Plant Stress 11 (2024) 100331

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Plant Stress
journal homepage: www.sciencedirect.com/journal/plant-stress

The potential of selenium to induce salt stress tolerance in Brassica rapa:

Evaluation of biochemical, physiological and molecular phenomenon
Saber Hussain a, b, Shakil Ahmed b, Waheed Akram a, c, Aqeel Ahmad c, Nasim Ahmad Yasin d, *,
Mei Fu a, **, Guihua Li a, Rehana Sardar b
a
Guangdong Key Laboratory for New Technology Research of Vegetables/Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou
510640, China
b
Institute of Botany, University of the Punjab, Lahore, Pakistan
c
Department of Plant Pathology, University of the Punjab, Lahore, Pakistan
d
RO-II Department, University of the Punjab, Lahore, Pakistan

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

Keywords: The efficacy of selenium (Se) to ameliorate salinity stress in Brassica rapa was elucidated using an in-vitro and in-
Antioxidant silico hybrid approach. Selenium was applied as a seed priming agent on the seeds grown in salt-spiked soils. The
Biomass priming treatment facilitated all the growth indicators and physiological parameters, including chlorophyll
Chlorophyll
synthesis, sugar contents, gas exchange attributes, etc. The advantageous effects of Se were accredited to the
Gene expression
ROS
alleviation of salinity stress through reducing the malondialdehyde (MDA), proline, electrolyte leakage, and
Molecular docking hydrogen peroxide (H2O2) level. Similarly, modulations in the expression of genes encoding antioxidative en­
Salt stress zymes enhanced reactive oxygen species (ROS) scavenging capacity along with a decrease in sodium (Na+) ions,
reciprocally enhancing potassium (K+) ions and resulting in a higher K+/Na+ ratio. Additionally, based on in
silico studies, a significant difference in the surface overlap of the stress-responsive proteins, including DREB,
SOS3, and STXBP1 of B. rapa, was observed, indicating the involvement of Se in the foiling interaction of NaCl
with the enzymes. In the case of the CAT macromolecule, ligands NaCl exhibited a -1.9-fold lower docking score
as compared to sodium selenite -4.9-fold, indicating a more overlapped interactive surface than sodium chloride.
The binding energies analysis recorded -3.3 kcal/mol of binding energy for the best pose of sodium selenite in the
case of DREB, while it was -3.5 and -3.5 kcal.mol-1 for SOS3 and 1 STXBP1, respectively. Ligand analysis
revealed a frequent existence of hydrogen bonds besides the hydrophobic interactions, salt bridges, and pi–pi
interactions representing high structural and functional stability. Results of the current study unveiled an in-vitro
and in-silico hybrid bioinformatics system to unveil the biochemical, physiological and molecular phenomenon
behind selenium-driven salt resilience in B. rapa.

Introduction growth by modulating the antioxidative system and nutrient orches­

Environmental stresses, especially salt stress, decrease agricultural
crop plants’ growth, development, and yield. About ~20% of the irri­
gated agricultural area, besides 6% of the global area, is salt-affected
(Kumar and Sharma, 2020). Natural geological progressions like
weathering of rocks, arid climate, and higher evaporation, as well as
anthropogenic activities, including the use of brackish water for irriga­
tion, and poor tillage operations, are the foremost causes of soil salini­
zation (Hussain et al., 2019). Salinity-induced stress reduces plant
tration. Researchers have warned that salinity may affect over half of the
arable land by 2050 (Nahar et al., 2022). If proper soil reclamation
measures are not adopted, salinity may become a conspicuous envi­
ronmental threat worldwide. Additionally, it has become very important
to effectively utilize salt-affected marginal areas for agricultural pur­
poses (Stavi et al., 2021). Salt toxicity augments the synthesis of reactive
oxygen species (ROS) which injure plant cells (Mangal et al., 2023).
Hence, salted plants exhibit leaf burns and necrotic lesions due to
reduced photosynthesis and degradation of photosynthetic pigments

* Corresponding author at: RO-II Department, University of the Punjab, Lahore, Pakistan.
** Corresponding author at: Vegetable Research Institute, Guangdong Academy of Agricultural Sciences, Guangzhou 510640, China.
E-mail addresses: nasimhort@gmail.com (N.A. Yasin), fumei@gdaas.cn (M. Fu).

