ORIGINAL PAPER

ZxSKOR is important for salinity and drought toleranceof Zygophyllum xanthoxylum by maintaining K+ homeostasis

Jing Hu1 • Qing Ma1 • Tanweer Kumar1 • Hui-Rong Duan1 • Jin-Lin Zhang1 •

Hui-Jun Yuan1 • Qian Wang1 • Sardar Ali Khan1 • Pei Wang1 • Suo-Min Wang1

Received: 17 July 2015 / Accepted: 21 February 2016

� Springer Science+Business Media Dordrecht 2016

Abstract As an important nutrient element, K? plays a

crucial role in plant stress resistance. It was reported that the

stelar K? outward rectifying channel (SKOR) is involved in

loading K? into xylem for its transport from roots to shoots.

Zygophyllum xanthoxylum, a succulent woody xerophyte,

could maintain stable K? concentration in leaves to adapt to

salt and arid environments. Here we characterized ZxSKOR

from Z. xanthoxylum, ZxSKOR expression patterns and Na?

and K? accumulation in Z. xanthoxylum treated with various

concentrations ofKCl andNaCl and-0.5 MPa osmotic stress

were investigated in order to assess the contribution of

ZxSKOR to K? homeostasis. The results showed that

ZxSKOR was predominantly expressed in roots and stems

rather than in leaves. Its expression levels in roots and stems

increased significantly accompanied by an increase in K?

concentration in leaves when plants were exposed to

5–10 mM KCl. Moreover, a positive correlation was identi-

fied not only between ZxSKOR expression in roots and K?

accumulation in shoots, but also between ZxSKOR expression

in stems and K? accumulation in leaves. Transcription levels

of ZxSKOR in roots and stems under high salinity

(100–150 mM NaCl) and osmotic stress (-0.5 MPa) were

2.0–2.8 times those in plants grown in the absence of NaCl or

osmotic stress. Concomitantly, the expression level of

ZxSKOR in roots under osmotic stress plus salt (-0.5 MPa

?50 mM NaCl) was significantly higher than that under

osmotic stress (-0.5 MPa) alone during 12–48 h of treat-

ment. We propose that ZxSKOR in roots and stems is well-

coordinated to mediate long-distance K? transport and per-

haps plays an important role in K? accumulation and home-

ostasis in Z. xanthoxylum under salt as well as drought stress.

Keywords Zygophyllum xanthoxylum � SKOR � Long-distance K? transport � Drought and salt tolerance

Abbreviations

AKT Arabidopsis K? transporter

ANOVA Analysis of variance

bp Base pair

FWC Field water capacity

GORK Guard rectifying K? channel

HKT High affinity K? transporter

KAT K? Arabidopsis channel

KIRC K? inward rectifying channel

KORC K? outward rectifying channel

MPa Megapascal

NHX Tonoplast Na?/H? antiporter

ORF Open reading frame

RT-PCR Reverse transcription polymerase chain

reaction

SKOR Stelar K? outward rectifying channel

SOS1 Plasma membrane Na?/H? antiporter

XPCs Xylem parenchyma cells

Introduction

Abiotic stresses such as drought and soil salinity are seri-

ous factors limiting the productivity of agriculture (Wang

et al. 2003; Naveed et al. 2014; Song and Wang 2015).

Electronic supplementary material The online version of thisarticle (doi:10.1007/s10725-016-0157-z) contains supplementarymaterial, which is available to authorized users.

& Suo-Min Wang

smwang@lzu.edu.cn

1 State Key Laboratory of Grassland Agro-ecosystems, College

of Pastoral Agriculture Science and Technology, Lanzhou

University, Lanzhou 730020, People’s Republic of China

123

Plant Growth Regul

DOI 10.1007/s10725-016-0157-z

These two stresses often coexist and are manifested in the

plant through osmotic stress, specific ion toxicity and ionic

imbalances (Munns 2002; Razmjoo et al. 2008), cell

membrane dysfunction, attenuation of metabolic activity

and finally plant growth inhibition and even death (Serrano

et al. 1999; Zhu 2001; Hu and Schmidhalter 2005). How-

ever, some xerophytic species from saline deserts have

acquired efficient measures to adapt to these hostile con-

ditions. Hence, understanding their physiological and

molecular adaptations to abiotic stresses could offer valu-

able information toward enhancing drought and salt toler-

ance of crops (Zhou et al. 2007).

The Zygophyllaceae, Zygophyllum xanthoxylum (Sup-

plementary Fig. S1), a C3 woody species with excellent

drought and salt tolerance, is a typical salt-accumulating

succulent plant distributed widely in the desert region of

northwest China (Liu et al. 1987; Dong and Zhang 2001).

