Saponin biopriming positively stimulates antioxidants defense, osmolytes metabolism and ionic status to confer salt stress tolerance in soybean

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Acta Physiologiae Plantarum (2020) 42:114
https://doi.org/10.1007/s11738-020-03098-w
ORIGINAL ARTICLE
Saponin biopriming positively stimulates antioxidants defense,
osmolytes metabolism andionic status toconfer salt stress tolerance
insoybean
MonaH.Soliman1· AwatifM.Abdulmajeed2· HaifaAlhaithloul3· BasmahM.Alharbi4· MohamedA.El‑Esawi5·
MirzaHasanuzzaman6· AmrElkelish7
Received: 24 May 2019 / Revised: 9 May 2020 / Accepted: 2 June 2020
© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2020
Abstract
Salinity is a deleterious factor that hinders plant production across the globe. Salinity reduces irrigation water quality. Plant
tolerance to salinity stress requires sophisticated adaptation at the physiological level and in metabolic pathways. Experiments
were set up to investigate saponin priming impact on the antioxidant metabolism and growth of soybean cultivated under
salt stress. Two concentrations of saponin, i.e., 5 and 10% were used for priming the seeds. Seed priming at 5% effectively
mitigated salinity stress mediating decline in growth and chlorophyll synthesis. Saponin (5%) improved chlorophyll and
carotenoid synthesis significantly and ameliorated the adverse effects of NaCl. Soybean seedlings primed with 5% saponin
exhibited the greater antioxidant enzyme activities and the biosynthesis of glutathione and ascorbic acid. Increased antioxi-
dant metabolism of primed seedlings accompanied by the reduced rate of lipid peroxidation and increased membrane stability
index. Additionally, saponin priming effectively augmented the relative water content of salt-stressed seedlings by improv-
ing the biosynthesis of proline, sugars, and glycine betaine. In conclusion, saponin priming (5%) proved beneficial through
modulation of the antioxidant system, osmolytes metabolism, and the significant reduction in sodium-ion accumulation.
Keywords Antioxidants· Growth· Lipid peroxidation· Osmolyte· Saponin· Soybean
Introduction
Salinity has been estimated to affect approximately 7% of
the land area worldwide (Ahmad etal. 2015; Fatma etal.
2016; Elkelish etal. 2019a). Higher levels of salt alter the
physiology of plants resulting in yield and growth reduc-
tions (Shabala and Cuin 2008), and this is a major constraint
to sustainable agricultural development (Wan etal. 2017;
Elkeilsh etal. 2019; El-Esawi etal. 2018a,b,c). Salinity has
been the major problem in the sustainable food production
of the arid and semiarid regions. It shall be pointed out that
global climate change has further aggravated the problem
compelling agriculturalists for the excess usage of saline
water for irrigation purposes thereby rendering more land
area as unproductive wasteland (Piscart etal. 2007; Nagaz
etal. 2012; Habib etal. 2020). Prolonged and high salt doses
induce hyperionic and hyperosmotic stress which causing
significant plant growth and development retardations by
interfering with physiological processes, i.e. nutrient imbal-
ance, membrane damage, enzyme inhibition, and metabolic
dysfunctions, including photosynthesis, hormone, and ions
Communicated by J. Gao.
* Amr Elkelish
amr.elkelish@science.suez.edu.eg
1 Botany andMicrobiology Department, Faculty ofScience,
Cairo University, Giza12613, Egypt
2 Biology Department, Faculty ofScience, University
ofTabuk, Umluj46429, SaudiArabia
3 Biology Department, College ofScience, Jouf University,
Sakaka2014, SaudiArabia
4 Biology Department, Faculty ofScience, University
ofTabuk, Tabuk71421, SaudiArabia
5 Botany Department, Faculty ofScience, Tanta University,
Tanta31527, Egypt
6 Department ofAgronomy, Faculty ofAgriculture,
Sher-e-Bangla Agricultural University, Sher-e-Bangla Nagar,
Dhaka1207, Bangladesh
7 Botany Department, Faculty ofScience, Suez Canal,
University, Ismailia, Egypt

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homeostasis which lead to plant death (Hasanuzzaman etal.
2018; Elkelish etal. 2019c; El-Esawi etal. 2019a; Soliman
etal. 2020). Photosynthesis inhibition due to soil salinity
has been reportedly shown to be associated with the altered
carbon and nitrogen metabolism (Ahmad etal. 2018a; Sal-
eem etal. 2020). Most deleterious products of environmental
stresses are the reactive oxygen species (ROS) having severe
impacts on the cellular macromolecules and hence the key
metabolic pathways (Cardeñosa etal. 2015; Hossain etal.
2017; Vwioko etal. 2017; El-Esawi etal. 2019b; Moustafa-
Farag etal. 2020). Stress generated ROS-mediated oxidation
of biomolecules including lipid bilayer membranes, nucleic
acids, proteins, and enzyme inhibitor, with an immediate
effect followed by growth inhibition and programmed cell
death (Shabala 2017; Soliman etal. 2018). Nevertheless, low
to moderate levels of ROS have some key roles and consen-
sus to have positive roles as essential translate signals into
suitable cellular communications in plant growth regulation
(Foyer etal. 2017; Bharti and Barnawal 2019; El-Esawi etal.
2020). However, their overproduction has adverse modifica-
tions to cell components causing oxidative damage, and the
functional integrity gets affected (Foyer and Noctor 2003;
El-Esawi and Alayafi 2019; Alhaithloul etal. 2019).
To combat the deleterious impact of excess ROS under
salinity stress plants tend to improve the indigenously exist-
ing defense mechanisms. Up-regulation of the tolerance
mechanisms help plants to withstand the stress triggered
oxidative damage (Jan etal. 2018; Abdelaal etal. 2019).
Key tolerance strategies involved include the antioxidant
systems, osmolyte accumulation, and ion sequestration
(Ahanger and Agarwal 2017; Soliman etal. 2019). Anti-
oxidant system mediating the neutralization of excess ROS
includes both non-enzymatic and enzymatic mechanisms
which effort together for prevention of the oxidative damage
(Nawaz etal. 2017; Elkelish etal. 2019b). Research evidence
is accumulating favoring the strengthening of the antioxidant
system due to various secondary metabolites and phytohor-
mones (Jan etal. 2018). Under adverse conditions, osmotic
adjustment is one of the important physiological features of
resilience to environmental stress. Proline, sugars, glycine
betaine and amino acids are accumulated in large quantities
as beneficial osmolytes to maintain the water content for
a greater lowering of the osmotic potential, therefore ena-
bling the security of the functional and structural stability
of plants (Zivcak etal. 2016; Zamin etal. 2019). Besides
this, the efficient uptake and assimilation of beneficial ions
like potassium and calcium, improving photosynthesis, and
accompanied with the compartmentalization of toxic ions
like sodium, are also key determinants of salt tolerance
potential (Tan etal. 2012; Ahanger and Agarwal 2017).
Seed priming is being utilized to augment seedlings emer-
gence and can effectively attenuate the salinity impact under
both optimal and adverse conditions (Singh etal. 2015; Jisha
and Puthur 2016). Seed priming is a beneficial strategy adapt-
ing glycophyte species to salt by inducing effective plant hor-
mones and antioxidant systems (Maiti and Pramanik 2013;
Gholami etal. 2015; Hassini etal. 2017). Plants provide meta-
bolic routes to the synthesis and accumulation of the secondary
metabolites providing plant protection against environmental
stressors (Tomar and Agarwal 2013; Joshi etal. 2018). Plant
secondary metabolites are considered as compounds without
a key role for primary growth in plants but have been con-
sidered very important for the interaction of plants with the
environment for imparting adaptation and defense mechanisms
(Nascimento and Fett-Neto 2010; Batiha etal. 2020). Thus,
the significance of secondary plant metabolites is undisputed.
Saponins are complex amphipathic glycosides and are
either steroidal or triterpenoid saponins, which is widely
distributed among plants (Vincken etal. 2007) and many
marine species like sea cucumbers and starfish (Faizal and
Geelen 2013). Saponins explore the biostimulant potential
as can be used as antifungal, stimulating seedling vigor and
contribute to the innate immunity of plants as photoprotect-
ants, ROS scavengers, and various commercial purposes
including surface-active agents and foaming (Kumar etal.
