Modulation of NaCl-induced osmotic, cytogenetic, oxidative and anatomic damages by coronatine treatment in onion (Allium cepa L.)

Abstract

Coronatine (COR), a bacterial phytotoxin produced by Pseudomonas syringae, plays important roles in many plant growth processes. Onion bulbs were divided four groups to investigate the effects of COR against sodium chloride (NaCl) stress exposure in Allium cepa L. root tips. While control group bulbs were soaked in tap water medium, treatment group bulbs were grown in 0.15 M NaCl, 0.01 µM COR and 0.01 µM COR + 0.15 M NaCl medium, respectively. NaCl stress seriously inhibited the germination, root lenght, root number and fresh weight of the bulbs. It significantly decreased the mitotic index (MI), whereas dramatically increased the micronucleus (MN) frequency and chromosomal aberrations (CAs). Moreover, in order to determine the level of lipid peroxidation occurring in the cell membrane, malondialdehyde (MDA) content was measured and it was determined that it was at the highest level in the group germinated in NaCl medium alone. Similarly, it was revealed that the superoxide dismutase (SOD), catalase (CAT) and free proline contents in the group germinated in NaCl medium alone were higher than the other groups. On the other hand, NaCl stress caused significant injuries such as epidermis/cortex cell damage, MN formation in epidermis/cortex cells, flattened cells nuclei, unclear vascular tissue, cortex cell wall thickening, accumulation of certain chemical compounds in cortex cells and necrotic areas in the anatomical structure of bulb roots. However, exogenous COR application significantly alleviated the negative effects of NaCl stress on bulb germination and growth, antioxidant defense system, cytogenetic and anatomical structure. Thus, it has been proven that COR can be used as a protective agent against the harmful effects of NaCl on onion.

Full text

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Modulation of NaCl‑induced
osmotic, cytogenetic, oxidative
and anatomic damages
by coronatine treatment in onion
(Allium cepa L.)
Dilek Çavuşoğlu
Coronatine (COR), a bacterial phytotoxin produced by Pseudomonas syringae, plays important roles in
many plant growth processes. Onion bulbs were divided four groups to investigate the eects of COR
against sodium chloride (NaCl) stress exposure in Allium cepa L. root tips. While control group bulbs
were soaked in tap water medium, treatment group bulbs were grown in 0.15 M NaCl, 0.01 µM COR
and 0.01 µM COR + 0.15 M NaCl medium, respectively. NaCl stress seriously inhibited the germination,
root lenght, root number and fresh weight of the bulbs. It signicantly decreased the mitotic index
(MI), whereas dramatically increased the micronucleus (MN) frequency and chromosomal aberrations
(CAs). Moreover, in order to determine the level of lipid peroxidation occurring in the cell membrane,
malondialdehyde (MDA) content was measured and it was determined that it was at the highest
level in the group germinated in NaCl medium alone. Similarly, it was revealed that the superoxide
dismutase (SOD), catalase (CAT) and free proline contents in the group germinated in NaCl medium
alone were higher than the other groups. On the other hand, NaCl stress caused signicant injuries
such as epidermis/cortex cell damage, MN formation in epidermis/cortex cells, attened cells nuclei,
unclear vascular tissue, cortex cell wall thickening, accumulation of certain chemical compounds in
cortex cells and necrotic areas in the anatomical structure of bulb roots. However, exogenous COR
application signicantly alleviated the negative eects of NaCl stress on bulb germination and growth,
antioxidant defense system, cytogenetic and anatomical structure. Thus, it has been proven that COR
can be used as a protective agent against the harmful eects of NaCl on onion.
e genus Allium, one of the largest monocot plant genera, has about 920 species1. Allium cepa L. is commonly
known as onion. It has been included in the Amaryllidaceae family and Allioideae subfamily in recent taxonomic
classications2. A biennial herb, Allium cepa L. has additional roots, yellowish leaves, and bulbs made from
concentric and enlarged eshy leaf bases. e outer leaf base forms the protective layer and is dry, thin and of
various colours. As the onion develops, the inner leaf bases thicken. A mature onion can be long, spherical or oval
in shape, and its size varies by variety3. Allium cepa L. is an important cultivated plant consumed as a vegetable.
Although this plant is mostly consumed as food, its antidiabetic, antioxidant and antimicrobial eects are also
widely used. is species contains various vitamins, minerals, sulfur amino acids, avonoids, phytosterols and
saponins4.
One of the most important environmental factors limiting the normal growth and development of plants
is salinity5,6. Plants are generally extremely sensitive to soil salinity during germination and early growth7,8.
