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Physiological functions of proline in
tobacco cultured cells under selenate stress
2020, 9
Mousumi Khatun
Graduate School of
Environmental and Life Science
(Doctor’s Course)
OKAYAMA UNIVERSITY, JAPAN
Ph
ysio
log
ical fu
nctio
ns o
f pro
line in
tob
acco
cu
lture
d c
ells
un
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te s
tress 2
02
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Mo
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i Kh
atu
n
Physiological functions of proline in
tobacco cultured cells under selenate stress
2020, 9
Mousumi Khatun
Graduate School of
Environmental and Life Science
(Doctor’s Course)
OKAYAMA UNIVERSITY, JAPAN
Physiological functions of proline in
tobacco cultured cells under selenate stress
A thesis Presented to Graduate School of Environmental and Life Science
Okayama University
In partial fulfillment of the requirement for the degree of
Doctor of Philosophy
Submitted by
Mousumi Khatun
Department of Biofunctional Chemistry
Graduate School of Environmental and Life Science
Okayama University, Japan
2020, 9
i
CONTENTS
Abbreviations used iii
List of figures iv
Chapter 1 General Introduction
1.1 Sources of selenium
1.2 Selenium uptake and metabolism in plants
1.3 Se toxicity in higher plants
1.4 Oxidative damage and ROS production by Selenium
1.5 Antioxidant defense mechanisms against oxidative damage
1.5.1 Ascorbate (AsA)
1.5.2 Glutathione (GSH)
1.5.3 Superoxide dismutase (SOD)
1.5.4 Catalase (CAT)
1.5.5 Ascorbate peroxidase (APX)
1.5.6 Peroxidase (POX)
1.5.7 Compatible solute
1.5.8 Proline
1.5.9 Mechanisms of Se tolerance
1
1
2
3
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4
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Chapter 2 Effects of exogenous application of proline in tobacco BY-2 cells under selenate stress
2.1 Abstract
2.2 Introduction
2.3 Materials and Methods
2.3.1 Tobacco BY-2 cell cultures
2.3.2 BY-2 cell growth assay
2.3.3 Assessment of cell death of BY-2 cells
2.3.4 Measurement of intracellular ROS
2.3.5 Determination of lipid peroxidation
2.3.6 Determination of antioxidant enzyme activities
2.3.6.1 APX activity
2.3.6.2 Catalase activity
10
11
13
13
14
14
15
15
15
ii
2.3.6.3 SOD activity
2.3.6.4 POX activity
2.3.7 Estimation of Se contents
2.3.8 Measurement of protein content
2.3.9 Statistical analysis
2.4 Results
2.4.1 Effects of exogenous proline on BY-2 cell growth
under selenate stress
2.4.2 Effects of exogenous proline on selenate-induced
cell death
2.4.3 Effects of exogenous proline on intracellular ROS
accumulation of BY-2 cells under selenate stress
2.4.4 Effects of exogenously applied proline on lipid
peroxidation of BY-2 cells under selenate stress
2. 2.4.5 Antioxidant enzyme activities of BY-2 cells under
selenate stress
2.4.6 Effects of exogenous application of proline on the
Se accumulation under selenate stress
2 2.4.7 Effects of ROS scavengers in BY-2 cells under
selenate stress
2.5 Discussion
2.6 Conclusion
Chapter 3 General summary
ACKNOWLEDGEMENTS
REFERENCES
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iii
Abbreviations used
APX, ascorbate peroxidase
AsA, ascorbic acid
CAT, catalase
DW, dry weight
FW, fresh weight
GSA, glutamate-5-semialdehyde
GSH, glutathione
H2DCF-DA, dichlorodihyrofluorescein diacetate
P5C, pyrroline-5-carboxylate
P5CDH, pyrroline-5-carboxylate dehydrogenase
POX, peroxidase
ROS, reactive oxygen species
SOD, superoxide dismutase
iv
List of figures
Fig. 2.1 Effects of exogenous proline (Pro) on BY-2 cell growth under
selenate stress
19
Fig. 2.2 Effects of exogenous application of proline on selenate-induced
BY-2 cell death
20
Fig. 2.3 Effects of exogenous proline (Pro) on intracellular ROS
accumulation and lipid peroxidation on BY-2 cells under selenate
stress
22
Fig. 2.4 Activities of antioxidant enzymes (A) SOD, (B) CAT, (C) APX, and
(D) POX in 4-day-old Se-unadapted tobacco BY-2 suspension
cells induced by proline and selenate
24
Fig. 2.5 Effects of exogenous application of proline on Se accumulation 25
Fig. 2.6 Effects of ascorbic acid (AsA) and glutathione (GSH) on 4-day-old
BY-2 cell growth under selenate stress
27
Fig. 2.7 Effects of ascorbic acid (AsA) and glutathione (GSH) on
intracellular ROS accumulation in BY-2 cells under selenate stress
28
1
CHAPTER 1
General Introduction
1.1 Sources of selenium
Selenium (Se) can be found in a wide variety of rocks, minerals, lunar and
volcanic materials, fossil fuels, soils, plant materials, and waters. However,
sedimentary rocks are the main sources Se in the nature (White et al. 2004).
Selenium is widely used in a number of industries, most commonly selenium
dioxide is employed as a catalyst in metallurgy and organic synthesis and as an
antioxidant in inks, mineral, vegetable, and lubricating oils. Selenium compounds
are released to the environment during the combustion of coal and petroleum
fuels, during the extraction and processing of copper, lead, zinc, uranium, and
phosphate, and during the manufacture of selenium based products. Selenium is
also released inadvertently into the environment from the agricultural use of
phosphate fertilizers, from the application of sewage sludge and manure to land,
and from the use of selenium-containing pesticides and fungicides.
1.2 Selenium uptake and metabolism in plants
The uptake and translocation of Se depends on several factors such as
plant species, concentration and form of Se, physiological conditions, other
substances, and translocation mechanisms of plants (Zhao et al. 2005, Li et al.
2008, Renkema et al. 2012). Usually, plants show a greater affinity for SeO42−
and selenite SeO32−. On the other hand, some organic forms of Se, e.g.
selenocysteine (Se-Cys) and selenomethionine (Se-Met), have higher
2
phytoavailability and are used as active components. In general, the order of
translocation of Se species have been reported as SeO42−> Se-Met > SeO3 2−/Se-
Cys in wheat and canola (Kikkert and Berkelaar, 2013). It is well-known that
selenium uptake in plants is mediated by transporters present in root cell
membrane. Selenite is found to be transported by phosphate transport
mechanism (Li et al. 2008) whereas, selenate through sulfate transporters and
channels (Feist and Parker, 2001, Zhang et al. 2003).