2013; Cheok etal. 2014).
Soybean (Glycine max L.) is a legume crop widely cul-
tivated in rainfed and irrigated agricultural conditions. It is
an important cash crop, and salinity significantly reduces its
productivity. The presence of high salinity imparts adverse
effects on the growth, nodulation, agronomic traits, quality,
and quantity of seed (Ha etal. 2018). The current investiga-
tion aimed at elucidating the probable role of saponin prim-
ing in improving salinity tolerance in soybean by examining
the ionic stress, osmotic stress, nitrogen content, antioxidant
system and oxidative stress, photosynthetic pigments, and
growth of soybean plants.
Materials andmethods
Saponin extraction
Saponins were extracted by adding 10g of quinoa hull meal
into a flask having 100ml of Milli-Q water. Flasks were
mechanically shaken for two hours and then the suspen-
sion was filtered two times to collect the filtrate. The extract
was considered as 10%, and 5% was prepared by diluting it
further.
Seed priming andstress treatment
Saponin priming treatment
Healthy, uniform soybean (Glycine max L.) seeds were sur-
face-sterilized with 5% NaOCl for 5min and then washed

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thoroughly with sterile water. Sterilized seeds were treated
with saponin as follows (a) distilled water (b) 5% saponin,
and (c) 10% saponin for 6h. After priming,seeds are dried
back before planting and were sown in pots having perlite and
sand and supplemented with half-strength Hoagland solution.
Seedlings were watered with full strength Hoagland’s solution
for 10days. Pots were kept in a completely randomized block
design with four replicates for each treatment in a greenhouse
with an average relative humidity of 70–75%, day/night tem-
peratures of 26 ± 2°C, and an average photoperiod of 18h
light/6h dark.
Salt stress treatment
After five days of germination, seedlings per pot were thinned.
The pots were separated in two sets, in which one set was
supplemented with 100mM NaCl. Therefore, the overall
treatments were as follows; (i) Control (primed with distilled
water), (ii) 100mM NaCl, (iii) Seeds primed with 5% saponin
(S1), (iv) Seeds primed with 10% saponin (S2), (v) 100mM
NaCl + S1, and (vi) 100mM NaCl + S2. Fifteen days old seed-
lings leaves were collected and analyzed for different physi-
ological parameters. Data presented in each treatment was
replicated five times.
Determination ofphotosynthetic pigments
Fresh 100mg leaf samples were extracted in 80% acetone and
the extract was centrifuged at 3,000g for 10min. The superna-
tant was made up, and the absorbances were read at 480, 645,
and 663nm against acetone (Arnon 1949).
Determination oflipid peroxidation, hydrogen
peroxide, andmembrane stability index (MSI)
The determination of lipid peroxidation was assayed as
reported by Heath and Packer (1968). While hydrogen perox-
ide (H2O2) is determined in fresh leaves after extraction with
0.1% TCA using the method of Sergiev etal. (1997). Finally,
membrane stability index (MSI) was measured by placing
0.1g leaf tissue in 10mL distilled water, followed by boiling at
40°C and measuring the electric conductivity (EC1), and then
boiled again at 100°C, and aEC (EC2) was recorded (Sairam
etal. 1997). MSI was determined as follows:
Determination ofrelative water content, proline,
glycine betaine, andtotal soluble sugars
Relative water content (RWC) was determined as reported by
Smart and Bingham (1974). Fresh leaves were punched for
(
MSI)=
[
1−
(
EC
1
∕EC
2)]
×
100
leaf discs from treated and normal plants to estimate the fresh
weight. The discs were permitted to acquire turgidity, and tur-
gid weight was reported. After that, the leaf discs were kept in
an oven at 80°C for 24h to record the dry weight. Calculations
were done using the following formula:
Proline was extracted by sulphosalicylic acid and deter-
mined in accordance to Bates etal. (1973). Glycine betaine
was estimated in the dry plant sample by 0.5% toluene using
the protocol of Grieve and Grattan (1983). After centrifuga-
tion at 5000g for 10min, 1ml supernatant was mixed with
1mL of sulfuric acid (2N). Out of this 0.5mL was mixed with
200μl of potassium tri-iodide solution flowed by cooling in
an ice bath. After that ice-cooled (2.8ml) distilled water and
5ml 1–2 di-chloroethane were added. The lower organic layer
absorbance was read at 365nm, and GB concentration was
calculated from a standard curve.
Sugar content was determined after homogenizing dry plant
samples in boiling ethanol. The extract was centrifuged at
5000g for 20min. Supernatants were used for measuring sug-
ars after reacting to its known volume with anthrone reagent.
Absorbance was taken at 585nm, and the concentration of
sugars was determined from a glucose standard curve(Yemm
and Willis 1954).
Assay ofantioxidant enzymes
Fresh leaves (0.5g) were macerated in 50mM potassium
phosphate buffer (pH 7.0) supplemented with 1% soluble pol-
yvinyl pyrrolidine, followed by centrifugation at 12,000rpm
for 15min at 4°C. The supernatant was utilized for the assays
of enzymes activity, and the protein in enzyme extracts was
quantified following Lowry etal. (1951).
For the assay of superoxide dismutase (SOD), the absorb-
ance was read at 560nm (Beyer and Fridovich 1987). SOD
activity was expressed as enzyme unit (EU) mg−1 protein.
Catalase (CAT) activity was determined by recording
the absorbance at 240nm for 2min (Lück 1965) and was
expressed as EU mg−1 protein.
For determining the APX activity, the method described
by Nakano and Asada (1981) was adopted and the change in
absorbance was recorded at 290nm for 3min.
Glutathione reductase (GR) activity was determined by
observing the change in absorbance at 340nm for 2min (Car-
lberg and Mannervik 1985) and was expressed as EU mg−l
protein.
RWC
=
Fresh weight −Dry weight
Turgid weight −Dry weight
×
100

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Determination ofnon‑enzymatic antioxidants
For estimation of ascorbic acid fresh leaf samples were
extracted using 6% TCA, and 2% dinitrophenyl-hydrazine
and 10% thiourea were added to the homogenate. The result-
ant mixture was boiled in a water bath for 15min, followed
by cooling and centrifugation at 1000g for 10min. Pellet
was dissolved in 80% H2SO4 and absorbance was read at
530nm. The ascorbic acid standard curve was utilized for
calculations (Mukherjee and Choudhuri 1983).
Reduced glutathione (GSH) was determined by extracting
100mg fresh leaf tissues in phosphate buffer and centrifuged
for 15min at 3000g. 500µl of supernatants were mixed with
1.0ml of 5, 5-dithiobis-2-nitrobenzoic acid. After 10min,
the optical density was read at 412nm (Ellman 1959). GSH
standard curve was utilized for calculations.
Nitrate reductase activity was assayed in accordance to
(Gharbi and Hipkin 1984). Fresh leaves are finely chopped,
and 0.2g was placed in 5mL of a composite assay buffer.
Assays were then concluded by putting the 0.2mL sulfanila-
mide and 0.2mL NEDA in a boiling water bath for 5min.
The absorbance was then read at 543nm.
Estimation ofleaf ion content
Na ion concentration was estimated using a flame photom-
eter and samples were digested in H2SO4/HNO3 (1/5, v/v).
Chloride was determined by the titration method. After
extraction in water, samples were titrated against silver
nitrate (0.02N) solution.
Determination ofnitrogen content
Estimation of nitrogen was done following the method of
(Zheljazkov and Nielsen 1996).
Statistical analysis
Data presented are means (± SE) of 3 replicates. Data as sta-
tistically analyzed employing Duncan’s Multiple Range Test
and using One Way ANOVA. Differences in means were cal-
culated by the least significant differences (LSD) (p = 0.05).