Today, approximately 23% of the total irrigated agricultural land is aected by high salinity due to the articial
irrigation methods used in modern agriculture9. Various morphological, anatomical, physiological, cytogenetic
and biochemical responses may occur in plants exposed to salt stress10–13. In addition, it causes osmotic and
oxidative stresses by increasing formation of reactive oxygen species (ROS) including free radicals, hydrogen
peroxide and singlet oxygen14–16. ROS induce a series of responses such as membrane degradation, lipid per-
oxidation, protein denaturation, antioxidant enzyme inactivation and DNA mutation16,17. Plants can cope with
OPEN
Department of Plant and Animal Production, Plant Protection Program, Atabey Vocational High School, Isparta
University of Applied Sciences, Isparta, Turkey. email: cavusoglu.dilek@gmail.com
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salinity-induced osmotic and oxidative stress by operating various mechanisms, including ion partitioning,
upregulation of antioxidant activities and osmoregulation18–20. Moreover, they can provide osmotic compatibility
by increasing the biosynthesis of compatible solutes such as soluble sugars, proline and proteins5,21. To mitigate
and repair the detriment of salt stress, plants also have a wide range of the antioxidant defense system includes
antioxidant enzymes (such as catalase, peroxidase and superoxide dismutase) and non-enzymatic antioxidants
(such as salicylate, glutathione, ascorbate, carotenoids and tocopherols). erefore, it is extremely important
to increase the activities of antioxidant enzymes and the content of non-enzymatic compounds to improve the
tolerance of plants to salt stress7,22–26.
Phytotoxins are highly toxic substances synthesized by plants and plant pathogens. ese substances, which
are mostly produced as secondary metabolites in plants, are also called phytochemicals, plant allelochemicals
and plant poisons27–29. ese toxins, which accumulate on plant surfaces and tissues, can be found naturally in
roots, bark, leaves, fruits and owers28,30,31. Phytotoxins not only protect plants against various biotic and abiotic
stress factors, but also serve as growth enhancers and defense proteins, promoting plant growth and survival32–34.
Phytotoxins are used in agriculture as plant growth regulators and biochemical agents for plant and cell physiol-
ogy, as they generally have stimulant eects on plants at low concentrations35–37.
Coronatine (COR), a non-host-specic phytotoxin, is produced by several members of the Pseudomonas
syringae group of pathovars38–40. e structure of COR is an amide of coronafacic acid and coronamic acid; it
is a methyl cyclopropyl amino acid derived from isoleucine41,42. COR is a new biotic plant growth regulator,
structurally and functionally similar to jasmonates (JAs) such as jasmonic acid, jasmonoyl-isoleucine and methyl
jasmonate43–45. Although the activities of COR and JAs are similar, they are not the same46. Studies have shown
that COR are biologically more eective than JAs in the production of secondary metabolites such as protease
inhibitors, glyceollin, sakuranetin, momilactone A, alkaloid, nicotine, volatile substances and taxol47–51. It has
been determined that the physiological eects of COR, a bacterial phytotoxin, vary depending on the plant
species46 and tissue type52. It plays important roles in many plant growth processes such as seed germination53,
seedling growth54, ethylene emission55, auxin synthesis56, anthocyanin production57, alkaloid accumulation51,
tendril coiling58, chlorosis in leaf tissues59, leaf senescence52, photosynthesis60, stomatal opening61, reactive oxygen
species (ROS) production62, lipid peroxidation63 and antioxidant enzyme activity64. Moreover, the micro-doses
of exogenously applied COR can increase tolerance or resistance of plants to dierent abiotic stresses such as
salinity65, osmotic66, drought62,67, heat68 and chilling64,69.
Allium test is a fast, inexpensive and sensitive method. This test associated two aims: toxicity and
mutagenicity70. Mutagenicity is connected with the chromosome breakdown rate and toxicity is evaluated by
observing inhibition root growth. e Allium test sensitivity is on par with test systems using algae or human
lymphocytes. e results of many tests using a variety of biological organisms yielded results similar to those of
the Allium test. is has made the mentioned test a reliable scanning test71,72. Moreover, the Allium test has been
proven to be an eective test for genetic monitoring of environmental pollutants in joint studies conducted by
WHO (World Health Organization), USEPA (US Environmental Protection Agency) and UNEP (United Nations
Environment Programme)73.
Although there are very few studies, made in some plant species, on the eects of exogenous COR on the
physiological and biochemical parameters examined in the current study under salt stress, unfortunately, there
is no study on the eects on cytogenetic parameters and root anatomical structure. On the other hand, the
eects of COR on all parameters examined in onion have never been studied and therefore the role of COR in
salt stress tolerance of onions has been reported for the rst time. us, the current study focused on improving
the negative eects of osmotic and oxidative stresses induced by NaCl on germination and seedling growth and
reducing genotoxicity and anatomical damage in onion plant with exogenous COR application.
Materials and methods
Test plant, salt and applied chemical dose. Allium cepa L. bulbs, commonly known as onion, were
used as plant material. e concentration used in the experiments of COR purchased from Sigma-Aldrich Com-
pany, United Kingdom was 0.01µM. Salt (NaCl) concentration used was 0.15M. ese levels were designated
by a preliminary study. Experimental research on plant samples, including the supply of plant material, complies
with institutional, national and international guidelines and legislation.