https://doi.org/10.1016/j.stress.2023.100331
Received 14 September 2023; Received in revised form 25 November 2023; Accepted 24 December 2023
Available online 28 December 2023
2667-064X/© 2023 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
S. Hussain et al.
(El-Serafy et al., 2021). Plant leaves deficient in chlorophyll content
develop poor roots and shoots.
Turnip (Brassica rapa L.) is an important horticultural crop
commonly cultivated for human consumption and fodder use. Oil
extracted from seeds of many B. rapa cultivars is a good source of biofuel
and nutritious cooking oil. Turnip roots have a higher concentration of
gluconasturtiin and IGSLs (Krumbein et al., 2005). These glucosinolates
have supreme pharmaceutical worth in administering various types of
cancer and other diseases. Salt stress exerts harmful consequences on
root growth, shoot growth and yield of B. rapa crop (Cheng et al., 2022).
Cultivation of salinity-tolerant cultivars seems an easy approach to
ingeniously consume salt-affected areas (Collinge and Sarrocco, 2022).
Therefore, several breeding and agricultural research organizations
have been developing salinity-tolerant high-yielding crop cultivars
(Chen et al., 2021).
Seed priming is an economical, bio-safe, and easy technique to as­
suage environmental stress in crop plants. Seed priming has shown
positive effects on metabolomics, proteomics, and the growth of plants
subjected to abiotic stress. Priming usually involves seed submersion in a
solution for a specific duration to induce pre-germination physiological
and metabolic progressions (Siregar et al., 2020). Pure water as well as a
solution of growth regulators, osmoregulators, acids, nutrients, and
minerals, may be used as priming agents (Habib et al., 2020). Exogenous
application of several elicitors assists plants to alleviate stress through
induction of stress related physiochemical and molecular changes
(Ahmad et al., 2019). Selenium (Se) is an important inorganic plant
elicitor which has shown stress ameliorative potential. Selenium-primed
seeds showed increased seed germination percentage, better photosyn­
thesis, improved antioxidative system, and elevated vegetative growth
in subsequently developed plants under assorted abiotic stresses (Sardar
et al., 2022). In the same way, the foliar spray of Se increased plant
growth under salinity regimes (Tisarum et al., 2019). Exogenous treat­
ment of Se enhances plant stress tolerance, increases photosynthesis of
photosynthetic pigments, activates antioxidant machinery, and pro­
motes plant nutrition and stress tolerance (Hawrylak-Nowak et al.,
2018).
Salt is an inevitable pollutant that negatively affects soil health and
fertility, reducing plant nutrient use efficiency. Consequently, it be­
comes indispensable to elucidate the influences of salt-induced stress
and find suitable eco-friendly techniques for mitigating relevant stress in
plants. The ameliorative function of Se in alleviating a range of envi­
ronmental stresses has been well documented (Zwolak, 2020). Various
metabolites, phytohormones, and bioactive compounds adjust cellular
activities in plants subjected to environmental stresses. However, the
exact function and mechanism of such signaling molecules in the
development of stress tolerance is unknown. Likewise, the interaction of
salt stress with stress-responsive enzymes and other metabolites is
poorly recognized. Therefore, computational modeling (molecular
docking), which is a well-recognized novel tool, may be employed to
analyze the interaction among micro molecules (ligands) with the
macromolecules (enzymes or proteins) (Choudhury et al., 2022). The
structural design developed by computational modeling enables one to
assume the possible interaction among the targeted protein and ligand
complex (Perveen et al., 2023).
The influences of various priming agents may vary in terms of their
concentrations and plant species under abiotic stress. The appropriate
concentration of a specific priming agent may advance plant growth and
stress tolerance. There is a scarcity of research on the potential appli­
cation of Se seed priming in alleviating salt stress and improving
nutrient uptake in B. rapa. Considering its beneficial role in abiotic stress
alleviation, it was assumed that seed priming with this dynamic element
might perk up plant nutrition and growth by mitigating salt stress in
B. rapa. It was also hypothesized that Se seed priming may enhance plant
growth through improving antioxidative system of plants subjected to
salinity stress. Henceforth, this study was intended to investigate salinity
stress effects on seed germination, biosynthesis of ROS, antioxidative
2
Plant Stress 11 (2024) 100331
system, nutrient orchestration, growth, and biomass production of Se
primed and non-primed B. rapa plants under greenhouse conditions. The
current research work spotlights the potential of Se in improving the
antioxidant machinery and computational analyses to elucidate the
growth of B. rapa exposed to salt stress.
Methodology
Seed priming and plant growth conditions
This study was performed during September-October 2022 (average
33 ◦ C / 23 ◦ C Day and night temperature; 12 hrs photoperiod). For
surface sterilization, mature and uniform seeds of B. rapa L. var. purple
top white globe were submerged in a solution of sodium hypochlorite
(0.5%) for 3 min, followed by rinsing thrice with distilled water. Af­
terward, seeds were primed for 12 hrs by placing them in 0, 75, 100 and
125 μmol. L− 1 Se solution prepared by sodium selenite (Na2SeO3)
because seed priming with these Se concentrations significantly affected
seed germination and plant growth (Hussain et al., 2022). For seed
priming, sterilized seeds were subdivided into four sets. Distilled water
was used for the hydropriming of the first set of seeds. The second, third
and fourth sets of seeds were primed by using 75, 100 and 125 μmol L− 1
of Na2SeO3 solution, respectively. The seed priming process was
accomplished under partial shade at room temperature. Seeds primed
with 0, 75, 100 and 125 μmol. L− 1 Se were nominated as control, Se1,
Se2 and Se3 treatment, respectively. Five seeds from each treatment
were evenly planted in 25 cm plastic pots with 7 kg of soil from the 0–30
cm depth, pH 8.26, and saline to carbonate ratio 271 S/s. There were 5
replicates for each treatment. The pots were placed under natural con­
ditions in a wire house located in the Botanical Garden, University of the
Punjab (74⁰21–00-E, 31⁰35–00-N) Lahore, Pakistan and watered every
alternate day. Nutrient absorption was also ensured by adding 100 mL of
Hoagland solution to each treatment. Salinity stress was applied to
allotted plants at 20 days of leaf development. Three amounts of sodium
selenite (Se1, Se2, and Se3) were used, with the final three treatments
(Se1+NaCl, Se2+NaCl, and Se3+NaCl) as positive controls and NaCl
stress alone as negative. For this purpose, NaCl solution was gradually
added to the pots to achieve the required level of 200 mM NaCl because
this concentration reduced germination as well as plant growth (Hus­
sain et al., 2022). After five days, foliage tissues from the fully stretched
leaves were collected and immediately stored in liquid nitrogen.
Estimation of plant growth characteristics
Soil particles were gently washed away from the plants when they
were collected by submerging them in water. The root and shoot lengths
were analyzed with a scale. Roots and shoots were isolated and placed
over blotting paper. Fresh root and shoot weights of air-dried plant
samples were evaluated separately to record their fresh mass. Afterward,
these roots and shoot samples were dried in an oven at 80 ◦ C for three
days to acquire constant dry biomass.
Assessment of mineral nutrients
All the mineral nutrients, including calcium (Ca), zinc (Zn), phos­
phorus (P), magnesium (Mg), sodium (Na) chlorine (Cl), and potassium
(K), were quantified. Plant samples were combusted to evaluate the
quantities of minerals from plant ash, according to AOAC (1990). Ash of
the plants was mixed with 10 mL of HCl/H2O/HNO3 (3:2:1) and placed
over a hot plate for digestion. The residual solution of every crucible was
blended with 10 mL of deionized water and heated until a colorless
solution was produced. This colorless mixture was passed by Whatman
filter paper. Potassium and Ca from the solution were quantified through
the flame photometry method, as described by Baruah et al. (1998). The
amount of Mg from the solution was estimated by the standard colori­
metric method (Piper, 1950). The methodology of Thimmaiah (1999)
S. Hussain et al.
was used to estimate P and Fe from the solution.
Estimation of chlorophyll from foliage of plants
The chlorophyll content from the 200 mg prewashed foliage sample
was isolated with the help of acetone (80%), and spectrophotometric
value was taken at 646 nm and 663 nm for the estimation of Chl a, Chl b,
and total chlorophyll involved combining the value of chl a and chl b,
respectively, according to Lichtenthaler and Wellburn (1983).
Assessment of total soluble sugars
For the evaluation of total soluble sugars, 1 mL of plant extract was
mixed with 9 mL of ethanol (80%) in a test tube. Subsequently, these
tubes were positioned on a cold bath, and 20 mL of anthrone reagent was
progressively incorporated. Afterward, tubes were placed over a boiling
water bath for 7.5 min and cooled in an ice bath. Absorbance from this
cool solution was taken at 630 nm, as described by Franscistt et al.
(1971).
Estimation of hydrogen peroxide and malondialdehyde level
For evaluation of H2O2, a reaction mixture containing 1 mL of 0.1%
trichloroacetic acid (TCA) was homogenized with 0.25 mm supernatant
of plant extract in the presence of 1 mL of 100 mM K-phosphate buffer
and reagent including 4 mL, 1 M potassium iodide (KI) in fresh double-
distilled water (w/v). The blank contained TCA (0.1%) in the absence of
plant extract. The solution was kept in the dark area for 60 min, after
which spectrophotometric value was observed at 390 nm and compared
with a standard curve of identified concentrations of H2O2 represented
by Velikova et al. (2000).
For the assessment of lipid peroxidation, the level of malondialde­
hyde (MDA) produced from the plant samples treated with the thio­
barbituric acid (TBA) method was analyzed according to Heath and
Packer (1968). For this purpose, 2 g of ground plant sample (0.25 mm)
was mixed with 2 mL of 0.5% TCA. This mixture was centrifuged at 10,
000 g for 20 min. Then, 1 mL supernatant was homogenized with 5 mL of
20% TCA in the presence of 0.5% TBA. This solution was placed over a
boiling water bath for 0.5 h and quickly cooled. The mixture was
centrifuged at 10,000 g for 10 min. The MDA level from the resulting
supernatant was evaluated by taking absorbance at 532 nm. In addition,
electrolyte leakage (EL) content and proline concentration were
measured according to Bates et al. (1973).
Estimation of the activities of antioxidant enzymes
The plant sample (2 g) treated with liquid nitrogen was ground in the
presence of a 20 mL extraction buffer containing 0.1 M phosphate buffer
at pH 7.8. Besides, 0.5 mM EDTA was added to evaluate the peroxidase
(POX), catalase (CAT), superoxide dismutase (SOD) and ascorbate
peroxidase activity (APX) activity.
For the evaluation of SOD activity, the inhibition of the photo­
chemical reduction of nitroblue tetrazolium (NBT) was determined. For
this purpose, two mM riboflavin (0.2 mL) was mixed with 6 mL of the
reaction mixture. The reaction mixture was prepared by adding NBT
(75 µM), methionine (13.33 mM), enzyme extract (0.2 mL), EDTA
(0.1 mM), sodium carbonate (50 mM), and phosphate buffer (50 mM) at
pH 7.8. The tubes containing the solution were placed under two 15 W
fluorescent tubes for fifteen minutes. Afterward, the spectrophotometric
value was observed at 560 nm (Cakmak, 1994).
The CAT activity was anticipated by evaluating the reduction of
H2O2. For this purpose, 1 mL H2O2 (75 mM) was mixed with 3 mL
phosphate buffer (0.1 M) at pH seven along with 100 µL of diluted
enzymatic extract in a 6 mL reaction mixture. The reduced spectro­
photometric value at 240 nm was analyzed for 60 s, and CAT activity
was estimated by measuring the quantity of H2O2 utilized (Chance and
3
Plant Stress 11 (2024) 100331
Maehly1955).
To estimate POX, a 1 g plant sample was extracted with 10 mM so­
dium phosphate buffer at pH 6.0. This mixture was centrifuged at
12,000 g for 15 min at 4 ◦ C. The reaction mixture contained a 6 mL
solution containing ten mM sodium phosphate buffer, 0.1 M H2O2 and
0.25% guaiacol, and 0.2 mL enzyme extract (Hammerschmidt et al.,
1982).
The APX activity was analyzed by estimating the reduction in optical
density attributable to ascorbic acid at 290 nm. Potassium phosphate
buffer (50 mM) at pH 7.0, ascorbic acid (0.5 mM), EDTA (0.1 mM),
H2O2 (0.1 mM), enzyme (0.1 mL) and water were mixed to make a final
volume of 3.0 mL in which 0.1 mL of H2O2 was added to begin the re­
action. The decline in absorbance was recorded and compared with
known concentrations of ascorbic acid (Chance and Maehly1955).
Determination of gas exchange attributes
The gas exchange parameters, including stomatal conductance (Gs),
internal CO2 concentration (Ci,), and transpiration rate (E), carbon di­
oxide assimilation rate (A) was estimated from the completely turgid
topmost leaves by using an Infra-Red Gas Analyzer at 9:30 am to 11:30
am. The water-use efficiency was measured by employing the formula
(A/E).
Extraction of RNA and real-time quantitative PCR (RT-qPCR) analysis
The methodology used by Ali et al. (2018) was employed for the
assessment of RT-qPCR. For this purpose, total RNA from the 200 mg
foliage samples of treated plants was isolated with the help of the Plant
RNA MiniPrep Kit (Zymo Research: California, America) as per the in­
struction manual. For the removal of genomic DNA and cDNA produc­
tion, Prime script™ RT reagent with genome DNA (gDNA) eraser kit
(Takara Co. Ltd. Japan) was used. Afterward, cDNA was examined for
RT-qPCR through the iCycleriQTM Real-time detection system (Bio-Rad:
California, America) via using CYBR Premix Ex Taq II (Takara Co. Ltd.,
Japan). The primers suggested by Zhang et al. (2020) were used to
evaluate the expression level of stress-responsive genes including SOD,
CAT, APX, and POD (Supplementary Table 1).
Interaction analysis of sodium selenite with stress responsive proteins
Target receptors
Three proteins (DREB, SOS3, STXBP1) were selected for molecular
docking investigation based on their role in the process of stress alle­
viation. The three-dimensional model of target proteins of B. rapa did
not exist in the protein database. The complete amino acid sequence
protein was retrieved from UniProtKB.
Homology modeling
Protein amino acid sequences were uploaded to the trRosetta server
for homology modeling. trRosetta predicted structure was refined by
ModRefiner at http://zhanglab.ccmb.med.umich.eduModRefiner (Mul­
ligan, 2021). The server improves the physical realism and structural
precision of protein models by a two-step atomic-level energy minimi­
zation and fabricates a PDB. The predicted 3D structure of target pro­
teins was further evaluated by PROCHECK (https://www.ebi.ac.uk/thor
ntonsrv/software/PROCHECK), and Ramachandran plots were ob­
tained. The 3D model with the best scoring was used for molecular
docking for each protein. The extended Sequence of proteins has been
downloaded from the NCBI database. The receptors, namely, the crystal
or NMR structures of targeted proteins were not available at the RCSB
Protein Data Bank or NCBI MMDB databases; thus, the
three-dimensional structures were obtained from the SWISS-MODEL
database (https: https://swissmodel.expasy.org/ (accessed on 05
October 2022), a database of protein structures predicted using ho­
mology modeling.
S. Hussain et al. Plant Stress 11 (2024) 100331

