Plant Growth Regul
DOI 10.1007/s10725-016-0157-z
ORIGINAL PAPER
ZxSKOR is important for salinity and drought tolerance
of 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
Ó 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 of KCl and NaCl 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 identified 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
Electronic supplementary material The online version of this
article (doi:10.1007/s10725-016-0157-z) contains supplementary
material, which is available to authorized users.
& Suo-Min Wang
[email protected]
1
State Key Laboratory of Grassland Agro-ecosystems, College
of Pastoral Agriculture Science and Technology, Lanzhou
University, Lanzhou 730020, People’s Republic of China
?50 mM NaCl) was significantly higher than that under
osmotic stress (-0.5 MPa) alone during 12–48 h of treatment. We propose that ZxSKOR in roots and stems is wellcoordinated to mediate long-distance K? transport and perhaps plays an important role in K? accumulation and homeostasis in Z. xanthoxylum under salt as well as drought stress.
Keywords Zygophyllum xanthoxylum SKOR Longdistance 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 serious factors limiting the productivity of agriculture (Wang
et al. 2003; Naveed et al. 2014; Song and Wang 2015).
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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). However, some xerophytic species from saline deserts have
acquired efficient measures to adapt to these hostile conditions. Hence, understanding their physiological and
molecular adaptations to abiotic stresses could offer valuable information toward enhancing drought and salt tolerance of crops (Zhou et al. 2007).
The Zygophyllaceae, Zygophyllum xanthoxylum (Supplementary 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 significantly 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). Therefore, maintaining K? homeostasis in leaves is a key protective strategy of Z. xanthoxylum to survive in harsh
environments. However, the molecular mechanism concerning 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 endodermis 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 concerned, numerous studies have focused on the K? channels
AKT2 and KAT2 which involved in both K? loading in
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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? conductance 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 xerophytes, 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 patterns together with K? and Na? accumulation under different 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°050 N, 105°340 E; 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 temperature 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
MgSO47H2O, 0.1 mM Ca(NO3)24H2O, 0.5 mM Fe-
Plant Growth Regul
citrate, 92 lM H3BO3, 18 lM MnCl24H2O, 1.6 lM
ZnSO47H2O, 0.6 lM CuSO45H2O and 0.7 lM (NH4)6Mo7O244H2O. 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 (Supplementary 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 NH4NO3 in the culture solution) and then harvested. (2) Modified 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 supplemented 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 maintain a constant ion concentration.
Cloning of ZxSKOR
Total RNA was extracted with a Trizol Kit (Sangon Biotech Co., Ltd, Shanghai, China) according to the manufacture’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 (Supplementary 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 manufacture’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 International, USA).
Semi-quantitative RT-PCR
Total RNA was extracted with a Trizol Kit (Sangon Biotech Co., Ltd, Shanghai, China) following the manufacture’s instructions. First strand cDNA was synthesized
from 4 lg of total RNA with MMLV-reverse transcriptase
(Takara, Biotech Co., Ltd, Dalian, China). Semi-quantitative RT-PCR was performed with the specific primers P7
and P8 (Supplementary Table S1), which yielded a RTPCR product of 472 bp. The PCR amplification was performed 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 fragment 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. Experiments 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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Plant Growth Regul
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 spectrophotometer (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 degenerate primers P1 and P2 (Supplementary Table S1) by RTPCR. 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 estimated molecular mass of 97 kDa and a theoretical isoelectric point of 7.0 (data not shown).
Multiple sequence alignment revealed that ZxSKOR
shared high similarity with other SKOR previously characterized 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 (Supplementary Fig. S3). Furthermore, the analysis of hydrophobicity 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 putative 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
123
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, respectively (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 9.3fold 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 (Nicotiana 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)
Plant Growth Regul
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
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 obvious 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 concentrations (Fig. 3c). A similar relationship was observed
between shoot K? concentration and transcript level of
ZxSKOR in roots (Fig. 3d).
(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, however, 150 mM NaCl significantly up-regulated ZxSKOR
expression at 12 h, which reached its peak value at 48 h
(Fig. 4c, d).
Expression of ZxSKOR under salt treatments
To evaluate the expression pattern of ZxSKOR under saline
condition, seedlings were treated with various concentration 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)
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
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Plant Growth Regul
Fig. 3 Tissue K? and Na? concentration in Z. xanthoxylum and their
relationships with expression of ZxSKOR. a K? and b Na? concentration 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
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.
ankyrin repeat domain in plants is likely to mediate protein–protein interactions (Sentenac et al. 1992; Véry 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 voltagesensing domain in voltage-gated channels (Gaymard et al.
1998; Gierth and Mäser 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 residues in ZxSKOR just upstream from the P domain (Supplementary 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
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 (Supplementary 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
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Plant Growth Regul
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
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.
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 (Supplementary Fig. S3). Moreover, the transcripts of ZxSKOR
were observed in all tissues under normal condition (containing 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.
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 (Mäser 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
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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
ZxSKOR plays a crucial role in improving salt
resistance in Z. xanthoxylum
(Fig. 4); simultaneously, Z. xanthoxylum shoots accumulated a significantly higher Na? concentration (by 47 %
under 150 mM NaCl) than that under 50 mM NaCl (Supplementary 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? concentration 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-induced 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
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? concentration 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
123
Plant Growth Regul
(Guo et al. 2012). This possibly causes membrane depolarization 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? penetration 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? concentration 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, significantly 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? homeostasis 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 Specialized Research Fund for the Doctoral Program of Higher Education
of China (Grant No. 20130211130001).
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