Germination and growth procedure. Germination experiments were carried out in the dark in an incu-
bator with a temperature of 20°C and no light. Onion bulbs of approximately the same size and healthy appear-
ance were selected and divided into four main groups (Table1).
Twenty bulbs from each test group were placed in plastic tubs and le to germinate in the incubator for seven
days. Bulbs reaching a root length of 10mm were considered germinated. On the last day of the experiment,
Table 1. Main groups and administration doses.
Main groups Administration doses
Group I/control Tap water
Group II 0.15M NaCl
Group III 0.01µM COR
Group IV 0.01µM COR + 0.15M NaCl
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the number of root and germination percentages of the bulbs were determined, and root lengths (mm) and
fresh weight (g seedling−1) were measured. For statistical evaluation, all experiments were arranged in triplicate.
Procedure for determining cytogenetic dierences. For cytogenetic examinations, 1–1.5cm long
pieces of bulb roots germinated for a few days were cut with a razor blade and kept in saturated paradichloroben-
zene for 4h. en, these fractions were xed in 3/1 ethanol-acetic acid solution and stored in 70% ethanol. ese
pieces were hydrolyzed in 1 N HCl for 17min at 60°C, stained with Feulgen for 1–1.5h, crushed on a slide in a
drop of 45% acetic acid and covered with a coverslip74. One day later, balm was applied around these coverslips
and made into permanent preparations. Mitosis stages and mitosis abnormalities seen in root tip meristem cells
of onion bulbs were photographed at 100× magnication with a digital camera mounted on a light microscope.
MI was calculated by counting a minimum of 30,000 cells (10,000 cells for per slides) from each of the 4 main
groups and CAs as % of 2000 dividing cells (for per slide) counted.
Procedure for determining antioxidant capacity. A quantity (0.2g) of germinated bulb roots were
weighed and homogenized with 5mL of 50mM chilled sodium phosphate buer (pH 7.8). e homogenates
were centrifuged at 10,000rpm for 20min and the supernatant obtained was used for internal analysis of SOD
and CAT antioxidant enzymes.
To determine the SOD content in the root tip cells of germinated onion bulbs, 1.5mL 0.05M sodium
phosphate buer (pH 7.8), 0.3mL 130mM methionine, 0.3mL 750μM nitroblue tetrazolium chloride, 0.3mL
0.1mM EDTA–Na2, 0.3mL 20μM riboavin, 0.01mL supernatant, 0.01mL 4% polyvinylpyrrolidone and
0.28mL deionized water were added in a test tube and a reaction mixture was prepared. en, the reaction was
started by keeping the tube containing this mixture under two pieces of 15 W uorescent lamps for 10min and
the reaction was terminated by keeping it in the dark for 10 minutes75. SOD activity was expressed as U mg−1
FW by measuring absorbance at 560 nm76.
To determine the CAT content in root tip meristem cells of germinated bulbs, a 2.8mL reaction mixture was
prepared containing 0.3mL of 0.1M H2O2, 1.5mL of 200mM sodium phosphate buer and 1.0mL of deion-
ized water. e reaction was started by adding 0.2mL of supernatant to this mixture and the decrease in 240nm
absorbance as a result of H2O2 consumption was measured with a UV–Vis spectrophotometer at 25°C and the
CAT activity was determined as OD240nmmin g−1 FW77.
Procedure for determining cell membrane injury. A quantity (0.5g) of the fresh roots of the germi-
nated onion bulbs were taken, homogenized with 5% TCA solution in a homogenizer and centrifuged for 15min
at 12,000rpm at 24°C. en, in a 20% TCA solution, 0.5% TBARS and the supernatant were transferred to a
dierent equal volume test tube and allowed to boil at 96°C for 25min. At the end of the period, these tubes
were placed in an ice bath and centrifuged at 10,000rpm for 5min. e absorbance was measured at 532nm, the
MDA content was calculated using the extinction coecient of 155 M−1 cm−1 and expressed as µmol g−1 FW78.
Procedure for determining free proline accumulation. A quantity (0.5g) of frozen root tips were
weighed and homogenized in 10mL of 3% aqueous sulfosalicylic acid solution and the homogenates were l-
tered into a test tube with lter paper. en, 2mL of acid-ninhydrin and 2mL of glacial acetic acid were added
to 2mL of ltrate and incubated at 100°C for 1h. is mixture was mixed with 4mL of toluene and the chromo-
phore containing toluene was separated from the hydrated phase. It was read spectrophotometrically at 520nm
absorbance using toluene as blank. e free proline content was calculated according to a standard curve and
expressed as µmol g−1 FW79.
Procedure for determining root anatomical dierences. Root tips of 1cm long were cut from ger-
minated onion bulbs to observe the anatomical damage and changes caused by NaCl and COR applications.