1.3 Se toxicity in higher plants
Selenium toxicity occurs in plants in increased concentrations of selenium.
High concentrations of Se in plant root can cause them to exhibit symptoms of
injury, stunting of growth, chlorosis, withering and drying of leaves, decreased
protein synthesis and premature death of the plant. Selenium causes toxicity by
two mechanisms, one of which is malformed selenoproteins and another by
inducing oxidative stress (Gupta et al. 2017). Substitution of SeCys/SeMet in
place of Cys/Met in protein chain causes malformation of selenoproteins, which
adversely affect protein functioning. It is well-reported that cysteine residues play
an important role in protein structure and function, and helps in formation of
disulfide bridges, enzyme catalysis, and metal binding site. Hence, replacement
of cysteine with SeCys is detrimental to protein structure and function as SeCys
is larger, reactive and more easily deprotonated than cysteine (Hondal et al.
2012), as seen in case of methionine sulfoxide reductase function that got
impaired after substitution with SeCys (Chatelain et al. 2013). At high doses, Se
acts as pro-oxidant and generates reactive oxygen species which cause
oxidative stress in plants. Many studies reported that ROS accumulation under
3
Se stress increased lipid-peroxidation, cell mortality in Arabidopsis and Vicia faba
(Lehotai et al. 2012, Mroczek-Zdyrska and Wójcik, 2012).
1.4 Oxidative damage and ROS production by Selenium
Reactive oxygen species (ROS) are produced as a natural byproduct of
the normal metabolism of oxygen and have important role in plant cellular
metabolism. The ROS is well-known second messengers in a variety of cellular
processes including tolerance to environmental stresses (Desikan et al. 2001,
Yan et al. 2007). However, during times of environmental stress, the levels of
ROS can increase dramatically and the generating stage is known as oxidative
stress. Enhanced level of ROS can cause damage to biomolecules such as
carbohydrates proteins, lipid, nucleic acid, and leading to cell death (Sharma et
al. 2012). At high concentration, Se acts as pro-oxidant and generates ROS
which cause oxidative stress in plants. Enhanced accumulation of ROS due to
Se toxicity leads to dysfunction of photosynthesis process, which is recognized
as one of the critical mechanisms of Se phytotoxicity (Gupta and Gupta, 2017).
Under Se stress, wheat seedling showed enhanced ROS and lipid peroxidation
which negatively affect seedling growth and development (Labanowska et al.,
2012). Several studies have reported that increased activity of antioxidant
enzymes indicating ROS accumulation under Se stress (Akbulut and Cakir, 2010;
Schiavon et al., 2012). Altogether, above studies indicate role of ROS in
imparting Se toxicity in plants.
1.5 Antioxidant defense mechanisms against oxidative damage
4
Selenium functions as a pro-oxidant at high doses, that is, selenium
induces ROS production, which triggers oxidative stress in plants. Plants have
enzymatic and non-enzymatic antioxidant defense systems, which play an
important role to detoxify the produced ROS. The non-enzymatic antioxidant
system includes ascorbate (AsA), glutathione (GSH), α-tocopherol, phenolic
compounds, alkaloids and non-protein amino acids. The enzymatic system
includes superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase
(APX), peroxidase (POX), and enzymes involved in the ascorbate-glutathione
(AsA-GSH) cycle. The increase in activity of these enzymes under various
stresses confers tolerance to plants. However, the details of the regulatory
mechanisms of the antioxidant defense system under selenate stress remain
unclear.
1.5.1 Ascorbate (AsA)
Ascorbate (AsA) is a vital antioxidant enzyme in plant tissues which is
synthesized in the cytosol of higher plants primarily from the conversion of D-
glucose to AsA. In plant cells, it is a key non-enzymatic antioxidant that can
directly scavenge ROS such as H2O2, O2•−, OH·, and 1O2 and plays an important
role in ascorbate-glutathione (AsA-GSH) cycle for scavenging H2O2 (Noctor and
Foyer, 1998; Asada, 199). Ascorbate plays important role in growth,
differentiation, and metabolism of plants. Many studies reported that the
exogenous application of AsA influences the activity of many enzymes and
minimizes the damage caused by oxidative stress through synergic function with
other antioxidants (Sharma et al. 2014).
5
1.5.2 Glutathione (GSH)
Glutathione (GSH) is another important non-enzymatic antioxidant in plant
cells that also either directly or indirectly scavenges a range of ROS such as
H2O2, OH·, and 1O2 (Noctor and Foyer, 1998; Asada, 1999). In plant tissues, GSH
is mainly found as reduced form and is localized in all cell compartments like
cytosol, endoplasmic reticulum, vacuole, mitochondria, chloroplasts,
peroxisomes as well as in apoplast (Mittler et al. 1992, Jimenez et al. 1998). The
GSH is a substrate for GPX and GST, which are also involved in the removal of
oxidative stress (Noctor et al. 2002a). GSH also helps the formation of
phytochelatins, which have an affinity to heavy metals and help to enhance the
heavy metal tolerance (Sharma and Dietz 2006 ) .
1.5.3 Superoxide dismutase (SOD)
The superoxide dismutase is a well-known metalloenzyme which provides
the first line of defense against the toxic effects of ROS within a cell (Gill et al.
2010). The SOD catalyzes the dismutation of O2•− into O2 and H2O2 and it can
play important role in protecting cells upon stresses. The SODs are classified into
three groups based on the metal ion in their active site, namely iron SOD (Fe
SOD), manganese SOD (Mn SOD), and copper-zinc SOD (Cu-Zn SOD). It is
well-reported that the enhanced activity of SODs minimizes oxidative stresses
and has a significant role in the adaptation of a plant to stressed environments
(Mobin and Khan 2007, Singh et al. 2008).
1.5.4 Catalase (CAT)
6
Catalases (CATs) are tetrameric heme-containing enzymes that use H2O2
as a substrate and convert it to H2O and O2, thus preventing cells from oxidative
damage (Gill et al. 2010). The CAT is the primary scavenger of ROS and mainly
presents in the peroxisomes, glyoxysomes, and related organelles where H2O2-
generating enzymes are located (Agarwal et al. 2009). Many studies showed that
CAT activity varies positively or negatively under various stress conditions
(Hasanuzzaman et al. 2011, 2014, Mostofa et al. 2017).