Research has shown that Z. xanthoxylum can accumulate a

large amount of Na? for osmotic adjustment even from low

salt soils (Wang et al. 2004). Further investigations

revealed that Z. xanthoxylum responded to salinity with

increased growth and, moreover, became more tolerant to

drought when the salinity was moderate (around, 50 mM

NaCl) (Ma et al. 2012; Yue et al. 2012). This enhanced

tolerance was closely related to high Na? concentrations in

leaves (Ma et al. 2012; Yue et al. 2012; Ma et al. 2014;

Yuan et al. 2014). For most glycophytes, elevated soil Na?

often induces K? deficiency due to the physiological and

biochemical similarity of the two ions (Maathuis and

Amtmann 1999; Kronzucker et al. 2006; Shabala and Cuin

2008; Horie et al. 2009; Zhang et al. 2010). However, in Z.

xanthoxylum, although Na? concentration in leaves sig-

nificantly increased under salt and drought treatments, K?

concentration was maintained at a relatively constant level

(Wu et al. 2011; Ma et al. 2012; Yue et al. 2012). There-

fore, maintaining K? homeostasis in leaves is a key pro-

tective strategy of Z. xanthoxylum to survive in harsh

environments. However, the molecular mechanism con-

cerning this strategy is still unclear.

Upon uptake from the soil (by root epidermal and hair

cells), K? is distributed to the other root cells and also

delivered to aerial organs via long-distance transport by

transporters/channels across the cell membrane (Boer

1999; Liu et al. 2006). Among these membrane proteins,

ion channels play an essential role in both K? uptake and

long-distance transport (Maathuis et al. 1997; Liu et al.

2006). The inward rectifying K? channel AKT1, expressed

predominantly in root hairs, epidermis, cortex and endo-

dermis of the mature roots is responsible for K? uptake

from the soil (Lagarde et al. 1996; Spalding et al. 1999). As

far as K? long-distance transport in the phloem is con-

cerned, numerous studies have focused on the K? channels

AKT2 and KAT2 which involved in both K? loading in

source leaves and unloading in sinks (Baizabal-Aguirre

et al. 1999; Marten et al. 1999; Deeken et al. 2000; Pilot

et al. 2001; Deeken et al. 2002; Ivashikina et al. 2003; Latz

et al. 2007). However, little research has been conducted

on the molecular mechanism of K? loading into the xylem

from XPCs. Outward rectifying, voltage-gated K? (Kout)

channels have been proven to dominate the K? conduc-

tance of most stelar cells: these channels are believed to

drive K? release into the upward-flowing xylem sap

(Wegner and Raschke 1994; Wegner and De Boer 1997;

Gaymard et al. 1998). SKOR, a Kout channel has been

identified in Arabidopsis thaliana and shown to function in

loading K? from XPCs into xylem process (Gaymard et al.

1998; De Boer and Volkov 2003; Pilot et al. 2003). A.

thaliana AtSKOR displays outward rectifying properties,

and its encoding gene is specifically expressed in the root

pericycle and xylem parenchyma cells (Gaymard et al.

1998; Lebaudy et al. 2007). A knockout mutant of A.

thaliana lacking AtSKOR had both lower shoot K? content

and lower xylem sap K? concentration than wild-type

plants, indicating that AtSKOR contributed to at least 50 %

of K? translocation toward shoots (Gaymard et al. 1998).

However, very little is known about this channel in xero-

phytes, especially, its role in salinity and drought tolerance.

Therefore, in order to elucidate the mechanism of K?

homeostasis in Z. xanthoxylum, we isolated the gene

encoding ZxSKOR and characterized its expression pat-

terns together with K? and Na? accumulation under dif-

ferent concentration of KCl (0–10 mM), NaCl (0–150

mM), osmotic stress (-0.5 MPa) or osmotic stress plus

NaCl (-0.5 MPa ?50 mM NaCl). The results indicated

that the high expression levels of ZxSKOR could increase

salinity and drought tolerance of Z. xanthoxylum by

maintaining K? homeostasis.

Materials and methods

Plant growth conditions and treatments

Seeds of Z. xanthoxylum were collected from Alxa League

(39�050N, 105�340E; elevation 1360 m) in Inner-Mongolia

Autonomous Region of China. After removal of the bracts,

plump seeds were surface sterilized for 10 min in 2.5 %

Na3ClO (v/v) and rinsed 4–5 times with distilled water.

