Role of Proline in Tobacco Cultured Cells Under
Arsenate Stress

2017, 9Mst. Nur-E-Nazmun Nahar

Graduate School ofEnvironmental and Life Science(Doctor’s Course)
OKAYAMA UNIVERSITY, JAPAN1

Role of Proline in Tobacco Cultured Cells Under
Arsenate Stress

A thesisPresented to Graduate School of Environmental and Life Science
Okayama University

In partial fulfillment of the requirement for the degree ofDoctor of Philosophy

Submitted by
Mst. Nur-E-Nazmun Nahar

Department of Biofunctional Chemistry
Graduate School of Environmental and Life Science
Okayama University, Japan
2017, 92

CONTENTS
List of figures8

Abbreviations used

10

Chapter 1

General Introduction

11

1.1

Toxicity of heavy metals

11

1.2

Sources of arsenic

11

1.3

Arsenic Transport and Hyperaccumulation in Plants

12

1.4

Arsenic toxicity

12

1.5

Cell death

13

1.6

DNA fragmentation

13

1.7

Reactive oxygen species (ROS)

14

1.8

Oxidative damage and antioxidant defense mechanism

14

1.9

Glutathione

15

1.10

Arsenate reductase

15

1.11

Superoxide dismutase

16

1.12

Compatible solutes

16

1.12.1 Proline

17

1.12.2 Arginine

18

1.12.3 Alanine

18

1.12.4 Betaine

18

1.13

Lipid peroxidation

19

1.14

Arsenic tolerance mechanisms

19

1.15

Purpose of the study

20

Chapter 2

Regulation of arsenate-induced changes by exogenous proline intobacco BY-2 cells

3

21

2.1

Abstract

21

2.2

Introduction

22

2.3

Materials and Methods

23

2.3.1 Culture of tobacco BY-2 cells

23

2.3.2 Measurement of BY-2 cell growth24

2.3.3 Estimation of cell death of BY-2 cells

24

2.3.4 Counting of BY-2 total cell number

25

2.3.5 Statistical analysis

25

Results

25

2.4.1 Effects of exogenous proline on BY-2 cell growth in the

25

2. 4

absence of arsenate
2.4.2 Effects of exogenous arsenate on BY-2 cell growth27

2.4.3 Effects of exogenous proline on the inhibition of BY-2 cell28

growth by arsenate
2.4.4 Effects of exogenous proline on arsenate-induced cell death31

2.4.5 Effects of exogenous proline on the reduction of BY-2 cell

33

number by arsenate
2.5
Chapter 3

Discussion

34

Mitigation of arsenate stress by exogenous proline in cultured

37

tobacco cells under arsenate stress
3.1

Abstract

37

3.2

Introduction

38

3.3

Materials and Methods

40

3.3.1 Culture of tobacco BY-2 cells

40

3.3.2 Estimation of proline content

40

4

3.4

3.3.3 Isolation of genomic DNA and gel electrophoresis

41

3.3.4 Detection of ROS

41

3.3.5 Measurement of GSH content

423.3.6 Extractions and measurements of arsenate reductase

42

3.3.7 Extractions and measurements of superoxide dismutase

43

3.3.8 Measurement of protein

43

3.3.9 Statistical analysis

43

Results

44

3.4.1 Proline content in response to application of exogenous

44

proline
3.4.2 DNA fragmentation

45

3.4.3 Intracellular ROS level

46

3.4.4 Effects of exogenous proline on glutathione content at 60

47µM AsO4- stressed BY-2 cells
3.4.5 Effects of exogenous proline on the activity of arsenate49

reductase in AsO4- stressed BY-2 cells

3.5
Chapter 4

3.4.6 Superoxide dismutase activity

50

Discussion

51

Comparison between the effects of proline and the effects of54

other osmolytes in BY-2 cells under arsenate stress
4.1

Abstract

54

4.2

Introduction

55

4.3

Materials and Methods

57

4.3.1 Culture of tobacco BY-2 cells

57

4.3.2 Measurement of BY-2 cell growth

58

5

4.4

4.3.3 Estimation of endogenous proline

58

4.3.4 Measurement of glutathione content

58

4.3.5 Extractions and measurements of arsenate reductase

58

4.3.6 Extractions and measurements of superoxide dismutase

59

4.3.7 Determination of lipid peroxidation

59

4.3.8 Measurement of protein

59

4.3.9 Statistical analysis

59

Results

60

4.4.1 Proline content in response to application of exogenous

60

proline
4.4.2 Effects of exogenous proline on glutathione content at 60

61µM AsO4- stressed BY-2 cells
4.4.3 Effects of exogenous proline on glutathione content at 40

62µM AsO4- stressed BY-2 cells
4.4.4 Effects of exogenous proline on the activity of arsenate64

reductase in AsO4- stressed BY-2 cells
4.4.5 Superoxide dismutase activity

65

4.4.6 Effects of exogenous arginine on BY-2 cell growth in the65

absence of arsenate
4.4.7 Effects of exogenous arginine on the inhibition of BY-2 cell67

growth by arsenate
4.4.8 Effects of exogenous alanine on BY-2 cell growth in the68

absence of arsenate
4.4.9 Effects of exogenous alanine on AsO4- induced BY-2 cell
growth670

4.4.10 Effects of exogenous glutamate on BY-2 cell growth71

4.4.11 Effects of exogenous glutamate on the inhibition of BY-2

72

cell growth by arsenate
4.4.12 Effects of exogenous betaine on BY-2 cell growth in the74

absence of arsenate
4.4.13 Effects of exogenous betaine on the inhibition of BY-2 cell75

growth by arsenate
4.4.14 Effects of exogenous proline on lipid peroxidation of76

tobacco BY-2 cells in absence and presence of arsenate
4.5

Discussion

78

Summary

84

Conclusions

88

Acknowledgements

89

References

90

7

LIST OF FIGURES
Figure 2.1

Effects of exogenous proline on BY-2 cell growth26

Figure 2.2

Effects of exogenous arsenate (As) on BY-2 cell growth27

Figure 2.3

Effects of exogenous pro (Pro) on 60 µM arsenate (As)-induced growth28

inhibition of BY-2 cells
Figure 2.4

Effects of exogenous proline (Pro) on 40 µM arsenate (As)-stressed BY-2

30

cells
Figure 2.5

Effects of exogenous proline (Pro) on arsenate (As)-induced BY-2 cell

32

death
Figure 2.6

Reduction of BY-2 cell number by arsenate (As) in the presence or34absence of proline (Pro)
Figure 3.1

Proline content in response to exogenous proline application

44

Figure 3.2

DNA fragmentation in arsenate (As) stress BY-2 cells in response to45

exogenous proline (Pro) application
Figure 3.3

Intracellular ROS levels in tobacco BY-2 suspension cells under arsenate

46(As) stress in the presence or absence of proline (Pro)
Figure 3.4

Glutathione content at 60 µM AsO4--stressed BY-2 cells in presence of48

lower concentration of exogenous proline (Pro)
Figure 3.5

Arsenate reductase (AR) activity of AsO4- unadapted tobacco BY-2

49

suspension cells induced by proline under AsO4- stress
Figure 3.6

Superoxide dismutase (SOD) activity of arsenate-unadapted tobacco BY-

50

2 suspension cells induced by different concentration of proline (Pro) under
AsO4- stress
Figure 4.1

Proline (Pro) content in response to exogenous proline application
860

Figure 4.2

Glutathione content at 60 µM arsenic (As)-stressed BY-2 cells in presence62

of higher concentration of exogenous proline (Pro)
Figure 4.3

Glutathione content at 40 µM arsenate (As)-stressed BY-2 cells in

63

presence of different concentration of exogenous proline (Pro)
Figure 4.4

Arsenate reductase (AR) activity of arsenate (As) unadapted tobacco BY-

64

2 suspension cells induced by proline (Pro) under AsO4- stress
Figure 4.5

Superoxide dismutase (SOD) activity of arsenate (As)-unadapted tobacco65

BY-2 suspension cells induced by higher concentration of proline (Pro)under AsO4- stress
Figure 4.6

Effects of exogenous arginine (Arg) on BY-2 cell growth66

Figure 4.7

Effects of exogenous arginine (Arg) on 60 µM arsenate (As)-stressed BY-

68

2 cells
Figure4.8

Effects of exogenous alanine (Ala) on BY-2 cell growth69

Figure 4.9

Effects of exogenous alanine (Ala) on 60 µM AsO4--stressed BY-2 cells

70

Figure 4.10

Effects of exogenous glutamate (Glu) on BY-2 cells growth72

Figure 4.11

Effects of exogenous glutamate (Glu) on 60 µM arsenate (As)-stressed BY-

73

2 cells
Figure 4.12

Effects of exogenous betaine on BY-2 cell growth74

Figure 4.13

Effects of exogenous betaine on 60 µM AsO4--stressed BY-2 cells

75

Figure 4.14

Effects of exogenous proline (Pro) on lipid peroxidation at 60 µM AsO4--

77

stressed BY-2 cells
Figure 4.15

A hypothetical flow-diagram showing the inhibition of proline catabolic
process by arsenite

981

ABBREVIATIONS USED

LS, Linsmaier and Skoog
ROS, reactive oxygen species
GSH, reduced glutathione
GSSG, oxidized glutathione
AR, arsenate reductase
SOD, superoxide dismutase
P5C, pyrroline-5-carboxylate
GSA, glutamate-5-semialdehyde
P5CDH, pyrroline-5-carboxylate dehydrogenase
PCs, phytochelatins

ATP, Adenosine tri-phosphate
O2-, superoxide anion
FW, Fresh weight
DW, Dry weight10

CHAPTER 1
General Introduction

1.1 Toxicity of heavy metals
Heavy metals are metallic elements which are toxic and have a high density (˂ 5.0 g/cm3),
specific gravity or atomic weight. Arsenic, chromium, cadmium, mercury, and lead have thegreatest potential to cause harm because of their extensive use, and widespread distribution
in the environment (Baird and Cann, 2012). The toxicity of heavy metals observed in plants
when they are present in excessive amounts due to a range of interactions at the cellularlevel. Enzymes are one of the main targets of heavy metal in plants, and prolonged exposure
to heavy metals results in remarkable decreases in enzyme activity (Tyler et al., 1989).
Heavy metals can pollute air, water, and soil quality, and cause toxicity to plants and animals(Stankovic and Stankocic, 2013). To maintain the level of essential metals within
physiological ranges, plants have evolved a variety of defense mechanisms that control the
uptake, accumulation and detoxification of metals.

1.2 Sources of arsenic
The natural source of arsenic is the earth crust. Arsenic can be introduced into theenvironment from various sources. The sources of arsenic include both natural andanthropogenic (e.g. use of insecticides, herbicides, and phosphate fertilizers) (Cozzolino etal., 2010; Verbruggen et al., 2009) processes. The involvement of arsenic with agricultural
practices began after the green revolution when the use of pesticides and fertilizers
increased rapidly. Arsenic also finds its way into the food chain (Meharg, 2004). One of themost widespread problems of naturally occurring arsenic is due to leaching into drinking
water aquifers, which have been reported in many countries including India and Bangladesh
11

(Kim et al., 2009). In Bangladesh, arseno pyrite has been identified as the prime source ofarsenic pollution (Fazal et al., 2001b).

1.3 Arsenic Transport and Hyperaccumulation in Plants
The processes of heavy metal transport have been recognized as a central mechanism ofmetal detoxification and tolerance. Transporters play a critical role in arsenic metabolism inplants. Arsenate is the dominant species in aerobic soils and readily enters plant roots via
phosphate transporters. A number of the aquaporin nodulin26-like intrinsic proteins are able
to transport arsenite, the predominant form of arsenic in reducing environments. Arsenic isprimarily taken up by plants via root and leaves. In most plants, arsenic is accumulated
through roots because its low mobility restricts its root-to-root translocation, except in arsenic
hyperaccumulators (Raab et al., 2005). A “hyperaccumulators” plant is defining as the plantthat could tolerate and accumulate ˃1 mg metal g-1 dry mass (Brooks et al., 1977). Threemain forms of arsenic in soil are available to plants, namely arsenate (AsO 4-), arsenite(AsO3-) and methylated arsenic. To understand how plant take up and metabolize arsenic is
important for the mitigation. Plant roots selectively take up arsenic species via distinct
pathways and transporters. Hyperaccumulators such as Pteris vittata L, Agrostis tenerrima
Trin have an extraordinary ability to absorb heavy metals from the soil under varying
concentration of heavy metals (Ma et al., 2001).

1.4 Arsenic toxicity
Arsenic is a highly toxic metalloid. It is naturally taken up by plants from the water and soil.
Arsenic toxicity to living cells causes at very low concentration and its toxicity symptoms inplants range from growth inhibition to ultimate cell death (Barrachina et al., 1995). Indeed,arsenic toxicity in humans has recently become evident on a very large scale in Bangladesh(Dhar et al., 1997). The acceptable level of arsenic safe drinking water is 0.01 mg/L (WHO,122003). Plants can respond to arsenic toxicity by a variety of mechanisms such as
hyperaccumulation, antioxidant defense system, and phytochelation (Garg and Singla, 2011).
Arsenic interacts with sulfhydryl (-SH) groups of enzymes and proteins, leading to inhibitionof several important cellular functions (Meharg and Hartley Whitaker, 2002). Plant growth
can severely have inhibited through exposure of arsenic by slowing or arresting expansion
and biomass accumulation. The toxicity of arsenic cause degradation of plant reproductive
capacity through losses in fertility, yield, and fruit production. Arsenic interferes with thecritical metabolic processes of plants that can lead to the death of plant (Garg and Singla,2011).