1.5.5 Ascorbate peroxidase (APX)
Ascorbate peroxidase is the first step of AsA-GSH cycle to scavenging of
H2O2 and plays the most essential role in scavenging ROS and protecting cells
in higher plants (Asada 1994). In the AsA-GSH cycle, APX oxidizes AsA to
produce monodehydroascorbate (MDHA) to detoxify H2O2 and
monodehydroascorbate reductase (MDAR) mediates regeneration of AsA from
MDHA using NADH. The APX family consists of five different isoforms including
mitochondrial (mAPX), thylakoid (tAPX) and glyoxisome membrane forms
(gmAPX), as well as chloroplast stromal soluble form (sAPX), cytosolic form
(cAPX) (Noctor and Foyer 1998). It has been demonstrated that APX activity is
enhanced in plants in response to during different abiotic stress conditions.
1.5.6 Peroxidase (POX)
The POXs are a large group of enzymes which catalyzes the oxidation of a
diverse group of organic compounds via the reduction of H2O2. Peroxidases are
found in all plants and animals, and are essential for living systems. The POXs
are classified as: Haem peroxidases (haem peroxidase, halo-peroxidase, etc.)
7
and Non-haem (glutathione peroxidase, manganese peroxidase, NADH
peroxidase, etc.). In plants, POXs are involved in many metabolic processes
such as auxin catabolism, lignin biosynthesis, and defense against pathogen
attack.
1.5.7 Compatible solute
Compatible solutes are a small group of chemically diverse organic
compounds and are also known as osmoprotectant. Compatible solutes are
highly soluble and do not interfere with cellular metabolism, even at high
concentrations (Okuma et al. 2000). It helps to scavenge ROS from the tissues
by maintaining osmotic balance and improving the redox homeostasis
(Krasensky and Jonak 2012). There are several classes of compatible solutes
such as betaines, sugars, sulfonium compounds, polyols, polyamines and amino
acids (Chen and Murata 2002, Cramer et al. 2011, Chen and Jiang 2010, Wani
et al. 2013). These molecules accumulate in cells and balance the osmotic
difference between the cells surroundings and the cytosol.
1.5.8 Proline
Proline is a compatible osmolyte which accumulate in plants under stress
conditions and protects subcellular structures and macromolecules under
stresses. Most plant species can accumulate proline in response to
environmental stresses including salt (Yoshiba et al. 1995), drought (Choudhary
et al. 2005), UV radiation (Saradhi et al. 1995), heavy metal ions (Chen et al.
2001), pathogens (Fabro et al. 2004), and oxidative stress. Proline accumulation
upon stresses depends on both its biosynthesis and inhibition of its degradation.
8
In plants, proline is synthesized from glutamate by the enzymes D1-pyrroline-5-
carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR).
It is well-known that proline plays multiple roles in plant stress tolerance.
Proline also plays role in protection against osmotic stress by stabilizing many
functional units such as complex II electron transport (Hamilton and Heckathorn,
2001) membranes, proteins and enzymes (Okuma et al. 2000, Sharma and
Dubey. 2005, Ashraf and Foolad. 2007). However, in some cases, exogenous
proline showed toxicity to plants and caused programmed cell death (Hellmann
et al. 2000). Although exogenous proline mitigates salt- and cadmium-induced
growth inhibition in Tobacco BY-2 cells (Haque et al. 2007, Islam et al. 2009) but
it enhances the sensitivity of BY-2 cells to arsenate (Nahar et al. 2017).
1.5.9 Mechanisms of Se tolerance
Selenium toxicity is associated with the incorporation of SeCys and SeMet
into proteins in place of Cys and Met, respectively (Gupta et al. 2017). The main
mechanisms appears to be the reduction of the intracellular concentration of
SeCys and SeMet, which otherwise would be incorporated into proteins and
result in detrimental effects on plant protein functions (Gupta et al. 2017). To
prevent SeCys incorporation into proteins, SeCys methyltransferase (SMT) from
the Se hyper-accumulator plants exhibited enhanced Se accumulation in the form
of methyl-SeCys, as well as enhanced Se tolerance. The expression of SMT also
resulted in increased rates of Se volatilization, with more volatile Se produced in
the form of DMD-Se (Pilon-Smits and Quinn, 2010). The SMT gene in E. coli
could confer tolerance to high concentrations of Se and reduce the nonspecific
incorporation of Se into proteins.
9
Excluding Se from proteins may be through the ability of Se accumulators
to discriminate against the incorporation of seleno-amino acids into proteins can
be an alternative pathway. For example, in the Se accumulator, A. bisulcatus,
cysteinyl-tRNA synthetase is unable to attach SeCys to the Cys tRNA, thereby
excluding SeCys from cellular incorporation into proteins. Selenium tolerance
may also be achieved by compartmentation into the vacuole as selenate and, in
the case of Se accumulator, as non-protein seleno-amino acids.
10
CHAPTER 2
Effects of exogenous application of proline in tobacco BY-2 cells
under selenate stress
2.1 Abstract
Selenium (Se) like other heavy metals causes oxidative damage to plants. Proline
is accumulated as a compatible solute in plants under stress conditions and
mitigates stresses. Selenate at 250 µM inhibited the growth of BY-2 cells while
exogenous proline at 10 mM did not mitigate the inhibition by selenate. Selenate
increased the number of Evans blue stained cells regardless of addition of 10 mM
proline. Selenate stress led to an accumulation of Se, ROS, and increased
antioxidant enzyme activities but did not significantly induce lipid peroxidation in the
BY-2 cells. Supplementation of proline increased Se accumulation and APX and
POX activities but did not affect either the ROS accumulation or the lipid
peroxidation in the BY-2 cells under selenate stress. Glutathione (GSH) and
ascorbic acid (AsA) reduced the accumulation of ROS whereas GSH mitigated the
growth inhibition but AsA did not affect the growth inhibition by selenate. These
results indicate that proline increases both antioxidant enzyme activities and Se
accumulation, which overall fails to ameliorate the growth inhibition by selenate and
that the growth inhibition is not accounted for only by ROS accumulation.
