ISSN (Print): 2957-4471, ISSN (Online): 2708-6364, Website: https://journal.esbangladesh.org
Ecol. J. (2022) 4 (1) : 79-89

OLOGICAEC
L

Ecology Journal

CIETY OFBSO

GLADESHAN

Research article

2019

EFFECTS OF SODIUM HYDROSULFIDE ON PHYSIOLOGICAL RESPONSES IN A
SALT-TOLERANT SOYBEAN GENOTYPE ‘AGS313’ UNDER SALINE CONDITIONS
Yuta Jinzenji, Tomohiro Kondo and Hirokazu Higuchi
Graduate School of Agriculture, Kyoto University, Kyoto, Japan.
Corresponding e-mail: kondo.tomohiro.0z@kyoto-u.ac.jp
Received: 17 May 2022, Revised: 28 May 2022, Accepted: 05 June 2022

ABSTRACT
Hydrogen sulfide (H2S) has been recently found to be a signaling molecule to environmental stress tolerance.
This study was conducted to investigate the effects of irrigation (Experiments 1 and 2) and foliar application(Experiment 3) of sodium hydrosulfide (NaHS), as a H2S donor, under NaCl stress. A Bangladeshi salt-tolerantsoybean genotype ‘AGS313’ was compared with a salt-sensitive Japanese variety ‘Fukuyutaka’. While the shoot
dry weight was improved by NaHS application in the experiment 1, the vegetative growth was not improved
by NaHS application in the experiments 2 and 3. NaHS irrigation decreased leaf Cl content, while NaHS foliar
application increased photosynthetic rate. These results showed that NaHS application had slightly positive effects
on soybean under NaCl stress conditions.
Keywords: Glycine max, oxidative stress, photosynthetic rate, sodium chloride, stomatal conductanceIntroduction
Salinity is an environmental stress that can greatly reduce
crop productivity. Salinity can occur under all climaticconditions, and is relatively more common in arid, semiarid, and coastal areas. The coastal area in Bangladesh is
the main soybean (Glycine max L.) production area, where
salinity threats economic crop production (Mannan et al.
2013). In Bangladesh, Mannan et al. (2012) evaluated thesalt tolerance of 170 soybean genotypes and ‘AGS313’,
the salt-tolerant genotype, has been introduced in salinity
area (Karim et al. 2012). Cultivation methods to reducesalinity have also been studied (Fujii and Higuchi 2021),
but are not yet fully established. Under salt stress, plants
are subjected to osmotic stress due to high salinity in therhizosphere and ionic stress due to the accumulation ofexcess Na and Cl (Munns and Tester 2008). As a result,reactive oxygen species (ROS) such as O2- and H2O2 are
generated. Excessive ROS accumulation damages toproteins, DNA, lipids, membrane tissues, and enzyme
activity.
In recent years, hydrogen sulfide (H2S) has gained
attention as a gaseous signaling molecule in the responseof plants to biotic and abiotic stresses, like nitric oxide andcarbon monoxide (da-Silva and Modolo 2017). H2S wasreported to be involved in physiological processes suchas blood pressure regulation, anti-inflammatory effects,
and vasorelaxation in mammalian studies (Wagner 2009).
As the effects of H2S, activation of photosynthesis (Aliet al. 2014, Chen et al. 2011, Duan et al. 2015), stomatal
opening and closure (Papanatsiou et al. 2015, Jin et al.
2017, Shen et al. 2020), seed germination (Zhang et al.
2008), root formation (Zhang et al. 2009, Jia et al. 2015,
Mei et al. 2019), and fruit maturation (Ge et al. 2017,
Munoz-Vargas et al. 2018) have already been reported.
Even under salt stress, the activation of antioxidant systems(Yu et al. 2013, Christou et al. 2013, Shan et al. 2014),
maintenance of K/Na (Chen et al. 2015, Lai et al. 2014,
Deng et al. 2016), activation of photosynthesis (Christou
et al. 2013, Mostofa et al. 2015), and accumulation ofosmotic regulators (Sun and Luo, 2014, Shi et al. 2013)
have also been reported.
To understand the effectiveness of H2S application on
soybean growth under saline stress conditions and thesaline stress mitigating mechanism of H2S application,
information on responses of leaf mineral contents andphysiological parameters, such as relative chlorophyll
content (SPAD), photosynthetic rate, stomatal conductanceand maximum quantum efficiency of PSⅡ photochemistry(Fv/Fm), would presumably be useful. Thus, the effects

Research article

Effects of sodium hydrosulfide on salt tolerance of soybeanof sodium hydrosulfide (NaHS) irrigation, as a H2S
donor, under different NaCl stress, such as 100 mM(Experiment 1) and 50 mM (Experiment 2) stress, andthose of foliar application by spray under 150 mM NaCl
stress (Experiment 3) on vegetative growth, leaf mineralcontents, and physiological responses, were investigatedin a salt tolerant soybean genotype ‘AGS313’. In theexperiments 2 and 3, salt-sensitive variety ‘Fukuyutaka’
was also used as a comparison with ‘AGS313’.