Results
Influence of NaCl stress and saponin priming on the length,
fresh and dry weights of soybean shoot is indicated in
Table1. Due to salinity stress, shoot length and fresh and
dry weight were declined by 39.63, 33.70, and 32.63% rela-
tive to control. Saponin (5%) primed seedlings showed an
increase of 19.26, 20.44, and 29.04% in length, fresh and dry
weight compared to the control plants while 10% saponin
did not impart any significant change in growth and biomass
accumulation. Salinity stressed seedlings primed with 5%
saponin showed an increase of 14.50, 17.12, and 12.58% in
length, fresh and dry weight over the NaCl stressed plants
without saponin. However, NaCl + 10% saponin treated
seedlings exhibited a further decline over the NaCl stressed
plants (Table1).
Soybean seedlings primed with saponin (5%) exhibited a
remarkable enhancement in carotenoid and chlorophyll syn-
thesis, and saponin also mitigated NaCl effects (Fig.1a, b).
Increase percentages in chlorophyll and carotenoid content
due to saponin (5%) priming were 16.42 and 12.50%, respec-
tively. Salt treatment reduced chlorophyll and carotenoid
contents by 45.26% and 25.71%, respectively, compared to
control. In NaCl + 5% saponin treated seedlings, the declines
were 31.21% and 14.28% reflecting the amelioration of NaCl
mediated adverse effect (Fig.1a, b). At 10% saponin inhibi-
tory impact on the synthesis of pigments was observed.
Seed priming with saponin (5%) significantly reduced
the oxidative damage induced by NaCl stress by decreas-
ing the new release of hydrogen peroxide thereby reduc-
ing the lipid peroxidation (MDA content) and improving
the MSI (Fig.2a–c). Salt treatment increased hydrogen
peroxide and lipid peroxidation by 55.93% and 40.80%,
respectively causing 37.51% decline in membrane stabil-
ity index. Compared to control seedlings, soybean plants
primed with 5% saponin showed a reduction of 25.36%
in hydrogen peroxide generation and 27.99% in lipid per-
oxidation and resulting in 5.75% improvement in MSI.
However, priming with 10% does not impart any positive
impact on these parameters but was observed to enhance
the generation of hydrogen peroxide. NaCl + 5% saponin
primed seedlings exhibited a decline of 7.27% in hydro-
gen peroxide and 18.05% in lipid peroxidation over the
NaCl stressed plants causing 14.09% amelioration in MSI
(Fig.2a–c). The 5% saponin priming enhanced glycine
betaine, proline, and sugars in soybean under normal and
saline conditions resulting in increased relative water con-
tent (RWC) in them (Fig.3a–d). Relative to control plants,
Table 1 Effect of saponin seed priming on the shoot length (cm),
shoot fresh weight (g/five plant) and shoot dry weight (g/five plant) in
soybean (Glycine max) under NaCl induced salinity stress
Data presented is mean (± SE) of three replicates and values denoted
by different letter are significantly different at P < 0.05
Treatments Shoot length Shoot fresh weight Shoot dry weight
Control 5.56 ± 0.115b 2.70 ± 0.200b 0.9046 ± 0.015b
NaCl 3.36 ± 0.183d 1.79 ± 0.114d 0.6094 ± 0.010d
S1 6.89 ± 0.261a 3.39 ± 0.099a 1.2749 ± 0.049a
S2 5.44 ± 0.383b 2.62 ± 0.132b 0.5891 ± 0.0011de
NaCl + S1 3.93 ± 0.207c 2.16 ± 0.106c 0.6971 ± 0.0064c
NaCl + S2 3.34 ± 0.166d 1.71 ± 0.044d 0.5892 ± 0.011de

Acta Physiologiae Plantarum (2020) 42:114
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proline content increased by 43.47%, glycine betaine by
20.55%, and sugars by 13.33% in seedlings primed with
5% saponin. Relative to control plants, NaCl treatment
improved the biosynthesis of sugars, glycine betaine, and
proline by 31.53, 33.92, and 32.30% respectively. Highest
increment in the biosynthesis of proline (52.29%), glycine
betaine (37.12%) and sugars (45.27%) was observed in the
seedlings primed with 5% saponin and treated with NaCl
(NaCl + 5% saponin) resulting in the maximal decrease of
17.45% in RWC over the NaCl stressed plants (Fig.3a–d).
Priming with 10% was not so beneficial in improving the
RWC. Relative to control, RWC increased by 5.31% in 5%
Fig. 1 Effect of saponin seed priming on the a total chlorophyll and b carotenoid content in soybean (Glycine max) under NaCl induced salinity
stress. Data presented are mean (± SE) of three replicates and bars denoted by different letter are significantly different at P < 0.05
Fig. 2 Effect of saponin seed priming on the a hydrogen peroxide, b
lipid peroxidation, and c membrane stability index in soybean (Gly-
cine max) under NaCl induced salinity stress. Data presented are
mean (± SE) of three replicates and bars denoted by different letter
are significantly different at P < 0.05

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primed seedlings while as declined by 32.72% due to NaCl
stress (Fig.3d).
Antioxidant enzymes activity and content of non-enzy-
matic antioxidants exhibited a significant enhancement in
seedlings primed with 5% saponin over the control plants
and 10% saponin does not prove beneficial (Fig.4a–f).
Activities of CAT, SOD, APX, and GR were stimulated by
19.62, 15.58, 32.75, and 10.32%, respectively by saponin
priming (5%). Salinity treatment imparted an enhance-
ment of 19.41, 31.86, 45.41 and 28.66%, respectively in
the activities of CAT, SOD, APX and GR, and seedling
primed with 5% saponin and treated with NaCl (NaCl + 5%
saponin) exhibited a further increment of 9.38% in SOD,
15.10% in CAT, 7.80% in APX and 8.27% in GR over the
NaCl treated plants. The 10% saponin was not so effective
under normal and saline conditions (Fig.4a–d). Ascorbic
acid (AsA) content declined by 23.96% while the reduced
glutathione (GSH) content was increased by 30.88% in
NaCl stressed plants over the control plants. Saponin (5%)
priming enhanced the AsA and GSH content by 13.53%
and 19.19%, respectively and also ameliorated the negative
impact of NaCl by increasing 12.39% in AsA content over
the NaCl treated plants (Fig.4e, f).
Priming of soybean seeds with 5% saponin significantly
augmented the activity of nitrate reductase and also amelio-
rated the negative effect of NaCl stress (Fig.5). Compared
to control plants, NR activity decreased by 50.71% due to
NaCl treatment and increased by 14.24% in saponin (5%)
primed seedlings. In NaCl + 5% saponin primed seedlings an
amelioration of 15.87% was observed over the NaCl treated
seedlings, however, seedlings primed with 10% saponin does
not show any apparent increase in NR activity under normal
and saline conditions (Fig.5).
Nitrogen level decreased by 37.73% and 50.21% in shoot
and root, respectively due to NaCl stress and was observed
to increase by 18.04% (shoot) and 16.93% (root) in seedlings
primed with 5% saponin.
Saponin (5%) priming declined the absorption of Na+ and
Cl− ions by 9.74% and 28.83%, respectively in the shoot, and
5.32% and 29.01%, respectively in the root (Table2). The
NaCl treated plants resulted in higher Na accumulation of
58.35 and 52.36% in shoot and root, respectively, while 5%
Fig. 3 Effect of saponin seed priming on a proline, b glycine betaine,
c soluble sugars, and d relative water content (RWC) in soybean
(Glycine max) under NaCl induced salinity stress. Data presented are
mean (± SE) of three replicates and bars denoted by different letter
are significantly different at P < 0.05

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Fig. 4 Effect of saponin seed priming on the activity of a superox-
ide dismutase, b catalase, c ascorbate peroxidase, and d glutathione
reductase the content of e ascorbic acid and f reduced glutathione in
soybean (Glycine max) under NaCl induced salinity stress. Data pre-
sented are mean (± SE) of three replicates and bars denoted by differ-
ent letter are significantly different at P < 0.05
Fig. 5 Effect of saponin seed
priming on the activity of nitrate
reductase in soybean (Glycine
max) under NaCl induced salin-
ity stress. Data presented are
mean (± SE) of three replicates
and bars denoted by different
letter are significantly different
at P < 0.05

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saponin primed and NaCl stressed (NaCl + 5% saponin) soy-
bean plants exhibited a decline of 16.32 and 19.94% in Na
accumulation in shoot and root, respectively, comparing to
salt-stressed plants (Table2). A similar trend was recorded
in the case of Cl ion. Supplementation of 10% saponin did
not show any remarkable impact on the accumulations of
Na and Cl ions.