They were then soaked in distilled water at room temper-

ature for 1 day and then at 4 �C for 1 day, then germinated

at 25 �C on filter paper wetted with distilled water in dark

for 2 days. Uniform seedlings were transferred to plastic

containers (5 cm3; 2 seedlings/container) filled with sand

and irrigated with modified Hoagland nutrient solution

containing 2 mM KNO3, 0.5 mM NH4H2PO4, 0.25 mM

MgSO4�7H2O, 0.1 mM Ca(NO3)2�4H2O, 0.5 mM Fe-

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citrate, 92 lM H3BO3, 18 lM MnCl2�4H2O, 1.6 lMZnSO4�7H2O, 0.6 lM CuSO4�5H2O and 0.7 lM (NH4)6-Mo7O24�4H2O. Solutions were renewed every 2 days. The

seedlings were grown in a greenhouse with day/night

temperatures of 28 �C/23 �C, a photoperiod of 16/8 h

(light/dark; the flux density was approximately

600 lmol m-2 s-1) and a relative humidity of about 65 %.

Three-week-old Z. xanthoxylum seedlings (Supplemen-

tary Fig. S2) were used for the following treatments. (1)

Modified Hoagland nutrient solution with or without KNO3

for 3 days (2 mM KNO3 was substituted by 1 mM NH4-

NO3 in the culture solution) and then harvested. (2) Mod-

ified Hoagland nutrient solution without KNO3 for 3 days

(2 mM KNO3 was substituted by 1 mM NH4NO3) and then

0, 0.1, 0.5, 1, 5 or 10 mM KCl was resupplied for 48 h. (3)

Modified Hoagland nutrient solution supplemented with

additional 0, 5, 25, 50, 100 or 150 mM NaCl for 48 h

before harvest. (4) Modified Hoagland solution supple-

mented with additional 50 or 150 mM NaCl, and harvested

at 0, 6, 12, 24 and 48 h after treatments. (5) Modified

Hoagland solution supplemented with additional sorbitol

where the osmotic potential was -0.5 MPa, and then

harvested at 0, 6, 12, 24 and 48 h after treatments. (6)

Modified Hoagland solution supplemented with -0.5 MPa

sorbitol and -0.5 MPa sorbitol plus 50 mM NaCl: plants

were harvested at 0, 6, 12, 24 and 48 h after treatments.

The treatment solutions were changed every day to main-

tain a constant ion concentration.

Cloning of ZxSKOR

Total RNA was extracted with a Trizol Kit (Sangon Bio-

tech Co., Ltd, Shanghai, China) according to the manu-

facture’s instructions (with minor modifications) from roots

of 3-week-old Z. xanthoxylum seedlings (Supplementary

Fig. S2) exposed to 5 mM KCl for 48 h. First-strand cDNA

was synthesized from 4 lg of total RNA as the template

with a cDNA synthesis Kit (Takara, Biotech Co., Ltd,

Dalian, China). The partial cDNA fragment was amplified

by PCR using degenerate primers P1 and P2 (Supple-

mentary Table S1). PCR amplification was programmed at

94 �C for 2 min; 30 cycles of 94 �C for 30 s, 50 �C for

50 s and 72 �C for 70 s; and a final extension at 72 �C for

10 min. PCR products were purified from agarose gels,

ligated into the pGEM-T vector (Promega, China) and

sequenced by Sangon (China). The 50- and 30-ends of

ZxSKOR were obtained with the Kit of RNA Ligase

Mediated Rapid Amplification of 50- and 30-cDNA Ends

(RLM-RACE) (Invitrogen, USA) according to the manu-

facture’s instructions and primers P3, P4, P5 and P6

(Supplementary Table S1), respectively. These fragments

were analyzed and then assembled to be the full-length of

the ZxSKOR cDNA.

DNA sequence and phylogenetic analysis

A sequence BLAST search was performed on the NCBI

platform (http://www.ncbi.nlm.nih.gov/BLAST). Sequence

multi-alignment analysis was run on the DNAMAN 6.0

software (Lynnon Biosoft, USA). Membrane spanning

domains were calculated by hydrophobicity plots using the

program TMpred available at http://www.ch.embnet.orgy/

software/TMPRED form.html. The specific primers were

designed with Primer 5.0 software (Premier Biosoft Inter-

national, USA).