1.5 Cell death
Cell death is essential for growth and development of eukaryotes, by maintaining tissue andorgan homeostasis in concert with proliferation, growth, and differentiation (Breusegem andDat, 2006). Treatment of tobacco cells in suspension cultures with arsenic caused cell death.
There are two main categories of cell death such as apoptosis and necrosis. The difference
between these two forms was based on the presence or absence of specific biochemical
and molecular hallmarks, such as DNA laddering, cytochrome c, caspase involvement, ATPdepletion, cytoplasmic swelling, and loss of membrane integrity (Pennell and Lamb, 1997).

1.6 DNA fragmentation
DNA fragmentation is an important hallmark of cell death. Arsenic caused DNA damage by
attacking on its bases and sugar moieties, consequently DNA fragmentation. It has alreadybeen documented that arsenic can replace phosphorous from phosphate group of DNA andinhibits DNA repair system which could lead to genomic template instability (Erturk et al.,2015). NaCl-induced apoptosis-like cell death along with genomic DNA degradation was also
observed in tobacco protoplasts (Lin et al., 2006).
13

1.7 Reactive oxygen species
Reactive oxygen species (ROS) are produced as a normal product of plant cellular
metabolism. Despite their destructive activity, ROS are well-described second messengers
in a variety of cellular processes including tolerance to environmental stresses (Desikan etal., 2001; Yan et al., 2007). The most common ROS include singlet oxygen (1O2), superoxide
radical (O2·-), hydrogen peroxide (H2O2) and hydroxyl radical (·OH). ROS are always formedby the inevitable leakage of electrons onto O2 from the electron transport activities ofchloroplasts, mitochondria, and plasma membranes or as a by-product of various metabolic
pathways localized in different cellular compartments. Production and removal of ROS must
be strictly controlled in order to avoid oxidative stress. When the level of ROS exceeds thedefense mechanisms, a cell is said to be in a state of “oxidative stress ˮ. However, theequilibrium between production and scavenging of ROS is perturbed under a number ofstressful conditions such as salinity, drought, toxicity due to metals, pathogens, and so forth.
Enhanced level of ROS can cause damage to biomolecules such as lipids, proteins and DNA.

1.8 Oxidative damage and antioxidant defense mechanism
In the cell, arsenate can be readily converted to arsenite that is more toxic. Arsenate andarsenite disrupt plant metabolism through distinct mechanisms. Arsenite is a dithiol reactive
compound that binds to and potentially inactivates enzymes containing closely spaced
cysteine residues or dithiol cofactors. Arsenic exposure generally induces the production of
ROS that can lead to the production of antioxidant metabolites and numerous enzymes
involved in antioxidant defense mechanisms. Oxidative carbon metabolism, amino acid andprotein relationships, and nitrogen and sulfur assimilation pathways are also impacted byarsenic exposure (Finnegan and Chen, 2012). Metal toxicity can cause a redox imbalance
and induce the increase of ROS concentration, activating the antioxidant defense
mechanisms of plants (Sharma and Dietz, 2009).
14

1.9 Glutathione
Glutathione (γ-glutamyl-cysteinyl-glycine) represents the major pool of reduced sulfur(Kurnert & Foyer 1993). Under normal conditions, glutathione is predominantly present in its
reduced form (GSH), with only a small proportion present in its fully oxidized state (GSSG).
Glutathione play roles in biosynthetic pathways, detoxification, antioxidant biochemistry andredox homeostasis. Glutathione is the precursor of phytochelatins (PCs) compounds that aresynthesized in response to heavy metals. Arsenate and arsenite are also known to have ahigh affinity for thiols such as glutathione (Jocelyn 1972). Glutathione is a key ROS
scavenger and major cellular redox buffer; it is a crucial part of stress signaling pathways
and has important roles in the regulation of the cell cycle. Glutathione is the main non-protein
thiols of the cell and is a non-enzymatic antioxidant that participates of free radical
scavenging and modulation of the cellular redox status and thiol-disulfide status of proteins(Cnubben et al., 2001; Foyer and Noctor, 2011).

1.10 Arsenate reductase
Arsenate reductase (glutaredoxin) (EC 1.20.4.1) catalyzes the following chemical reaction-

Arsenate reductase (AR) belongs to the family of oxidoreductases. This group of arsenate
reductases belongs to the low-molecular weight protein-tyrosine phosphatase family, as
does a group of glutathione/glutaredoxin type arsenate reductases. Arsenate can be
reduced to arsenite nonenzymatically, but the process is too slow to be physiologicallysignificant (Bhattacharjee et al., 1999). The thiol-linked reductases are required to confer
resistance to arsenate in both prokaryotes (Ji et al., 1994) and eukaryotes (Bobrowicz et al.,15

1997). The reduction of arsenate to arsenite is catalyzed by AR which is also considered asa mechanism involved in detoxification because arsenite can bind with PCs. Arsenate
reduction caused by AR and GSH serving as the electron donor (Ellis et al., 2006).

1.11 Superoxide dismutase (SOD)
It is a group of metalloisozymes that neutralized the highly reactive superoxide radical into
O2 and H2O2 so it can play a very important role in the protection of cells upon stress(Fridovich 1995).

O2˙- + O2˙- + 2H+

H2O2 + O2

Superoxide dismutase catalyzes the dismutation of the O2- into O2 or H2O2. Superoxide
dismutase constitutes the first line of defense against ROS within a cell (Alscher, 2002).
Superoxide dismutase is an antioxidant enzyme associated with metal cofactors. Based on
the metal co-factor used by the enzyme, SODs are classified into three groups: iron SOD(Fe SOD), manganese SOD (Mn SOD), and copper-zinc SOD (Cu-Zn SOD). During arsenic
stress, the up regulation of Cu/Zn SOD has been reported in rice seedlings (Shri et al. 2009).
The proteomic analysis of maize root reveals that Cu/Zn SOD is one of the highly responsive
enzymes to arsenic which involved in cellular homeostasis during redox disturbance(Requejo and Tena, 2005). Mylona et al. (1998) demonstrated that SOD activity increased in
response to low arsenic concentration but a high concentration of arsenic inhibits theaccumulation of SOD mRNA and leads to decline its activity.

1.12 Compatible solutes
Compatible solutes or osmoprotectants are small molecules that act as an osmolyte and helporganisms to survive at extreme osmotic stress. Compatible solutes are low molecularweights, highly soluble organic compounds that are usually non-toxic at high cellular16

concentrations. These solutes provide protection to plants from stress by contributing tocellular osmotic adjustment, ROS detoxification, protection of membrane integrity andenzymes/protein stabilization (Yancey,1994; Ashraf and Foolad, 2007). In plants, theaccumulation of compatible solute is increased. Examples of compatible solutes include
amino acids, glycine betaine etc. These molecules accumulate in cells and balance theosmotic difference between the cells surroundings and the cytosol. Compatible solutes havealso been shown to play a protective role by maintaining enzyme activity.

1.12.1 Proline
Proline is one of the most accumulated compatible solutes found in plants as well as in otherorganisms. Most plant species can accumulate proline in response to environmental stresses
including heavy metal stress. Proline is predominantly synthesized from glutamate. Basedon its known properties proline may be involved in plant heavy metal stress by different
mechanisms, i.e. osmo- and redox-regulation, metal chelation, and scavenging of free
radicals. However, in some cases, exogenous proline showed its toxicity to plants andcaused programmed cell death (Hellmann et al., 2000). Osmoregulation appears to be acommon element of plant reactions to various abiotic stress. Stress conditions up-regulate
the key enzyme ∆1-pyrroline-5-carboxylate synthetase (P5CS), for proline biosynthesis(Hare et al., 1999) that catalyzes the first two steps of proline biosynthesis from glutamate inplants (Delauney and Verma, 1993). Proline plays multiple roles in plant stress tolerance.

Spontaneous
P5C synthetase
Glutamatecyclization
Glutamate

Semialdehyde (GSA)

P5C reductase
Pyrroline-5-carboxylate

Proline(P5C)

Free proline accumulates in plants exposed to Cd (Sharma and Dietz, 2006). Exogenous
17

application of proline mitigates salt stress (Okuma et al., 2000, 2004; Hoque et al., 2007)
and cadmium stress (Islam et al., 2009) in BY-2 cells.

1.12.2 Arginine
L-arginine is an important and unique amino acid in plants. It is one of the most functionallydiverse amino acids in living cells and also serves as a precursor for the synthesis of protein,nitric oxide, creatine, polyamines, agmatine, and urea (Sidney and Morris, 2007). The
arginine metabolism plays important role in perception and adaptation of plant toenvironmental disturbances.

1.12.3 Alanine
The application of amino acids can result in a better plant development since thesemolecules can act as signals of several beneficial physiological processes of plants. Alanine,unlike proline, follows the separate catabolic pathway. Alanine inhibits arginine degradation
process and formation of urea from arginine.

1.12.4 Betaine
Glycine betaine (Betaine), a compatible solute, is a small organic metabolite that are very
soluble in water and non-toxic at high concentrations. Betaine is involved in the protection ofmacro components of plant cells, such as protein complexes and membranes, under stressconditions. Among the compatible solutes, betaine is particularly effective protectant against
abiotic stress (Sakamoto and Murata, 2000;2001). Exogenous application of betaine can
improve the tolerance of numerous plant species to various types of abiotic stresses. In
addition to its functions as an osmoprotectant, betaine contributes to stabilization andprotection of membranes, proteins and enzymes against stresses (Papageorgiou and
Murata, 1995; Ashraf and Foolad, 2007).

18

1.13 Lipid peroxidation
Lipid peroxidation is one of the first consequences of oxidative damage. Malondialdehyde(MDA), a lipid peroxidation product, has widely been used to assess the levels of free radicals
in living cells. The concentration of MDA was used as an indicator of lipid peroxidation. The
restriction on nutrient uptake can be associated to increased lipid peroxidation, which
reduces its functionality.

1.14 Arsenic tolerance mechanisms
Though arsenic is not a redox metal, there is significant evidence that exposure of plants toinorganic arsenic results in the generation of ROS. Plants have evolved mechanisms toprotect cells from the effects of ROS by using enzymatic antioxidants such as SOD, catalase(CAT), and ascorbate peroxidase and non-enzymatic antioxidants such as ascorbate,
glutathione, and α-tocopherol (Sairam et al., 2005; Gunes et al., 2009). Glutathione has adual role in response to metal stress, and it has been suggested that PCs production,
resulting in oxidized glutathione depletion, could itself cause oxidative stress (HartleyWhitaker et al., 2001; Verbruggen et al., 2009). One of the major types of arsenic
tolerance/resistance mechanisms that have been demonstrated in plants is complexation of
metals by PCs. Arsenate can be readily reduced to arsenite via arsenate reductase in aglutathione-dependent reaction (Mukhopadhyay et al., 2000) and arsenite can subsequently
complex with thiols, particularly PCs. One potential strategy for plants to protect from thetoxicity of arsenic to methylate inorganic arsenic to organic arsenic species (Lomax et al.,2012; Zhao et al., 2013).

19

1.15 Purpose of the study
To investigate the role of exogenous proline in plants, the present research was conductedwith the following objectives:(i) To investigate the effects of proline on arsenic-induced cell growth inhibition in
tobacco BY-2 cultured cells.
(ii) To clarify the protective mechanisms of proline in the components of theantioxidant defense systems against arsenic-induced oxidative damage.
(iii)

To compare the role of proline with other osmolytes such as arginine, alanine,glutamate, and betaine on BY-2 cell growth under arsenate stress.

20

CHAPTER 2
Regulation of arsenate-induced changes by exogenous proline intobacco BY-2 cells

2.1 ABSTRACT
Arsenic, a non-essential toxic element, causes toxicity to plants. Plants take up arsenic
mainly as arsenate. 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. However, in some cases, exogenous
proline showed its toxicity to plants and caused programmed cell death. It is reported thatproline ameliorates heavy-metal toxicity in plants. However, the role of proline in arsenatestressed BY-2 cells remains unclear. In this study, I investigated the effects of exogenous
proline on tobacco BY-2 cells cultured under AsO4- stress and found that proline depending
on its concentrations plays dynamic roles in BY-2 cells such as mitigation of arsenate stressin response to lower concentrations of proline (e.g., 0.05 mM), whereas sensitization of BY2 cells to arsenate in response to higher proline treatment (e.g., 10 mM). In this study, theeffects of exogenous proline, exogenous arsenate, and the co-treatment of arsenate andproline on BY-2 cells growth, cell death, and cell number were presented. Here, AsO4significantly inhibited the growth of BY-2 cells at 60 µM but not at either 40 µM or 50 µM.
Proline at 0.05 mM to 10 mM did not affect the cell growth but delayed it at 20 mM. Therefore,
for mitigating the arsenic stress, I examined the effects of a wide range (0.05, 0.1, 0.5, 1,
and 10 mM) of exogenous proline on the inhibition of cell growth by 60 µM arsenate andfound that proline at 0.05 mM and 0.1 mM alleviated the arsenate-induced cell growth
inhibition, but surprisingly accelerated the growth inhibition at 1 mM and 10 mM. Proline at
0.05 mM and 0.1 mM significantly decreased the number of Evans Blue stained cells but 10
21

mM proline boosted the number of stained cells. Proline at 0.05 mM and 0.1 mM increased
the total number of cells, whereas 10 mM proline decreased the total number of cells. These
results indicate that the effects of 0.05 mM and 0.1 mM proline on arsenate-stressed BY-2
cells reversed with the increase of proline concentration to 10 mM, and also suggests thatthe lower concentration of proline mitigates arsenate stress, whereas the higher
concentration of proline sensitizes BY-2 cells to arsenate. To insight into the issue that 10mM proline enhances the sensitivity of BY-2 cells to arsenate, I further investigated theeffects of 10 mM proline on BY-2 cells treated with 40 µM arsenate and found that 40 µM
arsenate did not inhibit cell growth in the absence of proline but inhibits it in the presence ofproline, suggesting that application of 10 mM proline enhances the adverse effects ofarsenate. Together, these results suggest that proline plays two distinct roles in BY-2 cells in
the presence of arsenate.