Table 1 protein-ligand docking tool, AutoDock Vina, by PyRx virtual screening

Influence of Se and NaCl on seed germination and vegetative growth of B. rapa. software individually, followed by the addition of the ligands to the
Treatments Growth traits workspace (Yang et al., Zhang, 2013). The binding sites were selected
Shoot Root Shoot Shoot Root Root around the detected cavity centered at X, Y, and Z for all four proteins
length Length FW DW FW DW Table 1. The five best-docked poses of all the ligands, in terms of docking
(cm) (cm) (g (g (g (g
score, were considered for further analysis, following the methodology
plant− 1) plant− 1) plant− 1) plant− 1)
provided by Choudhury et al. (2022).
Control 15.09 8.54 ± 7.54 ± 3.36 ± 3.05 ± 1.46 ±
0.32a 0.56abc 0.39ab 0.05bc 0.18bc
Statistical analysis
±
0.19cd
NaCl 10.21 5.58 ± 5.99 ± 2.57 ± 2.21 ± 0.99 ± The obtained data were evaluated through one-way ANOVA by
± 0.22 0.20cde 0.33bcd 0.23b 0.26cd 0.36cd means of Statistic 8.1 software. For the separation of the significance of
g treatments, Duncan’s multiple range test was used at p ≤ 0.05. The
Se1 16.10 6.6 ± 7.85 ± 3.26 ± 4.14 ± 1.85 ±
values symbolize means of 5 replications ± SD. PCA analysis, was per­
± 0.40bc 0.76bc 0.32ab 0.19a 0.27ab
0.42bc formed using Origin Pro 2021 to determine the effect of Se and NaCl on
Se2 18.20 7.08 ± 9.89 ± 4.16 ± 4.46 ± 2.18 ± the growth and development of turnip plants and correlation between
± 0.86b 0.48a 0.38a 0.47a 0.09a related parameters was studied.
0.42a
Se3 12.08 4.64 ± 5.64 ± 3.02 ± 2.04 ± 1.10 ±
± 0.30ef 0.39cd 0.39b 0.34d 0.21cd Results
0.32f
Se1þNaCl 13.09 4.96 ± 6.84 ± 3.21 ± 3.06 ± 1.13 ± Effect of selenium and salt stress on plant growth attributes
± 0.20def 0.74abc 0.35ab 0.25bc 0.23c
0.32ef
Se2þNaCl 17.09 5.98 ± 8.02 ± 3.41 ± 3.64 ± 1.80 ±
Statistical analysis revealed that the root and shoot length of B. rapa
± 0.25bcd 0.84b 0.41ab 0.42ab 0.27ab L. (turnip) plants significantly decreased under salinity stress. Salinity
0.62ab negatively affected turnip roots and shoot growth as the lowest growth
Se3þNaCl 14.08 4.46 ± 4.46 ± 1.18 ± 2.12 ± 0.55 ± was found in the case of plants treated with 200 mM NaCl, while control
0.46f 1.22d 0.43c 0.45cd 0.11d
±
plants exhibited pronounced root and shoot growth compared to those
0.52de
under stress. In short, the leave roots and shoot length were demon­
Data display means ± SD of 5 replications. Dissimilar letters denote significant strated by the plants under saline conditions. In contrast, Se priming
differences among the treatments (P ≤ 0.05). C = control, NaCl= 200 mM NaCl, alleviated salt stress. Regarding the priming interaction of Se, the
Se1= 75 μmol.L− 1 Se, Se2=100 μmol.L− 1 Se, Se3=125 μmol.L− 1 Se.
maximum roots and shoot growth were obtained by Se2-primed seeds
under stressed and non-stressed circumstances. Additionally, the sta­
Ligands tistical analysis showed that higher Se concentration (Se3), showed
The structure of the ligand [Na2SeO3 (CID 24,934)] was obtained harmful effects on root and shoot lengths of treated plants under stressed
from the NCBI PubChem compounds database (https://pubchem.ncbi. and non-stressed conditions as compared to Se1 and Se2 Table 1.
nlm.nih.gov/.