Semi-quantitative RT-PCR

Total RNA was extracted with a Trizol Kit (Sangon Bio-

tech Co., Ltd, Shanghai, China) following the manufac-

ture’s instructions. First strand cDNA was synthesized

from 4 lg of total RNA with MMLV-reverse transcriptase

(Takara, Biotech Co., Ltd, Dalian, China). Semi-quantita-

tive RT-PCR was performed with the specific primers P7

and P8 (Supplementary Table S1), which yielded a RT-

PCR product of 472 bp. The PCR amplification was per-

formed as follows: 94 �C for 2 min; 30 cycles of 94 �C for

30 s, 54 �C for 50 s and 72 �C for 50 s; and a final

extension at 72 �C for 10 min. ACTIN from Z. xanthoxylum

(GenBank accession No. EU019550) was used as the

internal control in the semi-quantitative RT-PCR. The

specific primers of ACTIN that amplified a 598 bp frag-

ment were A1 and A2 (Supplementary Table S1). PCR

amplification was programmed at 94 �C for 2 min; 28

cycles of 94 �C for 30 s, 54 �C for 50 s and 72 �C for 50 s;

and a final extension at 72 �C for 10 min. PCR products

were separated on 1.0 % (w/v) agarose gels containing

ethidium bromide and visualized by AlphaImager (Version

4.0.1) for subsequent analysis. The ratios of the quantity of

mRNA for ZxSKOR to that for ACTIN were calculated, and

the results reflected the relative expression levels. Experi-

ments were repeated at least three times to obtain similar

results.

Determination of Na1 and K1 concentrations

After treatments, plant roots were washed twice for 8 min

in ice-cold 20 mM CaCl2 to exchange cell wall-bound

Na?; stems or leaves were rinsed in deionized water to

remove surface salts (Maathuis and Sanders 2001; Wang

et al. 2007). Plants were separated into roots, stems and

leaves, and then dried in an oven at 80 �C for 48 h to

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123

obtain dry weights. Na? and K? were extracted from dried

plant tissues in 100 mM acetic acid at 90 �C for 2 h. Ion

concentration was determined with a flame spectropho-

tometer (2655-00; Cole Parmer Instrument Co., USA).

Statistical analysis

All the data are presented as means with standard errors

(SE). All statistical analyses including one-way ANOVA

and Duncan’s multiple range tests were performed by

statistical software (SPSS Ver.17.0, SPSS Inc., Chicago,

IL, USA).

Results

Isolation and characterization of ZxSKOR

A fragment of 1004 bp was initially isolated with degen-

erate primers P1 and P2 (Supplementary Table S1) by RT-

PCR. Nucleotide Blast search showed that the isolated

cDNA fragment shared high sequence homology

(72–82 %) with many known SKOR genes from other

plants, suggesting that a partial putative SKOR gene had

been isolated from Z. xanthoxylum. Specific primers

(Supplementary Table S1) were further designed based on

this fragment and 50-RACE and 30-RACE were performed.

Then a 50-RACE product of 980 bp and a 30-RACE product

of 1213 bp were amplified. Finally, a full-length cDNA

from Z. xanthoxylum designated as ZxSKOR was acquired,

which was 2881 bp long and contained an ORF of 2541 bp

nucleotides, encoding 847 amino acid residues with esti-

mated molecular mass of 97 kDa and a theoretical iso-

electric point of 7.0 (data not shown).

Multiple sequence alignment revealed that ZxSKOR

shared high similarity with other SKOR previously char-

acterized in higher plants, and its amino acid sequence

identity to GmSKOR from Glycine max and AtSKOR from

A. thaliana was 71.0 and 70.5 % respectively (Supple-

mentary Fig. S3). Furthermore, the analysis of hydropho-

bicity plot by the TMpred program predicted ZxSKOR had

six transmembrane segments, named S1–S6 (http://www.

sanger.ac.uk/Software/Pfam/), a P (pore) domain, a puta-

tive cyclic nucleotide-binding domain, and an ankyrin

domain (Supplementary Figs. S3, S4), as previously

reported SKOR from other plants. The highly conserved

domains were found in the S5, P domain, and S6. However,

there were also less conservative regions in the N-terminal

(1–94 amino acid) and the C-terminal (752–879 amino

acid) (Supplementary Fig. S3). Moreover, phylogenetic

analysis showed that ZxSKOR was grouped with outward

rectifying K? channels (SKORs), it shared less similarity

with inward rectifying K? channels such as ZxAKT1 (only

25.8 %) (Fig. 1). These data support the view that ZxSKOR

encodes an outward rectifying K? channel SKOR.

Responses of ZxSKOR and cation accumulation in Z.

xanthoxylum under various concentrations of KCl

treatment

In the control condition (2 mM K?), the transcripts of

ZxSKOR were observed in all tissues, and the level in roots

and stems was 3.3 and 1.5 times of that in leaves, respec-

tively (Fig. 2a, b). When plants were exposed to K?

deprivation for 3 days, the expression level of ZxSKOR

decreased significantly in all tissues compared to that under

control condition (2 mM K?) (Fig. 2a, b). These results

indicated that K? deprivation repressed the expression of

ZxSKOR significantly.

