise with 25 mM per 12 h) in modied Hoagland solution. Ten plants for H. brevisubulatum and two plants for wheat were pooled
in each replicate (n = 6). Values are means SD and bars indicate SD.

3.5. K+ levels in medium had small effect on Na+ accumulation in

H. brevisubulatum
To conrm whether high Na+ other than K+ accumulation in H.
brevisubulatum was caused by K+ deciency in medium, 5 mM K+

in the solution was replaced by various concentrations of K+

(0.0150 mM) in 100 mM NaCl treatment. Shoot Na+ concentration did not change signicantly with 0.01, 5 and 20 mM K+ , but
increased signicantly by 37% with 50 mM K+ . No signicant dif-

C.-M. Wang et al. / Plant Science 252 (2016) 358366

Na+ concentraon (mmolg DW-1)

(a)

(b)

3.0

1.2
a

2.5

0.6

0.0

b
2.0

0.0

K+ concentraon (mmolg DW-1)

ef

1.0 1.5 2.0

1.0
Shoot

Root
ef

de

0.5

14

21

28

35

Time (da y)

42

2.0
1.0

49

56

cdde efg

bc

cdedefg
cdef

cd

63

0.0

0.5

1.0

1.5
a

2.0

fg
a

1.0

0.5
0.0
36

fg efg
0

14

21

28
35
Time (da y)

42

36
24
12
0

24

49

56

b cc ca
0.0 0.5

cdc

63

70

1.0

100

-100

b
0mM-7d

100mM-7d

100mM-60d
c

-300

NaCl concntraon and treament me

Fig. 5. Net K+ ux of H. brevisubulatum test by MIFE between 50 and 390 s in measuring solution after 0 and 100 mM NaCl treatment (increased stepwise with 25 mM
per 12 h) in modied Hoagland solution for 7 d and 100 mM NaCl for 60 d. Roots
(with shoots retained) were incubated in the measuring solution to equilibrate for
15 min ahead. Steady-state ion uxes were then recorded until the values variation
amplitude is relatively stable. Ten plants for H. brevisubulatum were pooled in each
replicate (n = 6). Values are means SD and bars indicate SD.

b
def

300

70

2.0
1.5

500

0.0
2.5

30

K+/Na+ rao

0.5

e
de

3.0

(c)

d
ef ef ff
g ef

1.5

0.0
3.5

Mean net K+ ux between 50-390s in

measuring soluon (pmolcm-2s-1)

362

ed
1.5

2.0

18

Table 2
Selective absorption (SA) and transport (ST) capacity for K+ over Na+ of H. brevisubulatum exposed to 100 mM NaCl supplied with 0.01, 5, 20 and 50 mM K+ (NaCl
increased stepwise with 25 mM per 12 h) in modied Hoagland solution for 14 d.
Ten plants for H. brevisubulatum were pooled in each replicate (n = 6). Values are
means SD and bars indicate SD. The values were calculated from Fig. 6. SA = (K+ /Na+
in whole plant)/(K+ /Na+ in medium); ST = (K+ /Na+ in shoots)/(K+ /Na+ in roots).
K+ concentration (mM)

SA

ST

0.01
5
20
50

119.01 6.57 a
33.19 1.96 b
12.58 0.89 c

10.15 1.03 a
5.67 0.54 b
5.52 0.35 b
3.56 0.38 c

12
b

6
0
0

d
e
7

d
f
14

21

d
f
28

de
35

42

49

56

63

70

Time (day)

Mean net Na+ ux between 50-390s in

measuring soluon (pmolcm-2s-1)

Fig. 3. Time course of Na+ (a), K+ (b), K+ /Na+ ratio (c) in shoot (open circles) and
root (closed circles) of H. brevisubulatum during 060 days under 100 mM NaCl
(increased stepwise with 25 mM per 12 h) in modied Hoagland solution. The details
of 02 days were showed in inserted gures. Ten plants for H. brevisubulatum were
pooled in each replicate (n = 6). Values are means SD and bars indicate SD.