2.2 INTRODUCTION
Arsenic, a toxic metalloid, is widely distributed in the environment and causes physiologicaland structural disorders in plants (Sharma, 2012). Arsenic accelerates cell death and inhibits
plant growth (Stoeva and Bineva, 2003; Stoeva et al., 2005). Now a day, the reduction ofcrop yield by arsenic stress has been recognized as a threat to the sustainable foodproduction (Brammer and Ravenscroft, 2009; Panaullah et al., 2009). Arsenic occurs
predominantly as inorganic forms such as arsenate (AsO4-) and arsenite (AsO3-). Plants takeup arsenic mainly as arsenate (Tripathi et al., 2007).
The organic compatible solute, proline, has been reported to accumulate in plants
subjected to various abiotic stresses such as salt stress (Sakamoto and Murata, 2000).
Exogenous proline functions as a free radical scavenger and an enzyme protectant (Tsugane
et al., 1999; Hong et al., 2000). Okuma et al. (2004) reported that proline exhibits anantioxidant activity which was proved by the 1,1-diphenyl-2-picrylhydrazyl assay. Proline22improves plant metabolism and stimulates plant growth under stress conditions (Alia et al.,
1991; Fedina et al., 1993). Earlier studies suggest that exogenous application of proline
confers protection against metals (Islam et al., 2009) and that proline play diverse roles inplants and confer protection against a variety of abiotic stresses (Hare et al., 1998). However,in some cases, exogenous proline showed its toxicity to plants (Hellmann et al., 2000) andcaused programmed cell death by producing ∆1-pyrroline-5-carboxylate (P5C), anintermediate in biosynthesis and degradation of proline, is assumed to play a role in cell
death in plants (Deuschle et al., 2001).
Proline has been known to accumulate under metal stress in plants (Sharma and Dietz,
2006; Xu et al., 2009). In this study, I investigated the effects of exogenous proline on tobacco
BY-2 cultured cells under arsenate-stress conditions. I found that arsenate inhibited the BY2 cell growth. Exogenous proline at lower concentration mitigates arsenic induced growth
inhibition by decreasing Evans Blue stained cells and increasing the total number of cells,
whereas, that arsenate accelerated the inhibition of cell growth in the presence of higher
concentration of proline. I also found that arsenate boosted the number of dead cells anddecreased the total number of cells in the presence of higher proline. These results indicatedthat exogenous proline at higher concentration did not mitigate arsenate stress in BY-2 cells
but that proline enhances the sensitivity of BY-2 cells to arsenate.

2.3 MATERIALS AND METHODS

2.3.1 Culture of tobacco BY-2 cells
Suspension-cultured cells of tobacco (Nicotiana tabacum L., cv. BY-2) were used for thearsenic-unadapted cell lines (Murata et al., 1994a, b). The modified LS medium (Linsmaier
and Skoog, 1965) was used as a standard medium in which KH2PO4 and thiamine-HCl wereincreased to 370 and 1 mgL-1, respectively, supplemented with 3% sucrose and 1 µM 2,423

dichlorophenoxyacetic acid (Nagata et al., 1981). The LS medium supplemented with 40 µM,
50 µM, and 60 µM AsO4- were regarded as the standard arsenic medium of 40 µM, 50 µM,
and 60 µM AsO4-, respectively. The 0.05 mM, 0.1 mM, 0.5 mM, 1 mM and 10 mM proline
media were the 60 µM AsO4- media containing 0.05 mM, 0.1 mM, 0.5 mM, 1 mM and 10 mM
proline. The 0.5 mM, 1 mM and 10 mM proline media were the 40 µM AsO4- media containing
0.5 mM, 1 mM and 10 mM proline. The BY-2 cells were cultured and maintained as described
previously (Murata et al., 1994a, b). The cells were sub-cultured weekly and were incubatedon a rotary shaker at 100 rpm at 25°C in the dark.

2.3.2 Measurement of BY-2 cell growth
The growth of BY-2 cells was measured as described previously (Murata et al., 1994a, b).
To measure the BY-2 cell growth, the cells were incubated in the culture media for differentdays such as 2, 4, 6, 8, and 10 days. After incubation, the cells were collected by removing
the aqueous solution in a vacuum using a nylon sieve (pore size 45 µm), and the fresh weight(FW) of the cells was taken. Then the cells were dried in an oven at 70°C and the dry weight(DW) was measured. Both the FW and DW of the cells were taken at different days after
inoculation (DAI).

2.3.3 Estimation of cell death of BY-2 cells
Dead cells were quantified by the method described previously (Yano et al., 1998; Takatsuka
et al., 2004). Cells were stained with Evans Blue solution (0.05%) for 20 min andsubsequently washed with distilled water to remove the excess dye. Dye that had bound todead 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
24killed in such a way were used to define 100% cell death. The value obtained from 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 Counting of BY-2 total cell number
The number of total cells was counted using Haemacytometer (Burker-Turk, 0.0025 mm2,0.004 mm2) under the microscope.

2.3.5 Statistical analysis
Unless stated otherwise, the significance of differences between the mean values of allparameters was assessed by Tukey’s test. Differences at the level of p ≤ 0.05 wereconsidered as significant.

2.4 RESULTS

2.4.1 Effects of exogenous proline on BY-2 cell growth in the absence of arsenate
To investigate the roles of exogenous proline for the mitigation of arsenate stress in BY-2
cells, we examined whether exogenous proline shows any effects on BY-2 cells. We
measured the FW and DW of cells at 0, 2, 4, 6, 8, and 10 DAI in response to exogenous
proline in the absence of arsenate. We found that 0.05 mM, 0.1 mM, 0.5 mM, 1 mM and 10
mM proline did not change BY-2 cell growth compared with control but delayed it at 20 mM
as well as the cell growth curve was dramatically increased at 4 to 6 DAI and then steadily
increased up to 10 DAI (Fig.2.1A and B).

25

A

Fresh weight (g/Flask)

12
10
8
Control
0.05 mM Pro
0.1 mM Pro
0.5 mM Pro
1 mM Pro
10 mM Pro
20 mM Pro

6
4
2
0
0

2
4
6
8
Days after innoculation

10

B

Dry weight (g/Flask)

0.5
0.4
0.3
Control
0.05 mM Pro
0.1 mM Pro
0.5 mM Pro
1 mM Pro
10 mM Pro
20 mM Pro

0.2
0.1
0

1

2
4
6
8
Days after innoculation

10

Figure 2.1 Effects of exogenous proline (Pro) on BY-2 cell growth. A, Shows the cell growth
based on fresh weight and B, shows the cell growth based on dry weight in response to 0.05
mM, 0.1 mM, 0.5 mM, 1 mM, 10 mM and 20 mM Pro at 0, 2, 4, 6, 8, and 10 DAI. Averagesof cell growth from three independent experiments (n = 3) are shown. Error bars represent
SE. There were no significant differences (p<0.05) between control (untreated) cells andtreated cells in fresh weight or dry weight at each time point after inoculation.
26

2.4.2 Effects of exogenous arsenate on BY-2 cell growth
We tested the effects of arsenate on BY-2 cell growth. We measured the FW and DW of BY2 cells at 0, 4, 6, and 8 DAI in response to 40 µM, 50 µM, and 60 µM AsO4-. Compared with
control, arsenate did not affect the growth of BY-2 cells at 40 µM and 50 µM but significantly
inhibited it at 60 µM at all DAI (Fig.2.2A and B).

Fresh weight (g/ Flask)

A
Control
50 µM As

12

40 µM As
60 µM As

a

a a a

10
8

a a

a

a

a

6
4
2

a a a a

b

b

b

0
0

4

6

8

Days after inoculation

Dry weight (g/Flask)

B

Control
50 µM As

0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

40 µM As
60 µM As
a a a

a a a

b
a a a a

0

a a a

b

4
6
Days after inoculationb

8

Figure 2.2 Effects of exogenous arsenate (As) on BY-2 cell growth. A, Shows the cell growth
based on fresh weight and B, shows the cell growth based on dry weight in response to 40µM, 50 µM and 60 µM arsenate at 0, 4, 6 and 8 days after inoculation. Averages of cell27

growth from three independent experiments (n = 3) are shown.

2.4.3 Effects of exogenous proline on the inhibition of BY-2 cell growth by arsenate
To investigate whether exogenous proline recovered the inhibition of cell growth by arsenate,
we examined the effects of exogenous proline on the growth of BY-2 cells cultured at 40 µM
AsO4- and 60 µM AsO4-. The FW and DW of cells at 0, 4, 6, and 8 DAI were measured. At 60µM AsO4- stress condition, 0.05 mM proline significantly recovered the AsO4- induced
inhibition of BY-2 cell growth, whereas 0.1 mM and 0.5 mM proline did not affect the cell
growth but 1 mM and 10 mM proline significantly inhibited it (Fig. 2.3A and B). Moreover,
AsO4- at 60 µM induced more inhibition of cell growth in the presence of 1 mM and 10 mM
exogenous proline than in the absence of exogenous proline. In the presence of 40 µM AsO4-,
neither 0.5 mM proline nor 1 mM proline affected the cell growth but 10 mM proline inhibitedit (Fig. 2.4A and B). These results suggest that exogenous proline at lower concentration
mitigate the arsenate-induced growth inhibition of BY-2 cells but did not at 1 mM and 10 mM
and further enhances the sensitivity of BY-2 cells to arsenate.

Fresh weight (g/Flask)

A

12

Control
As + 0.05 mM Pro
As + 0.5 mMPro
As + 10 mM Pro

60 µM AsAs + 0.1 mM Pro
As + 1 mM Pro
a
a

10
8
a

6

4
2

aa aa a aa

c

bb

cd

e

cbc c

d

e

b
bc bc c cd
e

0

0

4
6
Days after inoculation
28

8

Dry weight (g/Flask)

B

0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

Control
As + 0.05 mM Pro
As + 0.5 mMPro
As + 10 mM Pro

60 µM AsAs + 0.1 mM Pro
As + 1 mM Pro
a

a

a
b
c

c
c cd

aa aa aaa

0dbc bccdd4
6Days after inoculation

b
c

e

bc
cd d

e

8

Figure 2.3 Effects of exogenous pro (Pro) on 60 µM arsenate (As)-induced growth inhibition
of BY-2 cells. Increment of cell growth in the presence of 0.05 mM proline and enhancementof arsenate-induced cell growth reduction (Fresh weight basis, A; dry weight basis, B) in thepresence of both the 1 mM and 10 mM Pro but not in the presence of 0.1 mM and 0.5 mM
Pro at 4, 6 and 8 days after inoculation. Averages of cell growth from three independent
experiments (n = 3) are shown. The error bars represent SE. For the same inoculation day,values indicated by the same letter do not differ significantly at 5% level of significance as
determined by Tukey’s test.

29

A

Control
As + 0.5 mM Pro
As +10 mM Pro

Fresh weight (g/Flask)

12

40 µM AsAs + 1 mM Proaa aa a

10
8

a

a a

a a aa6
42ba a a aa

b

b

0
0

4

6

8

Days after inoculation

Dry weight (g/Flask)

B

0.5

Control
As + 0.5 mM Pro
As +10 mM Pro

0.4

a aa a40 µM AsAs + 1 mM Pro

aa a aa aa a

0.3
0.2
0.1

b

b

b

a a aa a

0
0

4

6

8

Days after inoculation
Fig. 2.4 Effects of exogenous proline (Pro) on 40 µM arsenate-stressed BY-2 cells.
Reduction of BY-2 cell growth by arsenate (FW basis, A; DW basis, B) in the presence of 10
mM Pro but not in presence of 0.5 mM or 1 mM Pro at 4, 6 and 8 DAI. Averages of cell growthfrom three independent experiments are shown. Error bars represent SE. For the same
inoculation day, values indicated by the same letter do not differ significantly at 5% level ofsignificance as determined by Tukey’s test.

30

2.4.4 Effects of exogenous proline on arsenate-induced cell deathIn this study, we examined the cell death of BY-2 by arsenate in the presence and absenceof proline. Arsenate at 60 µM increased the number of Evans blue stained cells by 25%
compared with control. In the presence of AsO4-, 0.05 mM and 0.1 mM exogenous proline
significantly recovered the AsO4- induced cell death. On the other hand, arsenate boosted
the number of stained cells by 45% in the presence of 10 mM proline but not in the presenceof either 0.5 mM or 1 mM proline (Fig. 2.5A). Arsenate at 40 µM did not show any effect on
the cell death whereas that arsenate in the presence of 10 mM proline significantly increased
the Evans blue positive cells but not in the presence of 0.5 mM and 1 mM proline (Fig. 2.5B).
These results indicate that application of proline recovered the arsenate-induced cell deathat lower concentration but increase the number of dead cell by arsenate at higher
concentration.