After K? starvation for 3 days, the expression patterns

of ZxSKOR were also assayed when plants were resupplied

with various concentrations of KCl (0–10 mM). With

increase of the external KCl concentrations from 0.1 to

10 mM, the transcription levels of ZxSKOR in all tissues

were up-regulated and peaked at 5 mM KCl, then remained

constant (Fig. 2c, d): at the optimal 5 mM KCl, the

expression levels in roots and stems were 13.1- and 9.3-

fold higher than the respective controls (no additional KCl)

(Fig. 2c, d). This result indicated that ZxSKOR was induced

Fig. 1 hylogenetic analysis of ZxSKOR with closely related K?

channels from different plant species. Sources of K? channels and their

GenBank accession numbers are as follows: ZxSKOR (Zygophyllum

xanthoxylum), AtGORK (Arabidopsis thaliana, NP_198566.2), AtSKOR

(A. thaliana, NP_186934.1), VvSOR (Vitis vinifera, XP_002262949.1),

GmSKOR (Glycine max, XP_003544361.1), MtSKOR (Medicago

truncatula, XP_003616247.1), SsORK (Samanea saman, AJ299019.1),

PtSKOR (Populus trichocarpa, XP_002301665.1), NtTORK1 (Nico-

tiana tabacum, BAD81036.1), OsSKOR (Oryza sativa, EEE65456.1),

ZmZORK (Zea mays, AAW82753.1), HvAKT1 (Hordeum vulgare,

ABE99810.1), OsAKT1 (O. sativa, AAL40894.1), NtNKT1 (N.

tabacum, BAD81034.1), VvAKT1 (V. vinifera, XP_002282442.1),

ZxAKT1 (Z. xanthoxylum, ACX37089.1)

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123

and regulated by external KCl. Additionally, ZxSKOR was

preferentially expressed in roots and stems rather than in

leaves under 1–10 mM external K? (Fig. 2c, d).

Compared with control values, the K? concentrations in

roots, stems and leaves increased with the increase of

resupplied KCl concentration (0.1–10 mM) (Fig. 3a). For

Na?, however, additional KCl (0.1–10 mM) had no obvi-

ous impact on its concentrations in roots, stems or leaves

(Fig. 3b). Further analysis exhibited that there was a highly

significant positive correlation between K? concentration

in leaves and relative expression level of ZxSKOR in stems

of Z. xanthoxylum exposed to elevated external KCl con-

centrations (Fig. 3c). A similar relationship was observed

between shoot K? concentration and transcript level of

ZxSKOR in roots (Fig. 3d).

Expression of ZxSKOR under salt treatments

To evaluate the expression pattern of ZxSKOR under saline

condition, seedlings were treated with various concentra-

tion of NaCl (0, 5, 25, 50, 100 and 150 mM) for 48 h. With

the rise of external NaCl concentration, the transcript

abundance of ZxSKOR in leaves was relatively stable,

whereas it increased gradually to maximal levels in roots

and stems under 100 mM NaCl that were 2.0 and 2.8 times,

respectively, of that under control conditions (0 mM NaCl)

(Fig. 4a, b). Our data, therefore, suggested that the

expression of ZxSKOR in roots and stems could be induced

by salinity.

To determine the kinetics for salt-induced activation of

ZxSKOR in roots, its transcript levels were determined

under 50 and 150 mM NaCl over a 48 h period. 50 mM

NaCl had no effects on the expression of ZxSKOR, how-

ever, 150 mM NaCl significantly up-regulated ZxSKOR

expression at 12 h, which reached its peak value at 48 h

(Fig. 4c, d).

Effect of osmotic stress on expression of ZxSKOR

We also explored the transcription of ZxSKOR under

-0.5 MPa osmotic stress for 0, 6, 12, 24 and 48 h. The

expression level of ZxSKOR enhanced with the increase of

treatment time in all tissues, and reached peak values at

24 h in roots and at 48 h in stems and leaves (Fig. 5a, b):

these peak levels were 2.0, 2.6 and 3.6 times of those in

control plants. These results showed that the expression of

ZxSKOR was also induced by osmotic stress.