Molecular docking Fresh and dry weight of root and shoot

Prior to docking, the ligand was prepared to perform the followings:
conversions of 2D to 3D, energy minimization, the addition of hydrogen
atoms, and necessary structural optimizations using the Openbebal
program. Similarly, receptor molecules were prepared by adding polar
hydrogen atoms and eliminating water molecules on Discovery Studio
software. The molecular docking studies of active compounds were
performed using AutoDock Vina by PyRx virtual screening software. The
Grid box was set to cover the active site of the crystal structure with the
appropriate dimensions for all receptors. The score function and dock
function (S, kcal.mol− 1) were used for the evaluation of the binding
affinity of the sodium selenite with selected stress-related proteins used
as receptor molecules. The generated structures were visualized and
analyzed through PyMOL (Yuan et al., 2017). The binding efficiency of
the truncated variant to the substrate was determined using an online
Salinity declined (P ≤ 0.05) shoot fresh weight and fresh root weight;
while Se improved (P ≤ 0.05) this variable in the applied (turnip) plants.
Statistical analysis exhibited that higher biomass was recorded in plants
treated with Se2 (100 μmol L− 1) both in stressed and non-stressed
conditions, compared with respective control under salinity conditions.
Salinity decreased root as well as shoot dry weight in turnip plants.
Plants treated with Se showed significantly (P ≤ 0.05) increased root
and shoot dry weight under salinity conditions. Our results showed that
Se seed priming significantly enhanced plant biomass parameters,
particularly the seed priming with Se2 (under non-stressed regimes)
Table 1.
Improvement of Photosynthetic Content in Selenium Applied Plants
Salinity significantly reduced the chlorophyll content, including Chl
a, Chl b and total chl in turnip plants, compared to the level

Table 2
Influence of Se and NaCl on total soluble sugar and photosynthetic pigments of B. rapa.
1 1
Treatments Chlorophyll a Chlorophyll b Total Chlorophyll (mg g− FW) Total Soluble sugars (mg g− FW) Selenium
(mg g− 1 FW) (mg g− 1 FW) (mg g− 1DW)

Control 0.93 ± 0.02d 0.65 ± 0.06bc 1.58 ± 0.14cd 1.68 ± 0.22abc 0.40 ± 0.03c
NaCl 0.46 ± 0.06e 0.40 ± 0.06c 0.86 ± 0.03d 1.23 ± 0.12bc 0.08 ± 0.1d
Se1 1.72 ± 0.11b 0.79 ± 0.04abc 2.51 ± 0.4b 2.46 ± 0.70a 0.815 ± 0.03a
Se2 2.35 ± 0.05a 1.18 ± 0.20a 3.53 ± 0.58a 2.59 ± 0.18a 0.81 ± 0.01a
Se3 1.21 ± 0.11cd 0.52 ± 0.05bc 1.73 ± 0.34c 1.37 ± 0.19bc 0.67 ± 0.03b
Se1þNaCl 0.90 ± 0.07d 0.71 ± 0.03abc 1.61 ± 0.095cd 1.66 ± 0.26abc 0.46 ± 0.11c
Se2þNaCl 1.31 ± 0.10c 0.95 ± 0.07b 2.26 ± 0.18bc 2.24 ± 0.57ab 0.65 ± 0.02b
Se3þNaCl 0.27 ± 0.02e 0.55 ± 0.15bc 0.82 ± 0.14d 1.004 ± 0.024c 0.44 ± 0.03c

Data display means ± SD of 5 replications. Dissimilar letters denote significant differences among the treatments (P ≤ 0.05). C = control, NaCl= 200 mM NaCl, Se1=
75 μmol.L− 1 Se, Se2=100 μmol.L− 1 Se, Se3=125 μmol.L− 1 Se.

4
S. Hussain et al. Plant Stress 11 (2024) 100331

Table 3
Influence of Se and NaCl on the nutrition content of B. rapa.
Treatments K+ P Ca+2 Zn+2 Mg+2 Na+ Cl−
1 1 1 1 1 1 1
(mg g− DW) (mg g− DW) (mg g− DW) (mg g− DW) (mg g− DW) (mg g− DW) (mg g− DW)

Control 49.42 ± 0.32a 5.58 ± 0.30bc 26.42 ± 0.33c 0.14 ± 0.02b 12.5 ± 0.42a 11.11 ± 0.13de 6.56 ± 0.23cd
NaCl 13.36 ± 0.55 g 1.70 ± 0.16ef 6.42 ± 0.27 g 0.1 ± 0.01e 4.7 ± 0.28d 29.11 ± 0.14a 18.67 ± 1.2a
Se1 40.34 ± 0.42b 5.23 ± 0.31c 28.02 ± 0.41ab 0.97 ± 0.08a 9.45 ± .026c 7.43 ± 0.24ef 4.56 ± 0.45e
Se2 38.36 ± 0.47c 7.47 ± 0.35a 29.13 ± 0.32a 0.096 ± 0.001b 11.64 ± 0.23ab 5.98 ± 0.23f 4.76 ± 0.25e
Se3 39.41 ± 0.33bc 6.31 ± 0.47b 27.05 ± 0.40bc 0.87 ± 0.09b 10.84 ± 0.34 b 9.01 ± 0.10e 5.89 ± 0.45d
Se1þNaCl 14.87 ± 0.30f 3.20 ± 0.35d 16.87 ± 0.40e 0.017 ± 0.004d 1.68 ± 0.23f 18.12 ± 1.0b 9.89 ± 0.33b
Se2þNaCl 19.11 ± 0.33e 2.40 ± 0.05e 10.23 ± 0.48f 0.02 ± 0.004c 2.81 ± 0.16e 11.84 ± 0.50d 7.97 ± 0.67c
Se3þNaCl 25.78 ± 0.20d 1.07 ± 0.02f 18.48 ± 0.62d 0.02 ± 0.004c 3.54 ± 0.54e 13.86 ± 0.79c 6.98 ± 0.59cd

Data display means ± SD of 5 replications. Dissimilar letters denote significant differences among the treatments (P ≤ 0.05). C = control, NaCl= 200 mM NaCl, Se1=
75 μmol.L− 1 Se, Se2=100 μmol.L− 1 Se, Se3=125 μmol.L− 1 Se.