for each plant and the average values were recorded, using
a chlorophyll meter (SPAD-502, Konica Minolta Sensing
Inc., Japan). Fv/Fm was measured three times for each plant
and the average values were recorded, using a chlorophyll
fluorometer (OS-30P, Opti-Sciences, Inc., USA) under adark condition at 2100-2200 h. Photosynthetic rate andstomatal conductance were measured on May 10, 26, andJune 1. Photosynthetic rate was measured at 1000-1200 h,
using a photosynthetic rate measurement system (MIC100, Masa International Corp., Japan). The measurement
conditions of photosynthetic rate were as follows: PAR
1200 μmol/m2/s, stabilization time 3 sec, measurement
start CO2 concentration 400 ppm, and measurement
CO2 span 20 ppm. When the measurement was notfinished within 60 second, it was regarded as below thelower limit of measurement. Stomatal conductance wasmeasured using a SC-1 leaf porometer system (SC-1,
Meter Group, Inc., USA). The 3rd- 5th youngest leaves
were used for the measurements of photosynthetic rateand stomatal conductance. After the end of treatment,
the plants were sampled on July 4 and oven-dried at 70°C for 3 days. Then the shoot dry weight and root dry
weight were measured. To determine leaf Na, K, Ca,
Mg, and P contents, the leaves sampled on May 27 andJune 4 were oven-dried at 70 °C for 3 days and grounded.
After wet-ashing with nitric acid, Na, K, Ca, Mg, and P
contents were determined using an inductively coupledplasma spectrophotometer (ICPE-9000, Shimadzu Corp.,
Japan). To determine the Cl and NO3 contents in leaves,those were extracted from grounded leaves with 40 °C
water. The Cl concentration was measured using a Cl ion
electrode (CL-2021, DKK-TOA Corp., Japan) with ion/
pH meter (IM-22P, DKK-TOA Corp., Japan), and theNO3 concentration was determined using the method of
Cataldo et al. (1975). Soil water content at a depth of 0 to
5 cm was measured at 1500-1530 h on June 1, using a soil
moisture meter (EC5, Meter Group, Inc., USA).

Materials and Methods
Experiment 1
Thirty-two salt-tolerant soybean plants ‘AGS313’ weregrown in a greenhouse at Kyoto University (35.0 °N, 135.8°E). ‘AGS313’ was reported to be a salt tolerant soybean
genotype (Karim et al. 2012, Fujii and Higuchi, 2019).
The seeds were sown in plastic pots on March 26, 2021.
The diameter and height of the pots were 12 cm and 10
cm, respectively. It was filled with expanded vermiculite
for germination. After germination, the plants weretransplanted to 4 L plastic pots filled with 1: 1 mixtureof sandy soil and bark compost by volume. During theexperiment, 600 mL of nutrition solution containing
NH4-N: 0.7 mM, NO3-N: 8 mM, K: 4 mM, P: 0.7 mM,
Ca: 2 mM, Mg: 1 mM, S: 1 mM was applied three times a
week. One time of these applications, micronutrients (Fe:57 μM, Cu: 0.6 μM, Zn: 0.9 μM, Mn: 9.9 μM, B: 47.2 μM,
Mo: 0.08 μM) were added.
When the 5th leaf emerged, on May 10, the 32 plants weredivided into 4 groups with 8 replications. Each plant wasapplied with 600 mL of tap water (Control treatment) or
the solution, containing 100 mM NaCl (NaCl treatment),
100 mM NaCl + 0.1 mM NaHS (0.1 mM NaHS
treatment), or 100 mM NaCl + 0.2 mM NaHS (0.2 mM
NaHS treatment), twice a day on sunny days and 0 to 1
time a day on cloudy or rainy days instead of irrigationwater. To prevent severe NaCl damage due to a sudden
NaCl increase, NaCl concentration in the solutions wasincreased by 20 mM every day from May 10 to 14. From
May 14, 100 mM NaCl was applied for 18 days.

Experiment 2
Two soybean varieties, ‘AGS313’ and ‘Fukuyutaka’,
were sown on April 24, 2021, transplanted on June 10,
and cultivated in the same way as in the experiment 1.
When the 5th leaf emerged, on June 23, the 30 plants
were divided into 3 groups with 10 replications for eachvariety. Each plant was applied with 600 mL of tap water

Plant height, number of leaves, relative chlorophyll
content (SPAD), and maximum quantum efficiency of
PSⅡ photochemistry (Fv/Fm) were measured on May 10,
20, 26, and June 1. The SPAD was measured five times
80Research article

Effects of sodium hydrosulfide on salt tolerance of soybean(Control treatment) or solutions containing 50 mM NaCl
(NaCl treatment) or 50 mM NaCl + 0.1 mM NaHS (NaHS
treatment) twice a day on sunny days and 0 to 1 time aday on cloudy or rainy days instead of irrigation water.
NaCl concentration in the solutions was increased by
10 mM every day from June 23 to June 27. From June27, 50 mM NaCl was applied for 26 days. Plant height,
number of leaves, SPAD, Fv/Fm, and length and width ofmiddle leaflet at the 3rd youngest leaf were measured on
July 22. The area of middle leaflet at the 3rd youngest leaf
was estimated using the following calibration equations,which was established in advance for each variety. A, L,
and W denote area, length, and width of middle leaflet.