Root tips were washed with distilled water to remove residues on the surface of the onion roots. en, cross-
sections were taken from the root tips with a sharp razor blade and aer staining with 0.5% methylene blue for
2min, the stained samples of each group were examined with a research microscope at 500× magnication.
Evaluation of the obtained data. All data obtained from this study were analyzed with the help of SPSS
statistics V 23.0 (2015) package program and expressed as mean values by taking their standard deviations
(± SD). Statistical analysis of mean values was determined by Duncan’s multiple range test (DMRT) and p < 0.05
was considered highly signicant.
Ethical approval and informed consent. Not applicable: is study does not directly involve humans
or animals. Plant collectionpermits were not required because seed samples are commercial cultivars which can
bepurchased and no species are endangered or threatened.
Results and discussion
Eect of COR on the physiological parameters. Exogenous COR treatment was ineective on the
germination and growth of onion bulbs under normal conditions. at is, the germination percentage, root
length, root number and fresh weight of Group III bulbs germinated in medium containing COR alone showed
statistically the same values as the bulbs of the control group (Group I) germinated in tap water medium (Fig.1).
It has been found that COR decreases the seed germination53 and seedling growth46,57,80 at high concentrations,
while it stimulates63 or does not aect54,62,65 at low concentrations. Both the results of available research show-
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ing that COR applied in micro doses does not aect the germination and growth of onion bulbs in stress-free
environments and the results of above-mentioned researches have proven that this chemical has dierent eects
depending on the plant species, application dose and pretreatment form.
It has been known for a long time that salinity causes adverse eects even on the growth and development
of halophytes81,82. Salt stress has a negative eect on germination12,83,84 and seedling growth85–87 and has rep-
licated its negative eect on all physiological parameters examined of onion in this study (Fig.1). While the
germination percentage of Group I bulbs, known as control group, germinated in tap water medium at the end
of the experiment (seventh day) was 100 ± 0.0%, this rate was 22 ± 2.4% in Group II bulbs germinated in 0.15M
NaCl medium and thus salt stress reduced the germination of bulbs by 78%. In addition, root length, root
number and fresh weight of Group I (control) bulbs grown in tap water medium were 67.4 ± 2.5mm, 40.3 ± 2.3
and 13.7 ± 1.5g, respectively. ese parameters were determined as 12.6 ± 1.1mm, 11.5 ± 1.4 and 3.8 ± 0.7g in
Group II bulbs grown in 0.15M NaCl medium (Figs.1, 2). ese values were statistically signicant (p < 0.05).
Salt stress can exert its negative eect on germination by inhibiting water uptake of bulbs, by reducting growth
promotors (cytokinins and gibberellins) in bulbs and by increasing the growth inhibitors (abscisic acid, ABA)
in bulbs88–91. Due to the high osmotic pressure of the 0.15M NaCl medium, the fresh weight and water content
of the bulbs may have decreased due to the inability of the roots to receive sucient water (Fig.1). In addition,
Figure1. Eect of COR on some physiological parameters of Allium cepa L. Group I (control) was treated with
tap water; Group II was treated with 0.15M NaCl; Group III was treated with 0.01µM COR; Group IV was
treated with 0.01µM COR + 0.15M NaCl. e error bars indicate the standard deviation (± SD).
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this NaCl concentration may have caused a reduction in the root length and root number of bulbs as it inhibited
the mitotic activity in root tip meristematic cells (Fig.3). Addition of COR to 0.15M NaCl medium signicantly
increased the germination of onion bulbs. At this salt level, the germination of COR treated Group IV bulbs
reached 78 ± 2.8%. Exogenous COR application also showed a positive eect on the root length, root number
and fresh weight parameters. e root length, root number and fresh weight of Group II bulbs grown in 0.15M
NaCl medium were 12.6 ± 1.1mm, 11.5 ± 1.4 and 3.8 ± 0.3g, respectively. ese parameters were 38.9 ± 1.8mm,
27.9 ± 1.9 and 9.3 ± 1.1g in COR-treated Group IV bulbs grown at this salt level (Figs.1, 2). ese values were
statistically signicant (p < 0.05). Few studies have been conducted about the role of exogenous COR on the
fresh weight of seedlings grown under salt stress, but no studies about its eects on the germination percentage,
root elongation and root number have been conducted. Only, Xie etal.63,65 reported that 0.01µM COR treat-
ment enhanced the fresh weight of cotton seedlings grown under salt-stressed conditions; and these results
were agreement with ndings of the present study. COR may have attenuated the NaCl-induced inhibition on
the germination and seedling growth by increasing the water uptake of roots (Fig.1), by stimulating the mitotic
activity of root tip meristematic cells (Fig.3), by reducing lipid peroxidation in root tip meristem cells (Fig.4)
or by regulating the proline content and antioxidant enzyme activities of root cells (Fig.4).