C

A

Control

Cell death (%)

60

a

50

b

b

40
c

30
20

b
60 µM

c

d

As + 0.5 mM Pro

As + 0.05 mM Pro

As + 1 mM Pro

As + 0.1 mM Pro

As + 10 mM Pro

100
As (µM)0
Pro (mM) 060
60 60 60 60
0 0.05 0.1 0.5 1

31

60
10

D

B

Cell death (%)

Control

40
35
30
25
20
15
10
5
0

a
40 µM As

b

b

b
b

As + 0.5 mM Pro

As + 1 mM Pro

As + 10 mM Pro

Figure 2.5 Effects of exogenous proline (Pro) on arsenate (As)-induced BY-2 cell death. A,
Induction of cell death by the co-treatment of 60 µM arsenate. Arsenate at 60 µM induced
significant cell death which was recovered by 0.05 mM and 0.1 mM Pro, and enhanced in
the presence of 10 mM Pro. B, Induction of cell death by the co-treatment of 40 µM arsenate
and 10 mM Pro but not by the individual treatment of 40 μM arsenate. Averages of cell deathfrom 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. C and D, Evans Blue staining of arsenate-treated BY-2 cells in
the presence or absence of Pro. At 60 µM arsenate-stress condition, BY-2 cells showed more
Evans Blue positive cells compared with control. Pro at 0.05 mM and 0.1 mM decreased theEvans Blue stained cells at AsO4- stress but the stained cell was enhanced in the presenceof 10 mM Pro (Fig. 2.5C). At 40 µM arsenate-stress condition, BY-2 cells showed Evans
Blue-stained cells in the presence of Pro but not in the absence (Fig. 2.5D).

32

2.4.5 Effects of exogenous proline on the reduction of BY-2 cell number by arsenate
In our study, we monitored the effects of exogenous arsenate at 40 µM and 60 µM on the
total number of BY-2 cells with or without the application of proline. We found that arsenate
at 60 µM decreased the total number of cells by 3.5-fold compared with control. At 60 µM
arsenate, proline at 0.05 mM and 0.1 mM significantly increased the total number of cell andthat arsenate in the presence of 1 mM and 10 mM proline decreased the number of cells by
4.5- and 7.5-fold, respectively (Fig. 2. 6A). We also found that arsenate at 40 µM did notshow any effect on the cell number compared with control whereas that arsenate in thepresence of 10 mM proline significantly decreased the cell number but not in the presenceof 0.5 mM and 1 mM proline (Fig. 2.6B). These results indicate that application of proline atlower concentration recovered the reduction of cell number by arsenate but higher proline
level enhances the arsenate-induced decrease of cells number.

A

Cell number

600

a

500

b

400

b

300
200

c

c

100
0

33

c

d

Cell number

B
450
400
350
300
250
200
150
100
50
0

a

a

a

a

b

Figure 2.6 Reduction of BY-2 cell number by arsenate (As) in the presence or absence ofproline (Pro). A, the reduction of cell number by the co-treatment of 60 µM arsenate andincrement of cell number at 0.05 mM, and the reduction in cell number at 10 mM Pro. B,
Decrease of cell number by the co-treatment of 40 µM arsenate and 10 mM Pro but not by
the arsenate alone. Averages of cell number from three independent experiments (n = 3) are
shown.

2.5 DISCUSSION
Arsenic is one of the most hazardous elements in the environment and becomes a global
agricultural problem. Accumulation of arsenic in plants causes destruction of cellular
membranes interferes with plant metabolic processes and reduces plant productivity(Sharma, 2012; Singh et al., 2006). It was reported that proline ameliorates heavy-metal
toxicity in plants. However, whether proline mitigates AsO4- stress in BY-2 cells are to beinvestigated. In this study, we present the AsO4--induced growth inhibition of BY-2 cells. Here,the AsO4-- induced growth inhibition was recovered by lower concentration of exogenous
34

proline and the increasing rate of growth inhibition by that arsenate in the presence of higher
proline. We also demonstrate that the AsO4--induced increment of cell death and the
reduction of total cell number was recovered by lower proline but did not in the presence of
higher proline.
It is well-known that arsenate inhibits the growth of plants (Shri et al., 2009; Ali et al.,2014). In this study, we found that AsO4- significantly inhibited the growth of BY-2 cells at 60µM but not at either 40 µM or 50 µM (Fig. 2.2A and B). Therefore, for mitigating the arsenic
stress, we examined the effects of exogenous proline on the inhibition of cell growth by 60µM arsenate. Our results indicate that application of proline at lower concentration (0.05 mM)
ameliorated the arsenate-induced growth reduction. On the other hand, proline at higher
concentration (10 mM proline) did not recover arsenate-induced growth reduction but
surprisingly that arsenate enhances the cell growth reduction in the presence of 1mM and
10 mM proline (Fig. 2.3A and B). To insight into this issue, we further investigated the effects
of 0.5 mM to 10 mM proline on BY-2 cells treated with 40 µM arsenate. Arsenate at 40 µM
did not inhibit cell growth in the absence of proline but inhibits it in the presence of proline(Fig. 2.4A and B), suggesting that application of 10 mM proline enhances the adverse effects
of arsenate. In contrast to our proline sensitivity enhancement results, Singh et al. (2015)
reported that exogenous proline application ameliorated toxic effects of arsenate in Solanum
melongena seedlings. This difference may come from the major difference in endogenous
proline contents between BY-2 cells (approximately 3 mM) and eggplant seedlings (around
1 μg/g-FW; almost equivalent to about 10 μM). Choudhury et al. (2010) and Siddiqui et al.
(2015) reported that arsenate stress increased the proline contents. However, it is unclear
which inhibition of plant growth is due to accumulation of arsenate or proline or due toadditive or synergistic effect.
In plants, heavy metals can cause tissue damage, growth inhibition, and even death.
In this study, we found that arsenate induced the number of Evans Blue-stained (dead) cells
35

(Fig. 2.5A) and decreased the total number of BY-2 cells (Fig. 2.6A), which is consistent withthe previous results (Xue and Yi, 1014). Moreover, we found that exogenous proline at lower
concentration recovered the arsenate-induced cell death and increased the total number ofcells at arsenate stressed condition. On the contrary, proline at higher concentration boosted
the Evans Blue stained cells as well as the reduction of cell number by arsenate ispotentiated in response to exogenous proline. These results suggest that arsenate
decreased the number of death cell and increases the total number of cell in the presenceof lower proline level but increased the cell death and enhances the reduction of total cellnumber in the presence of higher proline.
There is no clear consensus about the role of proline in plants as well as themechanism by which proline mitigates heavy metal stresses in plants. Previous research
reported that proline mitigates stresses in plants. For example, application of proline
ameliorates salt stress (Hoque et al., 2007) and cadmium stress in BY-2 cells (Islam et al.,2009). However, the negative role of exogenous proline was also reported in some cases.
For instances, exogenous proline shows toxicity to plants (Hellmann et al., 2000; Deuschle
et al., 2001) and proline at 2 mM inhibits the growth of saltgrass (Distichlis spicata)(Rodriguez and Heyser, 1988). It was also reported that the accumulation of proline in plants
under stress condition is not associated with the mitigation of stress but it is just a symptom(Liu and Zhu, 1997) and did not show any protective value (Moftah and Michel, 1987). In thepresent study, our data showed that proline ameliorates arsenic stress at lower concentrationand higher proline did not mitigate arsenic stress in BY-2 cells but arsenate induced morestressing effects in the presence of higher proline. The previous reports with our findings
suggest that the mitigatory role of proline might depend on some conditions such as type ofstress, and plant species.
Together, we conclude that exogenous proline at lower concentration mitigatesarsenate stress but higher proline enhances the sensitivity of BY-2 cells to arsenate.
36

CHAPTER 3
Mitigation of arsenate stress by exogenous proline in cultured
tobacco cells under arsenate stress

3.1 ABSTRACT
Arsenic, one of the most toxic heavy metals, causes hazard to plant and human health.
Arsenic exposure generally induces the production of ROS causing progressive oxidative
damage and ultimately cell death that can lead to the production of antioxidants. Proline
alleviates the damaging effect of oxidative stress in plants by the up-regulation of theantioxidant defense system. In this study, we investigated the protective effects ofexogenously applied proline on cell growth, ROS production, glutathione content, andactivities of different antioxidant enzymes in cultured tobacco BY-2 cells exposed to arsenate(AsO4-) stress. This study describes the molecular mechanisms of lower proline in BY-2 cells
under arsenate stress. Previous evidences suggest that arsenic exposure induces thegeneration of ROS. Our results indicate that arsenate stress increased the ROS levels
compared with control and in the presence of arsenate, 0.05 mM and 0.1 mM proline
decreased the intracellular ROS level compared with arsenate stress. The mechanisms of
how proline mitigates stress in plants are not fully understood but appear to involve its
chemical properties and effects on redox systems such as the glutathione pool. Glutathione
is the major source of non-protein thiols in plant cells and functions as a key component of
the antioxidant network. The increased level of glutathione pool is generally regarded as aprotective response against oxidative stress. The reduced glutathione (GSH) content isassociated with the protection to oxidative stress in plants. In the presence of AsO4-, 0.05
mM proline did not show any effect on total glutathione (Total GSH) content compared witharsenate stress. Furthermore, 0.05 mM proline decreased the GSH and increased the37oxidized glutathione (GSSG) contents compared with arsenate stress. These results suggestthat during mitigation process proline maintains glutathione homeostasis by decreasing GSH
and increasing GSSG. Arsenate is readily reduced to arsenite through arsenate reductase
using GSH as a reductant. In this study, at AsO4- stress condition, compared with control,
0.05 mM proline did not show any effects on arsenate reductase activity. Moreover,compared with arsenate stress, 0.05 mM proline significantly increased the arsenate
reductase activity. These results suggest that proline mitigates arsenate stress by increasing
the arsenate reductase activity which accelerates the conversion of arsenate to arsenite,leading detoxification and sequestration of arsenite. In this study, we also found that the SOD
activity is increased at 60 µM arsenic stress compared with control. In the presence ofarsenate, proline at 0.05 mM did not show any effect on the SOD activity compared witharsenate stress. The above data indicate that 0.05 mM proline in most of the parameters
positively regulated, and recovered the stressing effects of arsenate. Therefore, themechanism of lower proline-induced stress mitigation is consistent with the previous reports,
which is important to understand the mitigatory roles of proline in BY-2 cells.

3.2 INTRODUCTION
Arsenic is an environmental toxin that is found naturally in the environment. The main two
forms of inorganic arsenic, arsenate and arsenite, are easily taken up by plant cell. In aerobic
environments, arsenic occurs mostly in its oxidized form, arsenate, and has been reported
to be taken up by plants via the phosphate transport system (Asher and Reay, 1979; Lee,
1982; Ullrich-Eberius et al., 1989; Meharg and Macnair, 1991, 1992; Meharg and HartleyWhitaker, 2002; Wang et al., 2002). Once taken up by the plant, arsenate can be readily
converted to arsenite, the more toxic of the two forms. Arsenic, when not detoxified, may
trigger a sequence of reactions leading to growth inhibition, and stimulation of secondary
metabolism, and causes membrane degradation and cell death (Meharg and Hartley38

Whitaker, 2002).
Arsenic is also known to induce oxidative stress directly by generating ROS during
conversion of its valence forms by inactivating antioxidant molecules through binding with
their sulfhydryl (-SH) groups (Sharma, 2012). Arsenic interferes with various metabolic
processes and thereby adversely affects the plant metabolism and leads to reduced plant
productivity.
In response to different stresses, plants accumulate large quantities of different typesof compatible solutes (Serraj and Sinclair, 2002). Proline is a well-known compatible solutethat accumulate under metal stress has been correlated with stress tolerance in plants.
Proline play significant functions under metal stress and it has been reported that proline
accumulation may be a part of stress signal influencing adaptive responses.
Both glutathione (Ogawa, 2005) and proline (Lehmann et al., 2010) perform multiple
functions in plants and originated from a common precursor L-glutamate (Moat et al., 2003).
Glutathione is a key component of the antioxidant network that functions as a major
intracellular antioxidant inside the cell (Cobbett, 2000). It is reported that glutathione canfunction as an intracellular signaling agent and responsive to changes in the extracellularenvironment (Fernandez et al., 1997). Previous reports suggest that arsenite form
complexes with thiol compounds in plants, due to its high affinity with sulfhydryl groups (Raabet al., 2005). The increased level of glutathione synthesis is considered a means of metalbinding capacity as well as a way to increased cellular defense against oxidative stress.
The reduction of arsenate to arsenite is a key step in the arsenic detoxification
pathways. Arsenate is readily reduced to arsenite through arsenate reductase using reduced
glutathione (GSH) as reductant (Duan et al., 2005; Mukhopadhyay et al., 2000). Though the
reduction mechanism is not well understood in plants, the reduction of arsenate to arsenite
occurs in a number of plant species (Pickering et al., 2000; Meharg and Hartley-Whitaker,
2002; Salt et al., 2002; Quaghebeur and Rengel, 2003).
39

Under environmental stresses, plants often produce ROS such as superoxide,hydrogen peroxide and hydroxyl radicals, causing damage to DNA, proteins and lipids. It isevident that arsenic exposure leads to the generation of ROS through the conversion ofarsenate to arsenite, a process that rapidly occurs in plants (Flora, 1999; Mascher et al.,2002). To minimize the harmful effects of ROS, plants have evolved an effective scavenging
system composed of antioxidant molecules and antioxidant enzymes (Meharg, 1994).
However, in some instances this defensive mechanism becomes inadequate to withstandagainst stressed conditions (Chandrakar et al., 2016).
Report suggest that during stress, proline decreased the denaturation of enzyme,
hence can be applied against arsenic stress which readily binds with thiol groups andinactivates enzymes (Chandrakar et al., 2016). Therefore, research on the protective role ofproline is a matter of scientific interest for its application against arsenic-stress.

