ZxAKT1 is essential for K+ uptake and K+/Na+ homeostasis ...caoye.lzu.edu.cn/upload/news/N20170220222756.pdf · in the succulent xerophyte Zygophyllum xanthoxylum, which exhibits

ZxAKT1 is essential for K+ uptake and K+/Na+ homeostasis inthe succulent xerophyte Zygophyllum xanthoxylum

Qing Ma1,†, Jing Hu1,†,‡, Xiang-Rui Zhou1,†,§, Hui-Jun Yuan1, Tanweer Kumar1, Sheng Luan2,3 and Suo-Min Wang1,*1State Key Laboratory of Grassland Agro-ecosystems, College of Pastoral Agriculture Science and Technology, Lanzhou

University, Lanzhou 730020, China,2Department of Plant and Microbial Biology, University of California at Berkeley, Berkeley, CA 73072, USA, and3NJU-NJFU Joint Institute for Plant Molecular Biology, College of Life Sciences, Nanjing University, Nanjing 210093, China

Received 6 November 2016; revised 14 December 2016; accepted 16 December 2016.

*For correspondence (e-mail [email protected]).†These authors contributed equally to this work.‡Present address: State Key Laboratory of Desertification and Aeolian Sand Disaster Combating, Gansu Desert Control Research Institute, Lanzhou 730070,

China.§Present address: College of Grassland Science, Gansu Agricultural University, Lanzhou 730070, China.

SUMMARY

The inward-rectifying K+ channel AKT1 constitutes an important pathway for K+ acquisition in plant roots.

In glycophytes, excessive accumulation of Na+ is accompanied by K+ deficiency under salt stress. However,

in the succulent xerophyte Zygophyllum xanthoxylum, which exhibits excellent adaptability to adverse envi-

ronments, K+ concentration remains at a relatively constant level despite increased levels of Na+ under salin-

ity and drought conditions. In this study, the contribution of ZxAKT1 to maintaining K+ and Na+

homeostasis in Z. xanthoxylum was investigated. Expression of ZxAKT1 rescued the K+-uptake-defective

phenotype of yeast strain CY162, suppressed the salt-sensitive phenotype of yeast strain G19, and comple-

mented the low-K+-sensitive phenotype of Arabidopsis akt1 mutant, indicating that ZxAKT1 functions as an

inward-rectifying K+ channel. ZxAKT1 was predominantly expressed in roots, and was induced under high

concentrations of either KCl or NaCl. By using RNA interference technique, we found that ZxAKT1-silenced

plants exhibited stunted growth compared to wild-type Z. xanthoxylum. Further experiments showed that

ZxAKT1-silenced plants exhibited a significant decline in net uptake of K+ and Na+, resulting in decreased

concentrations of K+ and Na+, as compared to wild-type Z. xanthoxylum grown under 50 mM NaCl. Com-

pared with wild-type, the expression levels of genes encoding several transporters/channels related to K+/

Na+ homeostasis, including ZxSKOR, ZxNHX, ZxSOS1 and ZxHKT1;1, were reduced in various tissues of a

ZxAKT1-silenced line. These findings suggest that ZxAKT1 not only plays a crucial role in K+ uptake but also

functions in modulating Na+ uptake and transport systems in Z. xanthoxylum, thereby affecting its normal

growth.

Keywords: Zygophyllum xanthoxylum, ZxAKT1, K+ uptake, K+/Na+ homeostasis, salinity.

INTRODUCTION

Drought and salinity are two major abiotic stresses restrict-

ing crop growth and agricultural production worldwide

(Tang et al., 2015). These two hostile stresses frequently

co-occur in agricultural ecosystems as the secondary salin-

ization induced by irrigation becomes increasingly severe,

especially in arid and semi-arid areas (Yan et al., 2013;

Tang et al., 2015). The xerophytic or halophytic species

widely distributed in arid and saline regions, however,

have evolved various protective strategies to ensure their

own survival and the establishment of their offspring

under these harsh environments (Flowers and Colmer,

2008; Ashraf, 2010; Burrieza et al., 2012; Ahmed et al.,

2013). One common mechanism is regulating K+, Na+

transport to re-establish cell osmotic and ionic homeosta-

sis during stress (Blumwald et al., 2000; Yuan et al., 2015;

Gu et al., 2016; Shabala et al., 2016).

Zygophyllum xanthoxylum, a perennial shrub widely

distributed in the desert areas of northwest China and

Mongolia, is a salt-accumulating succulent xero-halophyte

belonging to Zygophyllaceae with excellent resistance to

drought and salinity (Ma et al., 2012). This species plays a

vital role in sand-fixing, water and soil conservation in

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd

1

The Plant Journal (2016) doi: 10.1111/tpj.13465

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desert areas (Ma et al., 2012). Our previous studies indi-

cated that Z. xanthoxylum could absorb a large amount of

Na+ even from low salt soils (Wang et al., 2004), and mod-

erate concentrations of NaCl could stimulate the growth of

Z. xanthoxylum under well irrigated conditions and, more

interestingly, mitigate the deleterious impacts of drought

that are closely linked to the high Na+ accumulation in

leaves (Ma et al., 2012; Yue et al., 2012). In most glyco-

phytes, a high external concentration of Na+ would disturb

the ability to acquire K+ resulting from the competition

between Na+ and K+ for the major binding sites of K+ chan-

nels or transporters, and, consequently, triggers the imbal-

ance of cytoplasmic cations, thereby affecting many key

metabolic processes in cells (Shabala et al., 2016). How-

ever, although the concentration of Na+ in leaves is

tremendously increased under salt and drought conditions

in Z. xanthoxylum, the concentration of K+ only exhibited a

slight decrease under salt treatment and maintained

unchanged under drought stress (Wu et al., 2011; Ma

et al., 2012; Yue et al., 2012; Hu et al., 2016), suggesting

that, besides absorbing a great quantity of Na+ which is

efficiently accumulated in leaves, maintaining the stability

of K+ concentration in those leaves is another important

adaptive strategy for Z. xanthoxylum to survive in saline

and arid environments. Therefore, an efficient K+ transport

system at the plasma membrane of root cells is of crucial

importance for maintaining K+ acquisition and homeosta-

sis in Z. xanthoxylum under drought and salt conditions.

Numerous earlier studies found that K+ uptake exhibited

bi-phasic kinetics in response to increasing external K+

concentrations with a high- or a low-affinity component in

plants (Epstein et al., 1963; Maathuis and Sanders, 1994).

In the model plant Arabidopsis thaliana, the high-affinity

K+ transporter HAK5 and inward-rectifier K+ channel AKT1

(Arabidopsis K+ transporter 1) constitute important path-

ways for K+ acquisition in root cells (Maathuis et al., 1997;

Wang and Wu, 2013). It has been shown that AtHAK5 only

operates as a high-affinity K+ uptake system at external

concentrations <10 lM, while AKT1 operates as a low-affi-

nity K+ uptake system and plays a crucial role in K+ uptake

at external concentrations >500 lM. When concentrations

of external K+ are between 10 and 200 lM, both AtHAK5

and AKT1 contribute to K+ acquisition (Gierth et al., 2005;

Rubio et al., 2008). Given that the typical K+ concentration

in natural soils varies from 0.1 to 1 mM and potash fertiliza-

tion is a common practice in many crop producing areas

(Maathuis, 2009), it is likely that AKT1 plays a more impor-

tant role in K+ acquisition in natural environments.

AKT1 is expressed preferentially in roots hairs, epider-

mis, cortex and endodermis of mature roots, where cell

types specialized in K+ uptake (Lagarde et al., 1996; Fuchs

et al., 2005; Li et al., 2014). In Arabidopsis and rice, loss of

function of AKT1 led to a significant reduction of K+ uptake

and made the plants hypersensitive to K+ deficiency

(Hirsch et al., 1998; Spalding et al., 1999; Xu et al., 2006;

Li et al., 2014). Besides mediating K+ uptake, AKT1 was

also correlated with salt tolerance in plants. Golldack et al.

(2003) found that OsAKT1 transcript levels disappeared

from the exodermis and endodermis in roots of relatively

salt resistant (sodium-excluding) rice varieties whereas it

was expressed in salt-sensitive (sodium-accumulating)

variety under 150 mM NaCl. Interestingly, the expression

pattern of OsAKT1 in different varieties did not respond to

external K+ concentrations (Golldack et al., 2003). These

observations suggest that Na+ accumulation in the whole

plant, to some extent, is related to the expression of

OsAKT1 under salt stress (Golldack et al., 2003). Further-

more, Wang et al. (2007) found that tetraethylammonium

(TEA+), Cs+ and Ba2+, which inhibit the activity of AKT1

(Maathuis et al., 1997), significantly reduced 22Na+ influx

under higher NaCl concentration in the halophyte Suaeda

maritima. These results indicate that AKT1 not only medi-

ates K+ uptake in roots, but is also related to Na+ home-

ostasis under salt conditions. However, current knowledge

about this channel in xerophyte species, especially, its

roles in modulating K+ and Na+ accumulation and home-

ostasis remains unknown.

