Soil Science and Plant Nutrition

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Acclimation to NaCl and H2O2 develops cross

tolerance to saline-alkaline stress in Rice (Oryza
sativa L.) by enhancing fe acquisition and ROS
homeostasis

Rowland Maganizo Kamanga, Shohei Oguro, Mami Nampei & Akihiro Ueda

To cite this article: Rowland Maganizo Kamanga, Shohei Oguro, Mami Nampei & Akihiro Ueda
(2021): Acclimation to NaCl and H2O2 develops cross tolerance to saline-alkaline stress in Rice
(Oryza�sativa L.) by enhancing fe acquisition and ROS homeostasis, Soil Science and Plant
Nutrition, DOI: 10.1080/00380768.2021.1952849

To link to this article: https://doi.org/10.1080/00380768.2021.1952849

Published online: 19 Jul 2021.

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SOIL SCIENCE AND PLANT NUTRITION
https://doi.org/10.1080/00380768.2021.1952849

Acclimation to NaCl and H2O2 develops cross tolerance to saline-alkaline stress in

Rice (Oryza sativa L.) by enhancing fe acquisition and ROS homeostasis
Rowland Maganizo Kamangaa, Shohei Ogurob, Mami Nampeia and Akihiro Uedaa
a
Graduate School of Integrated Sciences for Life, Hiroshima University, Higashi-Hiroshima, Japan; bSchool of Applied Biological Science, Hiroshima
University, Higashi-Hiroshima, Japan

ABSTRACT ARTICLE HISTORY

Plants are known to develop cross-tolerance in which plants tolerate to one stress following prior Received 19 May 2021
exposure to another stress. In this study, we investigated this phenomenon in rice (Oryza sativa L.) to Accepted 4 July 2021
elucidate the underlying physiological mechanisms. Twenty-one (21) day-old seedlings were pre- KEY WORDS
exposed to various concentrations of NaCl, NaHCO3, and H2O2 for 7 days and then subjected to saline- Saline-alkalinity; salinity;
alkaline stress (SAS) for 14 days. The present findings report that pre-exposure to 10 mM NaCl and oxidative stress; cross-
10 µM H2O2 develops cross-tolerance to SAS by triggering systemic physiological processes involved in tolerance; acclimation
Fe uptake and ROS homeostasis. While the mechanisms of this tolerance vary among pre-treatments,
a consensus suggests the relevance of the following processes: (1) the ability to maintain a lower growth
medium pH, (2) ability to suppress root K+ losses, (3) maintenance of a stable acquisition of Fe and P and
(4) regulated generation of reactive oxygen species (ROS) via enhanced antioxidant enzyme activities.
Taken together, these results are consistent with the hypothesis that a crosstalk exists between salinity
stress, oxidative stress, and SAS, triggering adaptive physiological responses that develop tolerance to
SAS in rice, a phenomenon that offers a prospect for developing plants tolerant to multiple stressors.

1. Introduction
Saline-alkaline stress (SAS) is a serious environmental threat
that limits agricultural productivity worldwide. It is character­
ized by an inordinately high soil pH (Zhang et al. 2017), hyper­
osmotic stress, ionic toxicity (Na+), and oxidative stress. High pH
limits the bioavailability of some essential micronutrients, par­
ticularly iron, and most efforts aimed at increasing tolerance to
high pH in rice entailed increasing iron uptake (Masuda et al.
2008, 2009; Suzuki et al. 2008). Rice (Oryza sativa L.) is a cereal
crop that is extremely sensitive to salt stress (Munns and Tester
2008) and exhibits poor iron uptake capacities under high pH.
Therefore, SAS poses a much greater risk to rice growth than
sole salinity stress, necessitating the identification of agro­
nomic approaches that can increase the resistance of plants
under mixed SAS conditions.
Plants have remarkable abilities to acclimate to lethal stress
events when subjected to prior mild stress (Pandolfi, Mancuso,
and Shabala 2012). This is referred to as acclimation, which
describes the physiological ability of plants to adapt to stressful
events. Acclimation has been demonstrated as an effective and
quicker way to improve plant tolerance to various abiotic stres­
ses such as salinity stress (Kamanga et al. 2020; Sriskantharajah
et al. 2020; Umezawa et al. 2000). In a primed state, plants are
typified by faster and stronger responses triggered by systemic
defense systems that confer enhanced resistance.
Under SAS conditions, Na+ toxicity, osmotic stress, oxidative
stress, and high pH are key stress factors responsible for growth
inhibition and plant death (Yang et al. 2009; Zhang et al. 2019);
hence, the choice of acclimating agents should aim at those that
may enhance defense responses against these stress factors.
Studies have shown that plant leaves accumulate multiple folds
higher Na+ under SAS conditions compared to neutral salinity
stress (Cao et al. 2020; Jia et al. 2017). While it remains unclear, it
is speculated that high pH either results in uncontrollable uptake
of Na+ by roots or reduces the SOS1-mediated Na+ efflux in plant
roots because of a deficiency in the transmembrane proton
gradient required for SOS1 activity (Haruta and Sussman 2012;
Yang et al. 2019). Therefore, to address the latter, the ability to
maintain a lower growth medium pH is a prerequisite for the
regulation of leaf Na+ toxicity through root Na+ extrusion.
Moreover, rhizospheric acidification is also necessary for iron
uptake in plants utilizing strategy I, solubilizing ferric iron (Fe3+)
to utilizable ferrous iron (Fe2+) for uptake by plant roots.
The key components in a typical stress-response relation­
ship involve stress stimulus, perception of stress by signals,
expression of stress-induced genes, and resultant changes at
morphological, biochemical, and physiological levels (Pastori
and Foyer 2002). The signaling and response pathways have
been reported to overlap during exposure to different abiotic
stresses, including reactive oxygen species (ROS), hormones,
protein kinase cascades, and calcium gradients as common
elements (Atkinson and Urwin 2012). ROS are unstable mole­
cules inevitably produced by plants, both as a result of normal
aerobic metabolism and in response to abiotic stresses.
Meanwhile, more evidence suggests the beneficial roles of
ROS, such as stress signaling (Apel and Hirt 2004; Mittler
2017). Therefore, their fate relies on tight regulation of the