Discussion
Salinity is among the devastating stressors affecting food
crops worldwide. Increased accumulation of toxic salts not
only affects the growth of crops but also results in the conver-
sion of agricultural land into wastelands (Kreeb 1974; Zörb
etal. 2019). Therefore, to address this problem, researchers
have to develop effective management techniques and evolve
plants that are salt-tolerant to bring the salt-affected areas
under cultivation. Besides this improving the salt tolerance
potential of crop plants by adopting newer and cost-effective
methods can be worthwhile (Li etal. 2018; Huang etal.
2018). Seed priming with phytohormones, mineral ions, etc.
are value-added techniques and improve the stress resistance
potential in various crops (Jisha etal. 2013; Hussain etal.
2016, 2017). In the same direction, we conducted certain
experiments to test the efficacy of priming with saponin
extracted from the quinoa hull meal against the NaCl stress
in soybean. Priming of soybean with saponin affected the
growth in concentration-dependent manner where the lower
concentration improved the growth parameters under both
salinity and normal conditions. Greater salinity concentra-
tions reduced plant growth by reducing cellular division rate,
cell expansion, and proliferation (Taleisnik etal. 2009; Chat-
terjee and Majumder 2010). The current study reveals that
5% saponin reduced the adverse effect of NaCl and improved
growth parameters. Very rare research reports are availa-
ble discussing the growth regulation of plants under stress
owing to the triterpenoid applications. Higher concentrations
of plant extracts rich in triterpenoids have inhibitory impacts
on the growth of other plants (Wang etal. 2014). Tomar
etal. (2015) have reported that lower concentrations of Jat-
ropha extract improves germination and growth of wheat
seedlings significantly. We also observed that a higher per-
centage (10%) of saponin extract declined the growth while
as lower concentration (5%) resulted in improvement.
It has been reported that myo-inositol potentially reduces
the inter-nucleosome fragmentation under salt stress (Chat-
terjee and Majumder 2010) and saponins (5%) may have
prevented the DNA fragmentation and improved the activ-
ity of membrane proteins for maintaining the optimal water
content. Transgenic Medicago truncatula plants exhibiting
greater production of saponins show better growth under
salinity (Confalonieri etal. 2009). Improved growth in sapo-
nin primed seedlings in the present study may be attributed
by the greater uptake of nitrogen which affects the activities
of key enzymes and photosynthesis leading to greater accu-
mulation of osmolytes and hence the salt tolerance (Iqbal
etal. 2015).
In this study, 5% saponin priming was effective in reduc-
ing the accumulation of Na and Cl significantly under nor-
mal, and NaCl stressed conditions. Aggravation of saline
soil solution affects the expression and activity of transport
proteins and reduces the uptake of mineral nutrients from the
root through induction of membrane depolarization (Assaha
etal. 2017; Azeem etal. 2019). Salinity disturbs the mineral
relations of plants by decreasing N availability (Ahmad etal.
2013). Increasing oxidative damage is the result of excess
sodium, which causes ionic stress. To avoid the adverse
impacts of the Na mediated on plant growth, the expression
of several transport proteins such as NHX, HKT, HAK, SOS
is increased for better Na + exclusion and compartmentation
(Bharti etal. 2016). Reports addressing saponin’s role in ion
compartmentation are rare, and further studies in this direc-
tion are needed to reach a solid conclusion.
Salinity hampers water uptake by down-regulating the
expression of the tonoplast aquaporins (Boursiac etal.
2005). Saponin addition to NaCl treatment increased RWC.
Seed priming with saponins resulted in increased water
potential in salt-stressed quinoa causing greater growth
protection (Yang etal. 2018). The apparent effect on the
Table 2 Effect of saponin
seed priming on the content of
nitrogen (N), sodium (Na) and
chloride (Cl) (mg g−1 DW) in
soybean (Glycine max) under
NaCl induced salinity stress
Data presented are mean (± SE) of three replicates and values denoted by different letter are significantly
different at P < 0.05
Treatments Leaf Root
NNa Cl N Na Cl
Control 33.07 ± 2.44b 4.31 ± 0.46c 3.78 ± 0.49c 16.19 ± 0.81b 6.76 ± 0.20d 2.93 ± 0.255b
NaCl 20.59 ± 1.73e 10.35 ± 0.83a 5.88 ± 0.18a 8.06 ± 0.61e 14.19 ± 1.20a 3.69 ± 0.200a
S1 40.35 ± 2.47a 3.89 ± 0.15d 2.69 ± 0.49d 19.49 ± 1.57a 6.04 ± 0.17e 2.08 ± 0.412d
S2 30.86 ± 2.06c 4.33 ± 0.27c 3.66 ± 0.38c 15.62 ± 1.06c 6.45 ± 0.26de 2.75 ± 0.259b
NaCl + S1 24.72 ± 1.07d 8.66 ± 0.61b 4.92 ± 0.22b 10.07 ± 0.89d 11.36 ± 0.53c 2.68 ± 0.207bc
NaCl + S2 19.27 ± 2.13e 10.40 ± 0.50a 5.95 ± 0.28a 8.07 ± 0.36e 13.61 ± 0.37b 3.73 ± 0.062a

Acta Physiologiae Plantarum (2020) 42:114
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Page 9 of 13 114
RWC of cells and tissues osmolytes have been reported to
assist in scavenging of ROS (Couée etal. 2006). Proline,
sugar, and glycine betaine accumulation in saponin (5%)
primed soybean seedlings may contribute to higher water
use efficiency, water uptake, and photosynthesis potential.
Salinity enhances the compatible solutes accumulation, thus
allowing the protection of enzyme activity and cellular func-
tioning (Zivcak etal. 2016; Ahanger etal. 2017). Under
saline conditions, the expression of the osmolyte synthesiz-
ing proteins gets up-regulated with a significant decline in
the catabolizing enzymes (Ahmad 2010). Greater accumu-
lation of osmolytes protects the protein turn over and the
regulation of stress-protective proteins (Ahanger etal. 2014).
Improved glycine betaine improves metabolism by mediat-
ing the expressions of proteins included in blockage of ROS
mediated signaling (Einset and Connolly 2009).
Increased growth in saponin primed seedlings was
accompanied with greater chlorophyll and carotenoid pro-
duction in them. Reduced chlorophyll in NaCl treated seed-
lings has also been demonstrated by Rasool etal. (2013)
and Ahanger and Agarwal (2017) in Cicer arietinum and
Triticum aestivum respectively. Our results are in agreement
with those of Hafsi etal. (2017), reporting a decline in pig-
ment content and photosynthetic efficacy following expo-
sure to salt stress. Application of Jatropha extracts (10%)
to wheat increased pigment synthesis (Tomar and Agarwal
2013). Salinity drastically affects the synthesis and function-
ing of pigment-protein complex and triggers hindrances in
de novo synthesis of proteins and chlorophyll (Ahmad etal.
2018b). Priming with 5% saponin resulted in increased chlo-
rophyll synthesis causing obvious effects on growth through
enhanced metabolism. Saponin priming may have improved
the uptake of key ions like magnesium involved in chloro-
phyll synthesis and other ions counterbalancing the toxic
effects of salinity on the photosynthetic machinery. Recently,
Yang etal. (2018) reported that priming of quinoa with the
saponin (up to 25%) increased the chlorophyll level and the
photosynthetic attributes like stomatal conductance and pho-
tosynthetic rate under salinity stress.
In the current study soybean seedlings subjected to
salinity exhibited an apparent increment in the accumula-
tion of hydrogen peroxide reflecting in the declined mem-
brane functioning and 5% saponin priming was observed to
reduce the salinity mediated lipid peroxidation. Results of
the higher lipid peroxidation in salt-stressed plants mediated
by greater generation of hydrogen peroxide corroborate with
those reported by Ahmad etal. (2012) for mustard, Rasool
etal. (2013) for chickpea, Ahanger and Agarwal (2017) for
wheat, and Matrinze etal. (2018) for tomato. Salinity stress
triggers the production of ROS, resulting in peroxidation
of essential macromolecules, particularly lipids, thus affect-
ing the plasma membrane dysintegrity resulting in altered
membrane functioning (Ahmad etal. 2012; Kovács etal.