In this study, the ZxAKT1 gene was isolated from the

xerophyte Z. xanthoxylum and shown to be encoding an

inward-rectifying K+ channel. The expression patterns of

ZxAKT1 in response to KCl and NaCl were then investi-

gated. By using RNA interference, we also evaluated the

role of ZxAKT1 in K+, Na+ uptake and accumulation in

Z. xanthoxylum. The transcript levels of ZxSKOR, ZxNHX,

ZxSOS1 and ZxHKT1;1 in ZxAKT1-silenced lines and wild-

type (WT) were also studied. Our results indicate that

ZxAKT1 modulates K+ and Na+ transport and thus plays an

important role in maintaining K+, Na+ homeostasis in

Z. xanthoxylum.

RESULTS

Isolation and characterization of ZxAKT1

A DNA fragment of 404 bp was amplified from Z. xan-

thoxylum cDNA using degenerate primers P1 and P2

(Table S1) by RT-PCR. After performing 50- and 30-RACE, a3021 bp full-length cDNA was obtained, which contained a

50-untranslated region (UTR) of 163 bp nucleotides, a pre-

dicted open reading frame of 2607 bp nucleotides encod-

ing a protein of 869 amino acids, and a 30-UTR of 251 bp

nucleotides. Phylogenetic analysis revealed that this pro-

tein was highly homologous to inward-rectifying K+ chan-

nel AKT1, rather than KAT1, AKT2, AtKC1 and SKOR, from

Arabidopsis and other plant species (Figure S1). It shared

common features such as six transmembrane segments

(S1–S6), a pore domain (P) carrying the hallmark GYGD/E

motif of highly K+ selective channels, a putative cyclic

nucleotide-binding domain (cNBD), and an ankyrin domain

© 2016 The AuthorsThe Plant Journal © 2016 John Wiley & Sons Ltd, The Plant Journal, (2016), doi: 10.1111/tpj.13465

2 Qing Ma et al.

(A1–A5) (Figure S2), as previously reported for AKT1 pro-

teins from other plants (Sentenac et al., 1992; Gambale

and Uozumi, 2006). These results suggested that the

obtained cDNA encodes an AKT1-like inward-rectifying K+

channel. We therefore designated this gene as ZxAKT1.

The K+ transport activity of ZxAKT1 was tested in yeast

mutant strain CY162, which is defective in K+ uptake as a

result of deletions of two K+ transporters TRK1 and TRK2

(Anderson et al., 1992). AKT1 from Arabidopsis (AtAKT1)

was used as a positive control. Growth assays showed that

under high K+ conditions (100 mM), all tested yeast strains

showed similar growth (Figure 1a), while on the low K+

(0, 0.1 or 1 mM) medium, the growth of CY162 transformed

with empty vector was significantly depressed, and both

ZxAKT1 and AtAKT1 could rescue this growth defect

(Figure 1a).

To determine if ZxAKT1 is involved in Na+ uptake, we

expressed ZxAKT1, AtAKT1 and AtHKT1;1 (used as a posi-

tive control for Na+ uptake as it endows yeast cells with

increased Na+ sensitivity by mediating Na+ uptake; Uozumi

et al., 2000) in the yeast mutant strain G19, which exhibits

higher sensitivity to Na+ due to disruptions in Na+-extrud-

ing ATPases ENA1 to ENA4 (Quintero et al., 1996). All the

yeast cells grew well on the control medium (0 mM addi-

tional Na+) (Figure 1b). As expected, with increases of

external Na+ concentrations (10–100 mM), the expression

of AtHKT1;1 led to salt-hypersensitivity on G19 when com-

pared with control cells (transformation of empty vector)

(Figure 1b), while G19 cells that expressed ZxAKT1 and

AtAKT1 showed increased growth compared with the con-

trol cells (Figure 1b). These results suggest that ZxAKT1

could not function directly as a Na+ transporter in yeast.

(a)

(c)

(d)

(e)

(f)

(g) (h)

(j)(i)

(b)

Figure 1. Functional characterization of ZxAKT1 in yeast and Arabidopsis.

(a) ZxAKT1 complemented the K+-uptake-deficient yeast mutant strain CY162 on AP medium containing different K+ concentrations (0, 0.2, 1 or 100 mM). CY162

expressing AtAKT1 and empty vector were used as positive and negative controls, respectively.

(b) Expression of ZxAKT1 in yeast mutant strain G19. Yeast cells were plated on AP medium containing 1 mM K+ and various concentrations of Na+ (0, 10, 30,

50, 70 or 100 mM). G19 expressing AtHKT1;1 and empty vector were used as positive and negative controls, respectively.

(c) RT-PCR analysis of transgenic akt1 lines overexpressing ZxAKT1 (CZ1-4) and AtAKT1 (CA1-4).

(d–f) Phenotype comparison of wild-type Arabidopsis (WS), akt1 mutant, and transgenic lines (akt1/ZxAKT1 and akt1/AtAKT1) grown on MS medium or low K+

medium (0.5 and 0.1 mM K+) for 10 days.

(g, h) Primary root length and seedling fresh weight of different plant materials grown on MS or low K+ medium for 10 days. Values are means � SD (n = 5)

and bars indicate standard deviation (SD). Columns with different letters indicate significant differences at P < 0.05 (Duncan’s test).

(i, j) K+ concentrations in shoots and roots of different plant materials. K+ concentrations were determined after the plants were grown on MS and low K+

medium for 10 days. [Colour figure can be viewed at wileyonlinelibrary.com].


AKT1 affects K+/Na+ transport in Z. xanthoxylum 3

To further examine the possible function of ZxAKT1 in

planta, the coding sequence of ZxAKT1 and AtAKT1 was

transformed into an Arabidopsis akt1 mutant [ecotype

Wassilewskija (WS)], and two transgenic lines (CZ2 and

CA1, representing akt1/ZxAKT1 and akt1/AtAKT1, respec-

tively) were selected for phenotypic tests (Figure 1). The

akt1 mutant showed a low K+-sensitive phenotype and a

significant reduction in K+ content when grown on low

K+ (0.1 or 0.5 mM) medium (Figure 1d–j). The low

K+-sensitive phenotype of the akt1 mutant was rescued

in these two transgenic lines, which displayed a similar

phenotype as wild-type (WT) plants under low K+ condi-

tions (Figure 1d–f). K+ concentrations in shoots and roots

of WT and the two transgenic lines were significantly

higher than that of the akt1 mutants on MS medium or

at 0.5 mM K+ (Figure 1i, j). Although no significant differ-

ence in root K+ concentrations was observed among the

lines with less than 0.1 mM K+ concentration (Figure 1j),

the akt1 mutant displayed a significant decreased shoot

K+ concentration compared with WS and the two trans-

genic lines (Figure 1i). The akt1 mutant also showed a

Na+-sensitive phenotype under different NaCl concentra-

tions, and this phenotype was rescued by overexpres-

sion of ZxAKT1 and AtAKT1 (Figure S3a–d). With the

increase in external NaCl concentration, Na+ concentra-

tions in shoots and roots significantly increased in all

the lines, and the akt1 mutant possessed a significantly

higher shoot Na+ concentration (under 75 mM NaCl) and

root Na+ concentration (under 50 and 75 mM NaCl) than

the WT or two transgenic lines (Figure S3e,f). In addi-

tion, K+ concentrations showed a decrease trend in all

the lines, and the akt1 mutant showed significantly

reduced K+ concentrations in both shoots and roots in

comparison with WT and the two transgenic lines sub-

jected to different concentrations of NaCl (Figure S3g,h).

Taken together, these results demonstrate that ZxAKT1

has a similar function in K+ uptake as AKT1 in Arabidopsis.

The expression of ZxAKT1 was induced by KCl and NaCl

treatments

With increased external KCl concentrations, the expres-

sion of ZxAKT1 in roots and stems increased signifi-

cantly, peaking at 1 mM KCl, then decreased and

remained stable under 5 or 10 mM KCl, respectively,

while the expression level in leaves remained at a low

level under different concentrations of KCl (Figure 2a).

The highest expression level in roots and lowest expres-

sion level in leaves were always observed under different

concentrations of KCl (Figure 2a).

