Environmental and Experimental Botany 144 (2017) 11–24

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Environmental and Experimental Botany

journal homepage: www.elsevier.com/locate/envexpbot

Research Paper

Na+ induces the tolerance to water stress in white clover associated with T
osmotic adjustment and aquaporins-mediated water transport and balance
in root and leaf
⁎
Zhou Li1, Dandan Peng1, Xinquan Zhang, Yan Peng , Meng Chen, Xiao Ma, Linkai Huang,
Yanhong Yan
Department of Grassland Science, College of Animal Science and Technology, Sichuan Agricultural University, Chengdu 611130, China

A R T I C L E I N F O A B S T R A C T

Keywords: Aquapotins (AQPs) could be involved in Na+-regulated tolerance to water stress in white clover. White clovers
Aquaporins were subjected to water stress (−0.3 MPa) induced by polyethylene glycol 8000 for 16 days in the presence or in
Na+/K+ transportation absence of NaCl (30 mM) pretreatment. Na+ pretreatment significantly enhanced the tolerance to water stress in
Osmotic adjustment white clovers based on growth and physiological analyses. Na+-induced increases in endogenous Na+ content
Phytohormones
and transcript levels of NHX6, SOS1, and SKOR associated with ions compartmentalization could be responsible
Senescence
Transcription factors
for higher osmotic adjustment (OA) as opposed to the accumulation of more organic osmolytes (proline and
water soluble carbohydrates) in leaf and root. Na+ pretreatment not only further elevated endogenous ABA
content caused by water deficit, but also enhanced the accumulation of AQPs and the expression of genes
encoding AQPs under water stress. The increase in ABA content induced by Na+ could contribute to the en-
hancement of ABA-activated stress defense in relation to transcription factors and AQPs in root and leaf. Na+-
treated white clover exhibited significantly greater root growth and OA in root and leaf, but the significant
decline in transpiration rate in leaf under water stress, which provided a beneficial foundation for the AQPs-
regulated water transport leading to a favourable water status in white clover under water stress. These results
indicated that Na+ could play a positive role in activating ABA-regulated AQPs in response to water deficit in
mesophytic plants.

1. Introduction
The role of Na+ in plants exposed to salt stress and the potentially
toxic effect of Na+ accumulation have been extensively studied. A large
amount of Na+ in soil can decrease soil water potential leading to the
limitation of the water uptake in plant roots. In addition, the over-
accumulation of Na+ in cells not only cause ionic toxicity and ion
leakage, but also could disrupt the absorption of other irons resulting in
nutrition imbalance in plants (Hasegawa et al., 2000; Kawa et al., 2016;
Zhu, 2003). Despite these negative effects, increasing evidence has
demonstrated that plants could acquire stress tolerance after a short-
term exposure to relatively low Na+ (Glenn and Brown, 1998; Martínez
et al., 2005; Saha et al., 2010). Na+ in cells was transported into va-
cuole to maintain osmotic adjustment (OA), which is an important and
common regulatory mechanism of plants in response to water deficit
and salt stress (Hui and Jiang, 2010). Song et al. (2006) found that the
contribution of Na+ to water potential was over 50% in halophytic
species Suaeda physophora and Haloxylon ammodendron under field
condition and arid environment. Na+ accumulation rather than exclu-
sion in succulent xerophytes Haloxylon ammodendron and Zygophyllum
xanthoxylum could be one of the most effective strategies for the
adaptation to drought stress (Wang et al., 2004). The study of Glenn
and Brown (1998) has also showed that an appropriate amount of Na+
uptake in Atriplex canescens could enhance the ability to survive

Abbreviations: ABA, abscisic acid; AQPs, aquapotins; Chl, chlorophyll; CTK, cytokinin; EL, electrolyte leakage; GA, gibberellic acid; IAA, indole-3-acetic acid; LSD, least significance
difference; MDA, malondialdehyde; Mg-CHT, Mg-chelatase; NIPs, nodulin 26-like intrinsic proteins; OA, osmotic adjustment; PAO, pheophorbide a oxygenase; PIPs, plasma membrane
intrinsic proteins; PBGD, porphobilinogen deaminase; PEG, polyethylene glycol; Pn, net photosynthetic rate; POR, protochlorophyllide reductase; qRT-PCR, real-time quantitative
polymerase chain reaction; RGR, relative growth rate; RWC, relative water content; SIPs, small intrinsic proteins; TIPs, tonoplast intrinsic proteins; Tr, transpiration rate; WUE, water use
efficiency; WSC, water soluble carbohydrates
⁎
Corresponding author.
E-mail address: pengyanlee@163.com (Y. Peng).
1
These authors contributed equally to this work.