600

400

a
b

200
c
0
0mM-7d

100mM-7d

ferences in root Na+ concentrations were found among 5, 20 and

50 mM K+ treatments, even signicantly decrease occurred in these
three K+ levels compared with 0.01 mM K+ . Tissue K+ concentration
increased signicantly with the increase of K+ concentrations in
medium, and reached the maximum values with 50 mM K+ (Fig. 6b).
The selective absorption (SA) and selective transport (ST) values for K+ over Na+ were calculated and conrmed that SA values
decreased sharply, while the ST values remain relatively stable with
the addition of 550 mM K+ (Table 2).
3.6. Loss of Na+ content from the leaves
To assess the contribution of salt secretion from the leaves to
the decline of Na+ concentration in shoots during long-term salt
stress, Na+ and K+ contents on leaves surface was determined under
100 mM NaCl for 60 days. Na+ content washed from the leaves
were not signicantly different between 0 and 100 mM NaCl treatments accounting for only 2.52% of the entire plant Na+ content
with 100 mM NaCl (Fig. 7a). K+ content washed from the leaves
accounted only about 0.1% of the entire plant K+ content with 0
and 100 mM NaCl.

100mM-60d

4. Discussion

-200
NaCl concntraon and treament me

Fig. 4. Net Na+ ux of H. brevisubulatum test by MIFE between 50 and 390 s in measuring solution after 0 and 100 mM NaCl treatment (increased stepwise with 25 mM
per 12 h) in modied Hoagland solution for 7 d and 100 mM NaCl for 60 d. Roots (with
shoots retained) were incubated in the measuring solution to equilibrate for 15 min
in advance. Steady-state ion uxes were then recorded until the values variation
amplitude is relatively stable. Ten plants for H. brevisubulatum were pooled in each
replicate (n = 6). Values are means SD and bars indicate SD.

4.1. Rapid and higher Na+ accumulation in shoot was a rapid

response to short-term salt stress
Excessive Na+ induces osmotic stress and cytosolic toxicity,
resulting in growth inhibition for glycophytes, and Na+ also acts
as a competitor of K+ with similar binding sites in major metabolic
processes [31,32]. Therefore, limiting high Na+ concentration accu-

C.-M. Wang et al. / Plant Science 252 (2016) 358366

(b)
0.8
a

Shoot

0.6

Root
b

b
c

0.4
d

0.2
0
0.01

20

K+ concentraon (mmolg DW-1)

Na+ concentraon (mmolg DW-1)

(a)

363

3.0

a
c

2.0

1.0

0.0
0.01

50

20

50

K+ concentraon (mM)

K+ concentraon (mM)

Fig. 6. The inuence of K+ level (0.0150 mM) on Na+ (a) and K+ (b) concentration of H. brevisubulatum under 100 mM NaCl (increased stepwise with 25 mM per 12 h) in
modied Hoagland solution for 14 d. Ten plants for H. brevisubulatum were pooled in each replicate (n = 6). Values are means SD and bars indicate SD.

(b) 60

15
In whole plant
Washed from leaves
10

0
100
0
NaCl treatments (mM)

K+ content (molplant-1)

Na+ content (molplant-1)

(a)

40
b
20
c

0
0

c
100

NaCl treatments (mM)

Fig. 7. Na+ content loss from leaves of H. brevisubulatum under 0 and 100 mM NaCl (increased stepwise with 25 mM per 12 h) in modied Hoagland solution for 60 d. Ten
plants for H. brevisubulatum were pooled in each replicate (n = 6). Values are means SD and bars indicate SD.