CONTACT Akihiro Ueda akiueda@hiroshima-u.ac.jp Graduate School of Integrated Sciences for Life, Hiroshima University, 1-4-4 Kagamiyama, Higashi-
Hiroshima 739-8528, Japan
© 2021 Japanese Society of Soil Science and Plant Nutrition
2 R. M. KAMANGA ET AL.
equilibrium between their generation and detoxification.
However, this delicate balance is upset under abiotic stress,
resulting in oxidative stress, one of the prime suspected factors
of lethality under SAS conditions (Guo et al. 2014; Zhang et al.
2017). Therefore, plants acclimated to oxidative stress may
show better resistance to SAS by activating systemic defense
systems to counteract the stress. This illustrates the concept of
induced cross-tolerance in which priming of plants to one
stress type enhances tolerance to another stress type (Foyer
et al. 2016; Perez and Brown 2014), offering a prospect for
breeding crop plants resistant to multiple forms of stress.
While studies of simultaneous stress exposure and subse­
quent exposure to similar stress are widespread, little is known
regarding one-at-a-time exposure to different stresses. It is
speculated that the crosstalk between multiple stresses partly
involves the accumulation of ROS (Pastori and Foyer 2002;
Perez and Brown 2014). This recognition has led agronomists
to pursue chemical means to activate ROS-induced systemic
resistance. This includes the direct application of ROS, particu­
larly H2O2 (Fedina, Nedeva, and Çiçek 2009; Wahid et al. 2007),
and the application of salts such as NaCl (Kamanga et al. 2020;
Sriskantharajah et al. 2020) and NaHCO3 (Liu and Saneoka
2019), which are hypothesized to activate antioxidant defense
systems. Nevertheless, little is known about the physiological
mechanisms governing cross-tolerance between oxidative
stress, salinity stress, and SAS. We hypothesized that acclima­
tion to either or a combination of the four key saline-alkalinity
stress factors, namely, Na+ toxicity, osmotic stress, oxidative
stress, and high pH, may trigger inter-stress crosstalk that may
upsurge resistance of rice seedlings to alkaline stress.
2. Materials and methods
2.1. Plant materials and growth conditions
Seeds of rice (Oryza sativa L.) cultivar Hinohikari were incu­
bated, surface-sterilized, and germinated according to
Sriskantharajah et al. (2020). After one week, a slightly modified
Kimura B nutrient solution was added to the hydroponic cul­
ture following Chuamnakthong, Nampei, and Ueda (2019). The
nutrient solution was provided by immersing the plant roots
directly in the hydroponic solution. The experiment was con­
ducted in a greenhouse at Hiroshima University’s Faculty of
Biological Sciences Experimental Field. The hydroponic culture
temperature was maintained at 28°C, and the average day air
temperature was 30°C. After 18 days, seedlings were trans­
planted from a nylon mesh wire to 20 L buckets.
2.2. Treatment conditions
Twenty-six (26) day-old seedlings were subjected to three con­
centrations of each of the following three sets (N = 20) of pre-
treatments for 7 days: (1) NaCl (S) (2.5, 5.0, and 10 mM), (2) H2O2
(OX) (1.0, 5.0, and 10 µM), and (3) NaHCO3 (SA) (0.5, 1.0, and
2.5 mM). A fourth (4) set of plants was non-pre-treated and
maintained as controls (N = 40). Out of these, after 7 days accli­
mation period, half were subjected to saline-alkalinity (NPT)
while the remaining were maintained as control plants (no pre-
treatment and no saline-alkaline stress). Except for set 3, all sets
were maintained at a pH of 5.0–5.5 using 2.0 N HCl and 2.0 N
KOH. After 7 days, pre-treated (PT) and non-pre-treated (NPT)
plants were subjected to main SAS treatment (50 mM Na, pH
8.25) using a mixture of 0.75 mM Na2CO3, 25 mM NaHCO3, and
23.5 mM NaCl. Growth medium pH was monitored every
24 hours but was left unadjusted except for control conditions
adjusted to 5.0–5.5 daily. To evaluate whether pre-treatments
facilitate a quicker recovery after the disappearance of SAS con­
ditions, all PT plants were subjected to 50 mM Na, pH 8.25 for
14 days, at the conclusion of that all stressed plants were recov­
ered when exposed to 0 mM Na (1x Kimura B), pH 5.0–5.5 for
10 days. Shoot and root dry weight, chlorophyll concentration,
and membrane integrity were measured.
2.3. Determination of plant fresh and dry weight
Seedlings were harvested in replicates of 4–5 after 7 days of pre-
treatment period, after 14 days of main stress, and after 10 days
of the recovery period. Plant height and root length were mea­
sured using a plant-measuring ruler. Fresh weight (FW) was
measured immediately after dissecting plants into leaf blades,
leaf sheaths, and roots. Dry weights (DW) were obtained after
oven-drying the samples for at least 72 hours at 70°C.
2.4. Chlorophyll concentration (SPAD)
Leaf-blade chlorophyll concentration was determined using
a chlorophyll meter (SPAD – 502, Minolta Camera Co. Ltd.,
Japan) on three fresh fully expanded leaf blades of the same
positions in all plants according to Kamanga et al. (2020).
2.5. Determination of macro and micronutrient elements
Elemental analysis was performed on dried leaf blades and root
samples after 14 days of stress imposition using inductively
coupled plasma optical emission spectrometry (ICP-OES). For
this purpose, 100 mg of dried leaf blades and roots were
crushed using a freeze crusher (µT – 48, Taitec, Saitama,
Japan). Acid digestion was conducted using 2 mL of H2SO4
followed by repetitive cycles of 1 mL of H2O2 addition and
heating in a heat block at 150–200°C until the sample turned
colorless. An extra hour of heating was performed to remove
the remaining H2O2 after which the sample was filled to 25 mL
using Milli-Q water. ICP analysis was performed using iCAP –
6300 (Thermo-Scientific, USA) at wavelengths 766.4, 818.3,
177.4, 259.9, 249.7, and 213.8, for K, Na, P, Fe, B, and Zn,
respectively. Quantification was performed using various ele­
mental standard solutions prepared in 1% HNO3.
2.6. K+ leakage and Na+ efflux experiment
In order to determine plant roots’ ability to retain K+, leakage of
K+ was assessed by a method previously described in Pandolfi
et al. (2016) with slight modifications. Plants were pre-treated
with various pre-treatments for 7 days, at the conclusion of
which, four uniform seedlings of pre-treated and non-pre-
treated plants were transferred into 50 mL falcon tubes con­
taining 30 mL of 50 mM Na+, pH 8.5 (48 mM NaHCO3 + 1 mM
Na2CO3) for 24 h. Four more seedlings were transferred into