600 mL of 150 mM NaCl solutions was applied to the
NaHS treatment and 9 of 18 plants sprayed with tap water(NaCl treatment) twice a day on sunny days and 0 to 1
time a day on cloudy or rainy days. To the rest 9 plants
600 mL of tap water was applied (Control treatment).
Plant height, number of leaves, SPAD, Fv/Fm, and length
and width of middle leaflet at the 3rd and 4th youngest
leaves were measured on August 24, and the area of
those leaflets were calculated using the same calibration
equations in the experiment 2. Photosynthetic rate andstomatal conductance were measured on August 24
at 1100-1300 h using the same devices and methods inthe experiments 1 and 2. After the end of treatment, theplants were sampled on August 26 and dried at 70 °C for 3
days. Then the shoot dry weight and root dry weight were
measured. Leaf mineral contents and EL were measured in
the same way as in the experiment 2 using leaves sampled
on August 26.

AGS313: A = 0.62 LW + 1.42 r2 = 0.99 *** (n = 100)
Fukuyutaka: A = 0.64 LW + 1.83 r2 = 0.99 *** (n = 100)
Photosynthetic rate and stomatal conductance were
measured on July 21 at 1200-1330 h using the samedevices and methods in the experiment 1. After the end oftreatment, the plants were sampled on July 24 and ovendried at 70 °C for 3 days. Then the shoot dry weight androot dry weight were measured.

Statistical analysis
For the experiment 1, the data were analyzed by analysis ofvariance (ANOVA). The data with statistical differencesamong treatments as found by ANOVA were further
analyzed by Tukey’s multiple comparison tests (p<0.05).
For the experiments 2 and 3, the data were analyzed
by two-way ANOVA (variety × treatment). After that,statistical differences among treatments were subjected tofurther analysis using Tukey’s multiple comparison tests(p<0.05).

Leaf mineral contents were measured in the same way asin the experiment 1. The leaves were sampled on July 24.
On the same day, 50 mg of fresh leaves for each plant
was sampled. Fresh leaf was soaked to 10 mL of distilledwater and incubated in an incubator at 28 °C for 2 hours.
Then electrical conductivity (EC1) was measured using
a conductivity meter (LAQUAtwin, HORIBA Advanced
Techno, Co., Ltd., Japan). After the measurement, thesample was incubated at 100 °C for 20 min. After coolingto room temperature, electrical conductivity (EC2) was
measured again. Then electrolyte leakage (EL: EC1/EC2
× 100) was calculated.

Results
Experiment 1
Shoot dry weight was decreased by NaCl application and
0.1 mM NaHS application lessened the detrimental effectof the NaCl application (Table 1). Root dry weight wasalso decreased by NaCl application and not significantly
enhanced by NaHS application. Soil water content wasincreased by NaCl application. Plant height and numberof leaves were also decreased by NaCl application andnot significantly enhanced by NaHS application (Figure1). Photosynthetic rate and stomatal conductance weredecreased by NaCl application, and not enhanced by NaHS
application. SPAD was increased by NaCl application,and it was the highest in 0.1 mM NaHS on May 26. Fv/

Experiment 3
Two varieties, ‘AGS313’ and ‘Fukuyutaka’, were sownon July 20, 2021, transplanted on August 5, 2021, andcultivated in the same way as in the experiments 1 and 2.
When the 2nd leaf emerged, on August 12, the 27 plants
were divided into 2 groups with 9 and 18 plants for eachvariety, 10 mL NaHS solution was sprayed to leaves for
9 plants (NaHS treatment) and 10 mL of tap water was
sprayed for the rest 18 plants every day. From August 15,
81

Research article

Effects of sodium hydrosulfide on salt tolerance of soybean

Table 1. Effects of sodium chloride and sodium hydrosulfide application on shoot and root dry weights in ‘AGS313’ soybean
and soil water content 22 days after treatment.
Treatmentx
Control
NaCl
0.1mM NaHS
0.2mM NaHS

Shoot dry weight (g)27.4 a11.6 c17.8 b
14.6 bc

Root dry weight (g)
5.4 a
2.0 b
2.5 b
2.7 b

Soil water content (%)
21.1 b35.5 a34.2 a34.7 ax: Each plant was applied with 600 mL of tap water (Control) or solution, containing 100 mM NaCl (NaCl), 100 mM NaCl+ 0.1 mM NaHS (0.1mM NaHS), or 100 mM NaCl+ 0.2 mM NaHS (0.2 mM NaHS), twice a day on sunny days and 0 to 1 time a day on cloudy or rainy days insteadof irrigation water. Different letters within the same column indicate statistically different at p<0.05 by Tukey's multiple comparison tests (n=8).