mulation as a major salt tolerance strategy is adopted by most

monocotyledonous halophytes [35,33].
However, in present study, rapid and high Na+ was accumulated in the shoots, other than in roots of H. brevisubulatum, even
over 2-fold of wheat during short-term salt stress (Fig. 2a). Interestingly, although Na+ levels were higher in H. brevisubulatum, growth
rates of the shoots and root activity remained higher than in wheat
(Fig. 1a, Table 1). Li et al. found that Na+ could probably be compartmentalized into vacuole in H. brevisubulatum [10]. It was also found
that Na+ concentration in barley increased rapidly and sequestered
into the leaves during the rst several days [28], and tolerant barley genotypes accumulated signicantly higher Na+ in their leaves
compared with sensitive ones [34]. Similar results were also found
in Thellungiella halophila exposed to salt stress [35]. The most probable explanation is that the plants undergoing salt stress could
send the amount of required Na+ quickly to the shoot, as a cheap
osmoticum to achieve a rapid full osmotic adjustment [36], rather
than simply restricting Na+ inux at the beginning stage of salt
stress. This consequently provides an additional driving force for
water uptake by roots [37] and resumes growth of shoots (Fig. 1a).
In contrast, it was found that glycophytes like wheat (Fig. 2a) and
pea [38] could restrict xylem Na+ loading during the initial few days
of the salt stress treatments, but failed to prevent Na+ elevation over
longer term exposure to salinity.
An improved Na+ sequestering ability in the leaf vacuoles of H.
brevisubulatum could be achieved by tonoplast Na+ /H+ exchanger,
HbNHX1 [18,39]. In the present study, it is interesting to note that
rapid and higher Na+ accumulation was also found in the lower
(25 and 50 mM) NaCl treatments (Supplementary Figs. S3 and S4),
consistent with the result of RT-PCR identication with HbNHX1
not induced by salt stress [18]. It was proposed that Na+ transport
under mild salinity was mediated by AtSOS1 (Salt Overly Sensitive 1), which is located across the plasma membrane of xylem

parenchyma cells and could mediate Na+ loading into the xylem,
thereby controlling the long-distance Na+ transport from roots to
shoots [40,41]. Under higher saline conditions, AKT1 (Arabidopsis K+
transporter) regulated Na+ uptake in the roots [31] and its expression was not inuenced by outside K+ concentrations [39,42]. These
nding were consistent with the results in current study that Na+
concentrations in the roots were not inuenced by outside K+
levels from 5 to 50 mM (Fig. 6a). Our previous results in the saltaccumulating halophyte Suaeda maritima also supported that AKT
type K+ channels may be involved in Na+ uptake in the roots under
150 mM NaCl [43].

4.2. Na+ efux was the major mechanism for H. brevisubulatum

to maintain a low Na+ accumulation during long-term salt stress
Although high Na+ levels was rapidly observed in shoots of H.
brevisubulatum (Fig. 2a), once this was achieved to the limitation
of the vacuolar volume, it was better for the plants to reduce Na+
loading rate, increase Na+ retrieval from shoots to roots and exclude
Na+ to the soil [44,45]. In current study, Na+ concentration in shoots
reached its maximum values at 7th day of the treatments (Fig. 3a)
and then declined gradually. Consistently, Na+ concentration in
roots increased obviously from 7th day and reached the maximum values at 14th day (Fig. 3a). It was suggested that AtHKT1;1
(High-afnity K+ Transporter) located at the plasma membrane of
xylem parenchyma cells could mediated Na+ retrieval from the
xylem to the surrounding parenchyma cells [46,47], and thereby
regulated Na+ transport from shoots to roots [4850]. Consistently,
athkt1;1 mutant accumulated more Na+ in shoot and less Na+ in
roots compared with the wild type plants [49,50], indicating that
AtHKT1;1 could be a determinant to controlling Na+ unloading from
the xylem.

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C.-M. Wang et al. / Plant Science 252 (2016) 358366