http://dx.doi.org/10.1016/j.envexpbot.2017.09.011
Received 1 September 2017; Received in revised form 26 September 2017; Accepted 27 September 2017
Available online 28 September 2017
0098-8472/ © 2017 Elsevier B.V. All rights reserved.
Z. Li et al.
drought stress. In absence of K+, Na+ could replace K+ as a critical
inorganic solute in vacuoles for the maintenance of osmotic potential
and also could alleviate the symptoms of K+ deficiency in plants
(Subbarao et al., 2003). Zea mays plants after exposed to low level
salinity could achieve a significant improvement of salt tolerance be-
cause the salt-pretreated plants retained more K+ in roots and en-
hanced vacuolar Na+ sequestration ability in leaves contributing to
better OA in shoots (Pandolfi et al., 2016). In spite of these previous
findings, further understanding of Na+-induced tolerance to water
stress is imperative for mesophytic crops adapting to various abiotic
stresses.
Plants tolerance to water deficit involves complex traits including
phytohormones regulation. Endogenous hormonal metabolism and
balance are known to be highly related to whole-plant stress responses
(Vanstraelen and Benková, 2012). Abscisic acid (ABA) accumulation
induces stomatal closure resulting in the decrease in transpiration and
water loss, which is the primary line of drought defense in plants
(Wilkinson and Davies, 2002). Addition to the regulation of stomatal
closure, ABA promotes antioxidant defense to detoxify reactive oxygen
species (ROS) damage or triggers transcription factors to induce stress-
related genes expression closely associated with enhanced drought
tolerance in plants (Cutler et al., 2010; Lu et al., 2009). Cytokinin (CTK)
in plants could serve a positive regulator in alleviating stress-induced
senescence (Choi and Hwang, 2007; Lim et al., 2003). CTK-activated
antioxidant and carbohydrates metabolism could decrease water def-
icit-induced cellular oxidative damage and also can improve photo-
synthesis and osmotic OA contributing to better tolerance when plants
respond to water deficit (Merewitz et al., 2012, 2011a,b). Gibberellin
(GA) and auxin indole-3-acetic acid (IAA) are previously in relation to
plants growth (Vanstraelen and Benková, 2012). Differently, roles of
GA and IAA in regulating drought tolerance often show contradictory in
plants. The relationship between these two hormones and drought
tolerance depends on plant species, tissue maturity, and stress duration
and intensity (Li et al., 2016; Xing et al., 2016; Zawaski and Busov,
2014; Zhang et al., 2009). The complexity of stress mechanisms sug-
gests that crops can well adapt to water deficit depending on the cross
talk among different hormones (Peleg and Blumwald, 2011;
Vanstraelen and Benková, 2012). Previous studies have proved that the
alleviation of salt damage in plants is associated with changes of en-
dogenous hormones (Javid et al., 2011; Ma et al., 2016). However,
whether positive effects of the pretreatment with Na+ have a correla-
tion with hormones regulation from root to leaf under water stress still
deserves further investigation.
The tolerance to water stress regulated by ABA and CTK is also re-
lated to proteins synthesis in plants, such as dehydrins, metallothionein,
and aquaporins (AQPs) (Cerny et al., 2011; Li et al., 2016; Shatil-Cohen
et al., 2011; Wang et al., 2002). AQPs are a class of intrinsic membrane
proteins that exist in the plasma membrane and most of cell inner
membranes regulating a wide range of water, CO2, and other small
neutral molecules (H2O2, silicic acid, and arsenite) transport in animals
and plants under normal and stress conditions (Chrispeels and Agre,
1994; Groszmann et al., 2017; Maurel et al., 2015). It has been found
that plant AQPs are mainly divided into 4 different subclasses including
plasma membrane intrinsic proteins (PIPs), small intrinsic proteins
(SIPs), tonoplast intrinsic proteins (TIPs), and nodulin 26-like intrinsic
proteins (NIPs) (Maurel et al., 2015). These AQPs, known as water
channels, play critical roles in the regulation of cell-to-cell water
movement through the transcellular path (Javot and Maurel, 2002).
AOPs acting as ion channels have been studied in earlier study (Byrt
et al., 2016). AQPs also showed other important functions in adjusting
transpiration, photosynthesis, and shoot growth with respect to the
improvement of plant performance and recovery from severe water
stress (Maurel et al., 2016; Secchi et al., 2017). The study of Almeida-
Rodriguez and Hacke (2012) has revealed that drought stress induced
the expression of PIP2;3, PIP2;5, and TIP2;1 in stems of poplars (Po-
pulus), which is an important and feasible way to regulate water
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Environmental and Experimental Botany 144 (2017) 11–24
exchange between apoplast and symplast. Improved drought and salt
tolerance in Arabidopsis could be created by overexpressing a MaPIP1;1
gene in relation to the regulation of primary root elongation, water
uptake, and membrane stability (Xu et al., 2014). However, the over-
expression of a Arabidopsis plasma membrane aquaporin PIP1b could
significantly improve cell vigor and photosynthesis in tobacco (Ni-
cotiana tabacum) under favourable growth conditions, whereas fa-
cilitated faster wilting under drought stress (Aharon et al., 2003).
Therefore, functions of different AQPs still need to further investigation
when plants are exposed to water stress. In addition, Na+ directly or
indirectly affects AQPs accumulation or expression in plants. For ex-
ample, NaCl stress reduced the abundance of PIP2 in roots of Arabi-
dopsis, but increased the expression of FrPIP2;1 in leaf of red fescue
(Festuca rubra) (Chen et al., 2005; Diédhiou et al., 2009). It has been
reported that the promoter of NtAQP1 includes ABA response element
(CTAACCA), and the NtAQP1 could be activated by ABA in tobacco
(Siefritz et al., 2001). Many stress-mediated AQPs could also be regu-
lated by ABA in plants (Ding et al., 2016a; Li et al., 2008). In spite of
these studies, limited information is available about Na+-regulated
water transport and balance related to changes of AQPs and ABA in
plants under water-limited condition.
White clover (Trifolium repens), an important legume forage, is
widely cultivated all over the world and provides high quantity of crude
proteins for animal nutrition, but sensitive to water deficit due to
shallow root system and less adaptation to arid environment
(Annicchiarico and Piano, 2004). With a progressive decline in avail-
able irrigation water worldwide, drought-induced limitations of
growth, development, and productivity of crops will further expand
(Cattivelli et al., 2008). In order to better understand the possible re-
lationship between Na+ accumulation and ABA-regulated AQPs in re-
sponse to water deficit, the objectives of this study were to 1) examine
whether appropriate Na+ pretreatment could improve the tolerance to
water stress in mesophytic white clover via analyses of growth and
physiological changes; 2) explore whether OA and Na+/K+ transpor-
tation could be affected by Na+ pretreatment under water stress; and 3)
reveal whether alterations of phytohormones, stress-related genes, and
aquaporins contributing to the regulation of water transport and bal-
ance could be involved in Na+-induced tolerance to water stress in leaf
and root.
2. Materials and methods
2.1. Plant material and treatment
Seeds of white clover cv.‘Ladino’ were surface sterilized using 0.1%
mercuric chloride solution for 4 min and rinsed 3 times with distilled
water. 0.05 g seeds were germinated in each plastic pot (24 cm length,
20 cm width, and 15 cm deep) filled with sterilized quartz sands in a
controlled growth chamber (12 h photoperiod cycle, 23/19 °C day/
night temperature, 730 μmol photons m−2 s−1 photosynthetic active
radiation (PAR), and a relative humidity of 75%). Seven-day-old
seedlings were irrigated with Hoagland’s solution (Hogland and Arnon,
1950) until the second leaves fully expanded. The thirty-day-old plants
in the same size were carefully removed from quartz sands and then
subjected to either 0 (control) or 30 mM sodium chloride (NaCl) in
Hoagland’s solution for 3 days as the pretreatment. The pretreated and
untreated plants were subjected to Hoagland’s solution (control) or
water stress induced by polyethylene glycol (PEG) 8000 dissolving in
Hoagland’s solution for another 16 days, and osmotic potential of PEG
solution was maintained at-0.3 MPa (Vapro pressure osmometer,
Wescor, Inc. Logan, UT 84321, USA). The Hoagland’s solution and PEG
solution were refreshed every day to avoid the change of solution
concentration and aerated through aeration pumps (115 V, 60 Hz,
Tetra® Blacksburg, VA) to provide enough oxygen in solution. Hence,
there were four different treatments: (1) C (water-sufficient control) (2)
C + Na+ (water-sufficient control pretreated with 30 mM NaCl); (3) D
Z. Li et al.
(water stress); and (4) D + Na+ (water-stressed plants pretreated with
30 mM NaCl). Each treatment had four independent replicates. Leaves
and root tissues were taken at 0, 8 and 16 days under water-sufficient
and water-limited conditions for the subsequent measurements. Four
independent biological replicates from each treatment were used for the
analysis of all parameters. The optimum concentration of NaCl was
selected based on a preliminary test (0, 10, 20, 30, 40, and 50 mM)
when phenotypic changes could be obviously observed.
2.2. The measurement of growth and photosynthetic parameters
Mean relative growth rates (RGR) of leaf and root were calculated
as: RGR = (lnWf–lnWi)/Δt where Wf and Wi were final and initial dry
weights of leaf or root, respectively, and Δt was the time elapsed (d)
between the two measurements. Chlorophyll and carotenoid content
was calculated using the methods clearly described in the study of
Strain and Svec (1966). Net photosynthetic rate (Pn) and transpiration
rate (Tr) were measured using a photosynthetic apparatus (Li-Cor 6400,
Li-Cor, Inc., Lincoln, NE). One layer of leaves was placed in the leaf
chamber (400 μl L−1 CO2 and 800 μmol photon m−2 red and blue
light). Water use efficiency (WUE) was calculated following the for-
mula: WUE = Pn/Tr. The method of Salvucci and Anderson (1987) was
used for the measurement of rubisco activity. Briefly, 0.2 g leaves were
ground with 2 mL 100 mM Tris-HCl buffer containing 1 mM RDTA and
10 mM mercaptoethanol (pH 8.2). And then, homogenate was cen-
trifuged at 12,000 rmp for 10 min at 4 °C to get the supernatant. Re-
action buffer (0.3 mL each of 1 mM Tris-HCl buffer, 0.1 M MgCl2, 1 mM
EDTA, 50 mM ATP, 50 mM DTT, and 2 mM NADH) mixed with 0.1 mL
NaHCO3 (200 mM), 0.8 ddH2O, 0.1 mL GAPDH (15U), and 0.1 RuBP
(9 mM), and then mixture was incubated at 30 °C for 10 min. 0.1 mL of
the supernatant was added into the reaction mixture and the absor-
bance was measured at 340 nm.
2.3. The measurement of water status, root viability, and cell membrane
stability
OA in leaf and root was measured according to the method of Blum
(1989). Excised leaves or roots immediately frozen in liquid nitrogen
for 10 min. Frozen leaves or roots were thawed for 30 min at 4 °C, and
then pressed the cell sap for determination of the osmolarity (c) using