demonstrated by the plants under control circumstances Table 2. Salt-
induced toxicity exhibited injurious effects on the biosynthesis of
photosynthetic pigments. The least amount was observed in salted
plants. Quite the reverse, Se priming elevated the concentration of
photosynthetic pigments in applied plants as compared with the rele­
vant non-stressed and stressed regimes. Plants treated with Se2 (100
μmol L− 1) revealed upregulated Chl a content by 2.35±0.05a, Chl b
content by 1.18±0.20a and total Chl content by 3.53±0.58a respec­
tively, compared to those found in non-primed plants under stress. The
Se2 (100 μmol L− 1) treated plants mitigated the unfavorable conse­
quences of salt-induced toxicity and amplified Chl a, Chl b and total chl
content. Similarly, the damaging upshot of salt stress on the production
of Chl b was effectively alleviated, and consequently, the amount of Chl
b was augmented. Se1 (75 μmol L− 1) seed priming showed better results
in comparison with the Se3 (125 μmol L− 1) treatment showing negative
effects on the synthesis of chlorophyll content Table 2.
Role of selenium on total soluble sugar content
Biochemical attributes such as total soluble sugar significantly
decreased in turnip plants under salt stress. The decline in total soluble
sugar was detected in stressed situations at the vegetative stage.
Different treatments of selenium as a seed priming agent (Se1, Se2, and
Se3) showed beneficial effects on plants by enhancing the total soluble
sugar content of turnip plants under salinity and non-salinized circum­
stances Table 2. Sugar content evaluation showed better positive effects
when the various concentrations of selenium (Se1, Se2, and Se3) were
applied by the seed priming method. Statistical analysis revealed that
Se2-primed plants showed significant alleviation of total soluble sugar
content under stressed and non-stressed conditions. While Se1 showed
relative positive effects, the negative result was observed in the case of
Se3 treatment conditions under both stressed and non-stressed situa­
tions, as given in Table 2.
Effect of selenium and sodium chloride on mineral content
Salt-induced stress significantly declined nutrient uptake concen­
tration in both the root and shoot of plants as compared to controlled
conditions. It was found that salinity stress negatively affected the
nutrient uptake in the shoot and roots of the turnip. Seed priming with
Se2 alleviated salt stress and enhanced plant nutrition uptake under
salinity stress. Various seed priming treatments (Se1+NaCl, Se2+NaCl,
Se3+NaCl) recorded statistically similar or higher concentrations of
nutrients in turnip plants. Statistically, Se2 was found to be the most
effective treatment in terms of nutrient uptake in both controlled and
salinity-stressed turnip plants. In general nutrients, uptake was moni­
tored as very low in salinity-stressed plants without selenium-primed
seeds. Statistically, Se2 showed significantly more nutrient uptake in
both stressed and non-stressed conditions than Se3 after Se1 Table 3.
Alleviation of stress through non-enzymatic contents
MDA and H2O2 content increase significantly more under salinity-
stressed conditions than under the control treatment. Salinity signifi­
cantly produced the highest values (31.1% for MDA and 68.3% for H2O2
lower as compared to the control treatment) in this respect (Fig. 3A).
The MDA and H2O2 values gradually increased with increasing salinity
levels, with the maximum values being obtained under (200 mM NaCl).
The lowest levels of MDA and H2O2 were presented by Se primed seeds
plants under non-stressed conditions, but plants under saline conditions
significantly caused the maximum values in this respect. However, seed
priming of Se2 (100 μmol L− 1) significantly (P ≤ 0.05) decreased this
variable under salt stress and non-stressed conditions. Se2 primed (100
μmol L− 1) decreased proline and electrolyte leakage concentration
under salinity and selenium-treated non-stressed conditions (Fig. 3B). Se
primed plants presented the lowest proline and electrolyte leakage levels
under non-stressed conditions.
Modulations of antioxidant enzymes activities
Salinity affected the activity of antioxidant enzymes, including APX,
SOD, POD, and CAT. The utmost CAT activity was observed in the case
of plants treated with Se2 (100 μmol L− 1) at all vegetative growth,
whereas, Se1 (75 μmol L− 1) exhibited positive effects on CAT activity at
vegetative stages under salinity stress and non-stressed conditions and
statistically negative effects were recorded during the seed priming with
Se3 (125 μmol L− 1) (Fig. 2A).
Salinity significantly (P ≤ 0.05) decreased SOD activity in plants.
Seed priming of Se also affected (P ≤ 0.05) this variable. Statistical
analysis revealed that plants treated with Se2 level (100 μmol L− 1) had
the utmost values for SOD compared with an upper dose of Se3 (125
μmol L− 1) under salinity and controlled conditions. Plants also exhibited
positive effects on SOD activity at the vegetative stage during priming
with Se1 as compared to controlled and stressed conditions (Fig. 2A).
In addition, a maximal decrease in POD activity was observed in
salted plants. The selenium priming enhanced POD activity in salt-
stressed and non-stressed plants. Plants obtained from seeds primed
with higher Se3 concentration (125 μmol L− 1) did not illustrate any
affirmative effect on POD activity in all conditions (Fig. 2A)
Statistical analysis revealed that Se2 (100 μmol L− 1) priming
significantly enhanced APX activity in all treated plants (Fig. 2A). In
comparison, higher Se concentration (Se3) revealed a negative impact
and reduced the antioxidative activities.
Expression of defense-related antioxidative genes
The modulations in the transcriptomic level of genes involved in the
antioxidant systems of plants growing under salinity stress were evalu­
ated. It was found that salinity and Se2 modulated the expression level of
SOD, CAT, APX and POD. Although, Se2 priming enhanced the expres­
sion profile of genes encoding CAT activity as compared to the control.