11
2.2 Introduction
Selenium (Se) is a micro-nutrient for animals but not for most plants (Gupta
and Gupta et al. 2017, Hamilton 2004) and Se concentrations at higher than 100
µM can be hazardous for all biotas including plants and animals (Guerrero et al.
2014). In plants, lower concentration of Se has been shown to have positive effects
on stress tolerance. For example, exogenous application of selenium alleviates
cadmium-induced oxidative stress in tomato (Solanum lycopersicum L.) plants by
enhancing antioxidant system (Alyemeni et al. 2018) and salinity-induced damage
in rapeseed (Brassica napus) seedlings (Hasanuzzaman et al. 2011).
Selenium toxicity occurs in plants for its chemical similarity with sulfur.
Substitution of cysteine residues and methionine residue by selenocystein residue
and selenomethionine residue adversely affects the activities of proteins, which
result in mimic sulfur starvation such as withering, drying, and premature death of
leaves (Terry et al. 2000, Aggarwal et al. 2010, White 2016). Therefore, it is crucial
to understand the mechanisms of growth inhibition by selenium in plants in order to
develop approaches to alleviate Se stress.
Proline is a bioactive molecule that functions as an osmoprotectant. It is well-
known that proline accumulates in plants during various stresses (Bohnert et al.
1995, Sleator et al. 2002, Verslues et al. 2006, Amini et al. 2015). Heavy metal
toxicity and plant-pathogen interaction also cause proline accumulation in plants
(Fabro et al. 2004, Sharma et al. 2009). Supplementation of proline can improve
tolerance to a variety of environmental stresses in plants (Hayat et al. 2012). In the
case of tobacco Bright Yellow-2 (BY-2) cells, the growth inhibition by salt or
cadmium (Cd) was alleviated by supplementation of proline at 10 mM (Harinasut et
al. 1996, Okuma et al. 2000, Hoque et al. 2007, Islam et al. 2009). It has been stated
12
that proline plays role as an enzyme protectant and reactive oxygen species (ROS)
scavengers (Tsugane et al. 1999, Hong et al. 2000, Okuma et al. 2000, 2002).
However, growth inhibition by arsenate was alleviated by proline only at lower
concentrations in BY-2 cells (Nahar et al. 2017).
A variety of toxic metals can induce ROS accumulation in the plant tissues.
The ROS accumulation is responsible for damaging biological molecules such as
proteins, lipids, DNA, and carbohydrates (Noctor and Foyer 1998, Apel and Hirt
2004, Banu et al. 2009, Guo et al. 2009, Sharma et al. 2012). Selenium functions
as a pro-oxidant at high doses, that is, selenium induces ROS production, which
triggers oxidative stress in plants. In addition, ROS also acts as a key signaling
molecule to mediate many physiological processes in plants such as metabolism,
growth, and development, and response to different stress (Foyer and Noctor 2013,
Turkan 2018).
Plants have enzymatic and non-enzymatic antioxidant defense systems,
which play an important role to detoxify the produced ROS. In plant cells, ascorbic
acid (AsA) is a key non-enzymatic antioxidant that can directly scavenge ROS such
as H2O2, O2•−, OH·, and 1O2 and plays an important role in ascorbate-glutathione
(AsA-GSH) cycle for scavenging H2O2. Glutathione (GSH) is another important non-
enzymatic antioxidant in plant cells that also either directly or indirectly scavenges
a range of ROS such as H2O2, OH·, and 1O2 (Noctor and Foyer 1998, Asada 1999,
Polle 2001).
Tobacco is an annual crop, which is commercially cultivated in many
countries and tobacco BY-2 cells have been widely used in many biochemical
studies because they are highly homogenous and can grow rapidly in suitable
culture medium. Exogenous proline reduces intracellular ROS accumulation and
13
oxidative damage by increasing antioxidant enzyme activities in tobacco BY-2 cells
under Na and Cd stress (Hoque et al. 2007, Islam et al. 2009). Moreover, in some
cases, the exogenously applied proline is toxic to plants (Hellman et al. 2000).
However, it remains to be clarified whether the exogenous application of proline can
mitigate inhibition of growth by selenate in BY-2 cells. Therefore, the effects of
exogenous proline on growth, antioxidant enzyme activities, intracellular ROS
accumulation, and level of oxidative damage in tobacco BY-2 cultured cells under
selenate stress were investigated.
2.3 Materials and Methods
2.3.1 Tobacco BY-2 cell cultures
Tobacco (Nicotiana tabacum L., cv. BY-2) suspension-cultured cells were
employed as a cell line that is unadapted to selenium stress (Murata et al. 1994a,
b). The modified LS medium (Linsmaier and Skoog 1965) was used as a control
medium. Selenium medium was prepared by supplementation with 250 μM
Na2SeO4 in the control medium. In order to examine proline effects, the control
medium and the selenium medium supplemented with 5 mM or 10 mM proline were
used. The cell cultures were performed and managed, as previously described
(Murata et al. 1994a, b).
2.3.2 BY-2 cell growth assay
Two-gram cells were collected from 7-days old suspension culture and
inoculated in fresh medium. The medium was cultured on a rotary shaker (100 rpm,
25°C). The fresh weight (FW) of BY-2 was measured at 0, 4, 6, and 8 days after
14
inoculation as described previously (Murata et al. 1994a, b). The fresh BY-2 cells
were oven-dried at 70oC and the dry weight (DW) was measured after 24 h.
2.3.3 Assessment of cell death of BY-2 cells
Dead cells were estimated by the following procedure of (Nahar et al. 2017).
Cells were stained with Evans Blue solution (0.05%) for 20 min to load the dye. After
that the excess dye was washed out with distilled water. Dye-bounded dead cells
was solubilized in 1 mL of 50% methanol that contained 1% sodium dodecyl sulfate
followed by the incubation for 50 min at 50°C. Then the absorbance was measured
at 600 nm by a spectrophotometer. For calculation, the cells prepared in the same
way were subjected to two cycles of freezing at -20°C and thawing at room
temperature. The cells killed in such a way were used to define 100% cell death.
The value obtained from 2-day-old and 4-day-old cultured cells was defined as 0%
cell death. The death cells were calculated by comparing the absorbance of the
samples with the absorbance of 100% cell death.