30 mL deionized water as control plants. After 24 hours, solu­
tion was sampled for K+ analysis.
Efflux of Na+ from roots was assessed by a previously outlined
method (Pandolfi et al. 2016; Shabala et al. 2010) with some
modifications. Pre-treated and non-pre-treated plants were sub­
jected to 30 mL of 50 mM Na+, pH 8.5 (48 mM NaHCO3 + 1 mM
Na2CO3) for 24 hours, after which plants were transferred into
bathing medium containing 0.1 mM CaCl2, 0.5 mM KCl and
50 mM Na+ of pH 8.5 (48 mM NaHCO3 + 1 mM Na2CO3). After
1 hour, the solution was poured off and roots were rinsed with
10 mM CaCl2 three times. Then, the roots were transferred into
10 mL bathing medium containing 0.1 mM CaCl2 and 0.5 mM KCl
for 4 hours. The solution was then sampled and Na+ concentration
was measured. Both K+ and Na+ analysis were done using flame
photometry (ANA – 135, Tokyo Photoelectric, Tokyo, Japan).
2.7. Na+ uptake in excised leaves
In order to exclude contribution of Na+ exclusion from uptake
by roots, rice seedlings were pre-treated with various pre-
treatments for 7 days, thereafter uniform leaf blades were
excised under running water, and immersed into 30 mM Na+,
pH 8.0 at about the same depth (30 mm) for 3 days following
a method described in Shabala et al. (2010). Thereafter,
immersed cut ends of leaf blades were thoroughly washed
using deionized water, and dried at 65°C for 72 hours. Then,
50 mg of leaf blades was agitated on a shaker in 1.0 N HCl for
24 hours and measured for Na+ concentration using flame
photometry according to Kamanga et al. (2020).
2.8. Determination of membrane integrity
Membrane integrity was measured using electrolyte leakage
method and lipid peroxidation at the end of 14 days of main
stress and 7 days of recovery. Freshly harvested leaf blade disks
and roots (200 mg) were cut into 2 cm pieces and fully
immersed into 25 mL deionized water in 50 mL falcon tubes
and kept at room temperature for 24 hours. Electrical conduc­
tivity (EC) was then obtained (EC1) using an EC meter (CM-31P,
DKK Toa Co., Tokyo, Japan). Immediately, the samples were
killed by incubating at 100°C for 15 minutes, cooled and EC2
was obtained. Electrolyte leakage rate was calculated according
to Farooq and Azam (2006).
Lipid peroxidation was determined by obtaining malondial­
dehyde (MDA) concentration using the improved thiorbarbitu­
ric acid (TBA) reaction previously described in Hodges et al.
(1999). For this purpose, 100 mg of fresh leaf and root samples
were ground in presence of liquid nitrogen using mortar and
pestle and extracted with 3 mL 80% ethanol after incubating
under room temperature for 20 minutes and centrifuging at
3,000 x g for 10 minutes. Thereafter, two sets of 1 mL of the
supernatant were transferred into two glass tubes respectively,
one containing 1 mL TBA (-) solution (20% trichloro acetic acid
and 0.01% buthyl hydroxy toluene) and another containing
1 mL TBA (+) solution (20% trichloro acetic acid, 0.01% buthyl
hydroxy toluene and 0.65% thiobarbituric acid). MDA concen­
tration was calculated from the absorbances of the samples
measured spectrophotometrically (U-3310, Hitachi, Tokyo,
SOIL SCIENCE AND PLANT NUTRITION 3
Japan) using a 155 mM−1 cm−1 extinction coefficient according
to Hodges et al. (1999).
2.9. Measurement of root and leaf cell viability
Root and leaf cell viability were assessed using Evans Blue
staining procedure as previously described in Zhang et al.
(2017). In brief, fresh root tips and leaf blades were cut into
2 cm long pieces, 100 mg of which were infiltrated with 1.5 mL
of 0.25% aqueous solution of Evans Blue for 30 min under room
temperature. Upon absorption of the stain, leaf and root sam­
ples were rinsed with deionized water until no further blue dye
was eluted from the samples. To quantify the Evans Blue stain
absorbed, the stain was solubilized by incubating in 4 mL of 1%
(w/v) sodium dodecyl sulfate (SDS) prepared in 50% (v/v)
methanol at 50°C for 30 minutes, and measure absorbance at
600 nm using SDS as a blank. Stain absorption was then quan­
tified using Evans Blue standard solutions.
2.10. Determination of hydrogen peroxide concentration
H2O2 concentration was determined using ground frozen sam­
ples based on ferrous oxidation of xylenol orange (FOX) as
previously described in Suharsono et al. (2002). For this pur­
pose, 100 mg of leaf blade and root samples were homoge­
nized with 1 mL cold acetone. The homogenate was
centrifuged at 8,000 x g for 15 minutes at 4°C, and 100 µL of
the supernatant was added to 1 mL FOX reagent comprising
0.25 mM FeSO4, 0.25 mM (NH4)2SO4, 25 mM H2SO4, 125 µM
xylenol orange, and 10 mM sorbitol. The reaction mixture was
left at room temperature for 1 hour and H2O2 concentrations
were determined spectrophotometrically at 560 nm and quan­
tified using H2O2 standard solutions.
2.11. Antioxidant enzyme activity essays
To analyze antioxidant enzyme activities, 300 mg of frozen leaf
blades and root samples were ground in the presence of liquid
nitrogen using a mortar and pestle, extracted using 3 mL potas­
sium phosphate (KPB) buffer (pH 7.0), and centrifuged at
10,000 × g for 15 minutes at 4°C. Catalase (CAT, EC 1.11.1.6)
activity was measured as previously described (Mekawy,
Abdelaziz, and Ueda 2018; Takagi and Yamada 2013). One
unit of CAT activity was determined as mmol of H2O2 con­
sumed min−1 mg−1 protein using an H2O2 extinction coefficient
of 0.0436 mM−1 cm−1 at 240 nm. Ascorbate peroxidase (APX, EC
1.11.1.11) was assayed according to Mekawy, Abdelaziz, and
Ueda (2018). One unit of APX activity was determined as µmol
of ascorbate oxidized min−1 mg−1 protein using an extinction
coefficient of 2.8 mM−1 cm−1 at 290 nm. Glutathione reductase
(GR, EC 1.8.1.7) activity was assayed according to the outlined
method in Liu and Saneoka (2019). One unit of GR activity was
determined as µmol NADPH oxidized min−1 mg−1 protein using
an extinction coefficient of 6.22 mM−1 cm−1 at 340 nm.
Superoxide dismutase (SOD, EC.1.15.1.1) activity was assayed
based on its ability to inhibit photochemical reduction of nitro
blue tetrazolium (NBT), as outlined in Beauchamp and Fridovich
(1971). One unit of enzyme activity was defined as the quantity
of enzyme that caused 50% inhibition of NBT photoreduction
4 R. M. KAMANGA ET AL.