Eect of COR on the cytogenetic parameters. It has been reported that the exogenous application
of various growth-regulating agents during germination and seedling growth under normal conditions causes
cell disruptions, mitotic disorders and chromosomal abnormalities87,92,93. e cytogenetic results of this study
are very important as there are no available reports on the eects of COR on mitotic index (MI) micronucleus
(MN) frequency and chromosome aberrations (CAs) in root meristem cells of seedlings grown in both normal
and saline conditions. Figure3 shows the eects of exogenous COR administration on MI, MN frequency and
CAs in root meristem cells of Allium cepa L. bulbs. e MI, MN frequency and CAs in roots of the control
group (Group I) bulbs germinated in tap water medium were 7.1 ± 1.0%, 0.7 ± 0.7% and CAs 1.1 ± 0.3%, respec-
tively. ese parameters were 6.9 ± 0.9%, 0.9 ± 0.8% and CAs 1.2 ± 0.5% in roots of Group III bulbs germinated
in medium containing COR alone. at is, exogenous COR treatment was ineective on MI, MN frequency and
CAs in the root cells of onion bulbs germinated under normal conditions.
e increase or decrease in MI is an important indicator in determining the cytotoxicity level of a chemical94.
Salt stress has both inhibitory and cytotoxic eects on mitotic activity95–97, and it is well known that high salin-
ity inhibits mitotic activity in root tip cells and causes chromosomal abnormalities98,99. Salt stress, as expected,
seriously reduced the mitotic activity expressed as MI in root tips of the bulbs. e MI (1.8 ± 0.6%) in root tip
meristems of Group II bulbs germinated in the media containing 0.15M NaCl decreased approximately 75% as
compared with Group I (control) bulbs (7.1 ± 1.0%) germinated in tap water medium. Moreover, 0.15M salinity
induced a drastic increase in MN frequency and CAs in the roots of bulbs. e MN frequency and CAs in root
tips of the control (Group I) bulbs were 0.7 ± 0.7% and 1.1 ± 0.3%, respectively. ese parameters were 13.1 ± 1.8%
and 25.4.1 ± 2.1% in Group II bulbs at 0.15M NaCl concentration. In other words, 0.15M NaCl caused an
increase more than 18-fold in MN frequency and 23-fold in CAs according to the control (Group I). In summary,
0.15M salinity caused a signicant decrease in the MI and a signicant increase in the MN formation and CAs.
However, the addition of COR to the 0.15M NaCl medium signicantly alleviated the adverse eects of salt
stress on the MI, MN formation and CAs. MI, MN frequency and CA of root cells of Group II bulbs grown in
0.15M NaCl medium were 1.8 ± 0.6%, 13.1 ± 1.8% and 25.4 ± 2.1%, respectively. ese parameters were 5.3 ± 0.7%,
Figure2. e germination situations at the end of seventh day of Allium cepa L. bulbs. Group I was treated
with tap water, Group II was treated with 0.15M NaCl, Group III was treated with 0.01µM COR, Group IV was
treated with 0.01µM COR + 0.15M NaCl.
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6.2 ± 1.3% and 8.1 ± 1.4% in Group IV bulbs treated with COR (Fig.3). ese results showed that the damage
of sodium chloride stress on mitotic division of Allium cepa L. can be repaired by exogenous COR application.
Normal and abnormal mitotic stages observed as a result of microscopic examination of meristem cells of bulb
roots are shown in Figs.5 and 6. Common and notable abnormalities were metaphase with chromosome encir-
cleds (Fig.6a), nuclear budding (Fig.6b), trilobulated nucleus with micronucleus (Fig.6c), stickiness metaphase
(Fig.6d), metaphase/anaphase with chromosomal losses (Fig.6e,f), aberrant prophase/anaphase (Fig.6g,h),
chained telophase/anaphase (Fig.6i,j), telophase/anaphase with polar slip (Fig.6i,k,l), telophase/anaphase with
vagrant chromosome (Fig.6m,n) and alignment telophase/anaphase (Fig.6o,p). Chromosomal or chromosomal
breaks that remain in the anaphase stage and cannot combine with both nuclei in the telophase stage lead to the
formation of MN100,101. Nuclear budding is morphologically uniform to MNs with the exception that they are
participate in the nucleus102. Formation of MN and formation of cellular budding may be concluded with loss
of genetic materials103. During the S phase of mitosis, the suppressive eect of a nuclear poison 214 on DNA
synthesis causes the formation of lobed nuclei as a nuclear deformation104. DNA depolymerization, partial dis-
solution of nucleoproteins and increased chromosomal contraction and condensation can lead to the formation
of stick chromosomes in metaphase. Chromosomal stickiness is an indicator of toxic eects that are irrevers-
ible and result in cell death105. Chromosomal losses are alteration typically associated to the malfunction of the
mitotic spindle106. Vagrant chromosome with anaphase/telophase derives from unevenly sized or irregularly
Figure3. Eect of COR on some cytogenetic parameters of Allium cepa L. Group I (control) was treated with
tap water; Group II was treated with 0.15M NaCl; Group III was treated with 0.01µM COR; Group IV was
treated with 0.01µM COR + 0.15M NaCl. e error bars indicate the standard deviation (± SD).