Figure 1. Effects of sodium hydrosulfide irrigation on plant height (A), number of leaves (B), SPAD (C), Fv/Fm (D), photosynthetic
rate (E), and stomatal conductance (F) in ‘AGS313’ soybean under 100 mM NaCl. Bars indicate standard error (n=8), and different
letters within the same date indicate statistically different at p<0.05 by Tukey’s multiple comparison tests.

Fm was not affected by the application of NaCl or NaHS
throughout the experiment.

Leaf Cl content was increased by the 22 day-applicationof 0.2 mM NaHS. Leaf K, Ca, Mg, and NO3 contents were
not affected by the 22 day-application of NaCl or NaHS.
Leaf P content was increased by the 22 day-applicationof NaCl. Twenty-two days after the application, leaf K/
Na was the highest in control and the lowest in 0.2 mM
NaHS application.

Leaf Na content was not affected by the 17 dayapplication of NaCl or NaHS (Table 2). Leaf Cl content
was increased by NaCl application and suppressed by
the 17 day-application of 0.2 mM NaHS. Leaf K content
was decreased by NaCl application. Leaf Ca, Mg, P, andNO3 contents and K/Na were not affected by the 17 dayapplication of NaCl or NaHS. Leaf Na content was thehighest in 0.2 mM NaHS application and the lowest incontrol 22 days after application, on June 4 (Table 3).

Experiment 2
Vegetative growth, such as plant height, number of leaves,area of middle leaflet at the 3rd youngest leaf, shoot dry
82Research article

Effects of sodium hydrosulfide on salt tolerance of soybean

Table 2. Effects of sodium chloride and sodium hydrosulfide application on leaf mineral contents in ‘AGS313’ soybean 17 days
after treatment.
Treatmentx
Control
NaCl
0.1mM NaHS
0.2mM NaHS

Na

Cl

K

Ca

Mg

P

-1

-1

-1

-1

-1

-1

NO3

(mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g-1D.W.)
0.79
7.2 c
23.8 a
13.8
4.77
4.81
24.4
1.29
12.1 a17.2 b
20.7
6.24
5.76
25.0
0.67
10.9 ab20.3 ab
15.7
4.98
5.98
19.2
1.09
10.0 b
18.3 ab
23.6
7.95
5.82
23.9

K/Na
33.4
19.5
34.0
21.2

x: Each plant was applied with 600 mL of tap water (Control) or solution, containing 100 mM NaCl (NaCl), 100 mM NaCl+ 0.1 mM NaHS (0.1 mMNaHS), or 100 mM NaCl+ 0.2 mM NaHS (0.2 mM NaHS), twice a day on sunny days and 0 to 1 time a day on cloudy or rainy days instead ofirrigation water. Different letters within the same column indicate statistically different at p<0.05 by Tukey's multiple comparison tests (n=8).

Table 3. Effects of sodium chloride and sodium hydrosulfide application on leaf mineral contents in ‘AGS313’ soybean 22days after treatment.
Treatmentx
Control
NaCl
0.1mM NaHS
0.2mM NaHS

Na

Cl

K

Ca

Mg

P

-1

-1

-1

-1

-1

-1

NO3

(mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g-1D.W.)
2.68 b
6.8 c
17.6
10.9
3.72
3.75 b
12.6
4.43 ab13.9 b
19.9
13.7
4.77
5.32 a10.6
4.20 ab13.2 b
19.5
10.3
3.84
4.58 ab
11.4
5.34 a16.0 a
18.9
13.7
4.65
4.71 a
12.3

K/Na
7.9 a
4.9 ab
4.8 ab
3.9 b

x: Each plant was applied with 600 mL of tap water (Control) or solution, containing 100 mM NaCl (NaCl), 100 mM NaCl+ 0.1 mM NaHS (0.1 mMNaHS), or 100 mM NaCl+ 0.2 mM NaHS (0.2 mM NaHS), twice a day on sunny days and 0 to 1 time a day on cloudy or rainy days instead ofirrigation water. Different letters within the same column indicate statistically different at p<0.05 by Tukey's multiple comparison tests (n=8).

weight, and root dry weight, were decreased by NaCl
application, and NaHS application did not mitigate thedetrimental effect of NaCl (Table 4).

application in ‘AGS313’ and increased in ‘Fukuyutaka’.
Leaf Ca content was not affected by variety and application.
Leaf Mg content was increased by NaCl application. Leaf
P content was increased by NaCl application in ‘AGS313’
and was not affected by the application of NaCl or NaHS
in ‘Fukuyutaka’. Leaf NO3 content was not affected by theapplication. Leaf K/Na was decreased by NaCl applicationin both varieties.