at 560 nm. Protein concentration in the enzyme extract was

measured using the Bradford reagent (Bradford 1976) with
bovine serum albumin as a standard.

2.12. Statistical analyses

The experiment was arranged in a completely randomized
design (CRD). Each measurement was performed using four to
five biological replicates. Inferential statistics were performed
using analysis of variance (ANOVA) at a significance level of
0.05. Multiple comparison tests were performed using Tukey’s
test. All data were analyzed using R Studio.

3. Results
3.1. Effectiveness of pre-treatments on plant growth
After 7 days of pre-treatments, no significant differences in
growth were recorded among pre-treated and non-pre-treated
plants (Figure 1A). At the end of 14 days of main stress, shoot
and root dry weights were considerably decreased in NPT plants
(Figure 1B). However, pre-treatment with 5 mM and 10 mM NaCl,
and all concentrations of H2O2 (1, 5, and 10 µM) significantly
increased shoot DW. Interestingly, acclimation to mild NaHCO3
(SA) pre-treatments was not effective (Figure 1B), decreasing
growth except for 0.5 mM NaHCO3, which slightly increased
both shoot and root growth. Overall, 10 mM NaCl, 10 µM H2O2,
and 0.5 mM NaHCO3 were the most effective in their respective
groups; hence, these pre-treatments were chosen for subse­
quent experiments and will be the main focus of this study.