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shaped nuclei in daughter cells with unequal chromosomes107. Spindle disorders lead to anaphase/telophase
with fault polarization, which is highly correlated with the incidence of the aforementioned abnormalities other
than vacuole nuclei108.
Eect of COR on the biochemical parameters. Reactive oxygen species (ROS) are dangerous cytotoxic
molecules, but also act as intermediate signaling molecules to regulate the expression of genes associated with
antioxidant defense mechanisms. Plants have antioxidant systems to deal with the damage caused by ROS15,109,110
and these systems protect plants from the negative eects of oxidative stress. One of these systems includes anti-
oxidant enzymes such as SOD and CAT
111,112. Depending on the activity of these enzymes, salt stress tolerance of
plants may vary113,114. In this study, SOD and CAT contents in roots of the control group (Group I) bulbs germi-
nated in tap water medium were 45 ± 1.8 and 0.9 ± 0.6, respectively. ese parameters were 46 ± 2.1 and 0.7 ± 0.2
in roots of Group III bulbs germinated in medium containing COR alone. at is, exogenous COR treatment
was ineective on SOD and CAT activities in the root cells of onion bulbs germinated under normal conditions
Figure4. Eect of COR on some biochemical parameters of Allium cepa L. Group I (control) was treated with
tap water; Group II was treated with 0.15M NaCl; Group III was treated with 0.01µM COR; Group IV was
treated with 0.01µM COR + 0.15M NaCl. e error bars indicate the standard deviation (± SD).
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(Fig.4). ese data obtained indicate that COR did not trigger an additional ROS formation in Allium cepa L.
roots compared to the control group. ese ndings were consistent with the ndings of researchers who showed
that low concentrations of COR did not aect SOD and CAT activity in the leaves and roots of cotton63,65,
tobacco62, chickpea66 and maize54 grown under normal conditions. On the other hand, NaCl exposure triggered
a drastic increase in SOD (108 ± 3.7) and CAT (4.8 ± 1.5) levels in the roots of Group II bulbs. Really, SOD and
CAT contents of NaCl-treated root cells approximately risen up to 2.4 and 5.3 folds of their own control (Group
I) levels (SOD 45 ± 1.8; CAT 0.9 ± 0.6), respectively (Fig.4). Parallel results were obtained from studies with plant
species such as Polygonum equisetiforme115, Astragalus gombiformis116, Mentha aquatica117, Mentha pulegium118
and Chrysanthemum morifolium119. Signicant increases in SOD and CAT levels in Group II were reliable signs
of ROS formation caused by NaCl. Moreover, increases in MN frequencies, CAs (Fig.3) and MDA levels (Fig.4)
are an important indicator of NaCl-induced oxidative stress. Oxidative stress causes adverse eects on cell mem-
branes, nucleic acids and other important components of cells. Stimulation of antioxidant enzyme activity can
help protect the plant from oxidative damage120. CAT and SOD enzymes are enzymatic scavengers of ROS in
plants121. Of these, SOD converts the superoxide radical to molecular oxygen and H2O2122. CAT catalyzes the
degradation of H2O2 to H2O and O217,123 thereby increasing membrane stability124. Nevertheless, COR addition
to NaCl solution contributed to the suppression of oxidative stress. SOD (64 ± 2.6) and CAT (2.3 ± 0.8) contents
of Group IV treated with COR were signicantly lower than (SOD 108 ± 3.7; CAT 4.8 ± 1.5) Group II in 0.15M
salinity (Fig.4). e decrease of CAT and SOD enzyme contents in the roots of Group IV bulbs showed that
exogenous COR application helped to the ght against ROS in onion plant and increased salt tolerance. How-
ever, Xie etal.63,65 reported that 0.01µM exogenous COR application increased SOD and CAT contents in the
root of cotton seedlings grown in 150mM NaCl medium; and these results were not similar to the ndings of the
present study. ese limited research results revealed that the eect of exogenous COR application on SOD and
CAT activities may vary depending on the plant species and the degree of exposure to stress.