In the absence of NaCl, the expression level of ZxAKT1

was higher in roots than that in leaves and stems

(Figure 2b). The addition of 50 mM NaCl signifi-

cantly increased the transcription of ZxAKT1 in roots by

4.5-fold, but did not affect the expression level in leaves

and stems (Figure 2b). Along with the increase of exter-

nal NaCl, the expression level of ZxAKT1 in roots signifi-

cantly increased and reached peak levels at 50 or 100 mM

NaCl, which was 4.8-fold higher than that under control

condition (no additional NaCl). The expression level of

ZxAKT1 then significantly decreased under 150 mM NaCl,

but was still 2.5-fold higher than that under control con-

ditions (Figure 2c). We further investigated the expression

of ZxAKT1 in roots of plants exposed to 50 mM NaCl over

a 120 h period. The expression levels of ZxAKT1

increased with the elongation of treatment time, and

reached their peak values at 48 h, and then displayed a

decreasing trend (Figure 2d).

Taken together, these results revealed that ZxAKT1 was

preferentially expressed in roots, and the transcript was

induced not only by KCl treatment but also by NaCl treat-

ment.

ZxAKT1-silencing triggered a significant inhibition of the

growth of Z. xanthoxylum

To further explore the function of ZxAKT1 in planta,

ZxAKT1 was silenced by RNA interference (RNAi). Fourteen

transgenic lines carrying the RNAi construct were identi-

fied by PCR amplification. Since the mRNA amount of

ZxAKT1 was upregulated by salt and reached the highest

transcript level under 50 mM NaCl at 48 h (Figure 2c), the

expression level of ZxAKT1 in four randomly selected

transgenic lines treated with 50 mM NaCl for 48 h was

assessed. As expected, the expression level of ZxAKT1 in

the transgenic lines was reduced by different degrees in

comparison with WT (Figure S4), among which line 7 dis-

played the highest silencing efficiency and line 10 dis-

played the lowest silencing efficiency of ZxAKT1

(Figures S4 and 3a). These two lines were selected for fur-

ther physiological assays.

Phenotypically, the silencing of ZxAKT1 induced a signif-

icant inhibition on the growth of Z. xanthoxylum (Fig-

ure 3b). In line 7, especially, the fresh weight, dry weight,

shoot height and root length were significantly decreased

by 65, 62, 52 and 79% under control condition, respec-

tively, and by 66, 63, 50 and 78% under 50 mM NaCl,

respectively, in comparison with those of the WT

(Figure 3c–f).

ZxAKT1 silencing altered K+, Na+ accumulation and uptake

Under control conditions, K+ concentrations in roots, stems

and leaves of line 7 were significantly decreased by 40, 43

or 30%, respectively, compared with that of the WT, while

no significant difference in Na+ concentration was

observed between ZxAKT1-silenced lines and WT (Fig-

ure 4). The addition of 50 mM NaCl significantly increased

the Na+ concentration but hardly affected K+ concentra-

tions in any tissues of the WT or line 7 (Figure 4). Under

50 mM NaCl, line 7 accumulated less Na+ in roots, stems


4 Qing Ma et al.

and leaves by 30, 30 and 25% than that of WT, respectively

(Figure 4a–c). Meanwhile, K+ concentrations in all tissues

were also significantly reduced by 35, 41 and 20% in roots,

stems and leaves, respectively, compared with that in the

WT plant (Figure 4d–f).Further analysis showed that the net K+ uptake rate in

line 7 was sharply reduced by 42% compared with that of

the WT, but no significant difference in net Na+ uptake rate

was observed between line 7 and WT under control condi-

tions (Figure 5a). The addition of 50 mM NaCl significantly

decreased the net K+ uptake rate, while it increased the net

Na+ uptake rate in both WT and ZxAKT1-silenced lines (Fig-

ure 5). Under 50 mM NaCl, line 7 exhibited decreased net

K+ and Na+ uptake rate by 64 and 40% compared with the

WT (Figure 5b), respectively.

ZxAKT1 silencing downregulated the expression level of

ZxNHX in leaves, and ZxSKOR, ZxSOS1 and ZxHKT1;1 in

roots of Z. xanthoxylum under 50 mM NaCl

To further explore the possible molecular mechanisms

underlying the above-described observations, the expres-

sion levels of genes involved in K+ and Na+ uptake and

transport were analyzed in WT and ZxAKT1-silenced line 7

under control conditions or with 50 mM NaCl.

As shown in Figure 6(a), the K+ outward-rectifying chan-

nel gene ZxSKOR was preferentially expressed in roots

and to very low levels in leaves. Under control conditions,

the expression level of ZxSKOR in roots of line 7 was the

same as that of the WT (Figure 6a). The addition of 50 mM

NaCl significantly increased the expression level of

Figure 2. The expression patterns of ZxAKT1 in Z. xanthoxylum under various KCl or NaCl treatments.

(a) Expression of ZxAKT1 in roots, stems, and leaves of Z. xanthoxylum under different concentrations of KCl. Three-week-old plants were deprived of K+ (see

Experimental procedures) for 3 days, then treated with various KCl concentrations (0, 0.1, 0.5, 1, 5 or 10 mM) for 48 h.

(b) Tissue-specific expression of ZxAKT1 in Z. xanthoxylum under salt treatment. Three-week-old plants were treated without (�) or with (+) 50 mM NaCl for 48 h.

(c) Expression of ZxAKT1 in roots of 3-week-old seedlings treated with 0, 5, 25, 50, 100 or 150 mM NaCl for 48 h.

(d) Expression of ZxAKT1 in roots of 3-week-old plants treated with 50 mM NaCl over a 120 h period. ZxACTIN was used as an internal control. Values are

means � standard deviation (SD) (n = 3) and bars indicate SD. Columns with different letters indicate significant differences at P < 0.05 (Duncan’s test). [Colour

figure can be viewed at wileyonlinelibrary.com].



ZxSKOR in roots by approximately three-fold in the WT

but did not significantly affect the expression in roots of

line 7 (Figure 6a), suggesting that ZxAKT1-silencing

induced a significant downregulation of ZxSKOR in roots

of Z. xanthoxylum under the salt treatments.

The significantly higher expression levels of the plasma

membrane Na+/H+ antiporter gene ZxSOS1 and high-affi-

nity K+ transporter gene ZxHKT1;1 were also observed in

roots rather than leaves under both control condition and

50 mM NaCl treatment (Figure 6b,c). In the absence of

NaCl, there was no significant difference in the expression

levels of ZxSOS1 and ZxHKT1;1 in roots between line 7

and WT (Figure 6b,c). The addition of 50 mM NaCl signifi-

cantly upregulated their expression in WT roots, while it

did not induce any changes in their expression in roots of

line 7 (Figure 6b,c). Under 50 mM NaCl, the transcripts of

those two genes were lower by 60 and 58% in roots of line

7 compared with that of WT, respectively (Figure 6b,c).

Although the expression levels of ZxSOS1 and ZxHKT1;1

were much lower in leaves than in roots, the expression

levels of these two genes in leaves of both WT and line 7

were also induced by 50 mM NaCl, and the expression

levels were significantly lower in line 7 than that of WT

under control conditions or 50 mM NaCl treatment (Fig-

ure 6b,c). These results indicated that ZxAKT1 silencing

affected the expression level of genes involved in Na+

transport in Z. xanthoxylum (especially in roots) when cul-

tured in 50 mM NaCl.

The expression level of tonoplast Na+/H+ antiporter gene

ZxNHX was significantly higher in leaves than that in roots

under both control conditions and 50 mM NaCl (Figure 6d).

Although the expression levels of ZxNHX in the leaves of

WT and line 7 was significantly induced by the addition of

50 mM NaCl, the increasing trend in line 7 (67%) was signif-

icantly lower than that in WT (140%) (Figure 6d). These

results indicated that ZxAKT1 silencing triggered a signifi-

cant downregulation of the expression level of ZxNHX

under 50 mM NaCl in leaves of Z. xanthoxylum.

DISCUSSION

Transcriptional regulation of AKT1 gene might be a vital

mechanism in response to various K+ concentrations and

salt conditions in Z. xanthoxylum

Similar to previous reports on AKT1 from A. thaliana

(AtAKT1, Lagarde et al., 1996), rice (OsAKT1, Fuchs et al.,

(a)

(c)

(d) (f)

(e)

(b)Figure 3. The growth phenotype of WT and

ZxAKT1-silenced lines (7 and 10) under control (no

additional NaCl) and 50 mM NaCl for 7 days.