3.2. Acclimation to NaCl and H2O2 avert chlorosis under

SAS conditions
At the end of the pre-treatment period, chlorophyll concentra­
tion was significantly reduced by NaHCO3 pre-treatment,
whereas no differences were observed between controls, NaCl,
and H2O2 PT plants (Figure 1C). After 14 days of main stress,
chlorophyll concentration was significantly reduced in NPT and
NaHCO3 PT seedlings. However, the chlorophyll concentration
was unchanged in NaCl and H2O2 PT plants (Figure 1C).
3.3. NaCl and H2O2 pre-treatments suppressed rise of
growth medium pH
During the onset of stress, growth medium pH generally pro­
gressively increased, reaching 8.45, by the end of 14 days of
stress from the initial 8.0, in NPT plants (Figure 2). In compar­
ison to NPT, pre-treatments with NaCl were the most effective
in suppressing growth medium pH (Figure 2A), followed by H2
O2 pre-treatments (Figure 2B). Strikingly, PT with 1.0 and
2.5 mM NaHCO3 had considerably higher growth medium pH
of up to 8.7, by the end of the 14-day stress period, whereas
0.5 mM NaHCO3 did not significantly change it (Figure 2C).
3.4. Pre-treatments regulate accumulation of K+, Na+ and
p in roots and leaf blades
Significant reductions in K concentration were observed in
both leaf blades and roots, which were particularly
Figure 1. Vegetative growth and physiological data showing dry weight after
a 7-day acclimation period (A) and dry weight (B) and leaf chlorophyll concentra­
tion shown as SPAD units (C) after 14 days of exposure to main saline-alkaline
stress (SAS) treatment (B). plants were pre-treated with various concentrations of
NaCl (S) (2.5, 5.0, and 10 mM), H2O2 (OX) (1.0, 5.0, and 10.0 µM), and NaHCO3 (SA)
(0.5, 1.0, and 2.5 mM) for 7 days and subjected to 50 mM Na, pH 8.25 for 14 days.
the data represents means ± standard errors from five biological replicates.
different letters indicate significant differences by tukey’s multiple comparison
tests at P < 0.05.
pronounced in roots (Figure 3A). However, in leaf blades,
PT with 10 µM H2O2 and 0.5 mM NaHCO3 significantly
increased K concentration, whereas, all pre-treatments sig­
nificantly increased K concentration in roots (Figure 3A).
This was coincidental with a significant increase in Na con­
centration, especially in the roots. In leaf blades, PT with
10 mM NaCl significantly reduced Na accumulation, as did
NaHCO3, but H2O2 PT led to the highest accumulation of Na
(Figure 3B). However, in roots 10 mM NaCl and 10 µM H2O2
pre-treatments significantly reduced the Na concentration.
P concentration was significantly reduced in both leaves
 SOIL SCIENCE AND PLANT NUTRITION 5

Figure 2. Rhizosphere acidification data showing daily pH changes in the growth medium during a 14-day period of main saline-alkaline stress (SAS) treatment in
plants pre-treated with various concentrations of NaCl (A), H2O2 (B), and NaHCO3 (C) versus non-pre-treated seedlings. plants were pre-treated with various
concentrations of NaCl (S) (2.5, 5.0, and 10 mM), H2O2 (OX) (1.0, 5.0, and 10.0 µM), and NaHCO3 (SA) (0.5, 1.0, and 2.5 mM) for 7 days and subjected to 50 mM Na,
pH 8.25 for 14 days.

Figure 3. Macro and micronutrient analyses showing concentrations of potassium (A), sodium (B), phosphorus (C), iron (D), boron (E), and zinc (F) in leaf blades and
roots after 14 days of exposure to main saline-alkaline stress (SAS) treatment. plants were pre-treated with 0 (NPT), 10 mM NaCl (S10), 10 µM H2O2 (OX10), and 0.5 mM
NaHCO3 (SA0.5) for 7 days and subjected to 50 mM Na, pH 8.25 for 14 days. the data represents means ± standard errors from four biological replicates. different letters
indicate significant differences by tukey’s multiple comparison tests at P < 0.05.
6 R. M. KAMANGA ET AL.

Figure 4. Physiological experiments for studying K+ and Na+ homeostasis. K+ leakage in roots of 7-day pre-treated (PT) and non-pre-treated (NPT) seedlings subjected
to 50 mM Na, pH 8.50 for 24 h (A). Efflux of Na+ from roots of 7-day PT and NPT seedlings, subjected to 50 mM Na, pH 8.50 for 24 h and deionized water for 2 h (B).
Concentrations of Na+ (C) and chlorophyll (D) in excised leaves from 7-day PT and NPT plants subjected to 30 mM Na, pH 8.0 for 3 days. Plants were pre-treated with 0
(NPT), 10 mM NaCl (S10), 10 µM H2O2 (OX10), and 0.5 mM NaHCO3 (SA0.5) for 7 days and subjected to the above experiments. the data represents means ± standard
errors from four biological replicates. different letters indicate significant differences by tukey’s multiple comparison tests at P < 0.05.

and roots, whereas NaHCO3 and H2O2 PT significantly
increased it in roots (Figure 3C).
3.5. Stable micronutrient acquisition was critical for
tolerance to SAS conditions
All three pre-treatments significantly increased Fe concentrations
in both organs; however, this was most pronounced in roots
(Figure 3D). The same was observed for boron (B) in both leaf
blades and roots (Figure 3E). In leaf blades, Zn concentration was
significantly reduced by SAS treatment, except in plants accli­
mated using 0.5 mM NaHCO3, which accumulated nearly two-
fold higher Zn than NPT, NaCl, and H2O2 PT plants (Figure 3F),
whereas, Zn concentration was significantly increased by SAS
treatment in the roots of all PT plants (Figure 3F).
3.6. Root K+ leakage and Na+ efflux
Plant roots’ K+ leakage was extremely high under SAS conditions
regardless of pre-treatments (> 60-folds higher) (Figure 4A). The
efflux of Na+ was studied in a 4-hour period, and showed that
saline-alkaline stress triggered significantly higher Na+ efflux
(Figure 4B); however, this was more pronounced in pre-treated
plants, particularly by 10 mM NaCl in part, elucidating its lower
leaf blade and root Na+ concentrations.
3.7. Na+ uptake in excised leaves
When we eliminated the ability of the plant roots to control Na+
uptake, 10 mM NaCl and 10 µM H2O2 significantly reduced leaf
blade Na+ concentration, whereas NPT and 0.5 mM NaHCO3 PT
plants accumulated significantly higher amounts of Na+
(Figure 4C). Chlorophyll concentration in the excised leaves
showed significantly lower chlorophyll concentrations in NPT
and 0.5 mM NaHCO3 PT leaf blades (Figure 4D), which could be
attributed to the higher Na+ accumulation.
3.8. Oxidative stress, membrane integrity and cell
viability
In this study, leaves of NPT plants exhibited considerably higher
leakage of electrolytes (Figure 5A), whereas, no significant
differences were detected in the roots of PT plants, albeit
with higher values than leaf blades (Figure 5A). This was further
verified by measurement of MDA, a marker of lipid peroxidation
that showed significant reductions in leaf blades, particularly by
NaCl and H2O2 pre-treatments, whereas, only NaCl PT reduced
MDA levels in roots (Figure 5B). Cell viability was significantly
enhanced in the leaf blades of all PT plants (Figure 5C). In the
roots, cell viability was enhanced only by NaCl and H2O2 pre-
treatments (Figure 5C). The H2O2 concentration was signifi­
cantly reduced in leaf blades by 10 µM H2O2 and 0.5 mM
 SOIL SCIENCE AND PLANT NUTRITION 7