5
S. Hussain et al.
Fig. 1. Influence of selenium (Se) and sodium chloride (NaCl) on gas exchange parameters of B. rapa: (A)stomatal conductance (gs) (B), photosynthetic rate (A) (C),
rate of transpiration (E) (D), Intercellular CO2 concentration. Data display means ± SD of 5 replications. Dissimilar letters denote significant differences among the
treatments (P ≤ 0.05). C = control, NaCl= 200 mM NaCl, Se1= 75 μmolL− 1 Se, Se2=100 μmolL− 1 Se, Se3=125 μmolL− 1 Se.
Table 4
The homology modeling of proteins, binding energy, hydrogen bonds, ligand
efficiency, and contacting receptor residues.
Protein Hydrogen Bonds Contacting receptor residues
DREB1 3 ARG46, PRO48, TYR50, UNL1, TYR50, VAL53
SOS3 4 LEU62, SER209, UNL1, LEU62, PHE205
STRP 1 HIS46, UNL1, SER49, ALA99
Yet, the utmost level of gene expression was observed in plants under
salinity stress (Fig. 2B). Salinity stress as well as Se2 priming, upregu­
lated the relative mRNA level of APX. Furthermore, the maximum APX
expression level was recorded in the Se2-treated plants under salt stress
(Fig. 2B). The transcript level of POD-related genes showed 3.2-fold up-
regulation in the case of salt-stressed plants as compared to control
plants. The highest gene expression level was in salt-treated plants,
whereas 4.6 folds higher than in control plants (Fig. 3C). Salt-stressed
Se2-supplemented plants exhibited three folds higher expression levels
as compared to control plants. Salinity enhanced the expression level of
genes encoding SOD by 2.1 folds as compared to the control (Fig. 2B).
Under salinity stress, the plants that received the Se by seed priming
significantly showed 4.12 folds higher transcript levels than control
plants.
Gas exchange attributes and salinity stress
Salinity stress significantly decreased stomatal conductivity (gs)
(Fig. 1A) (p ≤ 0.05) photosynthetic rate (A) (Fig. 1B) and transpiration
rate (E) (Fig. 1C), while it also had a negative effect on intercellular CO2
(Fig. 1D). Statistical analysis revealed that seeds priming with selenium
(Se)in turnip plants increased stomatal conductance (gs) significantly (p
≤ 0.05), while it had positive effects on photosynthetic rate and tran­
spiration rate. The injurious impacts of salt-induced toxicity on stomatal
conductance, photosynthetic rate and Se on stomatal conductivity were
6
Plant Stress 11 (2024) 100331
more prominent than those on the other accession. Among the gas ex­
change factors, photosynthetic rate and stomatal conductivity declined
under salinity stress conditions as compared to controlled conditions.
Two levels (Se1 and Se2) were equally effective in affecting the gas
exchange attributes, but a higher Se dose (Se3) showed negative con­
sequences and decreased stomatal conductance and photosynthetic ac­
tivity both in stressed and non-stressed conditions. Analysis of variance
showed that selenium (Se2) priming significantly influenced the gas
exchange attributes under both stressed and non-stressed situations as
compared to the control (Fig. 1A-D).
Interaction analysis of sodium selenite with stress responsive proteins
The quality of the receptor’s models of stress-related proteins was
evaluated via the Ramachandran plot using the PROCHECK method. The
presence of not less than 89% residues in the most favorable region
confirmed the good quality of macromolecules (Supplementary
Figure 1). Sodium selenite was effectively docked in all the three stress
related proteins during the docking analysis (Fig. 4). Additionally, so­
dium selenite and stress-responsive protein complexes were further
authenticated using an interactive assessment to verify the types and
number of interactions formed among the docked (Fig. 4). Table 4
showcases the interaction results. All stress-responsive proteins docked
with sodium selenite were separately analyzed for the number and type
of interactions. The interaction analysis suggested good binding affinity
due to the existence of hydrogen bonds besides the hydrophobic in­
teractions, salt bridges, and pi–pi interactions (Fig. 4). Sodium selenite
formed 3, 4 and 1 hydrogen bonds with DREB, STS and STXBP1 proteins,
respectively Table 4.
Principal component analysis
This study employed principal component analysis (PCA) to analyze
the effects of Se treatments on the biochemical and morphological
S. Hussain et al.
characteristics of turnips during salt stress. Fig. 5 shows that PCA
described 58.5% of the overall variation using PC1 and 21.4% using
PC2. PCA analysis showed that the Se treatments significantly altered
biochemical and morphological characteristics. PCA scores were sub­
stantially higher and clustered to the right for Se2 (100 μmolL− 1)
treatments. PCA (Fig. 6) shows that supplementing with Se2 (100
μmolL− 1) is protective.
Discussion
Salt toxicity and other environmental stresses cause an injurious
effect on the morphological and physiological features of plants (Khan
et al., 2020). The yield of a crop depends upon the physicochemical
attributes of arable soil besides other prevailing environmental factors.
It has been observed that the continuous increase in soil alkalinity
resulting from salt stress is the most important cause of reduced plant
growth and yield.
The efficacy of biosafe selenium (Se) as a priming agent to increase
the salt tolerance of B. rapa has not been evaluated. Our study showed
that seedlings raised from the Se primed seeds improved root growth,
shoot growth, fresh weight and biomass production. B. rapa seeds
absorbed Se from the solution and transferred it into their endosperm
and embryo cells during imbibition, as revealed by Khaliq et al. (2015).
Afterward, the inorganic Se is then metabolically converted into organic
Se in the cells by cysteine synthase (Liu et al., 2011). Asthir et al. (2020)
also exposed the beneficial effects of Se priming on the growth of plants.
Selenium combines with different elements to make complexes which
are beneficial for plant growth (Fernandes et al., 2018). Our data
demonstrated that the increased biomass production in the case of Se2
treatment may be attributed to the enhanced root and shoot growth in
salt-stressed plants. It is assumed that the improvement in physiological
and metabolic attributes of Se-treated plants perhaps improved root and
shoot growth (Singh et al., 2018; Ikram et al., 2020). Tisarum et al.
(2019) also found that the application of Se at various growth phases of
plants assuaged salt toxicity and encouraged plant growth and biomass
production. Our results showing higher nutrient uptake in Se-treated
plants leading to improved gas exchange attributes are in harmony
with the study of Alyemeni et al. (2018). Turnip plants obtained from
the different concentrations of Se priming exhibited varying levels of salt
stress alleviation, as revealed by their growth and biomass production
Table 1. Selenium seed priming may offer a firm foundation for the
plant, resulting in improved growth and seed production (Moulick et al.,
2018).
Salt toxicity declines the photosynthetic activity and growth of
plants (Handaet al., 2019). Our results demonstrated that salinity
diminished the concentration of photosynthetic pigments, including
Chla, Chl b and total chlorophyll in turnip leaves. Nevertheless, seeds
priming with Se1 and Se2 significantly enhanced the Chla, Chl b and
total chlorophyll content of treated plants exposed to salt stress Table 2.
Selenium enhances the activity of glutathione peroxidase (GSH-Px) and
reduces the level free radicals to sustain stress. Substitution of Se by
sulfur enhances biosynthesis of protein and results in enhancement of
chlorophyll content through regulating the formation of porphyrins
(Yang et al., 2022; Khan et al., 2023). Many researchers have reported
the involvement of porphyrins in the biosynthesis of photosynthetic
pigments (Lidon et al. 2018; Zhao et al., 2022). Selenium enhanced the
concentration of photosynthetic pigments in treated tomato and sor­
ghum plants subjected to abiotic stresses (Zahedi et al., 2020). Selenium
augmented the synthesis of photosynthetic pigments by assisting in
electron transport and respiration during the respiratory chain
(Rasoolet al., 2020). For that reason, it is concluded that the increased
biosynthesis of photosynthetic pigments consequently enhanced the
photosynthetic activity in Se-applied turnip plants under salt stress.
Conversely, seed priming with higher Se concentration, i.e., Se3
(125 μmol L− 1), provoked negative effects on the biosynthesis of
photosynthetic pigments, photosynthetic activity, stomatal
7
Plant Stress 11 (2024) 100331
conductivity, and transpiration rate of developed plants. So, low con­
centrations of Se (100 μmol L− 1) assisting in improving photosynthesis
and gas exchange attributes are recommended for turnip seed priming to
mitigate salt stress.
The appropriate concentrations of osmolytes, such as total soluble
sugars, alleviate plant stresses through osmoregulation (Ozturk et al.,
2021; Sikder et al., 2020). These osmoprotectants help to maintain the
biosynthesis and degradation of proteins and chlorophyll and regulate
membranous integrity, as well as redox homeostasis and detoxification
of excessively synthesized ROS in stressed plants (Semida et al., 2021).
The synergistic interaction among the total soluble sugars and Se may
enhance the biosynthesis of plant metabolites responsible for the alle­
viation of salt stress by maintaining osmotic equilibrium. The osmotic
equilibrium sustains the cellular turgidity and guarantees the firmness
and protection of the cell membranes, consequently impeding oxidative
stress as well as photo-oxidation in plants under stress. Selenium prim­
ing during the current study increased the biosynthesis of total soluble
sugars Table 2, which may be attributed to the hydrolysis of starch into
sugars besides the elevated amylase activity. Additionally, Se might
have improved carbohydrate metabolism by enhancing the activity of
fructose 1, 6-bisphosphatase. Our data showed that salted B. rapa plants
accumulated more to mitigate stress. Likewise, Se2 exhibited an
augmented level of TSS, mineral content and higher activity of the
antioxidative enzymes to improve the growth of the plants (Nawaz et al.,
2015).
Salt-induced toxicity decreased the uptake of essential plant nutri­
ents Table 3. Se enhanced the uptake of nutrients, including K, P, Mg, Zn,
and Ca, in salted plants. Plants facing salinity stress showed poorly
developed root systems, consequently reducing nutrient uptake in salted
plants. However, Se-application improved the root architecture and
growth, which enhanced the uptake of essential plant nutrients. Both K
and Ca encourage growth by modulating the biosynthesis of stress-
responsive metabolites, improving the nitrogen assimilation and anti­
oxidative system of plants (Ahanger et al., 2017; Khan et al., 2016). The
improved physiochemical activities of plants facilitate them to mitigate
salt stress (Sarkar et al., 2018). Our results depicting the improved
nutrition by Se seed priming are harmonious with the conclusions of
Abedi et al. (2020). Se-treated plants exhibited decreased Na levels in
the roots and leaves of turnip plants. The beneficial impact of Se may be
due to the development of a complex between Se and Na at the root
level, which perhaps decreased Na translocation by reducing apoplectic
transport, as illustrated by Astaneh et al. (2018). Besides, Se might have
mitigated the negative effect of Na on the uptake and translocation of
Rubio et al. (2020) also found that the elevated activity of plasma
membrane H-ATPase enhanced K uptake and reduced Na uptake in
Se-applied plants under salt stress. Hassan et al., (2020) observed that Se
enhanced K concentration in Se-applied salted plants. On the other
hand, Hawrylak-Nowak et al. (2015) revealed that Se does not influence
the uptake of Na or K in treated plants. Hence, the exact mechanism
through which Se-treated turnip seedlings alleviated Na toxicity was not
elucidated. It is assumed that modification (s) in cellular procedures
resulting through the suitable concentration of Se may possibly support
the Na sequestration within root vacuoles. The ultra-structural modifi­
cations have shown the salinity stress alleviation strategy in plants
(Alkhatib et al., 2021). Consequently, the ultra-structural adjustments
may well elucidate the defensive role of Se in salted plants.
The augmented level of H2O2 in saline conditions causes an increase
in lipid peroxidation, which induces plant stress (Soleymanzadeh et al.,
2020). Also, the increased assembly of ROS such as H2O2, OH and
O−2 causes degradation of DNA, lipids and proteins (Kamran et al., 2019).
Accordingly, the integrity of the cellular membrane and the photosyn­
thetic activity are affected (Astaneh et al., 2019). During the present
study, salinity enhanced ROS content in plants. Yet, Se decreased ROS
synthesis in treated plants under stress (Fig. 1). The reduced lip­
oxygenase activity decreased the degradation of fatty acid and ROS level
in Se-primed seed plants under stress (El-Badri et al., 2021).
S. Hussain et al. Plant Stress 11 (2024) 100331