2017). Seed priming has been reported to improve the toler-
ance of many crop plants to different stresses like alkalinity
(Abdel Latef and Tran 2016) and cadmium (Jan etal. 2018).
However, the impact of saponins has not been worked out.
Salinity affects the polyunsaturated fatty acid composition
of membranes resulting in membrane dysfunction (Alqarawi
etal. 2014) and saponin at low concentrations may have
contributed to the maintenance of the ratio of polyunsatu-
rated to saturated fatty acids. A possible reason for improved
membrane functioning is the reduction of the generation of
ROS that triggers the peroxidation of unsaturated fatty acids
resulting in membrane instability, and hence leakage and
desiccation occur (Zhu etal. 2018; Wang etal. 2019). There-
fore, the present investigation favors the potentiality of exog-
enous saponin priming in the protection of membrane lipids
from the ROS mediated peroxidation. Abdel Latif and Tran
(2016) demonstrated that priming improves the antioxidant
potential to counteract the ROS mediated oxidative dam-
age. Seed priming with saponins improved the antioxidant
system by enhancing the activity of enzymes assayed and the
content of ascorbic acid and reduced glutathione, therefore,
strengthening the soybean seedlings against the NaCl medi-
ated oxidative damage (de Costa etal. 2013; Puente-Garza
etal. 2017). However, previous results supporting the pre-
sent research finding is rare.
Up-regulated antioxidant enzyme activity in plants results
in quick removal of toxic ROS (Agami etal. 2016; Ahanger
etal. 2017). The SOD, CAT, POD, and APX are all pro-
tective enzymes that contribute to the elimination and sub-
sequent neutralization of superoxide and hydrogen perox-
ide radicals (Qu etal. 2019). Improvement of antioxidant
enzymes in response to salinity has also been reported in
Cicer arietinum (Rasool etal. 2013) and tomato (Ahanger
etal. 2018). Improved SOD, CAT, and APX activity in sapo-
nin (5%) primed seedlings potentially reduced the super-
oxide and H2O2 for preventing the generation of hydroxyl
(OH−) radicals. Enhanced activities of CAT and APX in
saponin primed seedlings may have mediated growth main-
tenance by improved protection to delicate organelles like
chloroplast and mitochondria carrying important metabolic
pathways (Ahanger etal. 2014, 2017, 2018). Greater activity
of CAT prevents the excess accumulation of H2O2 prevent-
ing its diffusion through membranes so that membranes and
organelles get least affected (Bienert and Chaumont 2014;
Tamma etal. 2018). Ascorbic acid, glutathione, APX, and
GR are the essential components of scavenging pathways
involved in H2O2 detoxification in the AsA-GSH cycle (Noc-
tor and Foyer 1998; Qu etal. 2019) and increased activity of
enzymes of this cycle components in saponin primed seed-
lings significantly enhanced the cellular stability. Increased
activities of APX and GR contribute to the maintenance of
the NADP/NADPH ratio preventing the generation of super-
oxide radicals via the optimal flow of electrons to oxygen

Acta Physiologiae Plantarum (2020) 42:114
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114 Page 10 of 13
(Noctor and Foyer 1998). Plant cultivars maintaining higher
redox levels below critical cases elicit adaptive responses
and have greater stress withstanding ability (Iqbal etal.
2015; Shah etal. 2019). The antioxidant system was further
strengthened by the overproduction of glycine betaine, pro-
line, and sugars in primed seedlings.
Conclusion
Conclusively, the study reveals that the 5% level of saponin
is beneficial in ameliorating the negative impact of given
levels of salinity. Priming seeds with low (5%) saponin
improved antioxidant system for better counteraction of
NaCl induced oxidative damage, imparting membrane pro-
tection, photosynthetic pigment protection. Moreover, the
useful role of osmolytes was also proved in primed treat-
ments leading to enhancement of RWC. Saponin addition
affected the N content and NR activity in salt-affected seed-
lings. Saponin decreased ionic toxicity as induced by Na+
and Cl− ions. Thus, the saponin-induced improved growth
parameters were evident as a result of the improvement of
all the physiological and biochemical parameters examined
in the present study (Fig.6). However, there are not many
research findings existing at present to support the find-
ings of the present study. Many aspects remain unexplored.
Therefore, further physiological researches associated with
diversity studies (El-Esawi 2017; El-Esawi etal. 2016) are
required to improve crops and draw a supportive conclusion.
Author contributions statement MS, AE, AA, HA, BE,
MAE, and MH conceived and designed the experiments.
MS, MAE and AE conducted the experiments. MS, AE, AA,
MAEand HA analyzed the data and drafted the manuscript.
MS, AE, AA, BE, MAE, and HA supported the project. AA,
MAE, and MH polished the manuscript. All authors read
and approved the manuscript.
References
Abdel Latef AA, Tran L-SP (2016) Impacts of priming with silicon
on the growth and tolerance of maize plants to alkaline stress.
Front Plant Sci 7:243
Abdelaal KA, EL-Maghraby LM, Elansary H etal (2019) Treatment of
sweet pepper with stress tolerance-inducing compounds allevi-
ates salinity stress oxidative damage by mediating the physio-
biochemical activities and antioxidant systems. Agronomy 10:26.
https ://doi.org/10.3390/agron omy10 01002 6
Agami RA, Medani RA, Abd El-Mola IA, Taha RS (2016) Exogenous
application with plant growth promoting rhizobacteria (PGPR)
or proline induces stress tolerance in basil plants (Ocimum basili-
cum L.) exposed to water stress. Int J Environ Agri Res 2:78
Ahanger MA, Agarwal RM (2017) Salinity stress induced alterations
in antioxidant metabolism and nitrogen assimilation in wheat
(Triticum aestivum L) as influenced by potassium supplementa-
tion. Plant Physiol Biochem 115:449–460
Ahanger MA, Ashraf M, Bajguz A, Ahmad P (2018) Brassinosteroids
regulate growth in plants under stressful environments and cross-
talk with other potential phytohormones. J Plant Growth Regul.
https ://doi.org/10.1007/s0034 4-018-9855-2
Ahanger MA, Tomar NS, Tittal M etal (2017) Plant growth under
water/salt stress: ROS production; antioxidants and significance
of added potassium under such conditions. Physiol Molecul Biol
Plants 23:731–744
Ahanger MA, Tyagi SR, Wani MR, Ahmad P (2014) Drought toler-
ance: role of organic osmolytes, growth regulators, and mineral
nutrients. Physiological mechanisms and adaptation strategies in
plants under changing environment. Springer, Berlin, pp 25–55
Ahmad P (2010) Growth and antioxidant responses in mustard (Bras-
sica juncea L.) plants subjected to combined effect of gibberellic
acid and salinity. Archiv Agronomy Soil Sci 56:575–588
Ahmad P, Abass Ahanger M, Nasser Alyemeni M etal (2018a) Mitiga-
tion of sodium chloride toxicity in Solanum lycopersicum L. by
supplementation of jasmonic acid and nitric oxide. J Plant Inter-
act 13:64–72. https ://doi.org/10.1080/17429 145.2017.14208 30
Fig. 6 Saponin priming model
showing the regulation of salt
stress tolerance

Acta Physiologiae Plantarum (2020) 42:114
1 3
Page 11 of 13 114
Ahmad P, Alyemeni MN, Ahanger MA etal (2018b) Upregulation
of antioxidant and glyoxalase systems mitigates NaCl stress
in Brassica juncea by supplementation of zinc and calcium.
J Plant Interact 13:151–162. https ://doi.org/10.1080/17429
145.2018.14414 52
Ahmad P, Azooz MM, Prasad MNV (eds) (2013) Salt Stress in Plants.