3.3 MATERIALS AND METHODS

3.3.1 Culture of tobacco BY-2 cells
Suspension-cultured cells of tobacco (Nicotiana tabacum L., cv. BY-2) were used for thearsenic-unadapted cell lines (Murata et al., 1994a, b). The modified LS medium (Linsmaier
and Skoog, 1965) was used as a standard medium. The BY-2 cells were cultured andmaintained as described previously (Murata et al., 1994a, b). The culture and maintenanceof cells were described in Chapter 2.

3.3.2 Estimation of proline content
Proline was measured following the method of Bates et al. (1973). Aliquots of BY-2 cells were
washed with 0.2 M sorbitol. Cells were ground and homogenized using liquid N2 in 10 mL of
3% aqueous sulfosalicylic acid. The homogenate was centrifuged at 12,000 g for 15 min at40

40C. Two mL of the supernatant were reacted with 2 mL of acid ninhydrin (1.25 g ninhydrin
dissolved in 30 mL of glacial acetic acid and 20 mL 6M phosphoric acid) and 2 mL of glacialacetic acid for 1 h at 1000C, and the reaction was then terminated in an ice bath. The colored
reaction mixture was extracted with 4 mL of toluene and the absorbance was recorded at
520 nm. The proline content was obtained from the standard curve.

3.3.3 Isolation of genomic DNA and gel electrophoresis
Approximately 200 mg of fresh BY-2 cells was ground and homogenized with a mortar andpestleusing

liquidnitrogen

in

500

µL

of

extraction

buffercontaining

3%

cetyltrimethylammonium bromide, 1.4 M NaCl, 20 mM EDTA, 100 mM Tris-HCl, pH 8.0, and
0.2 % (v/v) β-mercaptoethanol. The mixture was incubated at 60 0C for 45 min. Aftercentrifugation at 11,000 g for 10 min at room temperature, the supernatant was transferred
to a new microtube and then mixed well with an equal volume of chloroform. Aftercentrifugation at 11,000 g for 10 min at room temperature, the aqueous upper phase wascollected. The DNA was precipitated with an equal volume of isopropanol, washed in 70%(v/) ethanol, dried, and suspended in sterile distilled water. The DNA was thenelectrophoresed on a 1% (w/v) agarose gel followed by visualization with ethidium bromide.

3.3.4 Detection of ROS
ROS in arsenate stressed BY-2 cells was measured using 2´, 7´-dichlorodihydrofluresceindiacetate (H2DCF-DA) as described previously (Biswas and Mano, 2015). BY-2 cells werecollected and washed with distilled water by centrifugation. Cells were then incubated in 20µM H2DCF diacetate in PBS at 37 0C for 30 min and washed two times with PBS. The
fluorescence was monitored using a fluorescence microscope.

41

3.3.5 Measurement of glutathione content
GSH contents were determined according to Baker et al. (1990) method. An aliquot of BY-2
cells was ground using liquid nitrogen (N2) and homogenized with extraction buffer (50 mM
KH2PO4, 5 mM EDTA, pH 8.0). The homogenate was centrifuged at 9500g for 10 min
followed by deproteinization with sulphosalicylic acid (4%) and the supernatant was collected.
In addition, for the measurement of GSSG content, 2-vinylpyridine (97%) andtriethanolamine (20%) were added to the supernatant. Then 50 µL of the supernatant was
taken in the wells and 100 µL of the reaction mixture (NADPH, DTNB, KH2PO4 andglutathione reductase) was added. The absorbance was recorded at 412 nm using a
microplate reader (Nippon Bio-Rad Laboratories (680), Tokyo, Japan). The GSH content wascalculated by subtracting the GSSG from the total GSH.

3.3.6 Extractions and measurements of arsenate reductase
For preparation of sample solution, approximately 0.5 g of fresh BY-2 cells was ground witha mortar and pestle that were chilled with liquid nitrogen and homogenized in 3 ml buffer
solution containing 50 mM MOPS ((3-(N-morpholino) propane sulfonic acid) and 50 mM
MES((2-(N-morpholino) ethane sulfonic acid), which was adjusted to pH of 6.5 with 1 M
NaOH. The homogenate was centrifuged at 10,000 ˣ g for 30 min at 4 0C. The supernatant
was filtered through Qualitative Advantec 2 filter paper. The resulting filtrate was collected
and passed through Sephadex PD-10 desalting columns (Duan et al., 2005). The final filtrate
was the extract containing arsenate reductase. All the above steps were carried out on ice.
Arsenate reductase activity was assayed using the methods described by Shi et al.
(1999) and Duan et al. (2005). The assay buffer was 50 mM MOPS and 50 mM MES, pH
6.5, with a total volume 1.5 mL containing 1.0 mM NADPH, 1 unit yeast glutathione reductase,
1 mM GSH, 10 mM sodium arsenate. The extract (100 µL) was pre-incubated at 30 0C for 5
min in a buffer containing yeast glutathione reductase and GSH. Small volumes of NADPH
42

and sodium arsenate were then added and mixed thoroughly to start the reaction. All steps
were carried out at 30 0C. Arsenate reductase activity was monitored by the decrease in
NADPH absorbance at 340 nm and the NADPH oxidation was calculated using a molar
extinction coefficient of 6200 for NADPH at 430 nm.

3.3.7 Extractions and measurements of superoxide dismutase
Approximately 200 mg of fresh BY-2 cells was harvested and ground with a mortar and pestle
that were chilled with liquid nitrogen and added 500 µL of phosphate buffer solution (pH 8.0).
SOD activity was measured by using SOD Test Kit (Wako). For each SOD activity
measurement, 100 µL of the sample solution was taken in microtube for sample (S) andsample. blank (S.BL). Hundred µL of distilled water was taken for blank (BL) and blank. blank(BL.BL). After that 1.0 mL of coloring reagent was added to each tube and 1.0 mL of enzyme
solution was added for sample (S) and blank (BL). One mL of blank reagent was added forsample. blank (S.BL) and blank. blank (BL.BL). After incubation at 370C for 20 min, 2.0 mL
reaction stopper was added to each tube. The absorbance was measured at 560 nm. The
activity was calculated (Inhibition rate %) using the following equation:
SOD activity (Inhibition %)= {(EBL-EBL.BL) -(ES-ES.BL)/ (EBL-EBL.BL)} χ 100

3.3.8 Measurement of protein
Protein determinations were carried out using the method of Bradford (1976) with BSA as
standard.

3.3.9 Statistical analysis
Unless stated otherwise, the significance of differences between the mean values of allparameters was assessed by Tukey’s test. Differences at the level of p ≤ 0.05 wereconsidered as significant.

433.4 RESULTS

3.4.1 Proline content in response to application of exogenous proline
To investigate whether exogenously applied proline induces the accumulation of proline
content under AsO4- stress, we measured the intracellular proline contents in BY-2 cells bothat arsenate stress and non-stress condition (Fig. 3.1). The proline content was significantly
increased at 60 µM AsO4- compared with control, showing that AsO4- stress induced theaccumulation of proline. Exogenously applied 0.05 mM proline showed remarkable increasein the accumulation of proline compared with control. In the presence of arsenate, 0.05 mM
proline led to significant increase in proline content which is similar with 60 µM AsO4- stress.
Proline content was significantly increased at 0.1 mM both at AsO4- stress and non-stresscondition. These results suggest that both under As-stressed and non-stressed conditions,proline content was increased in a concentration-dependent manner in response to theProline (mg/g FW)

application of exogenous proline.
40
35
30
25
20
15
10
5
0

a
b

b

b

c
d

Figure 3.1 Proline content in response to exogenous proline application. The Proline content
was increased both under 60 µM AsO4--stressed and non-stressed conditions in aconcentration-dependent manner in response to the application of exogenous proline.
Averages of proline contents from three independent experiments (n = 3) are shown.
44

3.4.2 DNA fragmentation
To investigate whether DNA fragmentation occurred in BY-2 cells under arsenate stress, DNA
was extracted from BY-2 cells and subjected to electrophoresis on agarose gel prior tostaining with ethidium bromide. No DNA fragmentation was detected in the arsenate stressed
cells irrespective of the presence or absence of proline (Fig. 3.2).

As (µM)

-

60

60

60

Pro (mM)

-

-

0.05

0.1

M

DNA migration
Figure 3.2 DNA fragmentation in arsenate (As) stress BY-2 cells in response to exogenous
proline (Pro) application. DNA was extracted and subjected to electrophoresis on 1 %agarose gel. Photographs shown are representative of four independent experiments. M, 1
kb DNA marker.

45

3.4.3 Intracellular ROS level
We investigated whether proline could inhibit arsenate-induced ROS accumulation in BY-2
cells (Fig. 3.3). Arsenate stress resulted in a significant increase in accumulation of
intracellular ROS in BY-2 cells compared with control. In the presence of arsenate, proline at
0.05 mM and 0.1 mM significantly inhibited the arsenate induced ROS accumulation in BY2 cells compared with arsenate stress (Fig. 3.3).

A

ii

i

B

iv

a

800

ROS Level (% of control)

iii

700
600
500
400

b

300
200

bc
c

100
0

Figure 3.3 Intracellular ROS levels in tobacco BY-2 suspension cells under arsenate (As)stress in the presence or absence of proline (Pro). Four-day-cultured cells were incubated in
As stress media with different concentration of Pro and DCF fluorescence was recorded
using fluorescence microscope. A, Typical photographs are shown: untreated cells as acontrol (i), 60 µM As (ii), As + 0.05 mM Pro (iii), and As + 0.1 mM Pro (iv). Bar = 10 µm. B,
46

DCF fluorescence intensity of BY-2 cells. The fluorescence intensity is shown as a ROS level.
Values represent the mean ±SE from four independent experiments. Bars with same letters
are not significantly different at P≤0.05.

3.4.4 Effects of exogenous proline on glutathione content at 60 µM arsenate- stressed
BY-2 cells
We examined the effects of proline on the total GSH, GSSG and GSH contents in BY-2 cells
at 60 µM (Fig. 3.4) arsenate stress. Arsenate stress significantly increased the total GSH
content in BY-2 cells (Fig. 3.4A) compared with control. In this study, exogenous proline at
0.05 mM did not show any effect on total GSH content compared with AsO4- stress. In thepresence of arsenate, proline at 0.05 mM decreased the GSH content and increased the
GSSG content compared with arsenate stress, whereas 0.1 mM proline did not show any
effect on total GSH, GSH and GSSG contents compared with arsenate stress (Fig. 3.4).
These results indicated that proline at 0.05 mM mitigates arsenate stress in BY-2 cells bydecreasing GSH and increasing GSSG contents.

A
Total GSH (µmole/g FW)

30

a

a

b

25
20
15
10
5
0

47

a

B
GSH (µmole/g FW)

30

a

a
b

25
c
20
15
10
5
0

C

GSSG (µmole/g FW)

6

a

5

b

4

bc

3

c
2
1
0

Figure 3.4 Glutathione content at 60 µM AsO4--stressed BY-2 cells in presence of lower
concentration of exogenous proline (Pro). Total GSH (A), reduced glutathione (GSH) (B), andoxidized glutathione (GSSG) (C) content in As-unadapted tobacco BY-2 cells induced by Pro
under AsO4-stress. Values represent the mean ±SD (n=3).
48

3.4.5 Effects of exogenous proline on the activity of arsenate reductase in AsO4stressed BY-2 cells
Fig. 3.5 shows effect of proline on the activities of arsenate reductase (AR) in tobacco BY-2
cells under AsO4- stress. In the presence of 60 µM AsO4-, compared with control, 0.05 mM
proline did not show any effects on AR activity. However, compared with arsenate stress,
0.05 mM and 0.1 mM proline significantly increased the arsenate reductase activity. These
results suggest that lower level of proline mitigates arsenate stress by increasing the AR
activity which accelerates the conversion of arsenate to arsenite.

AR activity (µmole/mg
Protein)

120

a

a

a

100
80

b

60
40
20
0

Figure 3.5 Arsenate reductase (AR) activity of arsenate unadapted tobacco BY-2
suspension cells induced by proline under AsO4- stress. Four-day-cultured cells were cotreated with 0.05 mM and 0.1 mM exogenous proline respectively at 60 µM AsO4- stressmedium. Means ± SD of three independent experiments are shown.

49

3.4.6 Superoxide dismutase activity
Arsenate stress significantly increased the superoxide dismutase (SOD) activity in tobacco
BY-2 cells represented in Fig. 3.6. The SOD activity in control condition is lower than the
AsO4- stress. In this study, proline at 0.05 mM and 0.1 mM did not show any effect on the

SOD activity (% Inhibition)

SOD activity in the presence of AsO4-.