Figure 5. Oxidative stress, membrane integrity, and cell viability parameters showing leakage of electrolytes (A), malondialdehyde concentrations (B), Evans Blue
staining (C), and concentrations of hydrogen peroxide (D) in leaf blades and roots after 14 days of main saline-alkaline stress (SAS) treatment. Plants were pre-treated
with 0 (NPT), 10 mM NaCl (S10), 10 µM H2O2 (OX10), and 0.5 mM NaHCO3 (SA0.5) for 7 days and subjected to 50 mM Na, pH 8.25 for 14 days. The data represents means
± standard errors from four biological replicates. different letters indicate significant differences by Tukey’s multiple comparison tests at P < 0.05.

NaHCO2 pre-treatments, whereas, the H2O2 concentration in
roots was significantly reduced by all pre-treatments
(Figure 5D).
3.9. Enzymatic ROS antioxidant capacities
CAT activity was significantly increased in the leaf blades of
NaCl and H2O2 PT plants, whereas it was significantly increased
in the roots of NaCl and NaHCO3 PT plants (Figure 6A). APX
activity was considerably increased in both the leaves and roots
of NaCl and H2O2 PT plants (Figure 6B). Strikingly, the activities
of APX were much higher in roots than in leaves (Figure 6B).
Furthermore, GR activity was significantly induced in leaf
blades; however, it was significantly induced only in PT plants
(Figure 6C). In roots, no GR activity was detected in any of the
samples. Finally, superoxide dismutase (SOD) activity was
assayed; no significant differences in SOD activity were
observed in the leaf blades among PT plants, except for H2O2
pre-treatment, which considerably decreased its activity
(Figure 6D). However, SOD activity was significantly increased
in the roots of PT plants. In NPT plants, SOD activity was con­
siderably suppressed (Figure 6D). In leaf blades, NPT and NaCl
PT plants had significantly higher protein concentrations,
whereas, in roots, NaHCO3 PT plants had the highest protein
concentration. Comparatively, the protein concentration was
significantly higher in leaf blades than in roots (Figure 6E).
3.10. H2O2 pre-treatment facilitates a quicker recovery
upon withdrawal of SAS conditions
The control plants grew approximately three times faster dur­
ing recovery than during the stress period (Figure 7
(A-Figure 7B)). However, in previously stressed plants, despite
subjecting them to similar recovery conditions, growth rates
did not significantly change. However, both shoot and root
growth rates were considerably increased in H2O2 PT plants
(Figure 7(A-Figure 7B)). Therefore, 10 µM H2O2 PT facilitates
quicker recovery and resumption of growth when stress is
concluded. Strikingly, in roots, NaHCO3 PT significantly reduced
the root mean growth rate during recovery (Figure 7B), sug­
gesting that irreparable damage was inflicted in NaHCO3 PT
and NPT plants. Chlorophyll concentration was recovered in PT
plants but not in NPT plants (Figure 7C). Furthermore, mem­
brane integrity was recovered in all plants, regardless of PT
(Figure 7D). These observations imply that during the recovery
period, some plants could physiologically recover, despite the
inability of some plants to resume rapid growth.
4. Discussion
Rice is extremely sensitive to salt stress, although it is one of
the prominent staples in the world. Hence, it remains impera­
tive to identify quicker, yet effective means to increase rice
8 R. M. KAMANGA ET AL.

Figure 6. Antioxidative enzyme activities showing activities of catalase (A), guaiacol peroxidase (B), ascorbate peroxidase (C), glutathione reductase (D), and superoxide
dismutase (E) and protein concentration (F) in leaf blades and roots after 14 days of exposure to main saline-alkaline stress (SAS) treatment. Plants were pre-treated
with 0 (NPT), 10 mM NaCl (S10), 10 µM H2O2 (OX10), and 0.5 mM NaHCO3 (SA0.5) for 7 days and subjected to 50 mM Na, pH 8.25 for 14 days. The data represents means
± standard errors from four biological replicates. Different letters indicate significant differences by Tukey’s multiple comparison tests at P < 0.05.

Figure 7. Effect of pre-treatments on recovery of plants after exposure to saline-alkaline stress. Comparative mean growth rates during and after stress in shoots (A) and
roots (B). Chlorophyll concentration (C) and membrane integrity assessed by the electrolyte leakage method in shoots (D) at the end of the 10-day recovery period.
‘Stress’ group represents mean growth rate of plants during the 14 day growth period under main saline-alkaline stress (SAS) (50 mM Na+, pH 8.25) after pre-treatment
with 0 (NPT), 10 mM NaCl (S10), 10 µM H2O2 (OX10), and 0.5 mM NaHCO3 (SA0.5) for 7 days. ‘Recovery’ group represents the growth rate of the same plants upon
completion of main saline-alkaline stress (SAS) and subjecting them to recovery under normal conditions (0 mM Na+, pH 5.5) for 10 days. The data represents means ±
standard errors from five biological replicates. different letters indicate significant differences by tukey’s multiple comparison tests at P < 0.05.