With large amount of Na+ retrieval from shoot to root, Na+

was efux from roots to external environment (Fig. 4). Then, Na+
concentrations in roots were consequently reduced from 14th
day (Fig. 3a). A plasma membrane-bound Na+ /H+ antiporter [39],
encoded by SOS1 at the epidermal cells of the root tips [40], is crucial
in mediating Na+ efux from roots to the environment [5153]. It
was noted that, regardless of halophytes or glycophytes, unidirectional Na+ efux accounted for large amounts of total Na+ inux
under salinity conditions [4,5456]. Na+ concentrations both in
shoots and roots (Fig. 3a) decreased with the increase of Na+ efux
(Fig. 4), suggesting that Na+ efux from roots was the main contributor to reduced Na+ concentration during the long-term salt
stress in H. brevisubulatum. The similar result showed that a better
ability of root cells to pump Na+ from cytosol to external medium
was also found in salt-tolerant varieties of barley [34], and accumulated approximately 30% less Na+ than in salt-sensitive varieties
after four weeks of salt stress [28].
4.3. Coordinated regulation of K+ and Na+ in H. brevisubulatum
under salinity stress
Maintaining a high-cytosolic K+ /Na+ ratio is one of the crucial
and essential mechanisms of salt tolerance [31,34,48]. The coordinated regulation of K+ and Na+ (not simply maintaining a high K+
and low Na+ ) may play an important role for salt tolerance in H.
brevisubulatum. As described above, Na+ uptake in H. brevisubulatum increased rapidly and was higher than in wheat during the
rst 168 h of salt stress (Fig. 2a). This was probably due to the
AKT1-type channels [57,58], the subfamily 1-type HKT transporters
[5961] on root epidermal cells and SOS1 on xylem parenchyma
cells. They together promoted the uptake and long distance transport of Na+ [44,45] into cell vacuoles of shoots to achieve a higher
osmotic potential. Na+ unloading into the xylem parenchyma cells
possibly depolarizes their plasma membrane, which in turn could
potentially activate the K+ channels, such as SKOR (Stelar K+ Outward Rectiers), to load K+ into xylem [45,62,63]. Therefore, K+
concentrations increased during 714 days in both in shoot and
root (Fig. 3b), following the rapid Na+ uptake during the rst 7
days (Fig. 3a). When Na+ content achieved its limitation of vacuolar volume, the subfamily 1-type HKT transporters mediated the
Na+ retrieval from shoots to roots [44,45]. Then, Na+ concentration
declined in the shoots and increased in the roots during 7 to14 days
of salt stress (Fig. 3a).
Once excessive Na+ accumulated in the root cells (Fig. 3a), K+
efux from epidermis cells was triggered due to salt-induced membrane depolarization [64,65] (Fig. 5), probably via the GORK(Guard
cell Outward Rectifying K+ channel) [66]. This consequently led
to a decrease in K+ concentration in both shoots and roots during 1428 days of treatment (Fig. 3b). Then, with the increase of
Na+ efux in roots (Fig. 4), Na+ concentration obviously decreased
during 2860 days (Fig. 3a) and K+ inux recovered, thereby K+ concentration and K+ /Na+ ratio increased progressively (Fig. 3b and
c). This was consistent with the MIFE results that K+ maintained
a higher efux at 7th day, but recovered inux at 60th day under
100 mM NaCl treatments compared with control (Fig. 5).
Accordingly, by integrating all the ndings as described above,
a probable pattern for elucidating salt tolerance mechanism of H.
brevisubulatum was proposed, in which the roles of K+ and Na+ coordinated regulations during short and long-term salt stress were
claried (Fig. 8). During the rst stage (07 days; Fig. 2a), Na+
was absorbed quickly into the epidermal cells of roots, probably
via AKT1 [57,58] or HKT (the subfamily 1-type HKT transporters)
[59,60], then was loaded directly into the xylem via SOS1 in the
plasma membranes of XPCs (Xylem Parenchyma Cell) and delivered
rapidly to the shoots motivated by transpiration stream [28,44,45].
Then, large amount of Na+ was rapidly sequestered into leaf vac-