90
80
70
60
50
40
30
20
10
0

a

a

a

b

Figure 3.6 Superoxide dismutase (SOD) activity of arsenate-unadapted tobacco BY-2
suspension cells induced by lower concentration of proline (Pro) under AsO4- stress.
Suspension cells of a 7-day-old culture were inoculated into different fresh media, and cells
were collected at 4-days after inoculation. Values represent the mean ±SD (n=3).

50

3.5 DISCUSSION
We have investigated the effect of exogenous proline on arsenate stressed BY-2 cells. We
found that proline at lower concentration mitigate AsO4- -induced growth inhibition by
suppressing Evans Blue stained cells and increasing the total number of cell. It was reported
that proline ameliorates heavy-metal toxicity in plants. However, the mechanisms of how
proline mitigates arsenate stress in plants are not fully understood but appear to involve its
chemical properties and effects on redox systems such as the glutathione pool and theactivities of antioxidant enzymes. Our recent study has indicated that exogenous proline
increased the intracellular accumulation of proline in a concentration dependent manner aswell as proline increased the inhibition of ROS production at arsenate stress. In our study,
the lower proline decreased the reduced glutathione and increased the oxidized glutathione
contents compared with arsenate stress. We also found that in the presence of arsenate,
exogenous proline increased the activity of arsenate reductase, and thus we elucidate therole of lower concentration of proline in BY-2 cells under arsenate stress.
There are significant evidences that arsenic exposure induces the generation of ROS
during the conversion of arsenate to arsenite (Hartley-Whitaker et al., 2001). Our results
indicate that arsenate stress increased the ROS levels compared with control. The present
data showed that in the presence of arsenate 0.05 mM and 0.1 mM proline decreased theintracellular ROS level compared with arsenate stress (Fig. 3.3B). These results suggest thatlower proline decreased arsenate toxicity by increasing the inhibition of ROS production.
A non-enzymatic antioxidant, GSH, constitutes an important plant defense systemagainst environmental stresses including heavy metals (Wingate et al., 1988). It was reported
that glutathione and glutathione metabolism is the primary detoxification strategy by whichplants tolerate arsenate stress (Mishra et al., 2008). Previous studies suggested that the
GSH content is either increase or decrease or maintains homeostasis in response to mitigate
stress-induced changes in plants and it was also reported that glutathione content is51

significantly increased in plants upon arsenic exposure (Srivastava et. al., 2007). Our dataalso showed that the total GSH content is increased in AsO4--stressed BY-2 cells (Fig.3.4A),
which indicates that glutathione is an important element that regulates stress-inducedchanges in plants. In this study, we also found that exogenous proline at 0.05 mM decreased
the GSH and increased the GSSG contents compared with arsenate stress (3.4B and C). It
was reported that the detoxification of heavy metals by GSH results in the rapid depletion of
GSH in the cytoplasm (Polhuijs et al., 1992). On the other hand, Agarwal et al. (2011)
reported that GSH content is increased in proline-treated bean (Phaseolus vulgaris) plantsthat mitigate selenium stress. Though it seems contradictory according to our findings but
the response of GSH and GSSG levels in terms of mitigation of stress is similar with theprevious one. It is well established that the detoxification of As and Cd requires GSH and
PCs in plants (Howden et al., 1995; Shi et al., 1996; Cobbet et al., 1998; Dhankher et al.,
2002; Verbruggen et al., 2009). Agarwal (2011) reported that selenium stress is mitigated byincreasing GSH level as well as we found that during mitigation process at 0.05 mM proline
maintains glutathione homeostasis by decreasing GSH content and increasing the GSSG
content.
Arsenic is detoxified by complexation with the thiol-rich peptide such as GSH. The GSH
is required for the reduction of arsenate to arsenite by arsenate reductase (Liu and Rosen,1997) and mitigates arsenic stress. Although arsenate can be reduced nonenzymatically inthe presence of GSH (Delnomdedieu et al., 1994), this process is too slow to be significant
biologically. In this study, compare with control, 0.05 mM proline did not show any effects on
arsenate reductase activity. Moreover, exogenous proline at 0.05 mM significantly increased
the arsenate reductase activity compared with arsenate stress (Fig. 3.5). These results
suggest that proline mitigates arsenate stress by increasing the arsenate reductase activity
which accelerates the conversion of arsenate to arsenite, leading detoxification andsequestration of arsenite.

52

The activity of antioxidant enzymes is important for stress mitigation. It is reported that
arsenic exposure induces the production of ROS that can lead to the production ofantioxidant metabolites and numerous enzymes involved in antioxidant defense
mechanisms (Finnegan and Chen, 2012). Reports suggest that antioxidant enzyme such as
superoxide dismutase and peroxidases plays prominent role in response to arsenate stressand the activity of SOD is increased at AsO4- stress condition (Armendariz et al., 2016). Ourdata also showed that the SOD activity is increase at arsenate stress condition (Fig. 3.6),
which indicates that SOD play an important role that regulates stress-induced changes inplants. Our previous reports have shown that proline increased antioxidant defense systems
and improve salt tolerance and Cd tolerance in BY-2 cells (Okuma et al., 2004; Hoque et al.,2007; Banu et al., 2009). On the other hand, Salim et al. (2009) did not found increased levelof SOD activity in BY-2 cell under Cd stress. In our study, in the presence of arsenate, proline
at 0.05 mM and 0.1 mM did not show any effect on the SOD activity compared with arsenate
stress (Fig. 3.6). Report also suggests that proline alleviates Cd toxicity in Solanum nigrum
by detoxifying ROS and increasing the activity of SOD (Xu et al., 2009). The above reports
with our findings suggest that the mitigatory role of proline might depend on some conditions
such as type of stress, and plant species.
However, the physiological bases of arsenic metabolism remain unclear and previous
reports indicate that proline is involved in plant heavy metal stress tolerance by different
mechanisms such as osmo and redox regulation, metal chelation and scavenging of radicals.
To combat arsenic stress, plants modulate a number of pathways that operate to keep thecellular concentration of metal ion to a minimum level via thiol-mediated complexation(Bleeker et al., 2006). In conclusion, based on reverse and forward studies, proline at lower
concentration decreased the production of ROS and ensures glutathione turnover during
arsenate stress as a tolerance mechanism in arsenate stressed tobacco BY-2 cells.
53

CHAPTER 4
Comparison between the effects of proline and the effects of other
osmolytes in BY-2 cells under arsenate stress
4.1 ABSTRACT
Arsenic is the most toxic metalloid widely concerned and widely distributed in theenvironment. Plants respond to arsenic toxicity by a variety of mechanisms and when notdetoxified, may trigger a sequence of reactions leading to growth inhibition, and stimulationof secondary metabolism. The report suggests that the accumulation of proline may be apart of stress signal inﬂuencing adaptive responses. In this study, we investigated the effects
of exogenously applied proline on cell growth, glutathione content, and activities of different
antioxidant enzymes in cultured tobacco BY-2 cells exposed to arsenate (AsO4-) stress. Here,
we also examine the effects of some stress-related organic molecules on the BY-2 cell
growth both under AsO4- stress and non-stress conditions to compare the results with proline
effects, as well as to clarify the proline-enhanced cell growth reduction by arsenate. In thepresence of arsenate, 0.5 mM and 1 mM proline did not show any effect on total GSH and
GSH and GSSG contents but 10 mM proline decreased it compared with arsenate stress.
Moreover, proline at 10 mM significantly decreased the arsenate reductase activity as well
as SOD activity. It has been reported that there is a considerable interconnection between
proline and arginine catabolic pathways when applied exogenously. Arginine is degraded toproline and then further catabolized following the metabolic pathway similar to proline. Here,
we found that 10 mM arginine did not affect BY-2 cell growth, but in the presence of arsenate,
like 10 mM proline, 10 mM arginine enhanced the cell growth reduction by arsenate. Alanine,unlike proline, follows separate catabolic pathway and report suggests that alanine removes
the feedback inhibition of proline biosynthesis pathway and enhances the accumulation of54

proline during stress condition. Here, exogenous alanine did not affect BY-2 cell growth at
0.5 mM to 10 mM. In the presence of AsO4-, neither 0.5 mM nor 1 mM alanine affected thecell growth but 10 mM alanine significantly recovered the AsO4--induced growth inhibition.
These results suggest that exogenous alanine mitigates arsenate stress and as alanine,
does not follow catabolic pathway similar to proline, arsenate did not show any inhibitory
actions. Glutamate is the precursor of proline biosynthesis in plants. During stress condition,
proline is synthesized from glutamate that functions as a protective molecule. In the present
study, glutamate at 0.5 mM to 10 mM did not show any effect on BY-2 cell growth and did
not increase arsenate induced cell growth reduction. Glycine betaine is a compatible solutethat accumulates in plants and mitigates stresses. Here, I found that exogenous betaine at
0.5 mM and 1 mM did not affect BY-2 cell growth and did not improve AsO4--induced cell
growth reduction. Unlike proline effects, the arsenate in the presence of glutamate andbetaine did not enhance the cell growth reduction, suggesting that arsenate, particularly inthe presence of proline, increased the sensitivity of BY-2 cells. In conclusion, exogenous
proline plays dynamic roles in BY-2 cells under arsenic stress, which depends on its level ofconcentration. Together, our results suggest that exogenous proline at higher concentration
does not alleviate arsenate toxicity but enhances the sensitivity of BY-2 cells to arsenate.

4.2 INTRODUCTION
Arsenic a toxic substance released into the environment, contributing to a variety of toxic
effects on living organisms. Arsenic toxicity has become one important constraint to crop
productivity and quality. Among inorganic and organic species of arsenic, arsenite is the more
toxic form due to its propensity to bind to sulfhydryl groups, which causes detrimental effects
on general protein functioning (Tripathi et al., 2007).
Glutathione, a non-enzymatic antioxidant, is a low molecular weight thiol implicated ina wide range of metabolic processes and constitutes an important plant defense systems
55

against environmental stress including heavy metal stress (Hossain et al., 2010, 2011). It is
one of the major antioxidant material in plants that found abundantly in all cell compartments.
It is reported that glutathione and glutathione dependent enzymes play an important role indetoxification of heavy metals (Hossain et al., 2012) such as arsenic. Glutathione acts as athiolate donor in plant heavy metal stress (Pickering et al., 2000). An increased level of GSH
concentration during heavy metal stress is always considered as a protective way in plants.
The reduction of arsenate to arsenite is regarded as another aspect of arsenic
detoxification in plants (Zhang et al., 2002) that required GSH as a reductant. Arsenate canalso be reduced by GSH alone but the process is usually very slow and incomplete, whereas
the enzymatic reduction under optimum condition was much faster. Arsenate reductase is a
specific catalyzer to reduce arsenate to arsenite in plants (Mukhopadhyay et al., 2000; Duanet al., 2005).
Under stress condition, plant produced ROS and within a cell, the SODs constitute thefirst line of defense against ROS (Alscher et al., 2002). To avoid the damages effectively, theanti-oxidative enzyme system would be activated. Several studies reported that these
enzymes (e.g. superoxide dismutase, SOD) are important to defend the plant oxidative
stress caused by arsenate (Cao et al., 2004; Srivastava et al., 2005).
Amino acids are the building blocks of proteins and are known to be induced
significantly upon heavy metal exposure (Davies et al.,1987). Amino acids and theirderivatives have been reported to chelate metal ions, thus conferring metal tolerance toplants. Plants have evolved a variety of adaptive mechanisms to respond to heavy metal
stress including arsenic stress. It is reported that accumulation of compatible solutes is themain adaptive mechanism in metal stress situation (Shah and Dubey, 1998; Mehta and Gaur,
1999; Sharma and Dietz, 2006; Ashraf and Foolad, 2007). When exposed to stressful
conditions, plants accumulate an array of metabolites, particularly amino acids. Proline, anamino acid, plays a highly beneficial role in plants exposed to various stress conditions
56

(Hayat et al., 2012). Proline is the most common compatible solutes that occur in a wide
variety of plants.
Arginine is one of the most versatile amino acids and it has multiple metabolic fates.
In addition to serving as a constituent of proteins, arginine is a precursor for biosynthesis ofpolyamines, urea, and proline as well as the cell signaling molecules glutamine and nitricoxide (Chen et al., 2004; Liu et al., 2006). The report suggests that Arginine follows proline
degradation pathway when applied exogenously (Thompson, 1980). Alanine, a nonessential
amino acid and the accumulation of alanine are increased at several stress conditions inplants. Glycine betaine or betaine, a quaternary ammonium compound, is regarded as oneof the most effective osmoprotectants in plants, bacteria and algae (Delauney and Verma,1993). Among the compatible solutes, betaine is a particularly effective protectant against
abiotic stress (Sakamoto and Murata, 2000, 2001, 2002).
However, the molecular mechanism by which proline, arginine, alanine, glutamate andbetaine regulated stress-related signals in plants remain to be investigated.

4.3 MATERIALS AND METHODS

4.3.1 Culture of tobacco BY-2 cells
Suspension-cultured cells of tobacco (Nicotiana tabacum L., cv. BY-2) were used for thearsenic-unadapted cell lines (Murata et al., 1994a, b). The modified LS medium (Linsmaier
and Skoog, 1965) was used as a standard medium. The 0.5 mM, 1 mM and 10 mM proline,
and arginine media were the 40 µM as well as 60 µM AsO4- media and containing 0.5 mM,
1 mM and 10 mM proline and arginine respectively. The 0.5 mM, 1 mM and 10 mM alanine,and glutamate media were the 60 µM AsO4- media containing 0.5 mM, 1 mM and 10 mM
alanine and glutamate respectively. The 0.5 mM and 1 mM betaine media were the 60 µM
AsO4- media containing 0.5 mM, and 1 mM betaine respectively. The BY-2 cells were cultured
57and maintained as described previously (Murata et al., 1994a, b). The culture andmaintenance of cells were described in Chapter 2.