Fig. 2. Influence of selenium (Se) and sodium chloride (NaCl) on enzymatic content and Quantitative real time PCR analysis of defense-related antioxidative genes
transcripts of B. rapa: (A), Catalase CAT, Superoxide dismutase SOD, Peroxidase POD, Ascorbate peroxidase APX (B) Relative expression of APX gene, Relative
expression of CAT gene, Relative expression of SOD gene, Relative expression of POD gene.

Fig. 3. Influence of selenium (Se) and sodium chloride (NaCl) on nonenzymatic content and growth of B. rapa (A) Malondialdehyde (MDA), Proline, Hydrogen
peroxide (H2O2), Electrolyte Leakage (EL), (B) vegetative growth. Data display means ± SD of 5 replications. Dissimilar letters denote significant differences among
the treatments (P ≤ 0.05). C = Control, NaCl= 200 mM NaCl, Se1= 75 μmol L− 1 Se, Se2=100 μmol L− 1 Se, Se3=125 μmolL− 1 Se.

Salt-stressed plants exhibited increased MDA, proline, and electro­
lyte leakage levels during the present research (Fig. 1). Our results
demonstrated that salinity modulated the activity of antioxidant en­
zymes. It seems defense strategy of B. rapa seedlings to alleviate salt
stress through scavenging the augmented level of ROS. Zaid et al. (2022)
also reported that higher activity of antioxidant enzymes scavenges the
increased production of ROS and preserve an optimal redox homeostatic
state. However, higher level of MDA caused increase in synthesis of ROS
which couldn’t be detoxified by the antioxidants. The further increase in
the antioxidant activity of these enzymes in Se treated plants enabled
plants to detoxify higher level of ROS and mitigate the salt induced
oxidative stress. Our findings are in consistent with the results of