(a) Real-time qPCR analysis of the relative expres-

sion level of ZxAKT1 in roots of WT and transgenic

ZxAKT1-silenced lines (7, 10). ZxACTIN was used as

an internal control.

(b) The growth phenotype of WT and ZxAKT1-

silenced lines (7 and 10).

(c–f) Fresh weight, dry weight, shoot height and

root length of WT and ZxAKT1-silenced lines (7 and

10) under control (no additional NaCl) and 50 mM

NaCl for 7 days. The scale bar in (b) equals 1 cm.

Values in (c–f) are means � standard deviation (SD)

(n = 5) and bars indicate SD. Columns with different

letters indicate significant differences at P < 0.05

(Duncan’s test). [Colour figure can be viewed at

wileyonlinelibrary.com].


6 Qing Ma et al.

2005), Puccinellia tenuiflora (PutAKT1; Wang et al., 2015)

and Suaeda salsa (Suaeda maritima subsp. salsa) (SsAKT1,

Duan et al., 2015), the transcript level of ZxAKT1 was more

abundant in roots than shoots (Figure 2a,b). In Arabidop-

sis, changes in external K+ availability do not significantly

affect AtAKT1 transcript levels in roots (Pilot et al., 2003),

and similar expression patterns were observed in the salt-

secreting plant Mesembryanthemum crystallinum (Su

et al., 2001) and the salt-excluding plant P. tenuiflora

(Wang et al., 2015). This finding suggests that the regula-

tion of external K+ on AKT1 in the above species is a post-

transcriptional process (Wang and Wu, 2013; Wang et al.,

2015). In Arabidopsis, the regulatory genes of the AKT1

channel (e.g. CIPK23, CBL10 and AtKC1) show significant

transcriptional changes under K+-deficiency stress (Cheong

et al., 2007; Wang et al., 2010; Ren et al., 2013), indicating

that the transcriptional regulation of AKT1 regulatory

genes, rather than AKT1 itself, may be a more important

mechanism for Arabidopsis responding to external K+

supply (Wang and Wu, 2013). However, in Z. xanthoxylum,

the expression of ZxAKT1 in roots increased significantly

with increase in external K+ concentrations (Figure 2a).

This finding was consistent with the expression pattern of

SsAKT1 in the salt-accumulating plant S. salsa (Duan et al.,

2015), demonstrating that in salt-accumulating species

such as Z. xanthoxylum and S. salsa, the transcriptional

regulation of AKT1 itself might be a crucial response mech-

anism to different K+ concentrations.

Some studies showed that the transcript level of AKT1

was significantly downregulated in roots under NaCl treat-

ments, such as AKT1 from M. crystallinum (Su et al., 2001)

and A. thaliana (Kaddour et al., 2009). However, the tran-

script level of ZxAKT1 was significantly upregulated by

NaCl, especially in roots (Figure 2b–d). It is worth noting

that under salt stress, the increase in Na+ content in leaves

of M. crystallinum and A. thaliana is always accompanied

by a very large decrease in K+ content (Adams et al., 1998;

Zhu et al., 1998). In contrast, although Na+ concentration in

Figure 4. Na+ and K+ concentrations in roots, stems

and leaves.

Na+ (a–c) and K+ (d–f) concentrations in roots,

stems and leaves of WT and ZxAKT1-silenced lines

(7, 10) under control (no additional NaCl) and

50 mM NaCl for 7 days. Values are means � stan-

dard deviation (SD) (n = 5) and bars indicate SD.

Columns with different letters indicate significant

differences at P < 0.05 (Duncan’s test). [Colour fig-

ure can be viewed at wileyonlinelibrary.com].



leaves of Z. xanthoxylum significantly increased under salt

conditions, K+ concentration only displayed a slight

decreased trend (Wu et al., 2011). Therefore, the upregu-

lated transcript of ZxAKT1 is responsible for K+ homeosta-

sis under salt conditions in Z. xanthoxylum.

The role of ZxAKT1 in K+ uptake and transport in

Z. xanthoxylum

In Arabidopsis and rice, AKT1 mediates root K+ uptake over

a wide range of external K+ conditions (Lagarde et al.,

1996; Hirsch et al., 1998; Xu et al., 2006; Li et al., 2014). In

the present study, ZxAKT1 is abundantly expressed in

roots (Figure 2), which is the precondition that ZxAKT1

mediates K+ uptake from soils in Z. xanthoxylum. The

complementation assays in both yeast and akt1 mutant

indicated that, similar to the AKT1 channel from Arabidop-

sis, ZxAKT1 is involved in K+ uptake (Figure 1). Further-

more, ZxAKT1-silencing triggered a significant decline in

net uptake of K+, resulting in decreased concentrations of

K+ in Z. xanthoxylum (Figures 4d–f and 5a). These results

were consistent with the observations that Arabidopsis

akt1 and rice osakt1 mutant plants display a decreased K+

uptake and reduced K+ content (Xu et al., 2006; Li et al.,

2014), demonstrating that ZxAKT1 mediates K+ uptake in

Z. xanthoxylum.

In higher plants, K+ uptake in roots displays typical

dual-affinity mechanisms: the high-affinity mechanism

mediates K+ uptake at low external K+ concentrations

(below 0.2 mM); while at relatively high external K+ con-

centrations (above approximately 0.5 mM), the low-affinity

mechanism is involved in K+ uptake (Epstein et al., 1963;

Maathuis and Sanders, 1996; Wang and Wu, 2013). It has

been shown that AKT1 contributes to both high- and

low-affinity K+ uptake, and accounts for a large portion

of the low-affinity uptake (Hirsch et al., 1998; Spalding

et al., 1999; Gierth et al., 2005; Rubio et al., 2008). In the

present study, ZxAKT1 expression levels were much

higher under high K+ concentrations (0.5–10 mM) than

that under low (0.1 mM) K+ concentration (Figure 2), sug-

gesting that ZxAKT1 plays a greater role in the low-affi-

nity system of K+ uptake.

The K+ outward-rectifying channel SKOR functions in

loading K+ from xylem parenchyma cells (XPCs) into

xylem sap toward the shoots (Wegner and Raschke,

1994; Wegner and De Boer, 1997; Gaymard et al., 1998;

Liu et al., 2006). Our recent investigation showed a posi-

tive correlation between ZxSKOR expression in roots and

K+ accumulation in shoots, indicating that ZxSKOR in

roots probably plays important roles in K+ accumulation

and homeostasis in Z. xanthoxylum under salt conditions

(Hu et al., 2016). In the present study, the transcript of

ZxSKOR in roots of line 7 was significantly downregu-

lated under 50 mM NaCl compared to that of WT (Fig-

ure 6a), thereby restricting the delivery of K+ from roots

to shoots and consequently reducing K+ accumulation in

stems and leaves of ZxAKT1-silenced lines than that of

WT (Figure 4d–f). It has been found that K+ deprivation

significantly repressed the expression of ZxSKOR in roots

of Z. xanthoxylum (Hu et al., 2016), and ZxAKT1-silen-

cing, to some extent, may trigger K+ deficiency in Z. xan-

thoxylum since ZxAKT1-silenced lines displayed

decreased K+ uptake (Figure 5a). This might be the rea-

son why ZxAKT1-silencing resulted in a significant reduc-

tion in the transcripts of ZxSKOR in roots under salt

treatment. These results suggest that ZxAKT1 not only

mediates K+ uptake in roots, but is also essential for

maintaining long-distance transport and homeostasis of

K+ in Z. xanthoxylum.

Figure 5. Net uptake rate of K+ and Na+.

Net uptake rate of K+ (a) and Na+ (b) in whole plants of WT and ZxAKT1-

silenced lines (7, 10) under control condition (no additional NaCl) or 50 mM

NaCl for 7 days. Values are mean � standard deviation (SD) (n = 5) and

bars indicate SD. Columns with different letters indicate significant differ-

ences at P < 0.05 (Duncan’s test).


8 Qing Ma et al.

ZxAKT1 is involved in modulating Na+ uptake, transport

and homeostasis in Z. xanthoxylum under salt conditions

Although ZxAKT1 could not mediate Na+ uptake in yeast

and the Arabidopsis akt1 mutant (Figure 1), the net uptake

rate and concentrations of Na+ in ZxAKT1-silenced plants

(line 7) were significantly lower than that of WT under

50 mM NaCl (Figures 4d–f and 5b), demonstrating that

ZxAKT1 might take part in modulating Na+ uptake and

transport under salt conditions.