2.3.4 Measurement of intracellular ROS
Intracellular ROS accumulation in BY-2 cells was analyzed by using 2´,7´-
dichlorodihydrofluorescein diacetate (H2DCF-DA) as described previously (Biswas
and Mano 2015). Four-day-old BY-2 cells were collected with brief centrifugation by
washing twice with phosphate-buffered saline (PBS). Then the collected cells were
incubated in PBS supplemented with 20 μM H2DCF-DA for 30 min at 30°C. After
that the incubated cells were washed two times with PBS and fluorescence images
were captured using a fluorescence microscope (Biozero, Keyence, Japan) and
analysed using Image-J software (NIH, USA).
15
2.3.5 Determination of lipid peroxidation
Lipid peroxidation was evaluated by measuring malondialdehyde (MDA)
contents, as previously described by (Okuma et al. 2004). One-gram of BY-2 cells
was grounded in mortar and pestle using liquid nitrogen (N2) and 2 mL of 5% (w/v)
trichloroacetic acid was added to homogenize the mixture. Then the homogenized
mixture was centrifuged at 12,000 g for 15 min. After that, the supernatant was
collected and diluted twice with 20% (w/v) trichloroacetic acid containing 0.5% (w/v)
thiobarbituric acid. Then the mixture was heated at 95°C for 25 min. After cooled in
an ice bath, the mixture was centrifuged at 7500 g for 5 min. At 532 nm, the
absorbance of the samples was measured and non-specific turbidity was
compensated by subtracting absorbance at 600 nm. MDA content was calculated
using extinction the coefficient of 155 mM-1cm-1.
2.3.6 Determination of antioxidant enzyme activities
2.3.6.1 APX activity
Ascorbate peroxidase (APX; EC 1.11.1.11) activity was determined by the
following procedure of (Ishikawa et al. 2005). The reaction buffer solution contained
50 mM KH2PO4 buffer (pH 7.0), 0.1 mM EDTA, 0.1 mM H2O2, and 0.5 mM AsA. The
reaction was started by adding the sample solution to the reaction buffer solution.
The oxidation of ascorbate was followed by a decrease in absorbance at 290 nm
for 60 s, where the extinction coefficient was 2.80 mM-1 cm-1.
2.3.6.2 CAT activity
Catalase (CAT; EC: 1.11.1.16) activity was analyzed by the following
procedure of (Islam et al. 2009). A reaction mixture was prepared which contained
16
50 mM Tris-HCl buffer (pH 8.0), 0.25 mM EDTA, 20 mM H2O2, and 25 μL of the
sample solution. The catalase activity was calculated from the decrease in
absorbance at 240 nm for 120 s when the extinction coefficient was 40 M-1 cm-1.
2.3.6.3 SOD activity
Superoxide dismutase (SOD) activity was measured by using an SOD Assay
Kit-WST (Dojindo Molecular Technologies, Inc., Kumamoto, Japan). For each SOD
activity measurement, 20 µL of the sample solution was added to the wells for
sample and blank 2. Twenty microliters of distilled water was added in the wells for
blanks 1 and 3. Two hundred microliters of WST working solution was added to
each well and mixed properly. Then, 20 µL of dilution buffer was added to the wells
for blanks 2 and 3. After that, 20 µL of enzyme working solution was added to the
wells for sample and blank 1 and then mixed thoroughly. The absorbance was
measured at 450 nm using a microplate reader after incubation at 37ºC for 20 min.
The SOD activity was calculated (inhibition rate %) using the following equation:
SOD activity (inhibition rate %)={[(A blank 1-A blank 3)-(A sample-A blank 2)]/(A
blank 1-A blank 3)}×100.
2.3.6.4 POX activity
Peroxidase (POX) activity was analyzed by the following procedure of
(Hoque et al. 2007). A reaction buffer solution was prepared which contained 50
mM KH2PO4 buffer (pH 7.0), 0.1 mM EDTA, 0.1 mM H2O2 and 10 mM guaiacol. The
reaction was started by the addition of the sample solution to the reaction buffer
solution. The activity was calculated from change in absorbance at 470 nm for 30 s
where an extinction coefficient is 26.6 mM M-1 cm-1.
2.3.7 Estimation of Se contents
17
Selenium contents were determined by the following procedure of (Ohno et
al. 2012) with modifications. Each sample was digested with 5 mL of HNO3 at 130-
150ºC and then treated with 1 mL of concentrated HCl. The Se was extracted with
distilled water, and the amounts of extracted Se were quantified by fluorometric
assay using 2,3-diaminonapthalene (DAN). Concisely, 2 mL of masking reagent
(EDTA, NH4OH, NH2OH.HCl, and methyl orange) was added to the cooled
extraction. The volume mixture was adjusted to 9 mL, and then freshly prepared
0.1% DAN (in 0.1 N HCl) was added in the mixture and incubate it 30 min at 50ºC.
After cooled the mixture, 5 mL of cyclohexane was added to the mixture and the
fluorescence of the cyclohexane solution was measured at 520 nm (excitation, 375
nm) using a fluorescence spectrophotometer. Selenium standard solution (Wako,
Japan) was used to measure the Se contents.
2.3.8 Measurement of protein content
Protein contents were determined as described previously (Bradford 1976).
2.3.9 Statistical analysis
The significance of differences between the mean values of all parameters
was assessed by Tukey’s test. Differences at the level of P ≤ 0.05 were
considered as significant.
2.4 Results
2.4.1 Effects of exogenous proline on BY-2 cell growth under selenate stress
The effects of selenate on BY-2 cell growth were examined by measuring the
fresh weight (FW) and dry weight (DW) of selenate-stressed BY-2 cells at 0, 4, 6,
18
and 8 days after inoculation (DAI). The FW of BY-2 cells cultured in the selenium
medium was significantly lower than that in the control medium at 4, 6, and 8 DAI
(Figure 2.1A). The DW of BY-2 cultured cells in the selenium medium was
significantly lower than that in the control medium at 4 and 6 DAI (Figure 2.1B).
These results indicate that selenate at 250 µM significantly inhibits the growth of
BY-2 cells.
In order to investigate whether exogenous proline mitigates the inhibitory
effects of selenate on the growth of BY-2 cells, the FW and DW of BY-2 cells at 0,
4, 6, and 8 DAI were measured. Application of proline at 5 mM and 10 mM did not
significantly ameliorate the selenate-induced growth inhibition in the BY-2 cells
(Figure 2.1), suggest that the mechanism of growth inhibition by selenium is
different from that by other metals such as Na and Cd.