Springer, New York
Ahmad P, Kumar A, Ashraf M, Akram NA (2012) Salt-induced
changes in photosynthetic activity and oxidative defense system
of three cultivars of mustard (Brassica juncea L.). Afr J Biotech
11:2694–2703
Ahmad P, Sarwat M, Bhat NA etal (2015) Alleviation of cadmium
toxicity in Brassica juncea L. (Czern & Coss.) by calcium appli-
cation involves various physiological and biochemical strate-
gies. PLoS ONE 10:e0114571. https ://doi.org/10.1371/journ
al.pone.01145 71
Alhaithloul HA, Soliman MH, Ameta KL etal (2019) Changes in
ecophysiology, osmolytes, and secondary metabolites of the
medicinal plants of mentha piperita and catharanthus roseus
subjected to drought and heat stress. Biomolecules 10:43. https
://doi.org/10.3390/biom1 00100 43
Alqarawi A, Hashem A, Abd_Allah E etal (2014) Effect of salinity
on moisture content, pigment system, and lipid composition in
Ephedra alata Decne. Acta Biol Hung 65:61–71
Arnon DI (1949) Copper enzymes in isolated chloroplasts. Polyphe-
noloxidase Beta vulgaris Plant Physiol 24:1–15
Assaha DV, Ueda A, Saneoka H et al (2017) The role of Na+ and
K+ transporters in salt stress adaptation in glycophytes. Front
Physiol 8:509
Azeem M, Qasim M, Waseem Abbasi M, etal (2019) Salicylic acid
seed priming modulates some biochemical parameters to improve
germination and seedling growth of salt stressed wheat (Triticum
aestivum L.) Pakistan J Botany https ://doi.org/10.30848 /PJB20
19-2(1)
Bates LS, Waldren RP, Teare ID (1973) Rapid determination of free
proline for water-stress studies. Plant Soil 39:205–207. https ://
doi.org/10.1007/BF000 18060
Batiha GE-S, Magdy Beshbishy A, Adeyemi OS etal (2020) Phyto-
chemical screening and antiprotozoal effects of the Methanolic
Berberis vulgaris and Acetonic Rhus Coriaria extracts. Mol-
ecules 25:550. https ://doi.org/10.3390/molec ules2 50305 50
Beyer WF, Fridovich I (1987) Assaying for superoxide dismutase
activity: some large consequences of minor changes in condi-
tions. Anal Biochem 161:559–566. https ://doi.org/10.1016/0003-
2697(87)90489 -1
Bharti N, Barnawal D (2019) Chapter Five - Amelioration of Salinity
Stress by PGPR: ACC Deaminase and ROS Scavenging Enzymes
Activity. In: Singh AK, Kumar A, Singh PK (eds) PGPR Amelio-
ration in Sustainable Agriculture. Woodhead Publishing, Cam-
bridge, pp 85–106
Bharti N, Pandey SS, Barnawal D etal (2016) Plant growth promoting
rhizobacteria Dietzia natronolimnaea modulates the expression
of stress responsive genes providing protection of wheat from
salinity stress. Sci Rep 6:34768
Bienert GP, Chaumont F (2014) Aquaporin-facilitated transmembrane
diffusion of hydrogen peroxide. Biochimica et Biophysica Acta
(BBA) General Subjects 1840:1596–1604
Boursiac Y, Chen S, Luu D-T etal (2005) Early effects of salinity on
water transport in Arabidopsis roots. Molecular and cellular fea-
tures of aquaporin expression. Plant Physiol 139:790–805
Cardeñosa V, Medrano E, Lorenzo P etal (2015) Effects of salinity and
nitrogen supply on the quality and health-related compounds of
strawberry fruits (Fragaria × ananassa cv. Primoris). J Sci Food
Agric 95:2924–2930. https ://doi.org/10.1002/jsfa.7034
Carlberg I, Mannervik B (1985) [59] Glutathione reductase. In: Meth-
ods in enzymology. Elsevier, pp 484–490
Chatterjee J, Majumder AL (2010) Salt-induced abnormalities on
root tip mitotic cells of Allium cepa: prevention by inositol
pretreatment. Protoplasma 245:165–172
Cheok CY, Salman HAK, Sulaiman R (2014) Extraction and quanti-
fication of saponins: A review. Food Res Int 59:16–40
Confalonieri M, Cammareri M, Biazzi E etal (2009) Enhanced
triterpene saponin biosynthesis and root nodulation in trans-
genic barrel medic (Medicago truncatula Gaertn) expressing
a novel β-amyrin synthase (AsOXA1) gene. Plant Biotechnol J
7:172–182. https ://doi.org/10.1111/j.1467-7652.2008.00385 .x
Couée I, Sulmon C, Gouesbet G, El Amrani A (2006) Involvement
of soluble sugars in reactive oxygen species balance and
responses to oxidative stress in plants. J Exp Bot 57:449–459
de Costa F, Yendo ACA, Fleck JD etal (2013) Accumulation of
a bioactive triterpene saponin fraction of Quillaja brasilien-
sis leaves is associated with abiotic and biotic stresses. Plant
Physiol Biochem 66:56–62. https ://doi.org/10.1016/j.plaph
y.2013.02.003
Einset J, Connolly EL (2009) Glycine betaine enhances extracellular
processes blocking ROS signaling during stress. Plant Signal
Behav 4:197–199
El-Esawi MA (2017) Genetic diversity and evolution of Brassica
genetic resources: from morphology to novel genomic technol-
ogies–a review. Plant Genetic Res 15:388–399
El-Esawi MA, Alayafi AA (2019) Overexpression of rice Rab7 gene
improves drought and heat tolerance and increases grain yield in
rice (Oryza sativa L). Genes (Basel) 10(1):56
El-Esawi MA, Alaraidh IA, Alsahli AA, Ali HM, Alayafi AA, Witczak
J, Ahmad M (2018a) Genetic variation and alleviation of salinity
stress in barley (Hordeum vulgare L). Molecules 23(10):2488
El-Esawi MA, Alaraidh IA, Alsahli AA, Alzahrani SM, Ali HM,
Alayafi AA, Ahmad M (2018b) Serratia liquefaciens KM4
improves salt stress tolerance in maize by regulating redox poten-
tial, ion homeostasis, leaf gas exchange and stress-related gene
expression. Int J Mol Sci 19(11):3310
El-Esawi MA, Al-Ghamdi AA, Ali HM, Alayafi AA, Witczak J,
Ahmad M (2018c) Analysis of genetic variation and enhance-
ment of salt tolerance in french pea (Pisum sativum L). Int J
Molec Sci 19(8):2433
El-Esawi MA, Al-Ghamdi AA, Ali HM, Alayafi AA (2019a) Azos-
pirillum lipoferum FK1 confers improved salt tolerance in chick-
pea (Cicer arietinum L.) by modulating osmolytes, antioxidant
machinery and stress-related genes expression. Environ Exp Bot
159:55–65
El-Esawi MA, Al-Ghamdi AA, Ali HM, Ahmad M (2019b). Overex-
pression of AtWRKY30 transcription factor enhances heat and
drought stress tolerance in wheat (Triticum aestivum L.). Genes
(Basel), 10(2): 163.
El-Esawi MA, Elkelish A, Soliman M etal (2020) Serratia marcescens
BM1 enhances cadmium stress tolerance and phytoremediation
potential of soybean through modulation of osmolytes, leaf gas
exchange, antioxidant machinery, and stress-responsive genes
expression. Antioxidants 9:43. https ://doi.org/10.3390/antio
x9010 043
El-Esawi MA, Germaine K, Bourke P, Malone R (2016) AFLP analysis
of genetic diversity and phylogenetic relationships of Brassica
oleracea in Ireland. CR Biol 339(5–6):163–170
Elkeilsh A, Awad YM, Soliman MH etal (2019) Exogenous application
of β-sitosterol mediated growth and yield improvement in water-
stressed wheat (g) involves up-regulated antioxidant system. J
Plant Res. https ://doi.org/10.1007/s1026 5-019-01143 -5
Elkelish A, Alnusaire TS, Soliman MH etal (2019) Calcium avail-
ability regulates antioxidant system, physio-biochemical activi-
ties and alleviates salinity stress mediated oxidative damage
in soybean seedlings. J Appl Botany Food Quality. https ://doi.