Oxidative stress caused by salt stress can promote excessive ROS production, which leads to lipid
peroxidation125,126, which can be determined by measuring the MDA level127. In this study, while the MDA
content in the roots of Group I bulbs, known as the control group, which germinated in tap water medium, was
4.5 ± 0.7, this parameter was measured as 4.7 ± 0.8 in the roots of Group III bulbs germinated in the medium
containing COR alone. In other words, MDA contents were found to be statistically the same in the roots of
Group I and Group III bulbs, and exogenous COR did not cause a signicant membrane damage in onion root
cells (Fig.4). Similar results were found in cotton63,65, chickpea66 and maize seedlings54 grown in stress-free, that
is, normal conditions. On the contrary, NaCl induced a marked increase in MDA (24.6 ± 1.7) content in the roots
of Group II bulbs. MDA (24.6 ± 1.7) content of NaCl-treated root cells approximately risen up to 5.5 folds of their
own control levels (4.5 ± 0.7). e destructive eect of NaCl-induced oxidative stress on membranes was markedly
triggered by increases in MDA content (Fig.4). ese ndings were consistent with the ndings of researchers
who showed that NaCl stress increased the lipid peroxidation in the roots of sweet pepper128, tomato129, mung
bean130 and mint117,131. On the other hand, COR addition to NaCI solution contributed to the suppression of
oxidative stress. Joint application of COR with NaCl lessened by 46% the MDA (13.2 ± 1.2) content of Group
Figure5. Normal mitosis phases in the roots meristem cells of Allium cepa L. grown in tap water and 0.01µM
COR medium (a) interphase, (b) prophase, (c) metaphase, 2n = 16 chromosomes, (d) anaphase, (e) late
anaphase, (f) telophase. Scale bar 10μm.
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IV according to that (24.6 ± 1.7) of Group II in 0.15M salinity (Fig.4). Xie etal.63,65 determined that 0.01µM
exogenous COR application decreased MDA content in the root of cotton seedlings grown in 150mM NaCl
medium; and these results were agreement with ndings of the this study. Haddadi etal.117 reported that MDA
content in tolerant genotypes to salt were lower than the sensitive genotypes. For this reason, the reduction of
MDA content by COR in Group IV may be a sign that Allium cepa L. provides tolerance to salinity.
Proline is one of the most widely produced osmolytes132, which plays an important role in maintaining
osmotic potential and turgor in plants exposed to high salinity133,134. Moreover, proline also performs the task of
protecting cells by stabilizing cellular membranes and proteins during dehydration135–137. Although this amino
acid is synthesized in plants through glutamate and ornithine, the glutamate pathway is the main source of proline
production under osmotic stress114. In this study, while the free proline content in the root of Group I (control)
bulbs germinated in tap water medium was 15.1 ± 1.7, this parameter was measured as 16.3 ± 2.1 in the root of
Group III bulbs germinated in medium containing COR alone, and these two values were statistically similar
(Fig.4). Ceylan etal.66 detected that 0.01µM COR application enhanced the proline content in roots of chickpea
plants grown in stress-free conditions; and this conclusion was not consistent with the nding of current research.
Whereas, NaCl triggered a drastic increase in the free proline level (54.5 ± 3.9) in the roots of Group II bulbs.
Figure6. Chromosomal abnormalites in the root meristem cells of Allium cepa L. grown in 0.15M NaCl
and 0.01µM COR + 0.15M NaCl medium, (a) metaphase with chromosome encircleds = arrows, (b) nuclear
budding = arrow, (c) trilobulated nucleus with micronucleus = arrow, (d) stickiness metaphase, (e) metaphase
with chromosomal losses = arrows, (f) anaphase with chromosomal loss = arrow, (g) aberrant prophase,
(h) aberrant anaphase, (i) anaphase with polar slip = arrow, (j) chained anaphase, (k) telophase with polar
slip = arrow, (l) anaphase with polar slip = arrow, (m) telophase with vagrant chromosomes = arrows, (n)
anaphase with vagrant chromosome = arrow, (o) alignment telophase = arrows, (p) alignment anaphase = arrows.
Scale bar 10μm.
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e free proline content (54.5 ± 3.9) of NaCl-treated root cells approximately risen up to 3.6 folds of their own
control (15.1 ± 1.7) level (Fig.4). Proline is known to accumulate under saline conditions115,117,138,139. However, it
is not known whether proline accumulation occurs as a result of the stress eect or stress tolerance114. Although
a positive correlation between abiotic stress tolerance and free proline accumulation has been reported136,140,141,
a negative correlation between proline accumulation and salt tolerance has also been reported137,142,143. As in
this study, a positive correlation was found between MDA and proline accumulation115. is shows that proline
eectively participates in scavenging the produced ROS and thus protects the cells from oxidative damage144.
On the other hand, joint application of COR with NaCl decreased the free proline content in the root of Group
IV bulbs. e free proline content of Group IV bulbs treated with COR was 33.4 ± 3.0 while this parameter was
54.5 ± 3.9 in Group II bulbs in 0.15M salinity (Fig.4). Unfortunately, there are no studies about the eects of
exogenous COR on free proline content in roots of plants exposed to NaCl stress. Khedr etal.145 determineted
that exogenous proline increased the protein content in Pancratium maritimum L. under saline conditions. e
reduce content of free proline in the roots of Group IV bulbs treated with COR may be due to the generation of
new proteins for oxidative stress tolerance.