A
19
Fig. 2.1 Effects of exogenous proline (Pro) on BY-2 cell growth under selenate
stress. Effects of 5 mM and 10 mM proline on BY-2 cell growth at 0, 4, 6 and 8 days
after inoculation (DAI) under 250 µM selenate stress based on fresh weight (A) and
(B) based on dry weight. Averages of cell growth from three independent
experiments (n=3) are shown. Error bars represent SE. For the same inoculation
day, bars with the same letters are not significantly different at P <0.05.
2.4.2 Effects of exogenous proline on selenate-induced cell death
We used Evans Blue to evaluate the cell death of BY-2 induced by selenate
in the presence and absence of proline. Under non-stress control condition, the
stained cells accounted for 2.8% and 8.8% in the absence of proline at 2 days and
4 days, respectively, and 4.9% and 9.5% in the presence of 10 mM proline at 2 days
and 4 days, respectively (Figure 2.2). When treated with selenate, the percentage
of stained cells was increased by 10.7% and 28.6% in the absence of proline at 2
days and 4 days, respectively, and by 14.5% and 34.2% in the presence of proline
at 2 days and 4 days, respectively (Figure 2.2). These results indicate that selenate
B
20
drastically induces cell death of BY-2 but that application of proline does not affect
the cell death.
Fig. 2.2 Effects of exogenous application of proline on selenate-induced BY-2 cell
death. Selenate at 250 µM significantly induced cell death and supplementation of
proline at 10 mM did not recover the selenate-induced cell death. Averages of cell
death from three independent experiments (n = 3) are shown. The error bars
represent SE. Values indicated by the same letter do not differ significantly at 5%
level of significance as determined by Tukey’s test.
2.4.3 Effects of exogenous proline on intracellular ROS accumulation of BY-2
cells under selenate stress
Intracellular ROS accumulation in the BY-2 cells was evaluated by using
2´,7´-dichlorodihydrofluorescein diacetate (H2DCF-DA). The application of 250 µM
selenate significantly induced the accumulation of intracellular ROS in the BY-2 cells
21
(Figure 2.3A). Exogenous proline at 10 mM did not affect the ROS accumulation
induced by selenate stress (Figure 2.3A).
2.4.4 Effects of exogenously applied proline on lipid peroxidation of BY-2 cells
under selenate stress
Effects of supplementation of proline on lipid peroxidation of selenate-
stressed BY-2 cells were investigated using thiobarbituric acid. Selenate at 250 µM
did not increase the MDA contents irrespective of the presence or absence of 10
mM proline in the culture medium (Figure 2.3B), suggesting that selenate stress
does not induce lipid peroxidation in the BY-2 cells.
22
Fig. 2.3 Effects of exogenous proline (Pro) on intracellular ROS accumulation and
lipid peroxidation on BY-2 cells under selenate stress. (A) Intracellular ROS level in
4-day-old Se-unadapted tobacco BY-2 suspension cells under selenate stress in
the presence or absence of proline. (B) Malondialdehyde (MDA) content in 4-day-
old Se-unadapted tobacco BY-2 suspension cells under selenate stress in the
presence or absence of proline. The percentage of fluorescence intensity is shown
as a ROS level. Averages from three independent experiments (n=3) per bar are
shown. Error bars represent SE. Bars with the same letters are not significantly
different at P <0.05.
2.4.5 Antioxidant enzyme activities of BY-2 cells under selenate stress
In order to investigate the mitigation effects of proline on the selenate-
stressed plants, activities of antioxidant enzymes SOD, CAT, APX, and POX in the
BY-2 cells under selenate stress were examined. The activities of SOD, CAT, APX,
B
23
and POX were significantly increased in the BY-2 cells in response to selenate
stress (Figure 2.4). Exogenous application of 10 mM proline significantly increased
the APX and POX activities of the selenate-stressed cells (Figure 2.4C, D) but did
not significantly increase the SOD or CAT activity of the stressed cells (Figure 2.4A,
B). These results indicate that selenate stress increases antioxidant enzyme
activities and that exogenous proline does not increase all antioxidant enzyme
activities of BY-2 cells under selenate stress.
A B
24
Fig. 2.4 Activities of antioxidant enzymes (A) SOD, (B) CAT, (C) APX, and (D) POX
in 4-day-old Se-unadapted tobacco BY-2 suspension cells induced by proline and
selenate. The activity of APX, CAT, and POX was expressed in unit’s mg-1 protein
and the SOD activity was expressed in % inhibition. One unit of activity was defined
as the μmol substrate metabolized per min. Averages from three independent
experiments (n=3) per bar are shown. Error bars represent SE. Bars with the same
letters are not significantly different at P <0.05.
2.4.6 Effects of exogenous application of proline on the Se accumulation
under selenate stress
To evaluate the effects of exogenous application of proline on Se
accumulation, we determined Se contents in BY-2 cells under selenate stress in the
presence or absence of proline. Selenate stress significantly increased Se contents
in BY-2 cells (Figure 2.5) and exogenous application of 10 mM proline enhanced
the Se accumulation in the selenate-stressed BY-2 cells (Figure 2.5).
D C
25
Fig. 2.5 Effects of exogenous application of proline on Se accumulation. Selenium
contents in 4-d-old Se-unadapted tobacco BY-2 suspension cells induced by proline
under Se stress. Averages from three independent experiments (n=3) per bar are
shown. “Nd” indicates the not detected. Error bars represent SE. Bars with the same
letters are not significantly different at P <0.05.
2.4.7 Effects of ROS scavengers in BY-2 cells under selenate stress
Ascorbic acid (AsA) and glutathione (GSH) are non-enzymatic antioxidants,
which are capable of scavenging ROS. To clarify whether these ROS scavengers
mitigate the growth inhibition by selenate stress, the effects of GSH and AsA were
examined in the BY-2 cells under selenate stress. Glutathione at 0.5 mM
significantly mitigated the selenate-induced growth inhibition whereas AsA at 20 mM
did not ameliorate the growth inhibition by selenate stress (Figure 2.6 A, B).
26
A
B
27
Fig. 2.6 Effects of ascorbic acid (AsA) and glutathione (GSH) on 4-day-old BY-2 cell
growth under selenate stress. (B) Shows the cell growth on the basis of fresh weight
and (B) shows the cell growth on the basis dry weight in response to 20 mM AsA,
0.5 mM GSH in presence or absence of selenate stress. Averages of cell growth
from three independent experiments (n=3) are shown. Error bar represents SE.