Photosynthetic rate and stomatal conductance weredecreased by NaCl application and were not improved
by NaHS application (Table 5). There were interactions
between variety and treatment of NaCl and NaHS
applications in SPAD, Fv/Fm, and EL. SPAD and Fv/Fmof ‘AGS313’ were not affected by the application of NaClor NaHS, while those of ‘Fukuyutaka’ were decreased by
NaCl application and not increased by NaHS application.
EL of ‘AGS313’ was not affected by NaCl application,
while that of ‘Fukuyutaka’ was increased by NaCl
application. NaHS application did not improve SPAD,
Fv/Fm and EL.

Experiment 3
Leaf number, area of middle leaflet at the 3rd and 4th
youngest leaf, shoot dry weight, and root dry weight weredecreased by NaCl application and were not improved by
NaHS foliar application by spray (Table 7). There was aninteraction between variety and treatment of NaCl and
NaHS applications in plant height, while it was decreased
by NaCl application and not improved by NaHS foliar
application in both varieties.

Leaf Na content of ‘AGS313’ was not affected by theapplications of NaCl or NaHS, while that of ‘Fukuyutaka’
was increased by NaCl application (Table 6). Leaf Cl
content of ‘AGS313’ was not affected by the applicationsof NaCl or NaHS while that of ‘Fukuyutaka’ wasincreased by NaCl application and was suppressed by
NaHS application. Leaf K content was decreased by NaCl

Photosynthetic rate was decreased by NaCl application,and improved by NaHS foliar application (Table 8).
Stomatal conductance was also decreased by NaCl
application and not improved by NaHS foliar application.
83

Research article

Effects of sodium hydrosulfide on salt tolerance of soybean

Table 4. Effects of sodium chloride and sodium hydrosulfide application on vegetative growth in ‘AGS313’ and ‘Fukuyutaka’soybean 28 days after treatment.
Leaf number
Plant height
Shoot dry
Root dryLeaf areay
Variety
Treatmentx(/plant)(cm)
weight(g)weight(g)(cm²)
AGS313Control
109.4
35.7
94.0
38.1
12.8
NaCl
89.0
27.1
84.0
27.1
9.5
NaCl+NaHS
93.8
28.0
81.1
26.7
7.9
Fukuyutaka

Control
NaCl
NaCl+NaHS

64.0
59.8
61.5

36.1
31.0
29.6

94.2
82.8
75.7

39.3
23.9
22.8

12.2
6.6
7.0

97.4 a61.8 b

30.3
32.2

84.2
86.4

30.6
28.7

10.1 a8.6 b

Variety［V］

86.7 a74.4 b
77.7 ab**35.9 a29.1 b
28.8 bn.s.

94.1 a83.4 b
78.4 bn.s.

38.7 a25.5 b
24.8 bn.s.

12.5 a
8.0 b
7.5 b
**

Treatment［T］

*

**

**

**

**

［V］×［T］

n.s.

n.s.

n.s.

n.s.

n.s.

Average
AGS313
Fukuyutaka

Control
NaCl
NaCl+NaHS

x: Each plant was applied with 600 mL of tap water (Control) or solutions containing 50 mM NaCl (NaCl) or 50 mM NaCl + 0.1 mM NaHS
(NaCl+NaHS) twice a day on sunny days and 0 to 1 time a day on cloudy or rainy days instead of irrigation water. y: Leaf area was measured at
middle leaflet of the third youngest leaf. **, *, and n.s. indicate significant differences at p<0.01, p<0.05, and not significant, respectively by twoway ANOVA. Different letters within the same column indicate statistically different at p<0.05 by Tukey's multiple comparison tests (n=10).

Table 5. Effects of sodium chloride and sodium hydrosulfide application on physiological responses in ‘AGS313’ and‘Fukuyutaka’ soybean 28 days after treatment.
Photosynthetic rate

Stomatal conductance

SPAD

Fv/Fm

Electrolyte
leakage (%)

39.9 a40.8 a40.9 a0.84 a0.84 a0.84 a9.6 a10.8 a
9.9 a

595
283
247

39.7 a30.3 b
29.3 b

0.84 a0.80 b0.81 b

14.8 b
28.8 a31.9 a17.3 a11.8 b

479
375

40.5
33.1

0.84
0.82

10.1
25.2

Variety［V］

18.0 a12.5 b
13.2 b
**

574 a
343 b
363 b
n.s.

39.8
35.5
35.1
**

0.84
0.82
0.82
**

12.2
19.8
20.9
**

Treatment［T］

**

**

**

**

**

［V］×［T］

n.s.

n.s.