When Na+ is taken up into roots, the surface area of

xylem vessels in contact with XPCs accommodates the

large quantities of Na+ that pass from roots to shoots

(Blumwald et al., 2000; Tester and Davenport, 2003; Apse

and Blumwald, 2007). SOS1 located at the plasma mem-

brane of XPCs in roots is mainly involved in the long-dis-

tance transport of Na+ from roots to shoots by loading Na+

from XPCs into the xylem (Shabala and Mackay, 2011;

Adolf et al., 2013; Ma et al., 2014). The HKT1 transporter

mediates Na+ unloading from xylem vessels to

parenchyma cells under salt treatment (Sunarpi et al.,

2005; Guo et al., 2012), and the opposite Na+ fluxes medi-

ated by ZxSOS1 and ZxHKT1 in roots are finely coordi-

nated to preserve the membrane integrity (Guo et al.,

2012). This would synergistically modulate Na+ transport

and homeostasis in Z. xanthoxylum (Yuan et al., 2015). In

the succulent leaves of Z. xanthoxylum, Na+ is compart-

mentalized by ZxNHX into the vacuoles to cope with envi-

ronmental stresses by using Na+ as an important

osmoregulatory substance (Wang et al., 2004; Wu et al.,

2011). A recent investigation indicated that ZxNHX also

plays a vital role in controlling the uptake, long-distance

transport, and homeostasis of Na+ at the whole plant level

via feedback regulation of the expression of ZxSOS1 and

ZxHKT1;1 in roots (Yuan et al., 2015). In this study, under

50 mM NaCl, the ZxSOS1, ZxHKT1;1 and ZxNHX transcript

were significantly lower in line 7 than that of WT (Fig-

ure 6), indicating that ZxAKT1-silencing triggered a signifi-

cant downregulation on the expression level of genes

Figure 6. Real-time quantitative PCR analysis.

The expression of ZxSKOR (a), ZxSOS1 (b), ZxHKT1;1 (c) and ZxNHX (d) in WT and ZxAKT1-silenced line 7 treated without (control) or with 50 mM NaCl for

48 h. ZxACTIN was used as an internal control. Values are means � standard deviation (SD) (n = 3) and bars indicate SD. Columns with different letters indicate

significant differences at P < 0.05 (Duncan’s test).



involved in Na+ transport and accumulation in Z. xanthoxy-

lum under salt conditions and consequently restrained Na+

transport from roots to shoots. This would in turn inhibit

Na+ uptake in roots (Yuan et al., 2015), and as a result,

ZxAKT1-silenced plants (line 7) displayed a reduced net

Na+ uptake rate (Figure 5b). Therefore, we propose that

ZxAKT1 not only mediates K+ uptake, but also functions in

modulating Na+ uptake and transport systems in Z. xan-

thoxylum under salt conditions.

Then, the question – How does ZxAKT1-silencing affect

the expression of genes involved in Na+ transport and

accumulation? – is worthy of further consideration. The

possible explanations may be as follows. ZxAKT1-silencing

reduced K+ uptake in roots and restricted the delivery of K+

from roots to shoots under salt treatments, and thus, led

to a significant decrease in K+ accumulation in leaves (Fig-

ure 4d). It has been reported that NHX1 and NHX2 in Ara-

bidopsis mediate K+ sequestration into the vacuoles of

leaves for osmotic adjustment and turgor regulation (Bassil

et al., 2011; Barrag�an et al., 2012; Andr�es et al., 2014).

ZxNHX may also mediate the vacuolar compartmentation

of both Na+ and K+, since ZxNHX-silencing triggers a sig-

nificant decrease in Na+ and K+ concentrations in Z. xan-

thoxylum (Yuan et al., 2015). Also, the overexpression of

ZxNHX and ZxVP1 enhances Na+ and K+ accumulation in

transgenic legumes under salinity and drought conditions

(Bao et al., 2014, 2015). Interestingly, recent research indi-

cated that K+-deficiency significantly reduces the tran-

scripts of wheat TaNHX2 in transgenic alfalfa under salt

stress (Zhang et al., 2015). Therefore, the decrease in leaf

K+ accumulation could result in a significant reduction in

the transcripts of ZxNHX in leaves of ZxAKT1-silenced

plants (Figure 6d), and consequently, the capacity for

sequestering Na+ into vacuoles of leaves would be weak-

ened in ZxAKT1-silenced plants, which in turn would

restrict Na+ long-distance transport from roots through

feedback regulation of ZxSOS1 and ZxHKT1;1 transcripts in

roots (Yuan et al., 2015).

ZxAKT1 is essential for normal growth and development

of Z. xanthoxylum

Our results showed that silencing of ZxAKT1 had strong

negative effects on plant growth under salt treatment and

even under normal conditions: the fresh weight, dry

weight, shoot height, and root length of ZxAKT1-silenced

lines were significantly lower than WT (Figure 3). These

observations might be partly caused by the significantly

reduced accumulation of K+ and Na+ (Figure 4), as K+ is

one of the crucial nutrient elements in plant growth and

development. Meanwhile, 50 mM NaCl could significantly

promote the growth of Z. xanthoxylum, which is strongly

related to Na+ accumulation (Ma et al., 2012; Yue et al.,

2012). These results indicate that ZxAKT1 is important for

maintaining growth and development of Z. xanthoxylum.

In conclusion, our results demonstrate that ZxAKT1 not

only mediates K+ uptake and plays a crucial role in regulat-

ing long-distance transport of K+, but is also involved in

modulating Na+ uptake and transport system through feed-

back regulation in Z. xanthoxylum, thereby affecting plant

growth.

EXPERIMENTAL PROCEDURES

Isolation of ZxAKT1 from Zygophyllum xanthoxylum

Seeds of Z. xanthoxylum were germinated and seedlings were culti-vated with modified ½ strength Hoagland nutrient solution (contain-ing 2 mM KNO3, 0.5 mM NH4H2PO4, 0.25 mM MgSO4.7H2O, 0.1 mM

Ca(NO3)2.4H2O, 50 lM Fe citrate, 92 lM H3BO3, 18 lM MnCl2.4H2O,1.6 lM ZnSO4.7H2O, 0.6 lM CuSO4.5H2O, 0.7 lM (NH4)6Mo7O24.4H2O)according to the described by Ma et al. (2012). Total RNA wasextracted from roots of 4-week-old seedlings exposed to 150 mM

NaCl for 48 h. A specific fragment of ZxAKT1 was amplified by RT-PCR using degenerate primers P1 and P2 (Table S1) designedaccording to the highly conserved regions of AKT1 genes from otherplant species. The 50- and 30-ends of ZxAKT1 were obtained usingthe RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) Kit (Invitrogen Co., Carlsbad, CA, USA) and primers P3 andP4 or P5 and P6, respectively (Table S1). These fragments wereassembled to obtain the full-length of the ZxAKT1 cDNA, which wasconfirmed by PCR with primers P7 and P8 (Table S1).

Multiple sequence alignment was performed with DNAMAN6.0software (Lynnon BioSoft, Vaudreuil, Canada). The phylogenetictree was generated using MEGA 6.0 software.

Real-time quantitative PCR analysis of ZxAKT1 responding

to different treatments

Four-week-old plants were used for the following treatments,respectively:

(i) Plants were treated with modified ½ strength Hoagland nutri-ent solutions deprived of KNO3 for 3 days, where 2 mM KNO3

was substituted with 1 mM NH4NO3. Different concentrationsof K+ treatments were then applied by adding 0, 0.1, 0.5, 1, 5or 10 mM KCl for 48 h.

(ii) Plants were treated with modified ½ strength Hoagland nutri-ent solutions supplemented with additional 0, 5, 25, 50, 100or 150 mM NaCl for 48 h.

(iii) Plants were treated with modified ½ strength Hoagland nutri-ent solutions supplemented with additional 50 mM NaCl, andharvested at 0, 3, 6, 12, 24, 48, 72, 96 or 120 h. The treatmentsolution was changed every day.

Total RNA was extracted and first strand cDNA was synthesizedfrom 2 lg of total RNA. Real-time quantitative PCR was performedon a thermal cycler (ABI PRISM 7500, Foster City, CA, USA) withthe primer pairs P9 and P10 (Table S1). ZxACTIN was used forRNA normalization, and the specific primers of ZxACTIN were A1and A2 (Table S1). SYBR Green PCR master mix (Takara, BiotechCo., Ltd, Dalian, China) was used for PCR reactions. The relativeexpression levels of ZxAKT1 were normalized to ZxACTIN and cal-culated using the 2�DDCt method (Duan et al., 2015). All reactionswere performed with three replicates.

Functional characterization of ZxAKT1 in yeast

The full-length coding sequences of ZxAKT1, AtAKT1 (AKT1 fromArabidopsis) and AtHKT1;1 (HKT1 from Arabidopsis) were each


10 Qing Ma et al.

inserted into yeast expression vector p426 containing the GAL1promoter.