growth and yield in such environments. Plants are adept at
acclimating to salt stress, characterized by quicker and stron­
ger responses to subsequent lethal exposure to the same
stress (Kamanga et al. 2020; Liu and Saneoka 2019;
Sriskantharajah et al. 2020). While acclimation studies are
widespread, the physiological processes governing responses
of plants to multiple exposures of different stresses are
unclear. Here, we have shown that plants pre-exposed to
lower concentrations of NaCl and H2O2 show improved
growth and physiological characteristics under SAS condi­
tions. Contrary to the hypothesis, the failure of NaHCO3 pre-
treatment to enhance growth suggests that the concentra­
tions used were too high for pre-treatment in rice, likely
causing physiological injuries. However, a similar concentra­
tion of 1 mM NaHCO3 in Rye (Secale cereale) effectively
improved tolerance to SAS conditions (Liu and Saneoka
2019). Overall, 10 mM NaCl and 10 µM H2O2 were the most
effective (Figure 1B). These latter results illustrate the concept
of cross-tolerance, wherein pre-exposure to one stress, such as
salinity or oxidative stress, promotes tolerance to another
stress, such as saline-alkaline stress, as previously described
(Foyer et al. 2016; Perez and Brown 2014). In light of a myriad
of abiotic stresses as currently faced, this critical attribute
offers a prospect for generating plants tolerant to multiple
abiotic stresses.
Under SAS conditions, the first line of defense entails acid­
ification of the rhizosphere through H+ ATPase-mediated pro­
ton efflux by plant roots (Li, Yang, and Zhang 2016). In the
present study, the failure of NaHCO3 PT plants to suppress rise
in growth medium pH (Figure 2C) may partly elucidate their
inability to effectively promote tolerance to SAS conditions
(Figure 1B). In contrast, plants pre-treated with NaCl and H2O2
maintained a much lower growth medium pH throughout the
growth period compared to the NPT plants, indicative of higher
root activity. Lower rhizospheric pH is also advantageous for
maintaining the transmembrane proton gradient required for
SOS-mediated efflux of Na+ by roots SOS1 (Cao et al. 2020;
Moriyama 2015). Experiments on Na+ efflux showed that all
pre-treated plants had higher root Na+ efflux than non-pre-
treated (Figure 4B) including NaHCO3. Therefore, whether
higher root Na+ efflux and resultant lower leaf blade Na+ accu­
mulation (Figure 3B) were as a result of lower growth medium
pH are difficult to untangle. Furthermore, when we eliminated
the contribution of Na+ uptake by roots using experiments on
Na+ accumulation in excised leaf blades, both 10 mM NaCl and
10 µM H2O2 PT plants accumulated significantly lower Na in the
leaf blades (Figure 4C). In H2O2 PT plants, notwithstanding
higher leaf blade Na+ accumulation, the deleterious effects of
Na+ were ameliorated and leaf photosynthetic competency
(Figure 1C) was preserved. Moreover, despite the ‘statistical’
significance of leaf blade Na+ accumulation among PTs, this
result suggests that the observed differences may not have
been ‘biologically significant.’
Na and K have similar physicochemical properties; hence,
they exhibit competitive uptake systems (Marschner and
Marschner 2012). Moreover, rice plant roots are leaky (Munns
and Tester 2008), potentially losing significant amounts of K+
under SAS conditions. Retention of root K+ has been cited as
a principal trait for salt tolerance (Chen et al. 2007). In the
SOIL SCIENCE AND PLANT NUTRITION 9
present study, pre-treatment with NaCl and H2O2 maintained
a higher root K concentration (Figure 3A). Separate experi­
ments revealed extremely high root K+ leakage under SAS
(Figure 4A), which could however not be alleviated by pre-
treatments. Therefore, while root K+ leakage was the primary
cause of K deficiency under SAS, its contribution to the
observed differences among various pre-treatments could not
be verified. Nonetheless, considering the extreme loss of K+ in
roots under SAS conditions, these results suggest that even the
slightest improvements in root K+ retention may be beneficial
under saline-alkaline stress, primarily for osmotic adjustments
and maintenance of root growth as also suggested by Chen
et al. (2007). Additional benefits of acclimation included
increased P uptake and accumulation especially in roots.
Uptake of P is driven by H+ gradient across the plasma mem­
brane, such that when the rhizospheric pH is lower than root
cytosolic pH, P uptake is promoted (Cao et al. 2020). Hence,
under saline-alkaline stress, maintenance of P uptake is an
extremely important attribute for plant growth and develop­
ment, and also dependant on plant roots ability to lower rhizo­
spheric pH through H+ ATPases mediated proton efflux.
However, further studies are required to elucidate the physio­
logical mechanisms underlying this enhanced P uptake.
Fe deficiency, which is a typical feature of plants growing
under high pH conditions, is characterized by chlorosis.
Although iron is a naturally abundant element, it forms insolu­
ble ferric compounds that are non-bioavailable (Marschner
1995; Nakanishi et al. 2004) for uptake under high pH condi­
tions. As shown in Figure 1C, NPT plant leaves were chlorotic,
which is a typical sign of Fe deficiency (Marschner and
Marschner 2012). However, in NaCl and H2O2 PT plants, leaves
were healthier (Figure 1C) and accumulated higher Fe concen­
trations (Figure 3D). Therefore, it is tempting to attribute the
high Fe concentration to the ability of plant roots to suppress
rise in growth medium pH, as observed in Figure 2A, B. Lower
growth medium pH is thought to increase the solubility of ferric
ions or support the reducing capacity of ferric Fe on the root
surface (Kobayashi and Nishizawa 2012). However, similar
increases in Fe concentration were also observed in NaHCO3