**

**

**

Variety

Treatmentx

AGS313

Fukuyutaka

Control
NaCl
NaCl+NaHS(μmol m ² s ¹)
18.9
16.4
16.6

(mmol m-² s-¹)
553
403
480

Control
NaCl
NaCl+NaHS

17.0
8.6
9.9

Average
AGS313
Fukuyutaka

Control
NaCl
NaCl+NaHS--

x: Each plant was applied with 600 mL of tap water (Control) or solutions containing 50 mM NaCl (NaCl) or 50 mM NaCl + 0.1 mM NaHS
(NaCl+NaHS) twice a day on sunny days and 0 to 1 time a day on cloudy or rainy days instead of irrigation water. **, *, and n.s. indicate significant
differences at p<0.01, p<0.05, and not significant, respectively by two-way ANOVA. Different letters within the same column indicate statisticallydifferent at p<0.05 by Tukey's multiple comparison tests (n=10).

84

Research article

Effects of sodium hydrosulfide on salt tolerance of soybean

Table 6. Effects of sodium chloride and sodium hydrosulfide application on leaf mineral contents in ‘AGS313’ and ‘Fukuyutaka’soybean 30 days after treatment.
Na

Cl

K

Ca

Mg

P

NO3

Variety

Treatmentx

AGS313Control
NaCl
NaCl+NaHS

1.29 a1.41 a1.34 a

5.1 a
6.5 a
6.1 a

13.0 a
9.3 b
9.2 b

18.6
21.3
22.3

5.21
6.08
6.10

2.56 c3.10 b
3.61 a

11.3
10.1
9.0

10.1
6.8
7.0

Fukuyutaka Control
NaCl
NaCl+NaHS
Average
AGS313
Fukuyutaka

0.72 b
1.51 a1.21 ab

7.3 c
19.3 a13.9 b

13.4 b
17.1 a16.8 a

18.8
22.3
21.0

4.73
6.32
6.13

3.37 a2.97 a3.22 a

13.7
15.3
15.0

20.1
14.6
15.4

1.35
1.15

5.9
13.5

10.5
15.8

20.7
20.7

5.80
5.73

3.09
3.19

10.1 b
14.7 a8.0 b16.7 aControl
NaCl
NaCl+NaHS
Variety［V］

1.00
1.46
1.27
n.s.

6.2
12.9
10.0
**

13.2
13.2
13.0
**

18.7
21.8
21.6
n.s.

4.97 b
6.20 a6.12 a
n.s.

2.96
3.04
3.42
n.s.

12.5
12.7
12.0
**

15.1 a10.7 b
11.2 b**Treatment［T］

**

**

n.s.

n.s.

**

**

n.s.

*

［V］×［T］

*

**

**

n.s.

n.s.

**

n.s.

n.s.

(mg g-1D.W.) (mg g-1D.W.) (mg g-1D.W.) (mg g-1D.W.) (mg g-1D.W.) (mg g-1D.W.) (mg g-1D.W.)

K/Na

x: Each plant was applied with 600 mL of tap water (Control) or solutions containing 50 mM NaCl (NaCl) or 50 mM NaCl + 0.1 mM NaHS
(NaCl+NaHS) twice a day on sunny days and 0 to 1 time a day on cloudy or rainy days instead of irrigation water. **, *, and n.s. indicate significant
differences at p<0.01, p<0.05, and not significant, respectively by two-way ANOVA. Different letters within the same column indicate statisticallydifferent at p<0.05 by Tukey's multiple comparison tests (n=10).

Table 7. Effects of foliar application of sodium hydrosulfide on vegetative growth in ‘AGS313’ and ‘Fukuyutaka’ soybean 13days after treatment under NaCl application.
x

Plant height(cm)

Leaf number(/plant)

Leaf areay of the
3rd youngest leaf
(cm²)

Leaf area of the
4th youngest leaf
(cm²)

Shoot dryweight(g)

Root dryweight(g)

Variety

Treatment

AGS313Control
NaCl
NaCl+NaHS

81.9 a63.4 b
63.4 b

10.8
7.4
7.4

74.9
46.5
51.3

64.0
39.4
42.7

4.5
2.3
2.6

0.5
0.3
0.4

Fukuyutaka

Control
NaCl
NaCl+NaHS

56.9 a51.1 b54.0 ab

14.8
9.8
11.4

63.3
46.0
46.0

61.6
40.4
54.8

5.3
2.9
3.4

0.7
0.5
0.5

69.6
54.0

8.6 b
12.0 a57.6 a51.8 b

48.7
52.3

3.2 b
3.9 a

0.4 b
0.6 a

Variety［V］

69.4
57.3
58.7
**

12.8 a
8.6 b
9.4 b
**

69.1 a46.3 b
48.7 b*62.8 a39.9 b
48.8 b
n.s.

4.9 a
2.6 b
3.0 b
**

0.6 a
0.4 b
0.4 b
**

Treatment［T］

**

**

**

**

**

**

［V］×［T］

**

n.s.

n.s.

n.s.

n.s.

n.s.