The yeast (Saccharomyces cerevisiae) strains CY162 [MATa,Dtrk1, trk2::pCK64, his3, leu2, ura3, trp1, ade2] defective in the K+

transporters TRK1 and TRK2 (Anderson et al., 1992) and G19[MATa, his3, leu2, ura3, trp1, ade2, ena1::HIS3::ena4] disrupted inthe ENA1-4 genes encoding Na+ export pumps (Quintero et al.,1996) were used for yeast complementation assays. Yeast trans-formations of the above constructed plasmids were performedusing LiCl as described by Chen et al. (1992). Positive transfor-mants were selected on Ura-selective medium [0.67% (w/v) yeastnitrogen base without amino acids, 0.077% (w/v) DO supplement-Ura, 2% (w/v) galactose, 100 mM KCl, and 1.5% (w/v) agar].

Yeast growth experiments were performed on arginine-phos-phate (AP) medium containing 8 mM phosphoric acid, 10 mM

L-arginine, 0.2 mM CaCl2, 2 mM MgSO4, 2% galactose, plus vita-mins and trace elements, and 1.5% (w/v) agar (pH = 6.5)(Rodr�ıguez-Navarro and Ramos, 1984). For growth tests of CY162transformed with plasmids, AP medium supplemented with threeconcentrations of K+ (0, 0.2, 1 or 100 mM) were used. For growthassays of G19 transformed with plasmids, AP medium with addedK+ (1 mM) and supplemented with various concentrations of NaCl(0, 10, 30, 50, 70, or 100 mM) were used. Yeast cells were platedon medium using 10-fold serial dilutions from OD600 = 0.6 toOD600 = 0.6 9 10�3.

Functional characterization of ZxAKT1 in Arabidopsis

Full-length coding sequences of ZxAKT1 and AtAKT1 were eachinserted into the vector pCAMBIA1301 containing the CaMV 35Spromoter. The two constructs were transformed into an Arabidop-sis akt1 mutant (Hirsch et al., 1998), using the floral-dip methodwith Agrobacterium tumefaciens (Clough and Bent, 1998). Trans-genic lines of T4 homozygous plants were used to examine thephenotype under low-K+ conditions or different NaCl treatments.The low-K+ medium was made by modification of MS medium,where KNO3 and KH2PO4 were replaced by NH4NO3 andNH4H2PO4, respectively (Xu et al., 2006). The final [K+] was 0.1 or0.5 mM by adding the according concentrations of KNO3. Themedium for NaCl treatments was made by adding different con-centrations of NaCl (0. 25, 50 or 75 mM) into MS medium. The K+

and Na+ concentrations were determined by atomic absorptionspectrophotometry (Varian 220, Palo Alto, CA, USA).

RNA interference silencing of ZxAKT1 in Z. xanthoxylum

Stable gene silencing via Agrobacterium-mediated transformationwas done using the pHANNIBAL vector (Wesley et al., 2001)designed for producing a hairpin RNA construct of ZxAKT1. A665 bp PCR fragment as the target of RNA interference (RNAi)located in the region encoding the long hydrophilic tail in theC-terminal end of ZxAKT1 (nucleotides from 1728 to 2352 bp) wasobtained by primer pairs P11 and P12 or P13 and P14 (Table S1,XhoI, KpnI, XbaI, ClaI restriction sites underlined). The whole NotIcassette bearing the RNAi construct was subcloned into the corre-sponding site of the binary vector pART27, under the control ofthe CaMV 35S promoter. The construct was introduced intoA. tumefaciens strain GV3101. The transformation was performedusing the procedure as detailed described by Ma et al. (2014) andYuan et al. (2015).

The transformed plants were screened by a PCR assay usingpHANNIBAL-specific primers and DNA obtained from leaves inorder to detect the presence of the RNAi construct. Positive plantswere selected to study the expression level of ZxAKT1 by RT-PCR performed with primer pairs P9 and P10 (Table S1). Two

ZxAKT1-silenced lines (7 and 10) with different reduced expres-sion levels of ZxAKT1 were chosen for further analysis. Individualplants of WT and each ZxAKT1-silenced line were propagatedfrom stem cuttings (ramets) as described by Ma et al. (2014) andYuan et al. (2015).

For physiological analysis, 4-week-old plants were treated withmodified ½ strength Hoagland nutrient solutions supplementedwithout (Control) or with additional 50 mM NaCl. After 7 days,roots was washed twice for 8 min in ice-cold 20 mM CaCl2 toexchange cell wall-bound Na+, and leaves and stems were rinsedin deionized water to remove surface salts (Maathuis and Sanders,2001). Fresh weight, shoot height and root length were measured.Finally, the tissues were immediately incubated in an oven at 80°Cfor 48 h to obtain dry weights.

Na+ and K+ were determined using a flame spectrophotometer(2655-00, Cole-Parmer Instrument Co., Vernon Hills, IL, USA)(Yuan et al., 2015). Net uptake rate (NUR) of Na+ and K+ in wholeplants was calculated according to the following equation (Wanget al., 2009): NUR = [D whole plant Na+ (or K+) content betweensalt-treated plant and BT plant]/root fresh weight/D time, where BTmeans before treatments.

To analyze the effect of ZxAKT1-silencing on the expressionlevel of ZxNHX, ZxSKOR, ZxSOS1 and ZxHKT1;1 in Z. xanthoxy-lum, 4-week-old seedlings were grown with modified ½ strengthHoagland nutrient solution supplemented without (Control) orwith additional 50 mM NaCl for 48 h. The expression level of eachgene was analyzed by real-time quantitative PCR performed on athermal cycler with primers described by Yuan et al. (2015). ZxAC-TIN was used for RNA normalization. The relative expressionlevels of ZxAKT1 normalized to ZxACTIN was calculated using the2�DDCt method (Duan et al., 2015). All reactions were performedwith three replicates.

Data analysis

All data were presented as means with standard errors. Data anal-ysis was conducted by one-way analysis of variance (ANOVA)using SPSS 13.0 statistical software (SPSS Inc., Chicago, IL, USA).Duncan’s multiple range test was used to detect a differencebetween means at a significance level of P < 0.05.

ACKNOWLEDGEMENTS

We are very grateful to Dr Owen Rowland from Calton University,Canada, for critically reviewing the manuscript and for valuablesuggestions. We also thank Professor Alonso Rodr�ıguez-Navarrofrom Centro de Biotechnolog�ıa y Gen�omica de Plantas, Universi-dad Polit�ecnica de Madrid, Spain, for providing Saccharomycescerevisiae strain G19. This work was supported by the NationalBasic Research Program of China (973 Program, grant no.2014CB138701) and the National Natural Science Foundation ofChina (grant nos 31501994, 31470503). The authors declare noconflicts of interest.

AUTHOR CONTRIBUTIONS

SMW conceived the project. SMW, SL and QM designed

the research. QM, JH and XRZ performed the experiments.

HJY and TK contributed to data analysis and discussion.

QM, JH and SMW wrote the manuscript.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article.



Figure S1. Phylogenetic analysis of ZxAKT1 with other shaker K+

channels from plants.

Figure S2. Sequence alignment of ZxAKT1 with AKT1 proteinsfrom other higher plants.

Figure S3. Functional characterization of ZxAKT1 in Arabidopsisunder salt treatments.

Figure S4. Semi-quantitative RT-PCR analysis of ZxAKT1 in roots,stems and leaves of wild type (WT) and ZxAKT1-silenced lines(1, 3, 7, 10) treated with 50 mM NaCl for 48 h.

Table S1. Primer sequences used in this study.

REFERENCES

Adams, P., Nelson, D.E., Yamada, S., Chmara, W., Jensen, R.G., Bohnert,

H.J. and Griffiths, H. (1998) Growth and development of Mesembryan-

themum crystallinum (Aizoaceae). New Phytol. 138, 171–190.Adolf, V.I., Jacobsen, S.E. and Shabala, S. (2013) Salt tolerance mechanisms

in quinoa (Chenopodium quinoa Willd.). Environ. Exp. Bot. 92, 43–54.Ahmed, M.Z., Shimazaki, T., Gulzar, S., Kikuchi, A., Gul, B., Khan, M.A.,

Koyro, H.-W., Huchzermeyer, B. and Watanabe, K.N. (2013) The influence

of genes regulating transmembrane transport of Na+ on the salt resis-

tance of Aeluropus lagopoides. Funct. Plant Biol. 40, 860–871.Anderson, J.A., Huprikar, S.S., Kochian, L.V., Lucas, W.J. and Gaber, R.F.