In order to assess further the response of ZxSKOR in Z.

xanthoxylum to osmotic stress and salinity occurred

simultaneously, plants were exposed to -0.5 MPa osmotic

stress or -0.5 MPa osmotic stress plus 50 mM NaCl over a

48 h period. Interestingly, the transcript abundance of

Fig. 2 Expression of ZxSKOR in Z. xanthoxylum under different

concentrations of KCl. Semi-quantitative RT-PCR analysis of

ZxSKOR mRNA in roots, stems and leaves of 3-week-old seedlings

a treated with (?) or without (-) 2 mM K? for 3 days and c were

grown in the modified Hoagland nutrient solution deprived of KNO3

for 3 days, and then treated with 0, 0.1, 0.5, 1, 5 and 10 mM KCl for

48 h. ACTIN was used as internal control. Experiments were repeated

at least three times. b, d show the relative expression level of ZxSKOR

(related to ACTIN) in various tissues. Values are mean ± SE (n = 3)

and bars indicate SE. Columns with different letters indicate

significant differences at p B 0.05, according to Duncan’s multiple

range test

Plant Growth Regul

123

ZxSKOR in roots was significantly higher under -0.5 MPa

osmotic stress plus 50 mM NaCl than that under -0.5 MPa

osmotic stress alone during 12–48 h of treatment (Fig. 5c,

d), indicating that the expression of ZxSKOR was induced

by co-occurrence of osmotic stress and salinity.

Discussion

Structural and evolutionary analysis of ZxSKOR

in Z. xanthoxylum

Structural analysis showed that the ZxSKOR possessed six

(S1–S6) transmembrane domains, a putative cyclic

nucleotide binding domain, and an ankyrin domain (Sup-

plementary Figs. S3, S4). The sequence and structural

pattern indicated that ZxSKOR belonged to the Shaker

family (Gaymard et al. 1998). It has been reported that the

ankyrin repeat domain in plants is likely to mediate pro-

tein–protein interactions (Sentenac et al. 1992; Very and

Sentenac 2003). Besides, in the ZxSKOR channel, S4

contains positively charged amino acids (Supplementary

Fig. S3). This segment has been proven to be a voltage-

sensing domain in voltage-gated channels (Gaymard et al.

1998; Gierth and Maser 2007). The P domain (located in

S5 and S6) forms the aqueous pore and controls permeation

(Doyle et al. 1998). This domain contained the universal

GYGD motif, a hallmark for the majority of K? selective

channels in plant and animal cells, which is well conserved

in the sequences of plant Shaker-type K? channels (Doyle

et al. 1998) and is also present in ZxSKOR (Supplementary

Fig. S3). Similar to AtSKOR, there were additional resi-

dues in ZxSKOR just upstream from the P domain (Sup-

plementary Fig. S5), which were the most distinctive

difference from the other types of plant K? channels

(Gaymard et al. 1998). In addition, within the Shaker

Fig. 3 Tissue K? and Na? concentration in Z. xanthoxylum and their

relationships with expression of ZxSKOR. a K? and b Na? concen-

tration in roots, stems and leaves of 3-week-old seedlings grown in

the modified Hoagland nutrient solution deprived of KNO3 for 3 days,

and then treated with 0, 0.1, 0.5, 1, 5 and 10 mM KCl for 48 h (two

plants were pooled in each replicate (n = 6)). c, d show the

relationship between K? concentration in leaves and relative ZxSKOR

expression level in stems (c) and K? concentration in shoots and

relative ZxSKOR expression level in roots of Z. xanthoxylum

(d) exposed to 0 (open triangle), 0.1 (open circle), 0.5 (filled

rectangle), 1 (filled square), 5(open square) and 10 (open diamond)

mM KCl treatments for 48 h (n = 3–6). Values are mean ± SE

(n = 3) and bars indicate SE. Columns with different letters indicate

significant differences at p B 0.05, according to Duncan’s multiple

range test

Plant Growth Regul

123

family in plants, which includes both KIRC and KORC;

KORC is further divided into SKOR-type and GORK-type

K? channels (Gambale and Uozumi 2006; Riedelsberger

et al. 2010). Phylogenetic analysis showed that ZxSKOR

was evolutionarily closer to SKOR, such as AtSKOR and

VvSKOR, than to KIRC or GORK-type channels, such as

ZxAKT1 or AtGORK (Fig. 1). Therefore, these results

showed that ZxSKOR encoded a SKOR-type K? channel.

ZxSKOR possibly mediates long-distance K1

transport in Z. xanthoxylum

Potassium (K?), as an essential plant nutrient, plays

important roles in enzyme activation, protein synthesis and

photosynthesis, osmoregulation during cell expansion,

stomatal movements and tropisms (Maser et al. 2002;

Gierth et al. 2005). In A. thaliana, AtSKOR functions in

loading K? into xylem sap toward the shoots (Gaymard

et al. 1998). Northern blot and RT-PCR experiments

showed that AtSKOR expression was restricted to roots

(Gaymard et al. 1998). The expression level of AtSKOR

was down-regulated significantly by K? deprivation (Pilot

et al. 2003), and researches showed that AtSKOR was

activated by external and intracellular K? (Gaymard et al.