pre-treated plants (Figure 3D) which otherwise did not show
different growth medium pH from NPT plants (Figure 2C), rais­
ing doubts on the causality between Fe concentration and
lower rhizospheric pH. Alternative causes for the variations in
Fe uptake could relate to differences in key enzyme activities
involved in Fe uptake (Nakanishi et al. 2004; Kobayashi and
Nishizawa 2012), however, further studies to elucidate the phy­
siological and molecular bases of this acclimation induced
increase in accumulation are imperative. In NaHCO3 pre-
treated plants (SA0.5), despite having higher leaf blade and
root Fe concentration (Figure 3D), leaf blades were relatively
chlorotic (Figure 1C), similar to non-pre-treated plants, incon­
sistent with our expectations. This might have been as a result
of considerably reduced chlorophyll concentration during the
7-day acclimation period (Figure 1C), such that enhanced Fe
uptake and accumulation during the main stress could not fully
recover chlorophyll concentration by the sampling time.
Several reports have implicated the lethality of SAS to ROS-
mediated damage. In an alkaline tolerant (ALT1) rice mutant,
Guo et al. (2014) credited tolerance to enhanced defense
10 R. M. KAMANGA ET AL.
against oxidative stress. Oxidative damage to membrane lipids
causes peroxidation, which is analytically deduced from the
concentration of malondialdehyde (MDA), an aldehyde by-
product of lipid peroxidation (Ayala, Muñoz, and Argüelles
2014). In the present study, NaCl and H2O2 pre-treatments
considerably alleviated SAS-induced membrane damage and
cell death (Figure 5(A–Figure 5C)). These results, in part, indi­
cate mitigated oxidative stress, owing to the enhanced ROS
scavenging capacity in PT plants. ROS generation in plants is
an inevitable consequence of aerobic metabolism. Until
recently, the traditional paradigm that considers ROS as dele­
terious to plant cells is gradually fading owing to recent
revelations of their critical role as secondary messengers
(Apel and Hirt 2004; Mittler 2017). We hypothesized that pre-
treatments would spark a mild generation of ROS, which
would eventually activate systemic antioxidant defense
responses. In agreement, H2O2 generation was significantly
reduced in roots in all PT plants, whereas only H2O2 and
NaHCO3 pre-treatments were effective in leaf blades. In NaCl
PT plants, despite having exceptional growth and lower Na
concentration in leaf blades, the higher H2O2 in tandem cau­
tions against the inevitable expectation that ROS results in
poor growth, harmonizing with notions earlier expressed in
Mittler (2017). Perhaps the crucial aspect is maintaining
a delicate balance between ROS generation and detoxifica­
tion. Given that NaCl PT plants had higher antioxidant activ­
ities of CAT, APX, and GR (Figure 6(A– Figure 6C), this balance
might have been attained, cushioning against vicious func­
tions of ROS (Apel and Hirt 2004). Better antioxidant enzyme
activities were also observed in H2O2 PT plants, in agreement
with earlier observations (Fedina, Nedeva, and Çiçek 2009;
Wahid et al. 2007). Moreover, all pre-treatments considerably
increased the SOD activity (Figure 6E). Since H2O2 and hydro­
xyl radicals are superoxide radical derivatives, SOD offers a first
line of defense against oxidative stress; hence, acclimation
provided nearly complete protection against oxidative stress.
Besides, H2O2 concentration (Figure 5A) did not essentially
correspond to the trends of electrolyte leakage, MDA concen­
tration, and cell viability (Figure 5(A–Figure 5C)) in some PTs,
in part cementing the above notions. Apparently, this may be
ascribed to different antioxidant enzyme capacities (Figure 6
(A–Figure 6C)) or capacities to repair ROS mediated damage.
Strategies adopted by plants to confront oxidative stress
mediated damage engage three lines of steps; avoidance of
ROS generation, detoxification of ROS (scavenging) and repair
of ROS mediated damage (Guo et al. 2014; Mittler 2017).
Hence, measurement of ROS generation alone may not suffi­
ciently predict resultant ROS mediated damage. Furthermore,
this result may point to involvement of other ROS, such as
superoxide and hydroxyl radicles that were otherwise not
assayed in the present study.
In conclusion, the present study reports that acclimation to
salinity stress or oxidative stress promotes cross-tolerance to
SAS conditions by fostering efficient Na+ homeostasis, Fe
acquisition, and ROS homeostasis. These processes are linked
to the ability of plant roots to supress excessive increases in
growth media pH, offering a first line of defense against SAS.
We further suggest crosstalk between salinity stress, oxidative
stress, and saline-alkalinity, likely functioning through
common signals and pathways that trigger adaptive physio­
logical responses, thereby developing cross-resistance to sal­
ine-alkalinity. Moreover, H2O2 pre-treated plants quickly
recover physiologically and resume rapid growth upon the
conclusion of stress (Figure 7A). This may be related to lower
accumulation of H2O2 in the leaf blades (Figure 5D), as a result
of higher antioxidant enzyme activities specifically CAT, APX
and GR (Figure 6(A–Figure 6C)) that detoxify cellular H2O2.
Rapid resumption of growth after withdrawal of a stressor is
apparently a fundamental attribute for recurrent stresses with
prolonged recovery periods. Therefore, pre-treatment with
optimal NaCl and H2O2 may be a recommended option for
quick improvements in tolerance to SAS conditions in rice.
This trait also offers a prospect for developing crop plants
tolerant to multiple stressors.
Acknowledgments
This research was supported by JSPS KAKENHI Grant Numbers 15KK0283
and 20KK0129 to AU.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
This work was supported by the JSPS KAKENHI Grant Number 15KK0283,
20KK0129.
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