(1992) Functional expression of a probable Arabidopsis thaliana potas-

sium channel in Saccharomyces cerevisiae. Proc. Natl Acad. Sci. USA,

89, 3736–3740.Andr�es, Z., P�erez-Hormaeche, J., Leidi, E.O. et al. (2014) Control of vacuolar

dynamics and regulation of stomatal aperture by tonoplast potassium

uptake. Proc. Natl Acad. Sci. USA, 111, E1806–E1814.Apse, M.P. and Blumwald, E. (2007) Na+ transport in plants. FEBS Lett. 581,

2247–2254.Ashraf, M. (2010) Inducing drought tolerance in plants: recent advances.

Biotechnol. Adv. 28, 169–183.Bao, A.K., Wang, Y.W., Xi, J.J., Liu, C., Zhang, J.L. and Wang, S.M. (2014)

Co-expression of xerophyte Zygophyllum xanthoxylum ZxNHX and

ZxVP1-1 enhances salt and drought tolerance in transgenic Lotus cor-

niculatus by increasing cations accumulation. Funct. Plant Biol. 41,

203–214.Bao, A.K., Du, B.Q., Touil, L., Kang, P., Wang, Q.L. and Wang, S.M. (2015)

Co-expression of tonoplast Cation/H+ antiporter and H+-pyrophosphatase

from xerophyte Zygophyllum xanthoxylum improves alfalfa plant growth

under salinity, drought and field conditions. Plant Biotechnol. J. 14, 964–975.

Barrag�an, V., Leidi, E.O., Andr�es, Z., Rubio, L., De Luca, A., Fern�andez, J.A.,

Cubero, B. and Pardo, J.M. (2012) Ion exchangers NHX1 and NHX2 medi-

ate active potassium uptake into vacuoles to regulate cell turgor and

stomatal function in Arabidopsis. Plant Cell, 24, 1127–1142.Bassil, E., Tajima, H., Liang, Y.C., Ohto, M.A., Ushijima, K., Nakano, R.,

Esumi, T., Coku, A., Belmonte, M. and Blumwald, E. (2011) The Ara-

bidopsis Na+/H+ antiporters NHX1 and NHX2 control vacuolar pH and K+

homeostasis to regulate growth, flower development, and reproduction.

Plant Cell, 23, 3482–3497.Blumwald, E., Aharon, G.S. and Apse, M.P. (2000) Sodium transport in plant

cells. Biochim. Biophys. Acta, 1465, 140–151.Burrieza, H.P., Koyro, H.-W., Tosar, L.M., Kobayashi, K. and Maldonado, S.

(2012) High salinity induces dehydrin accumulation in Chenopodium qui-

noa Willd. cv. Hualhuas embryos. Plant Soil, 354, 69–79.Chen, D., Yang, B. and Kuo, T. (1992) One-step transformation of yeast in

stationary phase. Curr. Genet. 21, 83–84.Cheong, Y.H., Pandey, G.K., Grant, J.J., Batistic, O., Li, L., Kim, B.G., Lee,

S.C., Kudla, J. and Luan, S. (2007) Two calcineurin B-like calcium sen-

sors, interacting with protein kinase CIPK23, regulate leaf transpiration

and root potassium uptake in Arabidopsis. Plant J. 52, 223–239.Clough, S.J. and Bent, A.F. (1998) Floral dip: a simplified method for

Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J.

16, 735–743.Duan, H.R., Ma, Q., Zhang, J.L., Hu, J., Bao, A.K., Wei, L., Wang, Q., Luan, S.

and Wang, S.M. (2015) The inward-rectifying K+ channel SsAKT1 is a

candidate involved in K+ uptake in the halophyte Suaeda salsa under sal-

ine condition. Plant Soil, 395, 173–187.

Epstein, E., Rains, D.W. and Elzam, O.E. (1963) Resolution of dual mecha-

nisms of potassium absorption by barley roots. Proc. Natl Acad. Sci.

USA, 49, 684–692.Flowers, T.J. and Colmer, T.D. (2008) Salinity tolerance in halophytes. New

Phytol. 179, 945–963.Fuchs, I., St€olzle, S., Ivashikina, N. and Hedrich, R. (2005) Rice K+ uptake

channel OsAKT1 is sensitive to salt stress. Planta, 221, 212–221.Gambale, F. and Uozumi, N. (2006) Properties of shaker-type potassium

channels in higher plants. J. Membr. Biol. 210, 1–19.Gaymard, F., Pilot, G., Lacombe, B., Bouchez, D., Bruneau, D., Boucherez,

J., Michaux-Ferri�ere, N., Thibaud, J.B. and Sentenac, H. (1998) Identifica-

tion and disruption of a plant shaker-like outward channel involved in K+

release into the xylem sap. Cell, 94, 647–655.Gierth, M., M€aser, P. and Schroeder, J.I. (2005) The potassium transporter

AtHAK5 functions in K+ deprivation-induced high-affinity K+ uptake and

AKT1 K+ channel contribution to K+ uptake kinetics in Arabidopsis roots.

Plant Physiol. 137, 1105–1114.Golldack, D., Quigley, F., Michalowski, C.B., Kamasani, U.R. and Bohnert,

H.J. (2003) Salinity stress-tolerant and-sensitive rice (Oryza sativa L.) reg-

ulate AKT1-type potassium channel transcripts differently. Plant Mol.

Biol. 51, 71–81.Gu, M.F., Li, N., Shao, T.Y., Long, X.H., Brestic, M., Shao, H.B., Li, J.B. and

Mbarki, S. (2016) Accumulation capacity of ions in cabbage (Brassica

oleracea L.) supplied with sea water. Plant Soil Environ. 62, 314–320.Guo, Q., Wang, P., Ma, Q., Zhang, J.L., Bao, A.K. and Wang, S.M. (2012)

Selective transport capacity for K+ over Na+ is linked to the expression

levels of PtSOS1 in halophyte Puccinellia tenuiflora. Funct. Plant Biol. 39,

1047–1057.Hirsch, R.E., Lewis, B.D., Spalding, E.P. and Sussman, M.R. (1998) A role

for the AKT1 potassium channel in plant nutrition. Science, 280, 918–921.

Hu, J., Ma, Q., Kumar, T., Duan, H.R., Zhang, J.L., Yuan, H.J., Wang, Q.,

Khan, S.A., Wang, P. and Wang, S.M. (2016) ZxSKOR is important for

salinity and drought tolerance of Zygophyllum xanthoxylum by main-

taining K+ homeostasis. Plant Growth Regul. 80, 195–205.Kaddour, R., Nasri, N., M’rah, S., Berthomieu, P. and Lachaal, M. (2009)

Comparative effect of potassium on K and Na uptake and transport in

two accessions of Arabidopsis thaliana during salinity stress. C. R. Biol.

332, 784–794.Lagarde, D., Basset, M., Lepetit, M., Conejero, G., Gaymard, F., Astruc, S.

and Grignon, C. (1996) Tissue-specific expression of Arabidopsis AKT1

gene is consistent with a role in K+ nutrition. Plant J. 9, 195–203.Li, J., Long, Y., Qi, G.N., Li, J., Xu, Z.J., Wu, W.H. and Wang, Y. (2014) The

Os-AKT1 channel is critical for K+ uptake in rice roots and is modulated

by the rice CBL1-CIPK23 complex. Plant Cell, 26, 3387–3402.Liu, K., Li, L. and Luan, S. (2006) Intracellular K+ sensing of SKOR, a shaker-

type K+ channel from Arabidopsis. Plant J. 46, 260–268.Ma, Q., Yue, L.J., Zhang, J.L., Wu, G.Q., Bao, A.K. and Wang, S.M. (2012)

Sodium chloride improves photosynthesis and water status in the succu-

lent xerophyte Zygophyllum xanthoxylum. Tree Physiol. 32, 4–13.Ma, Q., Li, X.Y., Yuan, H.J., Hu, J., Wei, L., Bao, A.K., Zhang, J.L. and Wang,

S.M. (2014) ZxSOS1 is essential for long-distance transport and spatial

distribution of Na+ and K+ in the xerophyte Zygophyllum xanthoxylum.

Plant Soil, 374, 661–676.Maathuis, F.J. (2009) Physiological functions of mineral macronutrients.