Bars with the same letters are not significantly different at P <0.05.
Furthermore, the effects of AsA and GSH on intracellular ROS accumulation in the
BY-2 cells under selenate stress were examined. Not only AsA at 20 mM but also
GSH at 0.5 mM significantly decreased the intracellular ROS accumulation in the
BY-2 cells under selenate stress, where neither AsA nor GSH changes the ROS
accumulation in the non-stressed BY-2 cells (Figure 2.7). These results suggest
that the growth inhibition by selenate stress is not accounted for by ROS
accumulation.
28
Figure 2.7 Effects of ascorbic acid (AsA) and glutathione (GSH) on intracellular
ROS accumulation in BY-2 cells under selenate stress. Intracellular ROS levels in
4-day-old Se-unadapted tobacco BY-2 suspension cells under selenate stress in
the presence or absence of AsA and GSH. The percentage of fluorescence intensity
is shown as a ROS level. Averages from three independent experiments (n=3) per
bar are shown. Bars with the same letters are not significantly different at P <0.05.
2.5 Discussion
Many studies reported that proline plays a defensive role against various
stresses in plants and cultured cells. Exogenous proline is known to alleviate Na-
and Cd-induced growth inhibition in BY-2 cells (Hoque et al. 2007, Islam et al. 2009,
Okuma et al. 2004) and exogenous proline at lower concentrations suppressed
growth inhibition of BY-2 cells by arsenate stress (Nahar et al. 2017). However, the
defensive role of proline against selenate stress in BY-2 cells remains to be
understood. Selenate toxicity was also evident from the inhibition of growth in
selenate-stressed BY-2 cells (Figure 2.1A, B) but proline did not mitigate the growth
inhibition by selenate (Figure 2.1A, B). Selenate stress may affect proline
metabolism. Proline content has been known to be involved in salt, drought, and
heavy-metal tolerance mechanisms in plants (Chen et al. 2001, Choudhary et al.
2005, Iqbal et al. 2015). Under stress conditions, the level of proline is maintained
by biosynthetic enzyme pyrroline-5-carboxylate synthetase (P5CS) as well as
degrading enzyme proline dehydrogenase (ProDH). Proline is catabolized to
cytotoxic glutamate-5-semialdehyde (GSA)/pyrroline-5-carboxylate (P5C) by
proline dehydrogenase and then GSA/P5C is converted to glutamate by pyrroline-
5-carboxylate dehydrogenase (P5CDH) (Deuschle et al. 2001, Kim et al. 2013,
29
Amini et al. 2015). Therefore, the reduction of cytotoxic GSA is critical to the stress
mitigation by exogenous proline, that is, inhibition of P5CDH activity by the stress
can impair the mitigation by proline. The present results in this study suggest that
selenate inhibits P5CDH, resulting in accumulation of cytotoxic GSA. However, it is
difficult to quantitate GSA and P5C because they are equilibrated mixture.
Both salt stress and arsenate stress increased the number of Evans Blue-
stained BY-2 cells (Banu et al. 2009, Nahar et al. 2017). However, the salt-induced
cell death was suppressed by supplementation of proline (Banu et al. 2009) but the
arsenate-induced cell death was aggravated by proline (Nahar et al. 2017). Proline
induced cell death by ProDH activation, which leads to GSA accumulation (Amini et
al. 2015). Hence, the difference in response to proline between salt-induced cell
death and arsenate-induced cell death may be due to proline metabolism such as
GSA accumulation. In this study, selenate stress induced cell death of BY-2 but the
cell death was not affected by exogenous proline (Figure 2.2), which may be
accounted for the proline metabolism.
Several studies have reported that selenate stress can increase intracellular
ROS accumulation in plants (Tamaoki et al. 2008, Akbulut et al. 2010, Schiavon et
al. 2012). Similarly, this study showed that intracellular ROS accumulation was
increased by selenate stress (Figure 2.3A). In plants, low concentration of Se
reduces the toxic effects of heavy metals by alleviating oxidative damages while
high dose of Se induces the oxidative stress (Malik et al. 2012, Chen et al. 2014,
Jozwiak et al. 2019). In this study, selenate stress increased antioxidant activities
(Figure 2.4) and drastically induced selenate uptake (Figure 2.5), resulting in
growth inhibition and cell death.
30
Islam et al. (2009) reported that Cd stress increased lipid peroxidation and
supplementation of proline decreased Cd-induced lipid peroxidation in BY-2 cells.
Several studies have reported that salt-induced lipid peroxidation is decreased by
the exogenous application of proline (Okuma et al. 2004, Demiral et al. 2004, Banu
et al. 2009). The present study showed that selenate stress did not affect the MDA
content and that exogenous proline also did not show any effect on MDA content
under selenate stress (Figure 2.3B). These results indicate that the level of
oxidative damage was not high enough to induce lipid peroxidation in selenate-
treated BY-2 cells and that selenate inhibited BY-2 cell growth not via increasing
oxidative damage.
It is well-known that proline plays an important role in protecting different
enzymes against stresses such as salinity stress (Tsugane et al. 1999, Hong et al.
2000, Okuma et al. 2000, 2002). Some previous studies reported that the
antioxidant defense system under salt and Cd stress was increased by exogenous
proline application in BY-2 cells (Okuma et al. 2004, Hoque et al. 2007, Banu et al.
2009). Hossain et al. (2010) reported that the ascorbate peroxidase (APX) activity
was increased in response to Cd stress on mung bean and supplementation of
proline also increased APX activity under Cd stress. It has been reported that the
APX, CAT, and POX activities are increased in response to salt stress and
supplementation of proline also increases APX, CAT, and POX activities under salt
stress on rice cultivars and suggest that proline detoxifies H2O2 by enhancing APX,
CAT, and POX activities under salt stress (Hasanuzzaman et al. 2014, Bhusan et
al. 2016). In this study, selenate stress increased activities of SOD, CAT, APX, and
POX (Figure 2.4). Exogenous application of proline increased the APX and POX
activities (Figure 2.4C, D) but did not affect either ROS accumulation (Figure 2.3A)
31
or the lipid peroxidation (Figure 2.3B) in the BY-2 cells under selenate stress. In
this study, proline enhanced APX and POX activities (Figure 2.4C, D) and also
accelerated selenate accumulation (Figure 2.5). Taken together with the result that
proline did not mitigate selenate stress (Figure 2.1 and 2.2), these results suggest
that the increment of APX and POX activities by exogenous proline is not sufficient
to suppress selenate stress due to selenate accumulation.