1998; Liu et al. 2006). In our study, ZxSKOR shared high

homology (70.5 %) with AtSKOR in A. thaliana (Supple-

mentary Fig. S3). Moreover, the transcripts of ZxSKOR

were observed in all tissues under normal condition (con-

taining 2 mM K?), but at significantly higher levels in

roots and stems than in leaves. The absence of K?

remarkably reduced the expression level of ZxSKOR in all

tissues (Fig. 2a, b). These findings were consistent with

observations on AtSKOR in A. thaliana (Pilot et al. 2003).

The addition of KCl (0.1–10 mM) dramatically triggered

ZxSKOR expression in roots and stems (Fig. 2c, d); and

more interestingly, the transcript level of ZxSKOR in stems

was positively correlated to K? accumulation in leaves

under various K? concentrations (0–10 mM) (Fig. 3c), a

similar positive correlation was also found between the

expression of ZxSKOR in roots and K? accumulation in

shoots (Fig. 3d). Taken together, our findings displayed

that ZxSKOR in roots and stems was well placed to

mediate long-distance K? transport in Z. xanthoxylum and

promote more K? accumulation in leaves.

Fig. 4 Expression of ZxSKOR in Z. xanthoxylum under different

concentrations of NaCl. Semi-quantitative RT-PCR analysis of

ZxSKOR mRNA a in roots, stems and leaves of 3-week-old seedlings

treated with 0, 5, 25, 50, 100 and 150 mM NaCl for 48 h, and c in

roots of 3-week-old seedlings treated with 50 and 150 mM NaCl over

a 48 h period. ACTIN was used as internal control. Experiments were

repeated at least three times. The relative expression level of ZxSKOR

b in roots, stems and leaves, and d in roots (related to ACTIN) under

different concentrations of NaCl. Values are mean ± SE (n = 3) and

bars indicate SE. Columns with different letters indicate significant

differences at p B 0.05, according to Duncan’s multiple range test

Plant Growth Regul

123

ZxSKOR plays a crucial role in improving salt

resistance in Z. xanthoxylum

Na?-induced depletion of tissue K? in most higher plants

has been cited as a contributor to salinity toxicity (Kinraide

1999; Qi and Spalding 2004; Rus et al. 2004; Ashley et al.

2006; Chen et al. 2014). Thus, to survive under salt stress,

it is necessary for plants to operate a highly effective K?

transport system to maintain adequate K? nutrition. In A.

thaliana, AtSKOR was significantly up-regulated in

response to salt stress (Maathuis 2006) and Garcia-Mata

et al. (2010) reported that the rise of K? content in shoots

was related to this up-regulation of AtSKOR under salt

stress. In our study, the expression levels of ZxSKOR in

roots and stems increased continuously to reach maximum

levels under 100 mM NaCl (Fig. 4a, b). Interestingly, the

addition of 5 and 50 mM NaCl significantly improved Na?

concentration in leaves and stems, but had no effect on K?

concentrations; although Na? concentration in leaves and

stems was remarkably enhanced under 150 mM NaCl (by

approximately 4.2-, and 5.1-fold, respectively), K? con-

centration declined smoothly and only by 26 and 22 %,

respectively (Wu et al. 2011). Moreover, the induction of

ZxSKOR mRNA in plants exposed to 150 mM NaCl was

far stronger than that of plants subjected to 50 mM NaCl

(Fig. 4); simultaneously, Z. xanthoxylum shoots accumu-

lated a significantly higher Na? concentration (by 47 %

under 150 mM NaCl) than that under 50 mM NaCl (Sup-

plementary Fig. S6a), while K? concentration in shoots

was only 16 % lower (Supplementary Fig. S6b). Therefore,

we propose that Z. xanthoxylum has the ability to transport

K? efficiently to leaves and maintain K? homeostasis by

up-regulating ZxSKOR, thus avoid K? deficiency caused by

the competition of the high external Na? concentration.

Chen et al. (2005) found that salt stress caused a severe

K? deficiency in shoots of barley. However, K? concen-

tration in the xylem sap increased when plants were treated

with NaCl, and Shabala et al. (2010) speculated that K?-

permeable voltage-sensitive channels (such as SKOR) were

involved in K? xylem loading in barley and operated in a

feedback manner to maintain a constant K?/Na? ratio in

the xylem sap. Similar results were reported for salt-in-

duced xylem K? increase in Populus euphratica species

(Chen et al. 2003). At the XPCs, SOS1 loads Na? into

xylem (Shi et al. 2002), whereas HKT mediates the reverse

Na? flux and unloads Na? from xylem vessels (Ren et al.