an sampling chamber of osmometer (Wescor, Logan, USA). The osmotic
potential was converted based on the formula:
MPa = −c × 2.58 × 10−3. OA was then calculated as the difference in
osmotic potential between stressed leaves and well-watered control
leaves. For the measurement the leaf relative water content (RWC), leaf
samples weighed for fresh weight (FW) and saturated with deionized
water at 4 °C for saturated weight (SW), then dry weight (DW) was
measured after oven-dried at 80 °C for 72 h. RWC was calculated by the
formula RWC (%) = [(FW–DW)/(SW–DW)] × 100 (Barrs and
Weatherley, 1962). Root viability was detected by using the method of
the triphenyltetrazolium chloride (TTC) reduction (Mcmichael and
Burke, 1994).
Electrolyte leakage (EL) determined as the percentage of Cm/Ci.
Fresh leaves or roots (0.2 g) were rinsed with ddH2O for 3 times and
immersed in 15 mL ddH2O for 24 h to measure the initial conductivity
(Ci) using a conductivity meter (YSI Model 32, Yellow Spring, OH).
Leaves then were autoclaved at 100 °C for 15 min to release all elec-
trolytes, and the conductance of the solution was measured as max-
imum conductance (Cm) after cooling to room temperature (Blum and
Ebercon, 1981). For measurement of malondialdehyde (MDA) content,
0.2 g of fresh tissue were homogenated with 2 mL of 50 mM PBS buffer
(pH 7.8) and centrifuged at 4 °C for 30 min at 12,000 rmp. 0.5 mL of
supernatant was added into 1 mL reaction solution containing 0.5% (w/
v) thiobarbituric acid (TBA) and 20% (w/v) trichloroacetic acid (TCA),
and then the mixture was incubated at 95 °C for 15 min. After cen-
trifuged at 8000 rmp for 10 min, the absorbance of reaction solution
13
Environmental and Experimental Botany 144 (2017) 11–24
was measured at 532 and 600 nm, and the concentration of MDA was
calculated by subtraction of OD600 from OD532 (Dhindsa et al., 1981).
2.4. The measurement of osmolytes in cells
For the determination of inorganic ions, 0.5 g of fine ground dry
leaves or roots tissue mixed with 30 mL concentrated HNO3 and HClO4
(5:1, V:V) and the mixture was heated at 180 °C until the mixture so-
lution turned into transparency liquid. After cooling to room tem-
perature, the reaction solution was set the volume to be 50 mL with
ddH2O. The concentration of Na+ and K+ were detected by using a
flame atomic absorption spectrophotometer (Analytik Jena AG, Jena,
Germany) (Zhang and Li, 2010). Free proline was quantified spectro-
photometrically according to the method of Bates et al. (1973). 0.2 g
fresh leaves or roots were homogenized in 3% aqueous sulfosalicylic
acid and then centrifuged at 10,000 rpm. The supernatant was used for
the estimation of the proline concentration. The reaction mixture (1 mL
of supernatant, 1.5 mL of acid ninhydrin, and 1.5 mL of glacial acetic
acid) boiled at 100 °C for 30 min. After termination of reaction in ice
bath, the reaction mixture was extracted with 5 mL of toluene, and
absorbance was read at 520 nm. Water soluble carbohydrates (WSC)
were quantified by using the method of Robyt and White (1987).
100 mg dry weight of tissue was extracted in 4 mL 80% methanol (v/v)
in a water bath (70 °C) for 40 min. The extract was then centrifuged at
5000 rpm for 10 min. 1 mL 5% phenol and 4 mL 98% sulphuric acid
were added into 1 mL supernatant, and the mixture was incubated in a
water bath (100 °C) for 10 min. Once the extract cooled to room tem-
perature, its absorbance was determined at 490 nm.
2.5. The measurement of endogenous phytohormones
To extract ABA, GA and IAA, 0.4 g of fresh leaves or roots were
ground in 3 mL of extraction solution (methanol: isopropanol, 1:4 (v/v)
with 1% of glacial acetic acid) and then incubated at 4 °C for 1 h in the
dark. The d6-ABA, d2-GA4, and d5-IAA were added as internal stan-
dards. The homogenate was centrifuged at 8000 rpm for 15 min at 4 °C.
2 mL of supernatant was evaporated to dryness and redissolved in
300 μl of methanol, and then filtered through a 0.22 μm PTFE filte
(Müller and Munnébosch, 2011). Waters Acquity UPLC- SCIEX Se-
lexION Triple Quad 5500 System mass spectrometer (Waters, Milford
MA, USA) was used for analyzing endogenous ABA, GA and IAA con-
tent. 5 μL of samples were injected into loop and then loaded onto an
Acquity UPLC BEH C18 column (1.7 lm, 50 9 2.1 mm; Waters, Wexford,
Ireland) at 40 °C. Mobile phase was H2O and acetonitrile both con-
taining 0.1% HCOOH at a flow rate of 0.6 mL min−1. Endogenous CTK
content was quantified by method of enzyme linked immunosorbent
assay (ELISA) (Beijing Fang Cheng Biological Technology Co. Ltd.,
China). In brief, 0.4 g fresh tissues were ground in 3 mL 0.1 M phos-
phate buffer (pH 7.4) and then centrifuged at 4 °C for 20 min. The su-
pernatant was used for measurement of CTK content. The procedure
was carried out according to the specification and the absorbance was
read at 450 nm after adding stop solution and within 15 min on a mi-
croplate reader (Synergy HTX, Bio Tek, USA).
2.6. The measurement of aquaporins
The method of Hachez et al. (2011) was used to extract the protein.
0.2 g of leaf or root samples were ground in 1.5 mL extraction buffer
containing 50 mM Tris/HCl (pH 8.0), 250 mM sorbitol, and 0.2 mM
EDTA. The mixture was centrifuged at 4 °C for 10 min at 5000 rpm, and
the supernatant was centrifuged for another 10 min at 10,000 rpm. The
second obtained supernatant was then centrifuged at 100,000 rpm for
30 min and the precipitate was resuspended in suspension buffer (5 mM
H2PO4, 330 mM saccharose, and 3 mM KCl, pH 7.8). The protein con-
centration of the extraction was detected by the method of (Bradford,
2015). 15 μg of equal denatured proteins were loaded in 12% sodium
Z. Li et al.
dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) for
the electrophoresis. The gel was washed in transfer buffer (25 mM Tris,
192 mM glycine, 20% methanol) for 2 times, and then the proteins were
transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore,
Billerica, MA, USA) at 65 V for 2 h. After that, the membrane was
blocked at room temperature in blocking solution (5% non-fatted dry
milk and Tris-Buffered saline with 0.1% Tween 20 (TBST)) for 1 h. The
membrane was incubated at room temperature overnight in TBST
blocking solution with primary antibodies (PIP1, SIP2, and TIP1). After
washing 3 times in TBST solution (each time for 10 min), the membrane
was incubated with horseradish peroxidase coupled goat antimouse IgG
as the second antibody (1: 100000) for 1 h at room temperature. The
immune-blotting signals were detected by using a chemiluminescent
substrate. The protein band was scanned and quantified using Gel-Pro
analyzer 4.0 (Media Cybernetics, Rockville, MD, USA).
2.7. Quantitative real-time PCR (qRT-PCR)
Transcript levels of genes were performed using real-time quanti-
tative polymerase chain reaction (qRT-PCR). For total RNA extraction,
0.2 g of leaves or roots was extracted by using RNeasy Mini Kit (Qiagen,
Germany) according to the manufacturer’s instructions. A revert Aid
First Stand cDNA Synthesis Kit (Fermentas, Lithuania) was used for
reverse-transcribing RNA to cDNA. Primer sequences of all genes were
shown in Table S1. The β-Actin was used as the internal control. Gene
expression levels were determined using an iCycler iQ qRT-PCR de-
tection system with SYBR Green Supermix (Bio-Rad). The conditions of
the PCR protocol for all genes were as follows: 5 min at 95 °C and 40
repeats of denaturation at 95 °C for 15 s, annealing at 57–61 °C (parti-
cular temperature for each gene was recorded in table S1) for 45 s,
following by heating the amplicon from 60 to 95 °C to obtain the
melting curve. The formula 2−ΔΔCt was used for calculating the tran-
script level of all genes (Xia et al., 2009).
2.8. Statistical analysis
The general linear model procedure was used for the analysis of
variance (SAS 9.1, SAS Institute, Cary, NC) for all parameters. The
significance of differences were tested using Fisher’s protected least
significance test (LSD) with p = 0.05.
Fig. 1. Effects of exogenously applied NaCl on (A) the change of phenotype and (B) relative growth rate in white clover under water-sufficient and water-limited conditions. Vertical bars
above columns indicate standard error of each mean (n = 4). Different letters above columns indicate significant difference for a comparison in leaf or root (P = 0.05). C, control;
C + Na+, control + NaCl; D, water stress; D + Na+, water stress + NaCl.
14
Environmental and Experimental Botany 144 (2017) 11–24
3. Results
3.1. Plants growth, photosynthesis, and leaf senescence affected by
exogenous Na+
Fig. 1A showed phenotypic changes of Na+-pretreated and un-
treated white clovers after 16 d of water-sufficient treatment and water
stress. The RGR of leaf between Na+-pretreated and untreated plants
did not show significant difference under water-sufficient condition,
but Na+-pretreated plants exhibited significantly higher leaf RGR than
untreated plants under water stress. The root RGR of plants pretreated
with Na+ increased by 7.2 and 20.9% than untreated plants under
water-sufficient and water-limited conditions, respectively (Fig. 1B).
Under water-sufficient condition, the pretreatment with Na+ could
significantly improve the synthesis of chlorophyll and carotenoid in
leaves. Similarly, exogenous Na+ effectively inhibited water deficit-
induced declines in chlorophyll and carotenoid content during water
stress (Fig. 2A and B). Exogenously applied Na+ could also significantly
improve Pn, WUE, and rubisco activity in leaves under both stress and
non-stress conditions (Fig. 2C, E, and F). Differently, exogenous Na+
induced the increase in Tr under non-stress condition, but the decrease
during water stress (Fig. 2D).
For genes relative expression, RubisCo was significantly up-regu-
lated by Na+ pretreatment at 8 d of water stress (Fig. 3). PBGD, POR,
and Mg-CHT were key genes associated with the synthesis of chlor-
ophyll, while PAO was involved in the degradation of chlorophyll. The
pretreatment with Na+ had no effects on changes of PBGD, Mg-CHT,
and PAO relative expression at 8 d of water-sufficient condition, but
transcript levels of PBGD and Mg-CHT significantly increased in Na+-
pretreated plants as compared to that in untreated plants under water
stress. In addition, the Na+-pretreated plant showed 2.4- and 3.8-times
higher POR transcript level than untreated plants under water-sufficient
and water-limited conditions, respectively. Water stress significantly
induced the increase in the expression of PAO in untreated plants, but
no significant change of PAO expression was observed in Na+-pre-
treated plants under water stress (Fig. 3).
3.2. Water status, cell membrane stability, and root viability affected by
exogenous Na+
The leaf RWC gradually decreased during water stress, but the
plants pretreated with Na+ maintained significantly higher leaf RWC as
compared to untreated plants at 8, 12, and 16 d of water stress
(Fig. 4A). In addition, exogenous application of Na+ had no significant
effects on MDA content and EL in leaves under optimal water condition,
but effectively decreased MDA accumulation and EL in response to
Z. Li et al. Environmental and Experimental Botany 144 (2017) 11–24