Both AsA and GSH are known as a vital component for the detoxification of
ROS in plants (Smirnoff et al. 2000, Foyer and Noctor 2011, Qian et al. 2014).
Exogenous application of AsA improves phytotoxic effects of selenium on rice by
reducing oxidative damage (Sharma et al. 2014). Jung et al. (2019) reported that
exogenous application of GSH alleviates heavy metal-induced oxidative damage
and enhances heavy metal stress resistance in rice plants. In this study, the
exogenous application of AsA and GSH significantly decreased the ROS
accumulation in the selenate-stressed cells (Figure 2.7). However, GSH application
significantly mitigated the growth inhibition by selenate (Figure 2.6) but AsA
application did not significantly alleviate the growth inhibition (Figure 2.6). It has
been reported that interference of GSH synthesis in plants by selenate or other Se
compounds may diminish plant defense against hydroxyl radicals and oxidative
stress (Terry et al. 2000). Hence, in this study, the exogenous application of GSH
might increase the GSH level in BY-2 cells and the increased GSH might suppress
the selenate toxicity. These results suggest that the application of proline does not
always mitigate growth inhibition by heavy metals and suggest that the growth
inhibition by selenate is not accounted for by ROS accumulation.
32
2.6 Conclusion
The presented results indicate that selenate induces cell death and inhibits
growth in BY-2 cells while selenate elevates antioxidant enzyme activities and that
proline enhances antioxidant enzymes such as APX and POX and also selenate
accumulation. It is concluded that proline improves antioxidant activities and
enhances selenate uptake and that the balance may account for growth inhibition
and cell death.
33
CHAPTER 3
General Summary
The naturally occurring metalloid selenium (Se) has emerged as a potential health
hazard for plants, animals, and humans, due to its extensive accumulation in the
soils over the past decades. It is a micro-nutrient for animals but not for most plants
and Se concentrations at higher than 100 µM can be hazardous for all biotas
including plants and animals. Selenium plays dual role in plants, for instance, it
reduces negative consequences of abiotic stresses and it can improve plant growth
and development when applied at relatively low concentrations but Se at excessive
concentrations leads to toxicity in plants, resulting in chlorosis and necrosis as well
as restricted growth and reduced protein biosynthesis. Proline is accumulated as a
compatible solute in plants under various stress conditions. Exogenous proline
scavenges free radicles, improves plant metabolism and stimulates plant growth
under stress conditions. It has been reported that exogenous application of proline
can improve tolerance to a variety of environmental stresses in plants. It was
reported that the growth inhibition by salt or cadmium (Cd) was alleviated by
supplementation of proline in the BY-2 cells. However, whether proline mitigates
selenate stress in BY-2 cells are to be investigated. In this study, I investigated the
effects of exogenous proline on tobacco BY-2 cells cultured under selenate stress
and I found that Selenate at 250 µM significantly inhibited the growth of BY-2 cells
while exogenous proline at 10 mM did not mitigate the inhibition by selenate. I also
found that selenate increased the number of Evans blue stained cells regardless of
addition of 10 mM proline. However, it has been reported that salt-induced cell death
was suppressed by supplementation of proline and arsenate-induced cell death was
34
aggravated by proline. Hence, the difference in response to proline between salt-
induced cell death and arsenate-induced cell death may be due to proline
metabolism such as GSA accumulation. In this study, the cell death by selenate was
not affected by exogenous proline which may be accounted for the proline
metabolism. To insight into the issue that proline did not mitigate selenate stress, I
further investigated the effects of proline on Se and ROS accumulation, antioxidant
enzyme activities. I found that selenate stress led to an accumulation of Se, ROS,
and increased antioxidant enzyme activities but did not significantly induce lipid
peroxidation in the BY-2 cells. Supplementation of proline increased Se
accumulation and APX and POX activities but did not affect either the ROS
accumulation or the lipid peroxidation in the BY-2 cells under selenate stress. Taken
together with the result that proline did not mitigate selenate stress, these results
suggest that the increment of APX and POX activities by exogenous proline is not
sufficient to suppress selenate stress due to selenate accumulation.
Further, I investigated whether the growth inhibition by selenate is accounted for
ROS accumulation or not. I investigated the effects of ROS scavengers, GSH and
AsA, I found that GSH reduced the ROS accumulation and significantly mitigated
the growth inhibition whereas AsA significantly decreased the ROS accumulation in
the selenate-stressed cells but did not affect the growth inhibition by selenate.
These results suggest that the application of proline does not always mitigate growth
inhibition by heavy metals and suggest that the growth inhibition by selenate is not
accounted for by ROS accumulation.
35
ACKNOWLEDGEMENTS
Thanks to the almighty Allah who keep me in good health to carry out my research
and writing dissertation to complete my doctoral course.
I would like to express my deepest gratitude and sincere appreciation to my
research supervisor Dr. Yoshiyuki Murata, Professor, Faculty of Agriculture,
Okayama University, Japan for your valuable suggestions and scholastic
guidance throughout the course of the study and writing of the thesis.
I also thankful to Dr. Yoshimasa Nakamura for your valuable discussion and
suggestions during the course of this study.
It’s my pleasure to acknowledge Dr. Shintaro Munemasa for his help and valuable
advice and intellectual suggestions during the research period that help me to set
the experiment properly
I also thankful to Toshiyuki Nakamura for your suggestions regarding scientific
meetings and use of laboratory equipment.
I would to extend my gratefulness to Professor Yoshinobu Kimura, Faculty of
Agriculture for his valuable comments.
I would like to express my heartiest appreciation to Mohammad Saidur Rhaman
for being the best companion, continuous support and suggestion for starting the
experiment properly. I am also grateful to my beloved son Safwan Shahir for his
special sacrifice.
I also thank all members of the laboratory of Chemistry of Bio-signaling, and
laboratory of Food Biochemistry for their support and making my student life
enjoyable.
Finally, I dedicate the thesis to my beloved parents Md. Motier Rahman and Mst.
Shamsunnahar.
36
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