Eect of COR on the anatomic parameters. Since the roots are the most vulnerable and rst part of
the plants, if this organ is exposed to external toxic agents, the most severe damage to the anatomical structure
is expected to occur in this part. Anatomic changes observed in root epidermis layer cells are associated with
deterioration in the characteristic structure of the cell membrane. Figure7 and Table2 show NaCl-induced root
anatomical damages of Allium cepa bulbs and the protective eect of COR against NaCl-induced structural dam-
Figure7. NaCl-induced root anatomical structure damages, (a) normal appearance of epidermis cells, (b)
normal appearance of cortex cells, (c) normal appearance of cell nuclei-oval = arrows, (d) clear vascular tissue,
(e) epidermis cell damage = arrow, (f) cortex cell damage = arrow, (g) attened cells nuclei = arrows, (h) unclear
vascular tissue, (i) micronucleus formation in epidermis cells = arrow, (j) cortex cell wall thickening (white)
and micronucleus formation in cortex cells (black), (k) accumulation of some chemical compounds in cortex
cells = arrow, (l) necrotic areas = arrow.
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ages. No damage was detected in the root anatomical structure of the control (Group I) bulbs germinated in tap
water medium and Group III bulbs germinated in the medium containing COR alone, as a result of microscopic
examinations. In the root anatomical structure of Group II bulbs germinated in 0.15M salinity determined
damages such as epidermis/cortex cell damage (Fig.7e,f), micronucleus formation in epidermis/cortex cells
(Fig.7i,j), attened cells nuclei (Fig.7g), unclear vascular tissue (Fig.7h), cortex cell wall thickening (Fig.7j),
accumulation of certain chemical compounds in cortex cells (Fig.7k) and necrotic areas (Fig.7l). is suggests
that these damages are occur as a result of the defense mechanisms of cells and tissues in order to minimize the
stress due to the exposure of Allium cepa L. to salt stress.
ECD epidermis cell deformation, CCD cortex cell deformation, MNE micronucleus formation in epidermis,
MNC micronucleus formation in cortex, FCN fattened cell nucleus, UVT unclearly vascular tissue, CWT cortex
cell wall thickening, NA necrotic areas, ACC accumulation of some chemical compounds in cortex cells. (−) no
damage, (+) little damage, (++) moderate damage, (+++) severe damage.
As seen in Table2, the addition of 0.01µM exogenous COR to 0.15M NaCl medium reduced to a minor
level the severity of these anatomical damages caused by salt stress observed in the root anatomical structure.
Epidermis and cortex cell damage may be indication that salt stress causes a toxicity severe enough to disrupt
cell wall integrity. Flattened cells nuclei formations can occur not only as a result of rupture of cell membranes,
but also as a result of DNA damage due to oxidative stress146. When plants are exposed to stress, they develop
mechanisms such as reduced substance transport, cortex cell wall thickening and accumulation of some chemi-
cal compounds in cells in order to tolerate the harmful eects of chemicals and stress147. As a result of these
mechanisms, anatomic changes occur in the plant and the harmful eects of chemical agents are reduced. No
data have been reported in the literature regarding the eect of COR on the root anatomy of plants grown under
both salt stress and normal conditions. erefore, the root anatomical ndings from this study are very important
as it is the rst to be reported.
Conclusion
e eects of exogenously applied COR on some physiological, cytogenetic, biochemical and anatomical param-
eters in the roots of Allium cepa L. bulbs germinated in saline (NaCl) conditions have been extensively investi-
gated. ere are no available literature data on the eects of COR application under salt stress conditions on all
parameters of Allium cepa L studied here. erefore, the results from this study are very important as it is the rst
time reported in onion. ese results showed that the application of COR at appropriate doses can signicantly
reduce sodium chloride stress on the germination and growth of onion bulbs by regulating osmoregulation,
mitotic activity and antioxidant capacity. Moreover, these results may help develop new hypotheses and con-
ceptual tools to increase salt tolerance in plants.
Data availability
e datasets used and/or analyzed during the current study are available from the corresponding author on
reasonable request.
Received: 16 December 2022; Accepted: 25 January 2023
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Acknowledgements
e cytogenetic and statistical analysis was performed Plant Physiology and Biochemistry Laboratory and
Cytogenetic Laboratory in Department of Biology at Süleyman DemirelUniversity. However, this research
received no specic grant from any funding agency in thepublic, commercial, or not-for-prot sectors.
Author contributions
D.Ç. designed the study, conducted preliminary experiments, performed seed germination analysis, cell division
analysis, anatomical and biochemical analysis conducted the statistical analyses. e author contributed to the
preparation of the manuscript, read and approved the nal version of the manuscript.
Competing interests
e author declares no competing interests.
Additional information
Correspondence and requests for materials should be addressed to D.Ç.
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