Fig. 2. Effects of exogenously applied NaCl on (A) chlorophyll (Chl) content, (B) carotenoid content, (C) net photosynthesis rate (Pn), (D) transpiration rate (Tr), (E) instantaneous water
use efficiency (WUE), and (F) rubisco activity in white clover under water-sufficient and water-limited conditions. Vertical bars above columns indicate standard error of each mean
(n = 4). Different letters above columns indicate significant difference for a comparison at a given day (P = 0.05). C, control; C + Na+, control + NaCl; D, water stress; D + Na+, water
stress + NaCl.

water deficit (Fig. 4B and C). Similarly, MDA content and EL in roots
did not significantly affected by exogenous Na+ pretreatment under
water-sufficient condition (Table 1). Na+-treated plants exhibited 18.6
and 17.4% significantly lower MDA content, and also showed 28.7 and
23.1% lower EL in leaves and roots than untreated plants at 16 d of
water stress, respectively (Fig. 4B,C and Table 1). The application of
Na+ significantly improved root viability throughout water-sufficient
cultivation and water stress (Fig. 4D).
3.3. The effects of applied Na+ on OA, osmolytes and Na+/K+
transportation in cells
OA was significantly enhanced by the pretreatment with Na+ in
leaves and roots, and Na+-treated plants exhibited 87.6 and 59.8%
significantly higher OA than untreated plants in leaves and roots at the
last day of water stress, respectively (Fig. 5). Exogenous supply of Na+
significantly enhanced the accumulation of endogenous Na+, but in-
hibited the absorption of K+ in leaves and roots under water-sufficient
and water-limited conditions (Fig. 6). Na+-treated plants had 49 and 18
times higher Na+ content than untreated plants in roots and leaves
under water-sufficient condition, and also exhibited 26 and 10 times
higher Na+ content as compared to untreated plants in roots and leaves
at 16 d of water stress, respectively (Fig. 6A). Water stress and Na+
pretreatment induced a significant decline in K+ content in roots and
leaves, and Na+-treated plants showed 29% lower K+ content in roots
compared to untreated plants under water-sufficient and water-limited
conditions (Fig. 6B). In leaves, 11% significantly lower K+ content also
was observed in Na+-treated plants relative to that in untreated plants
under water-sufficient and water-limited conditions (Fig. 6B). For
transcript levels of genes related to Na+/K+ transportation, all of these