Fig. 8. A proposed schematic pattern for K+ and Na+ transporters/channels to coordinate regulation of salt tolerance of H. brevisubulatum.
During the rst stage
(07 days), Na+ was absorbed quickly into the epidermal cells of roots, probably
via AKT1 or HKT, then was loaded directly into the xylem via SOS1 in the plasma
membranes of XPCs and delivered rapidly to the shoots motivated by transpiration
stream. Then, large amount of Na+ was rapidly sequestered into leaf vacuoles via
NHX, in order to achieve rapid full osmotic adjustment and avoid Na+ damage.
During the second stage, once Na+ achieved its limitation in vacuole, HKT could then
mediate Na+ retrieval from xylem into XPCs to limit Na+ accumulation in shoots, and
relatively larger amount of Na+ was accumulated in roots. Then in turn, this could
activate K+ channels, such as SKOR, to load the K+ into xylem through XPCs, and
nally promote the K+ uptake into the plants possibly via HAK or other K+ uptake
transporters or channels.
During the third stage, once Na+ was retrieved and
excessively accumulated in root cells, Na+ efux possibly via SOS1 on root epidermal cells was triggered. At the same time, K+ efux via K+ efux channels such as
GORK due to the salt-induced membrane depolarization from the cells was also
triggered, leading to a decrease in K+ concentrations in the plants. Then, with the
increase of Na+ efux, Na+ concentration decreased and K+ concentration recovered gradually by its uptake, resulting in a higher K+ /Na+ , which consequently was
sustained during long term of higher salt stress.

uoles via tonoplast Na+ /H+ exchanger (NHX) [18,39], in order to

achieve rapid full osmotic adjustment and avoid Na+ damage [36].
During the second stage, once Na+ achieved its limitation in vacuole, HKT could then mediate Na+ retrieval from xylem into XPCs to
limit Na+ accumulation in shoots [44,45,47,67], and relatively larger
amount of Na+ was accumulated in roots (Fig. 3a, days 714).Then
in turn, this could activate K+ channels, such as SKOR, to load the
K+ into xylem [45,62,63] through XPCs, and nally promote the
K+ uptake into the plants [28,48,50] possibly via HAK or other K+
uptake transporters or channels. During the third stage, once Na+
was retrieved and excessively accumulated in root cells (Fig. 3a,
days 714), Na+ efux possibly via SOS1 on root epidermal cells
was triggered [40,52,67,68]. At the same time, K+ efux via K+ efux
channels such as GORK due to the salt-induced membrane depolarization from the cells was also triggered [66], leading to a decrease
in K+ concentrations in the plants (Fig. 3b, days 1428). Then, with
the increase of Na+ efux (Fig. 4), Na+ concentration decreased and
K+ concentration recovered gradually by its uptake, resulting in a
higher K+ /Na+ (Fig. 3a, b, c, and Fig. 5), which consequently was
sustained during long term of higher salt stress.

C.-M. Wang et al. / Plant Science 252 (2016) 358366

4.4. Na+ loss from leaves makes a minor contribution to salt

tolerance of H. brevisubulatum
Although high Na+ level was maintained in shoots of H. brevisubulatum, Na+ loss from leaf surface was extremely minor of
Na+ amount in the entire plant (2.6%, Fig. 7) compared with saltsecreting halophytes, such as Spartina anglica (60%), Limonium
vulgare (33%), Glaux maritime (20%) and wild rice Porteresiacoarctata (over 50%) [69,70]. Moreover, although some salt hairs
were observed on the leaves [11], no salt-secreting structures was
observed in H. brevisubulatum [10], also no bicellular glands was
observed in any other species of the Pooideae family [71], indicating that Na+ secreting contributes little to salt tolerance of H.
brevisubulatum.
5. Conclusion
This study provided evidence that rapid Na+ inux occurred at
seedling stages with initial salt shock and Na+ efux and K+ inux
was enhanced to maintain a K+ and Na+ balance at tilling stages
during long-term salt stress in the monocotyledonous halophyte H.
brevisubulatum. A probable regulation pattern was hypothesized to
elucidate its K+ and Na+ co-ordinated mechanism against different
terms of salt stress. Although this seems a smart strategy, it is too
early to speculate that this mechanism could be adopted by other
wild plant species.
Acknowlegements
The authors would like to thank Professor S.-M. Wang, C.-J.
Li and Dr. P. Wang for their helpful discussion. We would also
like to thank the anonymous reviewers for their constructive suggestions regarding the manuscript. This research was supported
by the National Natural Science Foundation of China (31201841
and 31222053), and the Agricultural Science and Technology
Innovation Program of Chinese Academy of Agricultural Sciences
(CAAS-ASTIP-2014-LIHPS-08).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.plantsci.2016.08.
009.
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