org/10.5073/JABFQ .2019.092.036

Acta Physiologiae Plantarum (2020) 42:114
1 3
114 Page 12 of 13
Elkelish AA, Alhaithloul HAS, Qari SH etal (2019) Pretreatment
with Trichoderma harzianum alleviates waterlogging-induced
growth alterations in tomato seedlings by modulating physi-
ological, biochemical, and molecular mechanisms. Environ
Exp Bot. https ://doi.org/10.1016/j.envex pbot.2019.10394 6
Elkelish AA, MonaH S, Alhaithloul HA, El-Esawi MA (2019) Sele-
nium protects wheat seedlings against salt stress-mediated oxi-
dative damage by up-regulating antioxidants and osmolytes
metabolism. Plant Physiol Biochem https ://doi.org/10.1016/j.
plaph y.2019.02.004
Ellman GL (1959) Tissue sulfhydryl groups. Arch Biochem Biophys
82:70–77
Faizal A, Geelen D (2013) Saponins and their role in biological
processes in plants. Phytochem Rev 12:877–893
Fatma M, Masood A, Per TS, Khan NA (2016) Nitric oxide alleviates
salt stress inhibited photosynthetic performance by interacting
with sulfur assimilation in mustard. Front Plant Sci 7:521
Foyer CH, Noctor G (2003) Redox sensing and signalling associ-
ated with reactive oxygen in chloroplasts, peroxisomes and
mitochondria. Physiol Plant 119:355–364. https ://doi.org/10
.1034/j.1399-3054.2003.00223 .x
Foyer CH, Ruban AV, Noctor G (2017) Viewing oxidative stress
through the lens of oxidative signalling rather than damage.
Portland Press Limited
Gharbi AA, Hipkin CR (1984) Studies on Nitrate Reductase in
British Angiosperms. New Phytol 97:629–639. https ://doi.
org/10.1111/j.1469-8137.1984.tb036 27.x
Gholami M, Mokhtarian F, Baninasab B (2015) Seed halopriming
improves the germination performance of black seed (Nigella
sativa) under salinity stress conditions. J Crop Sci Biotechnol
18:21–26. https ://doi.org/10.1007/s1289 2-014-0078-1
Grieve CM, Grattan SR (1983) Rapid assay for determination of
water soluble quaternary ammonium compounds. Plant Soil
70:303–307
Ha J, Kim M, Kim MY etal (2018) Transcriptomic variation in
proanthocyanidin biosynthesis pathway genes in soybean
(Glycine spp.). J Sci Food Agric 98:2138–2146. https ://doi.
org/10.1002/jsfa.8698
Habib N, Ali Q, Ali S, etal (2020) Use of Nitric Oxide and Hydrogen
Peroxide for Better Yield of Wheat (Triticum aestivum L.)
under Water Deficit Conditions: Growth, Osmoregulation, and
Antioxidative Defense Mechanism. Plants (Basel) https ://doi.
org/10.3390/plant s9020 285
Hafsi C, Falleh H, Saada M etal (2017) Potassium deficiency alters
growth, photosynthetic performance, secondary metabo-
lites content, and related antioxidant capacity in Sulla car-
nosa grown under moderate salinity. Plant Physiol Biochem
118:609–617. https ://doi.org/10.1016/j.plaph y.2017.08.002
Hasanuzzaman M, Oku H, Nahar K etal (2018) Nitric oxide-induced
salt stress tolerance in plants: ROS metabolism, signaling, and
molecular interactions. Plant Biotechnol Rep 12:77–92. https
://doi.org/10.1007/s1181 6-018-0480-0
Hassini I, Baenas N, Moreno DA etal (2017) Effects of seed prim-
ing, salinity and methyl jasmonate treatment on bioactive
composition of Brassica oleracea var. capitata (white and red
varieties) sprouts. J Sci Food Agric 97:2291–2299. https ://doi.
org/10.1002/jsfa.8037
Heath RL, Packer L (1968) Photoperoxidation in isolated chloro-
plasts: I. Kinetics and stoichiometry of fatty acid peroxidation.
Arch Biochem Biophys 125:189–198
Hossain MS, ElSayed AI, Moore M, Dietz K-J (2017) Redox and
reactive oxygen species network in acclimation for salinity
tolerance in sugar beet. J Exp Bot 68:1283–1298
Huang Y, Zhao H, Gao F etal (2018) A R2R3-MYB transcription
factor gene, FtMYB13, from Tartary buckwheat improves
salt/drought tolerance in Arabidopsis. Plant Physiol Biochem
132:238–248. https ://doi.org/10.1016/j.plaph y.2018.09.012
Hussain M, Farooq M, Lee D-J (2017) Evaluating the role of seed
priming in improving drought tolerance of pigmented and non-
pigmented rice. J Agron Crop Sci 203:269–276
Hussain S, Khan F, Hussain HA, Nie L (2016) Physiological and bio-
chemical mechanisms of seed priming-induced chilling tolerance
in rice cultivars. Front Plant Sci 7:116
Iqbal N, Umar S, Khan NA (2015) Nitrogen availability regulates
proline and ethylene production and alleviates salinity stress in
mustard (Brassica juncea). J Plant Physiol 178:84–91
Jan S, Alyemeni MN, Wijaya L, etal (2018) Interactive effect of
24-epibrassinolide and silicon alleviates cadmium stress via the
modulation of antioxidant defense and glyoxalase systems and
macronutrient content in Pisum sativum L. seedlings. BMC plant
biology 18:146
Jisha KC, Puthur JT (2016) Seed priming with BABA (β-amino butyric
acid): a cost-effective method of abiotic stress tolerance in Vigna
radiata (L.) Wilczek. Protoplasma 253:277–289
Jisha KC, Vijayakumari K, Puthur JT (2013) Seed priming for abiotic
stress tolerance: an overview. Acta Physiol Plant 35:1381–1396
Joshi A, Kaur S, Dharamvir K etal (2018) Multi-walled carbon nano-
tubes applied through seed-priming influence early germination,
root hair, growth and yield of bread wheat (Triticum aestivum
L.): Multiwalled Carbon nanotube influence on Bread wheat. J
Sci Food Agricul. https ://doi.org/10.1002/jsfa.8818
Kovács J, Poór P, Kaschani F etal (2017) Proteasome activity profiling
uncovers alteration of catalytic β2 and β5 subunits of the stress-
induced proteasome during salinity stress in tomato roots. Front
Plant Sci. https ://doi.org/10.3389/fpls.2017.00107
Kreeb K (1974) Pflanzen an Salzstandorten. Naturwissenschaften
61:337–343. https ://doi.org/10.1007/BF006 00299
Kumar P, Pal M, Joshi R, Sairam RK (2013) Yield, growth and physi-
ological responses of mung bean [Vigna radiata (L.) Wilczek]
genotypes to waterlogging at vegetative stage. Physiol Molec-
ular Biol Plants 19:209–220. https ://doi.org/10.1007/s1229
8-012-0153-3
Li X, Wei J-P, Scott E etal (2018) Exogenous melatonin alleviates cold
stress by promoting antioxidant defense and redox homeostasis in
camellia sinensis L. Molecules 23:165. https ://doi.org/10.3390/
molec ules2 30101 65
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein meas-
urement with the Folin phenol reagent. J Biol Chem 193:265–275
Lück H (1965) Catalase. In: Methods of Enzymatic Analysis. Elsevier,
pp 885–894
Maiti R, Pramanik K (2013) Vegetable Seed Priming: a Low Cost,
Simple and Powerful Techniques for Farmers’ Livelihood. Int J
Bio-Resource Stress Manag 4:
Martinez V, Nieves-Cordones M, Lopez-Delacalle M etal (2018) Tol-
erance to stress combination in tomato plants: new insights in
the protective role of melatonin. Molecules 23:535. https ://doi.
org/10.3390/molec ules2 30305 35
Moustafa-Farag M, Almoneafy A, Mahmoud A etal (2020) Melatonin
and its protective role against biotic stress impacts on plants.
Biomolecules 10:54. https ://doi.org/10.3390/biom1 00100 54
Mukherjee SP, Choudhuri MA (1983) Implications of water stress-
induced changes in the levels of endogenous ascorbic acid and
hydrogen peroxide in Vigna seedlings. Physiol Plant 58:166–170
Nagaz K, Masmoudi MM, Mechlia NB (2012) Impacts of irrigation
regimes with saline water on carrot productivity and soil salinity.