15
Z. Li et al.
Fig. 3. Effects of exogenously applied NaCl on transcript level of RubisCO, PBGD, POR,
Mg-CHT, and PAO in leaf of white clover at 8 d of water-sufficient and water-limited
conditions. Vertical bars above columns indicate standard error of each mean (n = 4).
Different letters indicate above columns significant difference (P = 0.05). C, control;
C + Na+, control + NaCl; D, water stress; D + Na+, water stress + NaCl.
genes except HKT1 in roots did not affected by the pretreatment with
Na+ in leaves and roots under non-stressed condition (Fig. 7). In leaves,
water stress significantly up-regulated the expression of NHX6, H-A-
TPase, HKT8, HAL2, and SKOR with or without Na+ pretreatment, and
SOS1 was only up-regulated in leaves of Na+-pretreated plants in re-
sponse to water stress (Fig. 7A). In addition, exogenous application of
Na+ further up-regulated water stress-induced NHX6, H-ATPase, and
SKOR expression (Fig. 7A). In roots, exogenous Na+ pretreatment sig-
nificantly increased the transcript level of NHX6 under water-sufficient
and water-limited conditions and also enhanced SKOR expression under
water stress (Fig. 7B). For changes of organic osmolytes, the plants
Fig. 4. Effects of exogenously applied NaCl on (A) leaf relative water content (RWC), (B) malondialdehyde (MDA) content in leaf, (C) electrolyte leakage (EL) in leaf, and (D) root viability
in white clover under water-sufficient and water-limited conditions. Vertical bars represent least significance difference values at a given day of treatment (n = 4). C, control; C + Na+,
control + NaCl; D, water stress; D + Na+, water stress + NaCl.
16
Environmental and Experimental Botany 144 (2017) 11–24
pretreated with Na+ had no effects on the accumulation of free proline
and WSC in leaves and roots under non-stressed condition (Fig. S1 and
Table 1). Water stress enhanced the accumulation of free proline and
WSC in Na+-treated and untreated plants, and exogenous application of
Na+ significantly inhibited the synthesis of free proline and WSC in
leaves and roots under water stress (Fig. S1 and Table 1).
3.4. The effects of exogenously applied Na+ on changes of endogenous
phytohormones
Under water-sufficient condition, ABA and GA content were not
affected by exogenous Na+, whereas CTK and IAA content were ele-
vated by the pretreatment of Na+ in leaves (Fig. 8). Under water stress,
ABA remarkably accumulated in leaves of white clover, and the pre-
treatment with Na+ further enhanced the accumulation of ABA in
leaves when plants were exposed to 8 and 16 d of water stress (Fig. 8A).
Water stress significantly decreased the CTK and GA content regardless
of Na+ treatment in leaves (Fig. 8B and C). However, Na+-treated
plants exhibited 39.4, 34.0, and 26.4% significantly higher CTK content
than untreated plants in leaves at 0, 8, and 16 d of water stress
(Fig. 8B). With the pretreatment of Na+, plants maintained 19.7 and
42.2% significantly higher GA content in leaves at 8 and 16 d of water
stress (Fig. 8C). IAA content in leaves significantly declined under water
stress and did not affect by exogenous Na+ in response to water stress
(Fig. 8D). In roots, there were not significant differences in ABA, CTK,
GA, and IAA content between untreated and Na+-pretreated plants
under non-stressed condition, and water stress only significantly in-
creased the accumulation of ABA, CTK, and GA in roots of Na+-treated
plants (Table 1). IAA content in roots had no significant change in re-
sponse to water stress and exogenous Na+ pretreatment (Table 1).
Z. Li et al. Environmental and Experimental Botany 144 (2017) 11–24

Table 1
Changes in malondialdehyde (MDA), electrolyte leakage (EL), water-soluble carbohydrate (WSC), free proline, and endogenous phytohormones in root of white clover at 16 d of
treatment. Values are mean ± standard error (n = 4). Different letters in a horizontal column indicate a significant difference for each parameter (P = 0.05). C, control; C + Na+,
control + NaCl; D, water stress; D + Na+, water stress + NaCl.

Treatment

C C + Na+ D D + Na+

MDA (nmol g−1 DW) 83.53 ± 6.02 c 86.49 ± 4.66 c 146.61 ± 9.06 a 121.08 ± 5.08 b
EL (%) 42.37 ± 2.08 c 40.44 ± 4.57 c 95.59 ± 8.56 a 73.54 ± 7.53 b
WSC (mg g−1 DW) 51.63 ± 4.45 b 54.98 ± 4.67 ab 60.28 ± 3.49 a 42.86 ± 4.86 c
Free proline (mg g−1 DW) 0.0319 ± 0.0027 c 0.0334 ± 0.0025 c 0.6189 ± 0.0299 a 0.5251 ± 0.0459 b
ABA (μmol g−1 DW) 0.0166 ± 0.0014 b 0.0184 ± 0.0016 b 0.0165 ± 0.0022 b 0.0241 ± 0.0021 a
CTK (μg g−1 DW) 1.60 ± 0.22 b 1.65 ± 0.15 b 1.88 ± 0.22 b 2.72 ± 0.73 a
GA (μmol g−1 DW) 3.78 ± 0.25 ab 3.68 ± 0.45 ab 3.39 ± 0.16 b 3.95 ± 0.44 a
IAA (μmol g−1 DW) 0.153 ± 0.013 a 0.166 ± 0.012 a 0.179 ± 0.025 a 0.181 ± 0.025 a

Fig. 5. Effects of exogenously applied NaCl on (A) osmotic adjustment (OA) in leaf and (B) OA in root of white clover under water-sufficient and water-limited conditions. Vertical bars
represent least significance difference values at a given day of treatment (n = 4). C, control; C + Na+, control + NaCl; D, water stress; D + Na+, water stress + NaCl.