Average
AGS313
Fukuyutaka

Control
NaCl
NaCl+NaHS

x: Each plant was applied with 600 mL of tap water (control) or solutions containing 150 mM NaCl (NaCl) twice a day on sunny days and 0 to 1 timea day on cloudy or rainy days instead of irrigation water. Half plants applied with NaCl was foliar applied with 10 mL NaHS solution (NaCl+NaHS)
every day. y: Leaf area was measured at middle leaflet. **, *, and n.s. indicate significant differences at p<0.01, p<0.05, and not significant,respectively by two-way ANOVA. Different letters within the same column indicate statistically different at p<0.05 by Tukey's multiple comparison
tests (n=9).

85

Research article

Effects of sodium hydrosulfide on salt tolerance of soybean

SPAD and Fv/Fm were increased by NaCl application. EL
was not affected by the applications of NaCl or NaHS.

weight was improved by 0.1 mM NaHS application
while plant height, number of leaves, and root dry weight
were not significantly improved (Figure 1, Table 1). In
the experiments 2 and 3, there were no positive effects of
NaHS application on vegetative growth (Table 4, Table
7). Thus, NaHS had slightly positive effects on vegetative
growth in soybean under NaCl stress.

Leaf Na content was the highest at NaCl treatment
and the lowest at control in both varieties (Table 9).
Leaf Cl content in ‘AGS313’ was not affected by theapplications, while that in ‘Fukuyutaka’ was increased
by NaCl application and it was not decreased by NaHS
foliar application. Leaf K content was not affected by
the applications. Leaf Ca content in ‘AGS313’ was notaffected by the applications and that in ‘Fukuyutaka’ wasthe highest at NaCl treatment and the lowest at control.
Leaf Mg content was not affected by the applications.
Leaf P content was increased by NaCl application. Leaf
NO3 content was decreased by NaCl application. Leaf K/
Na was decreased by NaCl application and not increased
by NaHS foliar application.

In the experiment 1,17 day-application of 0.2 mM NaHS
improved leaf Cl content in ‘AGS313’ (Table 2). In
the experiment 2, 30 day-application of 0.1 mM NaHS
improved leaf Cl content in ‘Fukuyutaka’ (Table 6).
NaHS irrigation may have acted on root, inhibiting Cl
absorption from soil to root and Cl transport from root
to shoot. It is well known that regulation of Cl to shootis important for salt tolerance of soybean (Valencia etal. 2008, Onodera et al. 2019, Fujii and Higuchi 2019).
Soybean expressing the Cl-regulated genes, GmCLC1 andNcl, in the root increased salt tolerance by suppressing
Cl transport to shoot (Wei et al. 2016, Do et al. 2016).
NaHS was reported to be a signal substance to SOS1, agene that regulates Na transport, leading to maintenanceDiscussion
Vegetative growth was not greatly improved by NaHS
application in the 3 experiments except for some indexes
under saline conditions. In the experiment 1, shoot dry

Table 8. Effects of foliar application of sodium hydrosulfide on physiological responses in ‘AGS313’ and ‘Fukuyutaka’soybean 13 days after treatment under NaCl application.
Photosynthetic rate

Stomatal conductance

SPAD

Fv/Fm

Electrolyte
leakage (%)

635
129
105

36.3
39.9
40.9

0.83
0.84
0.84

4.2
8.2
6.3

16.9
7.2
8.2

669
114
83

36.1
37.7
39.3

0.83
0.85
0.84

7.2
9.6
8.8

9.6
10.8

290
289

39.1 a37.7 b

0.84 b
0.84 a

6.2 b
8.5 a

Variety［V］

15.9 a
6.5 c
8.4 b
n.s.

652 a
121 b
94 b
n.s.

36.2 b38.8 a40.1 a*0.83 b
0.84 a0.84 a
*

5.7
8.9
7.6
*

Treatment［T］

**

**

**

**

n.s.

［V］×［T］

n.s.

n.s.

n.s.

n.s.

n.s.

Variety

Treatmentx(μmol m ² s ¹)

(mmol m ² s ¹)

AGS313Control
NaCl
NaCl+NaHS

14.8
5.7
8.5

Fukuyutaka

Control
NaCl
NaCl+NaHS

Average
AGS313
Fukuyutaka

Control
NaCl
NaCl+NaHS

-

-

-

-

x: Each plant was applied with 600 mL of tap water (control) or solutions containing 150 mM NaCl (NaCl) twice a day on sunny days and 0 to 1 timea day on cloudy or rainy days instead of irrigation water. Half plants applied with NaCl was foliar applied with 10 mL NaHS solution (NaCl+NaHS)
every day. **, *, and n.s. indicate significant differences at p<0.01, p<0.05, and not significant, respectively by two-way ANOVA. Different letterswithin the same column indicate statistically different at p<0.05 by Tukey's multiple comparison tests (n=9).