J Saudi Soc Agricul Sci 11:19–27
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by
ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell
Physiol 22:867–880
Nascimento do NC, Fett-Neto AG (2010) Plant Secondary Metabolism
and Challenges in Modifying Its Operation: An Overview. In:

Acta Physiologiae Plantarum (2020) 42:114
1 3
Page 13 of 13 114
Fett-Neto AG (ed) Plant Secondary Metabolism Engineering.
Humana Press, Totowa, pp 1–13
Nawaz F, Naeem M, Akram A etal (2017) Seed priming with KNO3
mediates biochemical processes to inhibit lead toxicity in maize
(Zea mays L.). J Sci Food Agric 97:4780–4789. https ://doi.
org/10.1002/jsfa.8347
Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active
oxygen under control. Annu Rev Plant Biol 49:249–279
Piscart C, Webb D, Beisel JN (2007) An acanthocephalan parasite
increases the salinity tolerance of the freshwater amphipod Gam-
marus roeseli (Crustacea: Gammaridae). Naturwissenschaften
94:741–747. https ://doi.org/10.1007/s0011 4-007-0252-0
Puente-Garza CA, Meza-Miranda C, Ochoa-Martínez D, García-Lara
S (2017) Effect of invitro drought stress on phenolic acids, fla-
vonols, saponins, and antioxidant activity in Agave salmiana.
Plant Physiol Biochem 115:400–407. https ://doi.org/10.1016/j.
plaph y.2017.04.012
Qu DY, Gu WR, Zhang LG, etal (2019) Role of chitosan in the regula-
tion of the growth, antioxidant system and photosynthetic char-
acteristics of maize seedlings under cadmium stress. Russian J
Plant Physiol 1–12
Rasool S, Ahmad A, Siddiqi TO, Ahmad P (2013) Changes in growth,
lipid peroxidation and some key antioxidant enzymes in chickpea
genotypes under salt stress. Acta Physiol Plant 35:1039–1050
Sairam RK, Deshmukh PS, Shukla DS (1997) Tolerance of drought and
temperature stress in relation to increased antioxidant enzyme
activity in wheat. J Agron Crop Sci 178:171–178
Saleem MH, Ali S, Rehman M etal (2020) Influence of phosphorus on
copper phytoextraction via modulating cellular organelles in two
jute (Corchorus capsularis L.) varieties grown in a copper mining
soil of Hubei Province China. Chemosphere 248:126032. https
://doi.org/10.1016/j.chemo spher e.2020.12603 2
Sergiev I, Alexieva V, Karanov E (1997) Effect of spermine, atrazine
and combination between them on some endogenous protective
systems and stress markers in plants. Compt Rend Acad Bulg
Sci 51:121–124
Shabala S (ed) (2017) Plant stress physiology, 2nd edn. CABI,
Wallingford
Shabala S, Cuin TA (2008) Potassium transport and plant salt toler-
ance. Physiol Plant 133:651–669
Shah K, Chaturvedi V, Gupta S (2019) Climate Change and Abiotic
Stress-Induced Oxidative Burst in Rice. In: Advances in Rice
Research for Abiotic Stress Tolerance. Elsevier, pp 505–535
Singh H, Jassal RK, Kang JS, etal (2015) Seed priming techniques
in field crops -A review. Agricul Rev https ://doi.org/10.18805
/ag.v36i4 .6662
Smart RE, Bingham GE (1974) Rapid estimates of relative water con-
tent. Plant Physiol 53:258–260
Soliman M, Alhaithloul HA, Hakeem KR etal (2019) Exogenous nitric
oxide mitigates nickel-induced oxidative damage in eggplant by
upregulating antioxidants, osmolyte metabolism, and glyoxalase
systems. Plants 8:562. https ://doi.org/10.3390/plant s8120 562
Soliman M, Elkelish A, Souad T etal (2020) Brassinosteroid seed
priming with nitrogen supplementation improves salt tolerance in
soybean. Physiol Mol Biol Plants. https ://doi.org/10.1007/s1229
8-020-00765 -7
Soliman MH, Alayafi AAM, El Kelish AA, Abu-Elsaoud AM (2018)
Acetylsalicylic acid enhance tolerance of Phaseolus vulgaris L.
to chilling stress, improving photosynthesis, antioxidants and
expression of cold stress responsive genes. Botan Stud. https ://
doi.org/10.1186/s4052 9-018-0222-1
Taleisnik E, Rodríguez AA, Bustos D etal (2009) Leaf expansion in
grasses under salt stress. J Plant Physiol 166:1123–1140. https
://doi.org/10.1016/j.jplph .2009.03.015
Tamma G, Valenti G, Grossini E, etal (2018) Aquaporin membrane
channels in oxidative stress, cell signaling, and aging: recent
advances and research trends. Oxidative Med Cell Longev 2018
Tan D-X, Hardeland R, Manchester LC etal (2012) Emergence of natu-
rally occurring melatonin isomers and their proposed nomen-
clature: Naturally occurring melatonin isomers. J Pineal Res
53:113–121. https ://doi.org/10.1111/j.1600-079X.2012.00979 .x
Tomar NS, Agarwal RM (2013) Influence of treatment of Jatropha
curcas L. leachates and potassium on growth and phytochemical
constituents of wheat (Triticum aestivum L.). Am J Plant Sci
4:1134
Tomar NS, Sharma M, Agarwal RM (2015) Phytochemical analysis
of Jatropha curcas L. during different seasons and developmen-
tal stages and seedling growth of wheat (Triticum aestivum L)
as affected by extracts/leachates of Jatropha curcas L. Physiol
Molecular Biol Plants 21:83–92
Vincken J-P, Heng L, de Groot A, Gruppen H (2007) Saponins, clas-
sification and occurrence in the plant kingdom. Phytochemistry
68:275–297
Vwioko E, Adinkwu O, El-Esawi MA (2017) Comparative physiologi-
cal, biochemical, and genetic responses to prolonged waterlog-
ging stress in Okra and maize given exogenous ethylene priming.
Front Physiol 8:632
Wan H, Chen L, Guo J etal (2017) Genome-wide association study
reveals the genetic architecture underlying salt tolerance-related
traits in rapeseed Brassica napus L. Front Plant Sci 8:593
Wang C-M, Chen H-T, Li T-C etal (2014) The role of pentacyclic
triterpenoids in the allelopathic effects of Alstonia scholaris. J
Chem Ecol 40:90–98
Wang J, Islam F, Yang C etal (2019) Chapter32 - Use of Phytohor-
mones in Improving Abiotic Stress Tolerance in Rice. In: Hasa-
nuzzaman M, Fujita M, Nahar K, Biswas JK (eds) Advances in
Rice Research for Abiotic Stress Tolerance. Woodhead Publish-
ing, Cambridge, pp 651–675
Yang A, Akhtar SS, Iqbal S etal (2018) Saponin seed priming improves
salt tolerance in quinoa. J Agron Crop Sci 204:31–39
Yemm EW, Willis AJ (1954) The estimation of carbohydrates in plant
extracts by anthrone. Biochem J 57:508
Zamin M, Fahad S, Khattak AM etal (2019) Developing the first halo-
phytic turfgrasses for the urban landscape from native Arabian
desert grass. Environ Sci Pollut Res. https ://doi.org/10.1007/
s1135 6-019-06218 -3
Zheljazkov VD, Nielsen NE (1996) Effect of heavy metals on pep-
permint and cornmint. Plant Soil 178:59–66
Zhu H, Zhao S, Lu X etal (2018) Genome duplication improves the
resistance of watermelon root to salt stress. Plant Physiol Bio-
chem 133:11–21. https ://doi.org/10.1016/j.plaph y.2018.10.019
Zivcak M, Brestic M, Sytar O (2016) Osmotic adjustment and plant
adaptation to drought stress. In: Drought Stress Tolerance in
Plants, Vol 1. Springer, pp 105–143
Zörb C, Geilfus C-M, Dietz K-J (2019) Salinity and crop yield. Plant
Biol 21:31–38. https ://doi.org/10.1111/plb.12884
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