3.5. AQPs and genes encoding AQPs and stress-related transcription factors
affected by Na+ under water-sufficient and −limited conditions
Three types of AQPs were detected including SIP1, PIP2, and TIP1 in
leaves and roots (Figs. 9 and 10). There was no significant difference in
AQPs accumulation between Na+-treated and non-treated plants in
leaves and roots at 0 d. Both of water stress and exogenous Na+ pre-
treatment could significantly induce the accumulation of SIP1, PIP2,
and TIP1 in leaves and roots of white clover at 8 and 16 d (Fig. 9 and
10). Na+-treated plants had 1.7-fold higher PIP1 and TIP1than un-
treated plants in leaves at the last day of water stress (Fig. 9B and D).
SIP2 content in Na+-treated plants went up by 65.8 and 91.9% com-
pared to that in untreated plants at 8 and 16 d of water stress, respec-
tively (Fig. 9C). Similarly, the pretreatment of additional Na+ enhanced
the accumulation of three different AQPs in roots at 8 and 16 d of
water-sufficient and water-limited conditions. Water-stressed plants
with Na+ pretreatment exhibited 50.4, 45.4, and 44.8% significantly
higher PIP1, SIP2, and TIP1 content than water-stressed plants without
Na+ pretreatment in roots at 16 d, respectively (Fig. 10B–D).
Total of 13 genes encoding AQPs were detected at 8 d of treatment
in this study. Both of water stress and Na+ pretreatment had no sig-
nificant effects on transcript levels of PIP1;2 and PIP2;7 in leaves, and
PIP2;1 and TIP2;2 in roots (Fig. 11). Water stress induced remarkable
increases in PIP2;1 and PIP2;2 relative expression in leaves of Na+-
treated and untreated plants, and exogenous pretreatment with Na+
further improved water stress-induced the increase in transcript level of
PIP2;2 (Fig. 11A). Water stress significantly inhibited the expression of
SIP1;1, TIP1;1, and NIP2;1, but Na+-treated plants showed significantly
higher SIP1;1 transcript level than untreated plants in leaves accom-
panied by an increase of 189%. PIP1;1, SIP1;2, TIP2;2, and NIP5;1 were
not induced by water stress, but these genes were significantly up-
regulated in leaves of Na+-pretreated plants under water stress. Na+
pretreatment induced the increase in TIP2;1 expression under water-
sufficient and water-limited condition (Fig. 11A). In roots, the majority
of genes were not affected by Na+ pretreatment under non-stress
condition, but water stress significantly induced increases in the ex-
pression of PIP1;1, SIP1;2, and TIP1;1 (Fig. 11B). Na+-treated plants
had 57, 59, and 32% significantly higher PIP1;1, SIP1;1, and NIP1;2
expression in roots than untreated plants in response to water stress,
respectively (Fig. 11B).
The total of 13 stress-related transcription factors was analyzed in
leaves and roots at 8 d of treatment (Fig. S2). Water stress significantly
up-regulated many genes expression including BREB2, DREB5, bZIP11,
bZIP37, MYB48, WRKY2, WRKY56, WRKY108715, and ERF110, but
down-regulated DREB3 in leaves of Na+-treated and untreated plants
(Fig. S2A). Exogenous Na+ pretreatment further increased the expres-
sion of DREB5, bZIP11, MYB48, WRKY2, WRKY56, WRKY108715, and
ERF110 in leaves under water stress (Fig. S2A). Water stress had no
significant effects on the relative expression of DREB4 and bZIP107 in
leaves of untreated plants, while the transcript levels of them were
significantly up-regulated by Na+ pretreatment in response to water
stress. In addition, MYB112 was not affected by water stress and Na+
pretreatment in leaves (Fig. S2A). In roots, water stress and the pre-
treatment with Na+ had no significant effects on transcript level of
DREB4, DREB5, bZIP11, bZIP37, bZIP107, WRKY2, and ERF110 in this
study, but the transcript levels of DREB2, DREB3, and MYB48 sig-
nificantly decreased under water stress (Fig. S2B). The application of
Na+ inhibited the expression of WRKY56 under water-sufficient con-
dition; on the contrary, the transcript level of this gene was significantly
enhanced by exogenous Na+ pretreatment at 8 d of water stress.