Curr. Opin. Plant Biol. 12, 250–258.Maathuis, F.J. and Sanders, D. (1994) Energization of potassium uptake in

Arabidopsis thaliana. Planta, 191, 302–307.Maathuis, F.J. and Sanders, D. (1996) Mechanisms of potassium absorption

by higher plant roots. Physiol. Plant. 96, 158–168.Maathuis, F.J. and Sanders, D. (2001) Sodium uptake in Arabidopsis roots

is regulated by cyclic nucleotides. Plant Physiol. 127, 1617–1625.Maathuis, F.J., Ichida, A.M., Sanders, D. and Schroeder, J.I. (1997) Roles of

higher plant K+ channels. Plant Physiol. 114, 1141–1149.Pilot, G., Gaymard, F., Mouline, K., Ch�erel, I. and Sentenac, H. (2003) Regu-

lated expression of Arabidopsis shaker K+ channel genes involved in K+

uptake and distribution in the plant. Plant Mol. Biol. 51, 773–787.Quintero, F.J., Garciadebl�as, B. and Rodr�ıguez-Navarro, A. (1996) The SAL1

gene of Arabidopsis, encoding an enzyme with 30(20),50-bisphosphatenucleotidase and inositol polyphosphate 1-phosphatase activities,

increases salt tolerance in yeast. Plant Cell, 8, 529–537.


12 Qing Ma et al.

Ren, X.L., Qi, G.N., Feng, H.Q., Zhao, S., Zhao, S.S., Wang, Y. and Wu, W.H.

(2013) Calcineurin B-like protein CBL10 directly interacts with AKT1 and

modulates K+ homeostasis in Arabidopsis. Plant J. 74, 258–266.Rodr�ıguez-Navarro, A. and Ramos, J. (1984) Dual system for potassium

transport in Saccharomyces cerevisiae. J. Bacteriol. 159, 940–945.Rubio, F., Nieves-Cordones, M., Alem�an, F. and Mart�ınez, V. (2008) Relative

contribution of AtHAK5 and AtAKT1 to K+ uptake in the high-affinity

range of concentrations. Physiol. Plant. 134, 598–608.Sentenac, H., Bonneaud, N., Minet, M., Lacroute, F., Salmon, J.M., Gay-

mard, F. and Grignon, C. (1992) Cloning and expression in yeast of a

plant potassium ion transport system. Science, 256, 663–665.Shabala, S. and Mackay, A. (2011) Ion transport in halophytes. Adv. Bot.

Res. 57, 151–187.Shabala, S., Bose, J., Fuglsang, A.T. and Pottosin, I. (2016) On a quest for

stress tolerance genes: membrane transporters in sensing and adapting

to hostile soils. J. Exp. Bot. 67, 1015–1031.Spalding, E.P., Hirsch, R.E., Lewis, D.R., Qi, Z., Sussman, M.R. and Lewis,

B.D. (1999) Potassium uptake supporting plant growth in the absence of

AKT1 channel activity. J. Gen. Physiol. 113, 909–918.Su, H., Golldack, D., Katsuhara, M., Zhao, C. and Bohnert, H.J. (2001)

Expression and stress-dependent induction of potassium channel tran-

scripts in the common ice plant. Plant Physiol. 125, 604–614.Sunarpi, Horie, T., Motoda, J. et al. (2005) Enhanced salt tolerance medi-

ated by AtHKT1 transporter-induced Na+ unloading from xylem vessels

to xylem parenchyma cells. Plant J. 44, 928–938.Tang, X.L., Mu, X.M., Shao, H.B., Wang, H.Y. and Brestic, M. (2015) Global

plant-responding mechanisms to salt stress: physiological and molecular

levels and implications in biotechnology. Crit. Rev. Biotechnol. 35, 425–437.

Tester, M. and Davenport, R. (2003) Na+ tolerance and Na+ transport in

higher plants. Ann. Bot. 91, 503–527.Uozumi, N., Kim, E.J., Rubio, F., Yamaguchi, T., Muto, S., Tsuboi, A., Bakker,

E.P., Nakamura, T. and Schroeder, J.I. (2000) The Arabidopsis HKT1 gene

homologue mediates inward Na+ currents in Xenopus laevis oocytes and

Na+ uptake in Saccharomyces cerevisiae. Plant Physiol. 122, 1249–1259.Wang, Y. and Wu, W.H. (2013) Potassium transport and signaling in higher

plants. Annu. Rev. Plant Biol. 64, 451–476.Wang, S.M., Wan, C.G., Wang, Y.R., Chen, H., Zhou, Z.Y., Fu, H. and Sose-

beeb, R.E. (2004) The characteristics of Na+, K+ and free proline distribu-

tion in several drought-resistant plants of the Alxa Desert, China. J. Arid

Environ. 56, 525–539.Wang, S.M., Zhang, J.L. and Flowers, T.J. (2007) Low-affinity Na+ uptake in

the halophyte Suaeda maritima. Plant Physiol. 145, 559–571.Wang, C.M., Zhang, J.L., Liu, X.S., Li, Z., Wu, G.Q., Cai, J.Y., Flowers, T.J.

and Wang, S.M. (2009) Puccinellia tenuiflora maintains a low Na+ level

under salinity by limiting unidirectional Na+ influx resulting in a high

selectivity for K+ over Na+. Plant Cell Environ. 32, 486–496.Wang, Y., He, L., Li, H.D., Xu, J. and Wu, W.H. (2010) Potassium channel a--

subunit AtKC1 negatively regulates AKT1-mediated K+ uptake in Ara-

bidopsis roots under low-K+ stress. Cell Res. 20, 826–837.Wang, P., Guo, Q., Wang, Q., Zhou, X.R. and Wang, S.M. (2015) PtAKT1

maintains selective absorption capacity for K+ over Na+ in halophyte Puc-

cinellia tenuiflora under salt stress. Acta Physiol. Plant, 37, 1–10.Wegner, L.H. and De Boer, A.H. (1997) Properties of two outward-rectifying

channels in root xylem parenchyma cells suggest a role in K+ homeosta-

sis and long-distance signaling. Plant Physiol. 115, 1707–1719.Wegner, L.H. and Raschke, K. (1994) Ion channels in the xylem parenchyma

of barley roots (A procedure to isolate protoplasts from this tissue and a

patch-clamp exploration of salt passageways into xylem vessels). Plant

Physiol. 105, 799–813.Wesley, S.V., Helliwell, C.A., Smith, N.A. et al. (2001) Construct design for

efficient, effective and high-throughput gene silencing in plants. Plant J.

27, 581–590.Wu, G.Q., Xi, J.J., Wang, Q., Bao, A.K., Ma, Q., Zhang, J.L. and Wang, S.M.

(2011) The ZxNHX gene encoding tonoplast Na+/H+ antiporter from the

xerophyte Zygophyllum xanthoxylum plays important roles in response

to salt and drought. J. Plant Physiol. 168, 758–767.Xu, J., Li, H.D., Chen, L.Q., Wang, Y., Liu, L.L., He, L. and Wu, W.H. (2006) A

protein kinase, interacting with two calcineurin B-like proteins, regulates

K+ transporter AKT1 in Arabidopsis. Cell, 125, 1347–1360.Yan, K., Shao, H.B., Shao, C.Y., Chen, P., Zhao, S.J., Brestic, M. and Chen,

X.B. (2013) Physiological adaptive mechanisms of plants grown in saline

soil and implications for sustainable saline agriculture in coastal zone.

Acta Physiol. Plant, 35, 2867–2878.Yuan, H.J., Ma, Q., Wu, G.Q., Wang, P., Hu, J. and Wang, S.M. (2015)

ZxNHX controls Na+ and K+ homeostasis at the whole-plant level in

Zygophyllum xanthoxylum through feedback regulation of the expres-

sion of genes involved in their transport. Ann. Bot. 115, 495–507.Yue, L.J., Li, S.X., Ma, Q., Zhou, X.R., Wu, G.Q., Bao, A.K., Zhang, J.L. and

Wang, S.M. (2012) NaCl stimulates growth and alleviates water stress in

the xerophyte Zygophyllum xanthoxylum. J. Arid Environ. 87, 153–160.Zhang, Y.M., Zhang, H.M., Liu, Z.H., Li, H.C., Guo, X.L. and Li, G.L. (2015)

The wheat NHX antiporter gene TaNHX2 confers salt tolerance in trans-

genic alfalfa by increasing the retention capacity of intracellular potas-

sium. Plant Mol. Biol. 87, 317–327.Zhu, J.K., Liu, J. and Xiong, L. (1998) Genetic analysis of salt tolerance in

Arabidopsis. Evidence for a critical role of potassium nutrition. Plant Cell,

10, 1181–1191.



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ZxAKT1 is essential for K+ uptake and K+/Na+ homeostasis ...caoye.lzu.edu.cn/upload/news/N20170220222756.pdf · in the succulent xerophyte Zygophyllum xanthoxylum, which exhibits