2005; Byrt et al. 2007). When Na? in vacuoles of leaves

reaches its maximum concentration in plants exposed to

severe salt stress, transport activities of HKT overwhelm

SOS1 at the membrane of XPCs and thus, Na? is unloaded

Fig. 5 Time courses of ZxSKOR expression in Z. xanthoxylum under

-0.5 MPa osmotic stress and -0.5 MPa osmotic stress plus 50 mM

NaCl. Semi-quantitative RT-PCR analysis of ZxSKOR mRNA a in

roots, stems and leaves of 3-week-old seedlings subjected to

-0.5 MPa osmotic stress, and c in roots of seedlings treated with

-0.5 MPa osmotic stress and -0.5 MPa osmotic stress plus 50 mM

NaCl over a 48 h period. ACTIN was used as internal control.

Experiments were repeated at least three times. b The relative

expression level of ZxSKOR (related to ACTIN) under -0.5 MPa

osmotic stress (in roots, stems and leaves) and d -0.5 MPa osmotic

stress plus 50 mM NaCl (in roots). Values are mean ± SE (n = 3)

and bars indicate SE. Columns with different letters indicate

significant differences at p B 0.05, according to Duncan’s multiple

range test

Plant Growth Regul

123

(Guo et al. 2012). This possibly causes membrane depo-

larization of XPCs (Wegner and Raschke 1994; Roberts

and Tester 1995; Gaymard et al. 1998; Shabala et al. 2010)

or, in the case of Z. xanthoxylum, stimulates the expression

of ZxSKOR (Fig. 4), and subsequently triggers K? pene-

tration into the xylem via ZxSKOR, promoting an

enhancement of long-distance K? transport for maintaining

K? homeostasis in shoots. Therefore, it is proposed that the

strong transportation of K? to leaves mediated by ZxSKOR

is one of the strategies for Z. xanthoxylum to cope with salt

stress.

ZxSKOR plays a key role in maintaining K1

homeostasis in Z. xanthoxylum under osmotic stress

Maintaining adequate K? nutritional status is crucial for

plant adaptation to drought (Cakmak 2005; Shabala and

Pottosin 2014). It is worth noting that while Na? concen-

tration in leaves of Z. xanthoxylum significantly increased

by 64 % under moderate drought (30 % of FWC) and even

120 % under severe drought (15 % of FWC), compared

with the control (70 % of FWC), K? concentration in

leaves remained unchanged (Wu et al. 2011; Ma et al.

2012), implying that maintaining stable K? concentration

in leaves is especially important for Z. xanthoxylum to

adapt to drought. In our current study, the expression of

ZxSKOR in roots and stems was higher than in leaves under

normal conditions and -0.5 MPa osmotic stress, signifi-

cantly induced the transcript level in roots and stems

(Fig. 5a, b), demonstrating that ZxSKOR is a potential

candidate in maintaining stable K? concentration in leaves

under water stress. Surprisingly, under water stress, the

addition of 50 mM NaCl dramatically improved Na?

concentration in leaves by 232 %, while having no visible

impact on K? concentration (Ma et al. 2012). The amount

of ZxSKOR transcript in roots under -0.5 MPa osmotic

stress plus 50 mM NaCl was significantly higher than that

under -0.5 MPa osmotic stress over a 12–48 h period

(Fig. 5c, d). This provide an evidence that up-regulating

the transcript level of ZxSKOR is essential for K? home-

ostasis in leaves, which may be a key determinant for

50 mM NaCl mitigating the deleterious impacts of drought

stress on Z. xanthoxylum (Ma et al. 2012; Yue et al. 2012).

In conclusion, our results demonstrate that outward

rectifying K? channel ZxSKOR in Z. xanthoxylum is likely

to mediate long-distance K? transport from roots to shoots,

and might also be involved in improving the drought and

salt tolerance of Z. xanthoxylum by maintaining K?

homeostasis.

Acknowledgments We are very grateful to Professor Timothy J.

Flowers from University of Sussex, UK, for critically reviewing the

manuscript and for valuable suggestions. This work was supported by

the National Basic Research Program of China (973 Program, Grant

No. 2014CB138701), the National Natural Science Foundation of

China (Grant Nos. 31222053, 31470503 and 31501994), the Spe-

cialized Research Fund for the Doctoral Program of Higher Education

of China (Grant No. 20130211130001).

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