17
Z. Li et al.
Fig. 6. Effects of exogenously applied NaCl on (A) Na+ content and (B) K+ content in
white clover under water-sufficient and water-limited conditions. Vertical bars above
columns indicate standard error of each mean (n = 4). Different letters above columns
indicate significant difference for a comparison in leaf or root (P = 0.05). C, control;
C + Na+, control + NaCl; D, water stress; D + Na+, water stress + NaCl.
Exogenous Na+ pretreatment also significantly up-regulated MYB112
expression in roots under non-stress and stress conditions (Fig. S2B).
Fig. 12 showed that a model for Na+-regulated water transport and
balance involved in abscisic acid (ABA) and aquaporins (AQPs) in white
clover.
4. Discussion
Water loss, growth inhibition, and senescence are most common
symptom in plants under water stress. These negative effects cause
significant decrease in crops yield and quality (Bodner et al., 2015). In
this study, a short term of low Na+ pretreatment could significantly
alleviate water deficit-induced growth inhibition, leaf senescence, and
other physiological damages, as manifested by greater RGR, chlor-
ophyll, carotenoid content, and higher leaf RWC, as well as lower EL
and MDA content in leaf and root of white clover under water stress.
Na+ pretreatment also significantly promoted chlorophyll and car-
otenoid accumulation in leaf of white clover under non-stressed con-
dition. More importantly, Na+-pretreated plants maintained higher Pn
and WUE in leaf throughout water-sufficient treatment and water
stress. It has been reported that the low level application of Na+
(50 mM) had a positive function in promoting drought tolerance in
xero-halophyte Atriplex halimus associated with greater growth and
WUE, which was consistent with our findings (Martínez et al., 2005). In
addition, Na+ obviously improved root viability in white clover under
water-sufficient and water-limited conditions. Thereby, our results
supported that the appropriate Na+ could act as an important role in
maintaining better plant performance under stress and non-stress
18
Environmental and Experimental Botany 144 (2017) 11–24
Fig. 7. Effects of exogenously applied NaCl on transcript level of NHX6, VP1, H-ATPase,
HKT1, HKT8, SOS1, HAL2, and SKOR in leaf of white clover at 8 d of water-sufficient and
water-limited conditions. Vertical bars above columns indicate standard error of each
mean (n = 4). Different letters above columns indicate significant difference
(P = 0.05). C, control; C + Na+, control + NaCl; D, water stress; D + Na+, water stress
+ NaCl.
condition.
Rubisco is the key enzyme in calvin cycle, and its decrease or in-
activation limits carbon fixation of photosynthesis in plants under water
stress (Carmo-Silva et al., 2012; Parry et al., 2002). The porphobili-
nogen deaminase (PBGD), protochlorophyllide reductase (POR), and
Mg-chelatase (Mg-CHT) are important enzymes involved in chlorophyll
biosynthesis. The pheophorbide a oxygenase (PAO) mainly participates
in chlorophyll degradation (Hörtensteiner et al., 1998; Jespersen et al.,
2016). Na+ pretreatment significantly improved rubisco activity, Ru-
bisCO, PBGD, POR, and Mg-CHT expression in leaf of white clover in
response to water stress. However, the transcript level of PAO did not
affect by water stress in Na+-pretreated white clover in spite of water
deficit-induced a significant increase in PAO expression in untreated
plants. Current results suggested that positive regulatory roles of Na+
for the maintenance of growth and the inhibition of senescence in
mesophytic white clover could involve the improvement of rubisco
activity and chlorophyll biosynthesis.
In plants, one of the primary physiological changes in response to
water deficit is to accumulate organic and inorganic solutes in order to
decrease water potential in cells. The enhanced OA has become a cri-
tical strategy of plant adaptation to water stress with regard to water
uptake and balance (Patakas et al., 2002; Sanchez et al., 1998). WSC
and free proline are most abundant organic osmolytes contributing to
OA, while inorganic ions including Na+, K+, and Ca2+ have the same
function of OA in vacuoles (Shabala and Shabala, 2011). In this study,
OA was significantly enhanced by water stress in all plants, while Na+
pretreatment further improved OA in leaf and root conferring higher
leaf RWC in Na+-pretreated white clover during water stress. Water
stress induced large amount of WSC and free proline accumulation in
leaf and root, but did not obviously induce the accumulation of Na+
Z. Li et al.
Fig. 8. Effects of exogenously applied NaCl on (A) abscisic acid (ABA), (B) cytokinin (CTK), (C) gibberellins (GA), and (D) indole-3-acetic acid (IAA) content in leaf of white clover under
water-sufficient and water-limited conditions. Vertical bars above columns indicate standard error of each mean (n = 4). Different letters above columns indicate significant difference
for a comparison at a given day (P = 0.05). C, control; C + Na+, control + NaCl; D, water stress; D + Na+, water stress + NaCl.
and K+. In contrast, K+ content in root significantly declined in all
plants under water stress. Those results suggested that organic solutes
such as sugars and proline might be main osmolytes for OA in meso-
phytic white clover under water stress. However, the previous study has
found that more than 90% of the cell turgor in mannitol-stressed Ara-
bidopsis roots was recovered within 30 min by increased uptake of three
major inorganic ions (Na+, Cl−, and K+) based on the analysis of
combined ion-selective MIFE microelectrodes and a single-cell pressure
probe (Shabala and Lew, 2002). The role of Na+ in OA was over 15% of
total OA in the halophyte Atriplex halimus in response to water deficit
(Martínez et al., 2005). In addition, the xerophyte Zygophyllum xan-
thoxylum had the ability to accumulate larger quantities of Na+ than
K+ for OA even at low soil salinities. This could be an effective mean to
enhance plants adaptation to arid environments (Wang et al., 2004).
Interestingly, Na+ pretreatment decreased water stress-induced the
accumulation of WSC and free proline in white clover. Differently, more
than 10 times Na+ accumulated in cells of leaf and root along with Na+
pretreatment under water-sufficient and water-limited conditions, im-
plying that a large number of available Na+ accumulation in cells could
play a critical role in OA in Na+-pretreated white clover leading to
reductions in the synthesis of organic osmolytes in response to water
deficit. From the view of energy expenditure, the biosynthesis of a
proline or sucrose needs to consume much more ATP than accumulate a
Na+ in plants for OA (Gebre, 2001). Muñoz-Mayor et al. (2013) found
that although the tomato (Solanum lycopersicum) overexpressing HAL1
exhibited the better ability of Na+ exclusion, the transgenic plants in-
duced a greater utilizaiton of organic solutes for OA resulting in a
growth penalty because of the excessive energy cost under salt stress.
Based on earlier and our findings, the Na+ pretreatment might provide
many available Na+ ions for OA instead of going to the synthesis of
organic substances for osmotic balance in cells under water stress.
19
Environmental and Experimental Botany 144 (2017) 11–24
Thereby, the alteration may be propitious to save energy for the
maintenance of growth and stress defense when white clover suffers
from water stress.
A continual build-up of Na+ in cytoplasm is detrimental to plants.
Why did white clover accumulate so many Na+ ions in cells for OA or
other beneficial effects rather than the ionic toxicity in current study?
In plants, it is known that Na+ compartmentalization from cytosol into
the vacuole is not only a key detoxification mechanism, but also plays
an important role in maintaining cell osmotic potential through using
Na+ as a cheap osmolytes in vacuole (Apse and Blumwald, 2007; Wu
et al., 2011). The process of Na+ compartmentalization depends on
Na+/H+ antiporters in plants, such as plasma membrane Na+/H+
antiporter SOS1 and vacuolar/endosomal Na+/H+ antiporters NHX1-6
(Aharon et al., 2003; Pardo et al., 2006). The tonoplast H+-pumps H+-
ATPase and H+-PPase provide proton motive force across tonoplast for
Na+/H+ antiporter to drive Na+ transportation (Gaxiola et al., 2002).
Recent evidences showed that transgenic alfalfa (Medicago sativa) co-
expressing the ZxNHX and ZxVP1-1 encoding H+-PPase improved plant
growth and stress tolerance under salt and drought conditions asso-
ciated with the maintenance of ions homoeostasis and the osmor-
egulation (Bao et al., 2016). Similar results were found in transgenic
Lotus corniculatus overexpressing the ZxNHX and ZxVP1-1 under salt
and drought conditions (Bao et al., 2014). Our findings showed that
Na+ pretreatment further improved water stress-induced increases in
transcript levels of NHX6 and H-ATPase in leaf and also significantly up-
regulated SOS1 expression in leaf and NHX6 expression in root under
water stress. Those results might indicate that Na+-induced NHX6, H-
ATPase, and SOS1 expression could act important roles in Na+ com-
partmentalization for OA as opposed to ionic toxicity in cytosol in re-
sponse to water stress. In addition, previous studies have also proved
that the up-regulation of NHX and SOS1 had beneficial effects on K+
Z. Li et al. Environmental and Experimental Botany 144 (2017) 11–24

Fig. 9. Effects of exogenously applied NaCl on (A) aquaporins abundance, (B) plasma membrane intrinsic protein 1 (PIP1) abundance, (C) small basic intrinsic protein 2 (SIP2)
abundance, and (D) tonoplast intrinsic protein 1 (TIP1) abundance in leaf of white clover under water-sufficient and water-limited conditions. Vertical bars above columns indicate
standard error of each mean (n = 3). Different letters above columns indicate significant difference for a comparison at a given day (P = 0.05). C, control; C + Na+, control + NaCl; D,
water stress; D + Na+, water stress + NaCl.

uptake and intracellular Na+/K+ homeostasis under drought stress
(Bao et al., 2014; Liu et al., 2008; Ma et al., 2014). More genes related
to Na+/K+ homeostasis include HKT mainly controlling Na+ uptake
and Na+/K+ balance in shoots (Horie et al., 2009), HAL promoting
Na+ exclusion and K+ absorbalility in leaf (Muñoz-Mayor et al., 2013),
and SKOR regulating K+ transport from root to shoot (Hu et al., 2016).
The significantly higher transcript abundant of SOS1 in leaf as well as
greater NHX6 and SKOR expression in leaf and root of Na+-pretreated
white clover than untreated plants under water stress might partly
explain that K+ content did not dramatically decline in root and leaf
despite larger amount of Na+ accumulation in cells of white clover.
Significant increases in endogenous ABA, CTK, and GA content in-
duced by Na+ in leaf and root were observed under water stress in
current study. In addition, Na+ pretreatment also further increased the
expression level of abundant transcription factors in leaf and root in
response to water stress. It is well-known that ABA is the positive reg-
ulator of senescence, whereas elevated endogenous CTK content can
effectively suppress stress-caused senescence in plants (Burgess and
Huang, 2016). Exogenous application of CTK or the overexpression of
key genes related to CTK synthesis was found to be an effective way to
alleviate stress-induced leaf senescence in perennial grass species (Liu
and Huang, 2002; Ma et al., 2016; Merewitz et al., 2011a). It was also
reported that foliar application of CTK and GA3 significantly alleviated
drought-imposed adverse effects on growth and yield in maize (Zea
mays) (Akter et al., 2014). Although the accelerated senescence could
be induced by high ABA concentration, the truth is that many processes
of stress defense needs the accumulation of ABA in plants. The reg-
ulation and feedback regulation between transcription factors and ABA
signaling are important parts of acquired stress tolerance in plants
(Agarwal and Jha, 2010; Zong et al., 2016). ABA-induced DREB, bZIP,

20
Z. Li et al. Environmental and Experimental Botany 144 (2017) 11–24

Fig. 10. Effects of exogenously applied NaCl on (A) aquaporins abundance, (B) plasma membrane intrinsic protein 1 (PIP1) abundance, (C) small basic intrinsic protein 2 (SIP2)
abundance, and (D) tonoplast intrinsic protein 1 (TIP1) abundance in root of white clover under water-sufficient and water-limited conditions. Vertical bars above columns indicate
standard error of each mean (n = 3). Different letters indicate significant difference for a comparison at a given day (P = 0.05). C, control; C + Na+, control + NaCl; D, water stress; D
+ Na+, water stress + NaCl.