8
S. Hussain et al.
Fig. 4. Molecular Docking and interaction analysis of sodium selenite with different stress related proteins of B. rapa. (A) Dehydration-responsive element-binding
protein (DREB) (B) Salt overly sensitive 3 (SOS3) (C) Syntaxin binding protein 1 (STXBP1).
Shekari et al. (2017) who revealed that Se application decreased ROS
and MDA level through improving the antioxidative activity of enzymes.
Furthermore, seleno-proteins have demonstrated a significant role in
stress tolerance through regulating redox (Ingold et al., 2018). Hence,
seleno-proteins developed by Se priming perhaps decreased
salinity-induced oxidative injuries by detoxifying the ROS generated
during stress (Bybordi et al., 2016). In the same way, improving anti­
oxidative defense coordination in Se-treated plants decreased the
membranous damage and MDA content under salinity regimes (Kamran
et al., 2019). The augmented level of ROS generated by salt toxicity may
be declined through the activity of enzymatic antioxidants, including
SOD, POD, CAT and APX or by means of non-enzymatic antioxidative
systems (Ahmad et al., 2020; El-Badri et al., 2021). The elevated activity
of SOD is perhaps the foremost line of defense against ROS because this
enzyme impedes the development of the hydroxyl (OH− ) radical, which
may decline photosynthetic activity (Elkelish et al., 2019). Furthermore,
CAT detoxifies H2O2 in leaf tissues and reduces lipid peroxidation by
scavenging OH- radicals in cellular membranes (Shalaby et al., 2021).
What is more, POX plays a significant role in decreasing oxidative in­
juries in plants by scavenging H2O2 (Desoky et al., 2021). Similarly, APX
safeguards cellular membranes by detoxifying H2O2 through modulating
enzymatic and non-enzymatic defense systems (Ahmad et al., 2021).
Together CAT and APX sustain redox homeostasis by scavenging ROS
(Jiang et al., 2017). Our findings established that salinity stress
enhanced the activity of antioxidative enzymes in turnip plants. Elkelish
et al. (2019) also observed that salinity elevated the activity of POD in
9
Plant Stress 11 (2024) 100331
common beans and maize respectively. Besides, Se further augmented
the activity of antioxidant enzymes in stressed plants. Our results
advocate that the modulated activities of CAT, POX, SOD and APX
improved the stress mitigation potential of Se-treated plants. The
detoxification of ROS in Se-applied plants decreased the deterioration of
photosynthetic pigments and regulated NADP levels, which conse­
quently enhanced the photosynthetic activity. In the same way, Se
priming escorted in a decrease of ROS synthesis and increased root and
shoot growth through improving enzymatic and non-enzymatic anti­
oxidative systems (Fig. 2A). Our results regarding the seeds priming with
Se in the improvement of plant stress tolerance are in conformity with
the conclusions of Jóźwiak et al. (2019).
Our results are in agreement with the findings of Ulhassan et al.
(2018), who revealed that Se enhanced the antioxidative activity and
gene expression level of POD, SOD and APX in rapeseed plants (Fig. 2B).
In the same way, Luo et al. (2021) reported a higher expression level of
CAT in Se-applied plants. Additionally, Kang et al. (2022) found that
exogenously applied Se improved the antioxidant system of cucurbits
through the up-regulation of stress-responsive genes. Similarly, El-Badri
et al. (2022) observed that Se modulated the expression level of
stress-responsive genes in B. napus plants. In another study, rose plants
subjected to salinity stress enhanced the antioxidative activity and
expression level of CAT, SOD, POD and APX enzymes to scavenge the
higher level of ROS (Omidi et al., 2022). Hence, the upregulated activity
and higher expression level of antioxidant enzymes is perhaps a strategy
to mitigate salt stress.
S. Hussain et al.
Fig. 5. Principal component analysis (PCA) showing the relationship of different morphological attributes, with non-enzymatic and enzymatic, antioxidants of turnip
modulated by application of sodium selenite Na2SeO3 under salinity stress.
Plants often face salinity stress in tropical and subtropical regions.
Plants mitigate salt stress by upregulating their genes involved in the
physiochemical activities (Raja et al., 2020; Guo et al., 2016). It is a fact
that DREB proteins regulate the expression level of genes related to plant
tolerance against salt stress (Ali et al., Hadi, 2018; Eckardt, 2019).
Arabidopsis plants upregulate the expression of the DREB gene under
saline conditions to alleviate stress (Faraji et al., 2020). Similarly, DREB
genes have a crucial role in the salinity tolerance of upland cotton plants
by regulating the ABA pathway (Chen et al., 2007). Our results showed
that salinity alters the number and category of DREB genes.
The STXBP1 genes standardize the expression of salt stress-related
genes besides various stress-related pathways, which together improve
plant tolerance against abiotic stresses. Many studies have exposed the
significance of STXBP1 in abiotic stress mitigation in plants (Angamu­
thu and Piramanayagam, 2017; Mulligan, 2021).
Likewise, Yang and Guo (2018) found that SOS pathways genes
trigger salt stress in plants by sustaining ionic homeostasis. Moreover,
the SOS regulatory pathway was revealed to be the intention for
Na+/H+ replacement in the vacuolar membrane of B. rapa, which was
maintained by modulations in Na+/H+ exchange activity in the vacuolar
membrane of the mutants (SOS3) compared to the control type. During
the current study, SOS3 genes in B. rapa were significantly
over-expressed. The expression level of SOS3 was higher than that of
SOS1 and SOS2. The plasma membrane Na+/H+ exchanger revealed
that the SOS3 gene enhances salt tolerance in rice plants (El Mahi et al.,
2019).
Our results exhibiting a reduction in stomatal conductance, tran­
spiration rate, and net photosynthetic rate in stressed plants are
10
Plant Stress 11 (2024) 100331
incongruent with the findings of Jia et al. (2016). Marques et al. (2021)
also observed a diminution in gas exchange attributes of Triticum aesti­
vum under salt stress. It seems that the malformation of the stomatal
structure reduces gas exchange attributes in salted plants. Jalal et al.
(2022) have also revealed a reduction in gs and E in plants subjected to
salt stress. A number of studies have expressed the negative effects of
salinity stress on the expression level of the Rubisco protein and stomata
structure significantly decreased photosynthetic activity and biomass
production in plants (Alyemeni et al., al., 2018; Hassan et al., al., 2021).
Sehar et al., (2021) suggested that the intonation in the photosynthetic
activity is due to the enhanced level of CO2 diffusion into the intercel­
lular spaces due to the modified functions of stomata in Se-treated
plants. As well, the improvement in redox machinery of Se-applied
plants perhaps increased gas exchange parameters and photosynthetic
activity of turnip plants under salt regimes. Da-Silva et al. (2020) also
concluded that Se application amplified the stomatal conductivity, rate
of photosynthesis and internal CO2 concentration in cucumber plants.
Still, our results demonstrated a higher concentration of Se (Se3/
125 μmol L− 1) provoked injurious effects on the photosynthetic ma­
chinery of turnip plants, ensuing additional decline in stomatal con­
ductivity, transpiration rate, intercellular CO2 concentration,
chlorophyll content and net photosynthetic rate (Figs. 3A, B, C, D). For
that reason, a low dose of Se priming (Se2, 100 μmol L− 1) may perk up
gas exchange attributes in turnip plants and result in enhanced growth
under salt stress and control regimes.
Molecular docking is a very useful computational modeling tool to
predict the interactions of small molecules with an enzyme, channel
protein or receptor. It is extensively used in drug discovery/repurposing
S. Hussain et al.
Fig. 6. Correlations between morphometric and chemical variables in the studied in B.rapa: A) heatmap with marginal dendrogram and B) heatmap correlation
coefficients.
research. When a ligand binds to an enzyme’s active site, it may prevent
the binding of another, which depends on the affinities measured as
docking scores. The docking scores represent the energy liberated when
a compound docks/binds with an enzyme. A ligand with a higher
docking score (i.e., a more negative score) may prevent another with a
lower (i.e., a less negative) docking score from interacting with a re­
ceptor. Compared to other similar modeling algorithms, the AutoDock
Vina by PyRx has better accuracy (Pawar and Rohane, 2021). Of late,
molecular docking tools were employed to predict the possible in­
teractions of NaCl with such targets of B. rapa (Rahman et al., 2020;
Jose et al., 2022). In the present study, in vitro analysis revealed that
selenium may affect the antioxidant system of B.rapa; thus, it was
thought prudent to perform an in silico analysis. AutoDock Vina by
PyRx, used in the present study, is a very useful molecular docking
software that predicts the interactions of ligands and receptors with over
88% accuracy (Sunny and Jayaraj, 2022).
The present results indicate that selenium may potentially interact
with the active sites of the stress responsive proteins. The presence of
hydrogen bonding of the sodium selenite with all the receptors are
attributable to greater affinity of sodium selenate with stress responsive
proteins (Choudhury et al., 2022). When a ligand binds at the active site
of a receptor/enzyme, it may interfere with its activity, which may be
regarded as a direct effect of selenium, resulting in oxidative stress;
however, selenium was found to be potent in ameliorating the effect of
NaCl, by excluding it from the active site of the target enzymes. The
limitation of the present study is that crystal structures of the four target
enzymes were not available, and the modeling study was performed
based on structures predicted using homology modeling. In the future,
an illustration of the stress response (including the interaction effects)
would be much clearer for researchers with the help of such an inte­
grative approach.
11
Plant Stress 11 (2024) 100331
Conclusions
Salt-induced toxicity caused harmful effects on gas exchange pa­
rameters and nutritional content that led to diminished shoot and root
growth in addition to the chlorophyll content of the B. rapa plant. On the
other hand, seed priming with selenium (100 μmol L− 1) enhanced plant
growth under salinity stress and normal circumstances. Selenium
augmented plant nutrition which helped to adjust stomatal conductiv­
ity, intercellular CO2 concentration and photosynthetic activity, result­
ing in enhanced plant growth. Findings of the present study advocated
that B. rapa var. purple top white globe performed better against salinity
stress by applying selenium as a growth promotor and also modulations
in the expression of genes encoding antioxidative activities. The datasets
obtained from the computational analysis substantiate our findings,
thus, clearly indicating NaCl stress amelioration by Se supplementation.
Taken as a whole, this research work offers an excellent perceptive for
the researchers to expose the definite physiochemical mechanism
involved in the Se persuaded salt stress alleviation in B. rapa that will
serve as a roadmap for advanced studies at the cellular level. Moreover,
additional field experiments are required to assess the role of Se seed
priming on salinity stress mitigation and to examine if the tolerance
effect persists during the whole crop growing season or is just
temporary.
CRediT authorship contribution statement
Saber Hussain: . Shakil Ahmed: Conceptualization, Project
administration, Supervision. Waheed Akram: Data curation, Formal
analysis, Investigation, Project administration, Validation, Writing –
review & editing. Aqeel Ahmad: Formal analysis, Funding acquisition,
Investigation, Resources, Software. Nasim Ahmad Yasin: Conceptual­
ization, Formal analysis, Funding acquisition, Methodology, Project
administration, Resources, Supervision, Validation, Writing – original
draft, Writing – review & editing. Mei Fu: Funding acquisition,
S. Hussain et al.
Methodology, Validation. Guihua Li: Investigation, Software, Valida­
tion, Writing – review & editing. Rehana Sardar: Formal analysis,
Software, Writing – original draft, Writing – review & editing.
Declaration of competing interest
The authors declare that the research was conducted in the absence
of any commercial or financial relationships that could be construed as a
potential conflict of interest.
Data availability
Data will be made available on request.
Data availability statement
The original contributions presented in the study are included in the
article/Supplementary Material. Further inquiries can be directed to the
corresponding authors.
Funding
This research work was funded by the Modern Agriculture Industry
Technology Innovation Teams (Funding no: 2023KJ122, 2022-NBA-
00–014); Guangzhou Science and Technology Planning Project (Funding
no: 202102020442; 2023B03J1270); Project of the State Administration
of Foreign Affairs of China (QN2022030025L, QN2022030024L).
Supplementary materials
Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.stress.2023.100331.
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