86Research article

Effects of sodium hydrosulfide on salt tolerance of soybean

Table 9. Effects of foliar application of sodium hydrosulfide on leaf mineral contents in ‘AGS313’ and ‘Fukuyutaka’ soybean
13 days after treatment under NaCl application.
Na

Cl

K

Ca

Mg

P

-1

-1

-1

-1

-1

-1

Control
NaCl
NaCl+NaHS

1.06
2.58
1.71

6.1 a
7.8 a
8.1 a

11.7 a10.4 a10.0 a

4.04
3.52
3.86

4.47
6.58
6.43

12.2
12.8
13.1

13.3
11.5
11.6

11.6
7.4
8.8

Fukuyutaka Control
NaCl
NaCl+NaHS
Average
AGS313
Fukuyutaka

1.03
2.19
1.75

7.1 b
11.2 a10.4 a13.5 b
17.2 a16.2 ab

4.89
5.17
5.00

4.57
5.51
5.74

15.8
15.6
16.1

15.2
11.6
12.2

15.5
8.4
9.6

1.78
1.66

7.3
9.6

10.7
15.7

3.81 b
5.02 a5.83 a5.27 b

12.7 b
15.9 a

12.1
13.0

9.2 b
11.2 aControl
NaCl
NaCl+NaHS
Variety［V］

1.04 b
2.39 a1.73 ab
n.s.

6.6
9.5
9.3
**

12.6
13.8
13.1
**

4.46
4.34
4.43
**

4.52 b
6.05 a6.09 a
*

14.0
14.2
14.6
**

14.2 a11.6 b
11.9 bn.s.

13.5 a
7.9 b
9.2 b
*

Treatment［T］

*

**

n.s.

n.s.

**

n.s.

**

**

［V］×［T］

n.s.

**

**

n.s.

n.s.

n.s.

n.s.

n.s.

Variety

Treatmentx

AGS313

NO3(mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g D.W.) (mg g-1D.W.)

K/Na

x: Each plant was applied with 600 mL of tap water (control) or solutions containing 150 mM NaCl (NaCl) twice a day on sunny days and 0 to 1 time aday on cloudy or rainy days instead of irrigation water. Half plants applied with NaCl was foliar applied with 10 mL NaHS solution (NaCl+NaHS)
every day. **, *, and n.s. indicate significant differences at p<0.01, p<0.05, and not significant, respectively by two-way ANOVA. Different letterswithin the same column indicate statistically different at p<0.05 by Tukey's multiple comparison tests (n=9).

In the experiment 3, photosynthetic rate was enhanced
by NaHS foliar application, while stomatal conductanceand Fv/Fm were not (Table 8). On the other hand, NaHS
application enhanced photosynthetic rate, stomatal
conductance, and Fv/Fm (Zhang et al. 2010; Christou
et al. 2013, Jiang et al. 2019). Therefore, photosynthetic
rate was not enhanced by stomata and photosystem II as
factors in the experiment 3. A possible factor of enhancing
photosynthesis might have been the higher rate of CO2
fixation due to the activation of dark reaction systems
such as RuBisCO. NaHS application also increased
photosynthetic rate in rice and spinach by increasing theactivity of RuBisCO (Duan et al. 2015, Chen et al. 2011).
Further studies are needed to investigate the activity of thedark reaction system, such as the initial slope of the A/Cicurve (Kondo et al. 2017) and the activity of RuBisCO.
NaHS foliar application increased photosynthetic rate in
the experiment 3, while NaHS irrigation application did notincrease it in the experiments 1 and 2. In the experiments
1 and 2, where H2S may not have been transferred to theof K/Na (Deng et al. 2016). As well as Na regulation, for
Cl regulation, in soybean NaHS might have acted as asignal substance to express genes such as GmCLC1 andNcl, which suppress Cl absorption and transport. On theother hand, the NaHS foliar application in the experiment
3 did not reduce leaf Cl content (Table 9). H2S may nothave been transferred to the roots by foliar application andhad no effect on Cl absorption and transport. H2S may bedifficult to be transferred from leaf to root.
In the experiment 1, leaf Cl content was decreased by 17
day-application of 0.2 mM NaHS (Table 2), while it wasincreased by 22 day-application (Table 3). Appropriate
concentration of NaHS application was reported topromote root formation and elongation (Mei et al., 2019).
On the other hand, an excess H2S in the soil negatively
affects root activity in general. In the experiment 1, 22
day-application of NaHS concentration at 0.2 mM may
have been slightly excess and negatively affected root
activity, while 17 day-application of 0.2 mM may havebeen appropriate and inhibited Cl absorption and transport.
87

Research article

Effects of sodium hydrosulfide on salt tolerance of soybean

leaves. H2S might be as difficult to be transferred fromroot to leaf as it is from leaf to root. Further studies areneeded on the transferability of H2S in plants.

Duan B, Ma Y, Jiang M, Yang F, Ni L and Lu W. 2015.
Improvement of photosynthesis in rice (Oryza sativa
L.) as a result of an increase in stomatal aperture anddensity by exogenous hydrogen sulfide treatment.
Plant Growth Regulation, 75: 33-44.

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89