ERF, and WRKY expression made a greater contribution to improved
drought tolerance in various plant species (Ding et al., 2016b; Liu et al.,
2013; Yoshida et al., 2010; Zhao et al., 2010), which is agree with our
findings. The study of Xiong et al. (2014) revealed that the over-
expression of an OsMYB48-1 effectively enhanced drought tolerance in
rice (Oryza sativa) through regulating drought-induced ABA synthesis
and signaling. Interestingly, the OsMYB48-1 expression also could be
strongly activated by ABA. In Camellia sinensis, ABA up-regulated the
CsWRKY2 involved in defense response to the cold and drought stresses
(Wang et al., 2016). Our current results indicated that the alteration of
endogenous hormones might balance water stress-induced plant growth
inhibition and senescence in white clover. Importantly, Na+-alleviated
growth inhibition and senescence in white clover did not depend on the
decrease in ABA, but might be associated with the increases in CTK and
GA content under water stress. Na+-induced further enhancement of
ABA content could help to improve ABA-activated stress defense in
relation to transcription factors under water stress. Thereby, Na+-
treated plants exhibited the preferable tolerance to water stress partly
due to the improvement of hormone levels and transcription factors
expression in response to water deficit.
Increasing evidences have demonstrated that ABA also has an im-
portant role in the regulation of AQPs. For examples, ABA exhibited a
long-lasting effect on AQPs activities contributing to maintenance of a
favourable water status in maize (Parent et al., 2009); the OST1-de-
pendent phosphorylation of PIP2;1 was involved in ABA-triggered
stomatal closure in Arabidopsis thaliana (Grondin et al., 2015); Ammo-
nium supply significantly enhanced drought tolerance in rice seedling
associated with the interaction of root ABA accumulation and PIP/TIP
expression (Ding et al., 2016a). It was recently reported that the
AtPIP2;1 had the important function of non-selective cation channel
positively associated with Na+ entry into roots (Byrt et al., 2016).
Abundant PIPs expression in roots highly contributed to whole plant
water fluxes when rice responded to water stress (Grondin et al., 2016).
Increased endogenous ABA content, AQPs proteins, and transcript le-
vels of genes encoding AQPs induced by Na+ pretreatment were ob-
served in roots and leaves of white cover under water stress in our

21
Z. Li et al. Environmental and Experimental Botany 144 (2017) 11–24

Fig. 11. Effects of exogenously applied NaCl on transcript level of genes encoding
aquaporins in white clover at 8 d of water-sufficient and water-limited conditions.
Vertical bars above columns indicate standard error of each mean (n = 4). Different
letters above columns indicate significant difference (P = 0.05). C, control; C + Na+,
control + NaCl; D, water stress; D + Na+, water stress + NaCl.

study. These results were similar with the finding of Olaetxea et al.
(2015) who showed that rhizosphere humic acids (RHA) induced a
significant increase in ABA content in roots of cucumber (Cucumis sa-
tivus) companied by the up-regulation of many AQPs genes, which in-
dicated that RHA-regulated root PIPs were crucial to the enhancement
of the plant shoot growth through ABA-dependent and −independent
pathways. However, the enhancement of AQPs accumulation or re- Fig. 12. A model for Na+-regulated water transport and balance involved in abscisic acid
(ABA) and aquaporins (AQPs) in white clover. Osmotic adjustment, OA; Water use effi-
levant genes expression had contradictory results in the regulation of
ciency, WUE; net photosynthetic rate, Pn; transpiration rate, Tr.
drought tolerance in different plant species (Aharon et al., 2003; Li
et al., 2015; Wang et al., 2015). Peng et al. (2007) found that the
overexpression of a PgTIP1 diminished the drought tolerance of trans- WSC). Na+-induced increases in endogenous ABA, CTK, and GA might
genic Arabidopsis grown in shallow (10 cm deep) pots, but improved balance water stress-induced plant growth inhibition and senescence.
tolerance to water stress in transgenic plants grown in deep (45 cm) The increase in ABA content induced by Na+ could help to improve
pots. The possible mechanism for this contrasting response depended on ABA-activated stress defense in relation to transcription factors and
root growth and morphology. Based on current results, effects of Na+ AQPs-regulated water transport under water stress. These results pro-
pretreatment on enhancing AQPs genes expression and proteins accu- vide new evidence for the understanding of the Na+-induced adapta-
mulation could be a useful strategy for white clover surviving water tion to water deficit in mesophytic plant species.
stress through the enhancement of water absorption and transport from
roots to leaves. Na+-treated white clover exhibited significantly greater
root growth and OA in roots and leaves, but significant declined in Conflict of interest
transpiration rate in leaves under water stress, which might avoid water
stress-induced excess water loss and also could provide a beneficial The authors declare that the research was conducted in the absence
foundation for the AQPs-regulated water transport. These changes of any commercial or financial relationships that could be construed as
could lead to a favourable water status and growth in white clover in a potential conflict of interest.
response to water deficit.
In conclusion, the appropriate Na+ pretreatment is an efficient and
cheap measure to improve the tolerance to water stress in white clover Acknowledgements
via the suppression of leaf senescence and the maintenance of better
plant growth and water status under water stress. The Na+ pretreat- This research was supported by grant CARS-35-05 from Modern
ment not only elevated Na+ accumulation in cells, but also enhanced Agro-industry Technology Research System, and by grant NSFC
Na+ compartmentalization from cytosol into the vacuole for OA as 31372371 from National Natural Science Foundation of China.
opposed to the accumulation of organic osmolytes (free proline and

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Z. Li et al. Environmental and Experimental Botany 144 (2017) 11–24

Appendix A. Supplementary data 85, 10–16.

Grondin, A., Rodrigues, O., Verdoucq, L., Merlot, S., Leonhardt, N., Maurel, C., 2015.
Aquaporins contribute to ABA-triggered stomatal closure through OST1-mediated
Supplementary data associated with this article can be found, in the phosphorylation. Plant Cell 27, 1945–1954.
online version, at http://dx.doi.org/10.1016/j.envexpbot.2017.09.011. Grondin, A., Mauleon, R., Vadez, V., Henry, A., 2016. Root aquaporins contribute to
whole plant water fluxes under drought stress in rice (Oryza sativa L.). Plant Cell
Environ. 39, 347–365.
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