4.3.2 Measurement of BY-2 cell growth
The growth of BY-2 cells was measured as described previously (Murata et al., 1994a, b).
The measurement procedure of BY-2 cell growth was described in Chapter 2. Cultured cells
transferred to the different fresh media were used for the experiments at 4 d after inoculation.

4.3.3 Estimation of endogenous proline
Proline was measured following the method of Bates et al. (1973). The detailed of thismethod was described in Chapter 3. The proline content was obtained from the standard
curve.

4.3.4 Measurement of glutathione content
GSH contents were determined according to Baker et al. (1990) method. An aliquot of BY-2
cells was ground using liquid nitrogen (N2) and homogenized with extraction buffer (50 mM
KH2PO4, 5 mM EDTA, pH 8.0). The homogenate was centrifuged at 9500g for 10 min
followed by deproteinization with sulphosalicylic acid (4%) and the supernatant was collected.
The measurement procedure of the total GSH and GSSG content was described in Chapter
3. The GSH content was calculated by subtracting the GSSG from the total GSH.

4.3.5 Extractions and measurements of arsenate reductase
Arsenate reductase activity was assayed using the methods described by Shi et al. (1999),
Mukhopadhyay et al. (2000) and Duan et al. (2005). The detailed procedure for themeasurement of arsenate reductase activity was described in Chapter 3.

58

4.3.6 Extractions and measurements of superoxide dismutase
Approximately 200 mg of fresh BY-2 cells were harvested and ground with a mortar andpestle that were chilled with liquid nitrogen and added 500 µL of Phosphate buffer solution(pH 8.0). SOD activity was measured by using SOD Test Kit (Wako). The detailed explanationof SOD measurement procedure was described in Chapter 3. The activity was calculated(Inhibition rate %) using the following equation:
SOD activity (Inhibition %)= {(EBL-EBL.BL) -(ES-ES.BL)/ (EBL-EBL.BL)} χ 100

4.3.7 Determination of lipid peroxidation
Lipid peroxidation was assayed as described by Okuma et al. (2004) and Banu et al., (2008)
by measuring the malondialdehyde (MDA) content. An aliquot of BY-2 cells was groundedusing liquid Nitrogen (N2) and homogenized with 2 ml of 5% (w/v) trichloroacetic acid (TCA).
After centrifuging at 12,000g for 15 min, the supernatant was diluted with 1.2 ml of 20% (w/v)
trichloroacetic acid containing 0.5% (w/v) thiobarbituric acid. The mixture was heated at 950C
for 25 min and the reaction was stopped by placing the tubes in an ice bath. The samples
were centrifuged at 7,500g for 5 min. The absorbance of the supernatant was measured at
532 nm, and the value for non-specific turbidity at 600 nm was subtracted. MDA content wascalculated using extinction co-efficient of 155 mM–1 cm–1.

4.3.8 Measurement of protein
Protein determinations were carried out using the method of Bradford (1976) with BSA as
standard.

4.3.9 Statistical analysis
Unless stated otherwise, the significance of differences between the mean values of allparameters was assessed by Tukey’s test. Differences at the level of p ≤ 0.05 wereconsidered as significant.

594.4 RESULTS
4.4.1 Proline content in response to application of exogenous proline
To examine whether exogenous proline induces the accumulation of proline content under
AsO4- stress, we measured the intracellular proline contents in BY-2 cells both at arsenate
stress and non-stress condition (Fig. 4.1). The proline content was significantly increased at
60 µM AsO4- compared with control, showing that AsO4- stress induced the accumulation of
proline. Exogenously applied 0.5 mM, 1 mM and 10 mM proline showed a remarkableincrease in the accumulation of proline compared with control. Proline content wassignificantly increased at 0.5 mM to 10 mM both at AsO4- stress and non-stress condition.
These results suggest that endogenous proline content was increased in a concentrationdependent manner in response to the application of exogenous proline.

Proline (mg/g FW)

600
500
400

100

a

80
60
40
20

300

b

0

c

200
100

h

g

e

f

d

0

Figure 4.1 Proline (Pro) content in response to exogenous proline application. The proline
content was increased both under 60 µM arsenate (As) stressed and non-stressed
conditions in a concentration-dependent manner in response to the application of exogenous
proline. Averages of proline contents from three independent experiments (n = 3) are shown.
60

4.4.2 Effects of exogenous proline on glutathione content at 60 µM arsenate- stressed
BY-2 cells
We examined the effects of proline on the total GSH, GSH and GSSG contents in BY-2 cells
at 60 µM (Fig. 4.2) arsenate stress condition. Arsenate stress significantly increased the total
GSH content in BY-2 cells (Fig. 4.2A) compared with control. In this presence of arsenate,
exogenous proline at 0.5 mM and 1mM did not show any effect on total GSH, GSH and
GSSG content whereas, 10 mM proline decreased them compared with arsenate stress (Fig.
4.2A, B and C). These results indicated that proline decreased the glutathione pool andtherefore, sensitizes the BY-2 cells to arsenate.

Total GSH (µmole/g FW)

A
35
30

a

a

a

b

25

b

20
15
10
5
0

B

GSH (µmole/g FW)

30

25

a

a
b

a

b

20
15
10
5
0

61

GSSG (µmole/g FW)

C
6

a

5
4
3

b

b

b

2
c
1
0

Figure 4.2 Glutathione content at 60 µM arsenic (As)-stressed BY-2 cells in presence of
higher concentration of exogenous proline (Pro). Total GSH (A), reduced glutathione (GSH)(B), and oxidized glutathione (GSSG) (C) content in As-unadapted tobacco BY-2 cells
induced by Pro under AsO4-stress. Values represent the mean ±SD (n=3).

4.4.3 Effects of exogenous proline on glutathione content at 40 µM arsenate- stressed
BY-2 cells
We investigated the effects of proline on the total GSH, GSH and GSSG contents in BY-2
cells at 40 µM AsO4- (Fig. 4.3A, B and C). Arsenate stress significantly increased the total
GSH content in BY-2 cells (Fig.4.3A) compared with control. Exogenous application of 0.5
mM and 1 mM proline did not change the contents of total GSH, GSH and GSSG in BY-2
cells treated with arsenate but 10 mM proline significantly decreased them. Results indicatethat arsenate inhibits the growth of BY-2 cells by decreasing the glutathione pool in presenceof exogenous proline.

62

Total GSH (µmole/g FW)

A
a

35

a

a

30
25

b
b

20
15

10
5
0

B
GSH (µmole/g FW)

35

a

a
a

30
25

b
c

20

15
10
5
0

C
GSSG (µmole/g FW)

1.4
a

1.2
1

a

a

b

b

0.8

0.6
0.4
0.2
0

63

Figure 4.3 Glutathione content at 40 µM arsenate (As)-stressed BY-2 cells in presence ofdifferent concentration of exogenous proline (Pro). Total GSH (A), reduced glutathione (GSH)(B), and oxidized glutathione (GSSG) (C) content in As-unadapted tobacco BY-2 cells
induced by Pro under AsO4- stress. Values represent the mean ±SD (n=3)

4.4.4 Effects of exogenous proline on the activity of arsenate reductase in arsenatestressed BY-2 cells
Fig. 4.4 shows the effect of proline on the activities of arsenate reductase in tobacco BY-2
cells under AsO4- stress. In the presence of arsenate, compared with control, 0.5 mM and 1
mM proline did not show any effect on arsenate reductase activity, whereas, 10 mM proline
decreased it. At 60 µM AsO4-, proline at 10 mM did not show any effect on arsenate reductase

AR activity (µmole/mg Protein)

activity compared with arsenate stress.

140
120

a

a

a

100
80

b

60

b

40
20
0

Figure 4.4 Arsenate reductase (AR) activity of arsenate (As) unadapted tobacco BY-2
suspension cells induced by proline (Pro) under AsO4- stress. Four-day-cultured cells wereco-treated with 0.5 mM, 1 mM and 10 mM exogenous Pro respectively at 60 µM AsO4- stressmedium. Means ± SD of three independent experiments is shown.
64

4.4.5 Superoxide dismutase activity
Arsenate stress significantly increased the SOD activity in tobacco BY-2 cells represented inFig. 4.5. The SOD activity in control condition is lower than the AsO4- stress. In the presenceof AsO4-, proline at 0.5 mM to 10 mM did not show any effect on the SOD activity comparedwith control. On the other hand, exogenous proline at 0.5 mM, to 10 mM decreased theactivity of SOD compared with AsO4- stress.

SOD activity (% inhibition)

90

a

80
70
60

b

b

b

b

50
40
30
20
10
0

Figure 4.5 Superoxide dismutase (SOD) activity of arsenate (As)-unadapted tobacco BY-2
suspension cells induced by higher concentration of proline (Pro) under AsO4- stress.
Suspension cells of a 7-day-old culture were inoculated into different fresh media, and cells
were collected at 4-days after inoculation. Values represent the mean ±SD (n=3).

4.4.6 Effects of exogenous arginine on BY-2 cell growth in the absence of arsenate
To investigate the roles of exogenous arginine for the mitigation of arsenate stress in BY-2
cells, we examined whether exogenous arginine shows any effects on BY-2 cells. We
measured the fresh weight (FW) and dry weight (DW) of cells at 0, 4, 6, and 8 days after65

inoculation (DAI) in response to exogenous arginine in the absence of arsenate. We foundthat 0.5 mM, 1 mM and 10 mM arginine did not change BY-2 cell growth compared with
control. (Fig. 4.6A and B).

A

COT
1 mM Arg

0.5 mM Arg
10 mM Arg

Fresh weight (g/Flask)

12a a a a10a a a aa a a a8
6
4
2

a a a a00

4
6Days after inoculation

B

Dry weight (g/Flask)

COT
1 mM Arg
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

8

0.5 mM Arg
10 mM Arga a a aa a a aa a a aa a a a0

4
6Days after inoculation

8

Figure 4.6 Effects of exogenous arginine (Arg) on BY-2 cell growth. A, Shows the cell growth
based on fresh weight and B, shows the cell growth based on dry weight in response to 0.5
mM, 1 mM, and 10 mM Arg at 0, 4, 6, and 8 days after inoculation. Averages of cell growthfrom three independent experiments (n = 3) are shown. Error bars represent SE. Based on
p values obtained in the t-test, there were no significant differences (p<0.05) between control
66

(untreated) cells and treated cells in fresh weight or dry weight at each time point after
inoculation.

4.4.7 Effects of exogenous arginine on the inhibition of BY-2 cell growth by arsenate
To investigate whether exogenous arginine recovered the inhibition of cell growth byarsenate, we examined the effects of exogenous arginine on the growth of BY-2 cells cultured
at 60 µM AsO4- stress. The FW and DW of cells at 0, 4, 6, and 8 days after inoculation were
measured. In the presence of 60 µM AsO4-, 0.5 mM and 1 mM arginine did not affect the cell
growth but 10 mM arginine inhibited it (Fig. 4.7A and B). Moreover, AsO4- at 60 µM induced
more inhibition of cell growth in the presence of exogenous arginine than in the absence ofexogenous arginine. These results suggest that exogenous arginine, like proline, did not
mitigate the arsenate-induced growth inhibition of BY-2 cells but enhances the sensitivity of
BY-2 cells to arsenate.

A
Control
As + 0.5 mM Arg
As + 10 mM Arg

Fresh weight (g/Flask)

12

60 µM AsAs + 1 mM Arg
a

10
8

a

a

6
4
2

a a a a a

b

bc
bc c

b bc

bc
c

b b

0
0

4
6
Daya after inoculation

67

8

b

c

B

Dry weight (g/Flask)

Control
As + 0.5 mM Arg
As + 10 mM Arg
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

60 µM AsAs + 1 mM Arg
a

a

a

b

d

0

b

b bc
bc

bc
bc

a a a a a

b b

d

4
6
Days after inoculationc8

Figure 4.7 Effects of exogenous arginine (Arg) on 60 µM arsenate (As)-stressed BY-2 cells.
Reduction of BY-2 cell growth by arsenate (Fresh weight basis, A; dry weight basis, B) in thepresence of 10 mM Arg but not in the presence of 0.5 mM or 1 mM Arg at 4, 6 and 8 days
after inoculation. Averages of cell growth from three independent experiments (n = 3) are
shown. The error bars represent SE. For the same inoculation day, values indicated by thesame letter do not differ significantly at 5% level of significance as determined by Tukey’s
test.

4.4.8 Effects of exogenous alanine on BY-2 cell growth in the absence of arsenate
We tested the effect of exogenous alanine in BY-2 cells growth to find out whether exogenous
alanine mitigates arsenate induced growth inhibition of BY-2 cells. We measured the FW and
DW of cells at 0, 2, 4, 6, and 8 DAI in response to exogenous alanine in the absence ofarsenate. We found that 0.5 mM to 10 mM alanine did not show any effect on BY-2 cell
growth compared with control. (Fig. 4.8A and B).
68

A
Control
1 mM Ala

Fresh weight (g/Flask)

12

0.5 mM Ala
10 mM Ala

10
8

a aaa

a aaa

6d

8d

a aaa

6
4
2

a aaa

a aaa

0
0d

2d

4d

Days after inoculation

Dry weight (g/Flask)

B

0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

Control

0.5 mM Ala

1 mM Ala

10 mM Ala
aaa a

aaa a

aaa a

4d

6d

8d

aaa a
aaa a

0d

2d

Days after inoculation

Figure 4.8 Effects of exogenous alanine (Ala) on BY-2 cell growth. A, Shows the cell growthon the basis of fresh weight and B, shows the cell growth on the basis dry weight in response
to 0.5 mM, 1 mM, and 10 mM Ala at 0, 2, 4, 6, and 8 days after inoculation. Averages of cell
growth from three independent experiments (n = 3) are shown. Error bars represent SE.
Based on p values obtained in the t-test, there were no significant differences (p<0.05)
between control (untreated) cells and treated cells in fresh weight or dry weight at each time
point after inoculation.

69

4.4.9 Effects of exogenous alanine on AsO4- induced BY-2 cell growth
We examined the effects of exogenous alanine on AsO4- induced BY-2 cell cultured under
60 µM AsO4- stress. The FW and DW of cells at 0, 4, 6, and 8 DAI were measured. In thepresence of AsO4-, 0.5 mM and 1 mM alanine showed a similar effect on BY-2 cell growthcompared with 60 µM AsO4- stress but 10 mM alanine significantly recovered the AsO4induced growth inhibition (Fig. 4.9A and B). These results suggest that exogenous alanine
mitigate the arsenate-induced growth inhibition of BY-2 cells.

A

Control
As + 0.5 mM Ala
As + 10 mM Ala

60 µM AsAs + 1 mM Ala

Fresh weght (g/Flask)

10

a

a
a

8
6
4
a a a a a2c c c bc c c bb b b b

0
0d
B

4d
6d
Days after inoculation
Control
As + 0.5 mM Ala
As + 10 mM Ala

Dry weight (g/Flask)

0.4

8d

60 µM AsAs + 1 mM Ala

a
a

0.35

a

0.3
0.25

0.2
0.15
0.1

c c

a a a a a

c

b
d c c

b
c bc

bc b

0.05
0
0d

4d
6d
Days after inoculation
708d

Figure 4.9 Effect of exogenous alanine (Ala) on 60 µM arsenate (As)-stressed BY-2 cells.
Increment of BY-2 cell growth by 10 mM Ala (Fresh weight basis, A; dry weight basis, B) but
not in the presence of 0.5 mM or 1 mM Ala at 4, 6 and 8 days after inoculation under AsO4stress. Averages of cell growth from three independent experiments (n = 3) are shown. The
error bars represent SE. For the same inoculation day, values indicated by the same letterdo not differ significantly at 5% level of significance as determined by Tukey’s test.

4.4.10 Effects of exogenous glutamate on BY-2 cell growth
We examined the effect of exogenous glutamate to see whether glutamate has any effect on
BY-2 cell growth (Fig. 4.10). We measured the FW and DW of cells at 0, 2, 4,6, and 8 DAI in
response to exogenous glutamate in the absence of arsenate. We found that 0.5 mM to 10
mM glutamate did not show any effect on BY-2 cell growth compared with control (Fig. 4.10).

Fresh weight (g/Flask)

A

Control
1 mM Glu
10
9
8
7
6
5
4
3
2
1
0

0.5 mM Glu
10 mM Glu
aaa a

aaa a

6d

8d

aaa a

aaa a
aaa a

0d

2d

4d

Days after inoculation

71

B

Dry weight (g/Flask)

Control
1 mM Glu
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

0.5 mM Glu
10 mM Glua aa aa aa aa aa a6d

8d

a aa aa aa a0d

2d

4dDays after inoculation
Figure 4.10 Effects of exogenous glutamate (Glu) on BY-2 cells. A, Shows the cell growthon the basis of fresh weight and B, shows the cell growth on the basis dry weight in response
to 0.5 mM, 1 mM, and 10 mM Glu at 0, 2, 4, 6, and 8 days after inoculation. Averages of cell
growth from three independent experiments (n = 3) are shown. Error bars represent SE.
Based on p values obtained in the t-test, there were no significant differences (p<0.05)
between control (untreated) cells and treated cells in fresh weight or dry weight at each time
point after inoculation.

4.4.11 Effects of exogenous glutamate on the inhibition of BY-2 cell growth by arsenate
Figure 4.11 shows that in the presence of arsenate, exogenous glutamate at 0.5 mM, 1 mM
and 10 mM did not affect BY-2 cell growth as well as did not improve arsenate-induced cell
growth reduction. These results suggest that unlike proline effects, the arsenate in thepresence of glutamate did not enhance the cell growth reduction.

72

A

Fresh weight (g/Flask)

Control
As + 0.5 mM Glu
As + 10 mM Glu
10

a

a

8

a

6
4
2

b b b ba a a a ab b b bb b bb00d

B

Dry weight (g/Flask)

60 µM AsAs + 1 mM Glu

4d
6d
Days after inoculation

Control
As + 0.5 mM Glu
As + 10 mM Glu
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

a

8d

60 µM AsAs + 1 mM Glu

a

a

bbbb
a aaaa

0d

4d

bbbb

bbbb

6d

8d

Days after inoculatiion

Figure 4.11 Effects of exogenous glutamate (Glu) on 60 µM arsenate (As)-stressed BY-2
cells. In the presence of As, Glu at 0.5 mM, 1 mM and 10 mM (Fresh weight basis, A; dryweight basis, B) did not show any effect on BY-2 cell growth compared with AsO4- stress.
Averages of cell growth from three independent experiments (n = 3) are shown. The error
bars represent SE. For the same inoculation day, values indicated by the same letter do notdiffer significantly at 5% level of significance as determined by Tukey’s test.
73

4.4.12 Effects of exogenous betaine on BY-2 cell growth in the absence of arsenate
To investigate the roles of exogenous betaine for the mitigation of arsenate stress in BY-2
cells, we examined whether exogenous betaine shows any effects on BY-2 cells. We
measured the FW and DW of cells at 0, 4, 6, and 8 DAI in response to exogenous betaine
in the absence of arsenate. We found that 0.5 mM and 1 mM betaine did not change BY-2
cell growth compared with control but 10 mM betaine drastically decreased the cell growth(Fig. 4.12A and B).

Control
1 mM Betaine

Fresh weight (g/Flask)

A

10
9
8
7
6
5
4
3
2
1
0

a a a

a a a

a a a

a a a

a a a
aa aa

b

0

2

B

Dry weight (g/Flask)

0.5 mM Betaine
10 mM Betaineb4
6Days after inoculation

Control
1 mM Betaine
0.45
0.4
0.35
0.3
0.25
0.2
0.15
0.1
0.05
0

b

b

b

8

10

0.5 mM Betaine
10 mM Betaine
a

a
a

a

a

a

a

a

a

aaa

a
a

a
b

a aaa

0

2

b

b

4
6
8
Days after inoculation
74

b

b

10

Figure 4.12 Effects of exogenous betaine on BY-2 cell growth. A, Shows the cell growth
based on fresh weight and B, shows the cell growth based on dry weight in response to 0.5
mM, 1 mM, 10 mM and 20 mM betaine at 0, 2, 4, 6, 8 and 10 days after inoculation. Averagesof cell growth from three independent experiments (n = 3) are shown. Error bars represent
SE. Based on p values obtained in the t-test, there were no significant differences (p<0.05)
between control (untreated) cells and treated cells in FW or DW at each time point after
inoculation.

4.4.13 Effects of exogenous betaine on the inhibition of BY-2 cell growth by arsenate
Figure 4.13 shows that exogenous betaine at 0.5 mM and 1 mM did not affect BY-2 cell
growth as well as did not improve arsenate-induced cell growth reduction. These results
suggest that the arsenate in the presence of betaine did not enhance the cell growth
reduction.

A

Control
As + 0.5 mM Betaine

Fresh Weight (g/Flask)

12

60 µM AsAs + 1 mM Betaine
a

a

10
8

a

6
4
2

a a a a

b b b

0

4

b b b

b b b

0
6

Days after inoculation

75

8

B

Control

60 µM AsAs + 0.5 Betaine

As + 1 Betaine

Dry weight (g/Flask)

0.5

a
a

0.4

a

0.3
b b b

0.2
0.1

b b b

b b b

a a a a

0
0

4

6

8

Days after inoculation

Figure 4.13 Effects of exogenous betaine on 60 µM arsenate (As)-stressed BY-2 cells.
Exogenous betaine at 0.5 mM and 1 mM (Fresh weight basis, A; dry weight basis, B) showedsimilar effect on BY-2 cell growth at AsO4- stress. Averages of cell growth from three
independent experiments (n = 3) are shown. The error bars represent SE. For the same
inoculation day, values indicated by the same letter do not differ significantly at 5% level ofsignificance as determined by Tukey’s test.

4.4.14 Effects of exogenous proline on lipid peroxidation of tobacco BY-2 cells in
absence and presence of arsenate
We measured the MDA, a product of lipid peroxidation, contents in BY-2 cells to investigate
the protective effect of exogenous proline against AsO4- induced oxidative damage (Fig.
4.14). At 60 µM AsO4- stress, BY-2 cells did not show an increased level of MDA (Lipid
peroxidation) content compared with control (Fig.4.14A). Exogenous proline at 0.5 mM, 1
mM and 10 mM did not show any effect on MDA content in BY-2 cells in absence of AsO476

(Fig. 4.14A). In the presence of arsenate, 1mM and 10 mM exogenous proline significantly
decreased the MDA content in BY-2 cells compared with AsO4- stress only.

Aa

MDA (n mole/g FW)

15

a

a

a

a

12
9
6
3
0

B
MDA (n mole /g FW)

15

a

ab

b

12

c

9

d

6
3
0

Figure 4.14 Effects of exogenous proline (Pro) on lipid peroxidation at 60 µM AsO4--stressed
BY-2 cells. Exogenous Pro effect on MDA content in BY-2 cells cultured at 0.5 mM, 1 mM
and 10 mM Pro (A, in absence of arsenate, B, in presence of arsenate stress). Averages ofthree independent experiments (n = 3) are shown. The error bars represent SE. For the same
inoculation day, values indicated by the same letter do not differ significantly at 5% level ofsignificance as determined by Tukey’s test.

77

4.5 DISCUSSION
Arsenic, the most toxic metalloid, widely concerned and distributed in the environment.
Plants take up arsenic mainly as arsenate. Exposure to arsenate causes considerable stressin plants, including inhibition of growth (Stoeva and Bineva, 2003), physiological disorders(Stoeva et al., 2005) and finally death. It was reported that proline mitigates heavy-metal
toxicity by different mechanisms in plants. However, whether proline mitigates AsO4- stressin BY-2 cells are to be investigated. In the previous study, we have presented the AsO4-induced growth inhibition of BY-2 cells and the increasing rate of growth inhibition by that
arsenate in the presence of higher proline. In the present study, we examine that higher
proline decreased the glutathione content and did not show the effect on arsenate reductase
activity as well as decreased the activity of SOD. We also investigated the effects of somestress-related organic molecules on the BY-2 cell growth both under stress and non-stressconditions to compare the results with proline effects, as well as to clarify the prolineenhanced cell growth reduction by arsenate.
In the present study, the proline content was increased both under arsenic-stressedand non-stressed conditions in a concentration-dependent manner in response to theapplication of exogenous proline (Fig. 4.1). It was reported that GSH content is significantly
increased in plants upon arsenic exposure (Srivastava et. al., 2007). Our data also showedthat total GSH content is increased in AsO4--stressed BY-2 cells (Fig. 4.2A), which indicatesthat GSH is an important element that regulates stress-induced changes in plants. In thisstudy, we also found that the total GSH, GSH and GSSG contents are decreased in BY-2
cells under arsenate stress condition in presence of exogenous proline (Fig. 4.2A, B and C).
On the other hand, Agarwal et al, (2011) reported that GSH content is increased in prolinetreated bean (Phaseolus vulgaris) plants that mitigate selenium stress. Previous reportssuggest that the increased level of glutathione pool is generally regarded as a protective
response against oxidative stress (May and Leaver, 1993; Noctor and Foyer, 1998) and78

glutathione-deficient plants are hypersensitive to arsenate (Li et al., 2006). To further clarify
with this, we investigated the effect of proline at 40 µM AsO4- stress (Fig. 4.3). In the presenceof 40 µM AsO4-, neither 0.5 mM nor 1 mM proline affected the glutathione content in BY-2
cell but 10 mM proline decreased total GSH, GSH and GSSG compare with AsO4- stress.
Though it seems contradictory according to our findings but the response of GSH level interms of mitigation of stress is similar to our findings. It is well established that the
detoxification of As and Cd requires GSH and PCs in plants (Howden et al., 1995; Shi et al.,
1996; Cobbet et al., 1998; Dhankher et al., 2002; Verbruggen et al., 2009). Agarwal (2011)
reported that selenium stress is mitigated by increasing GSH level as well as we found that
GSH level is decreased and did not mitigate stress. It is reported that the decreased level of
GSH hampers the cell division as well as GSH-deficient plants are sensitive to heavy metal
stresses such as copper and cadmium (Xiang et al., 2001). It is also reported that GSH
content is enhanced in some metal-tolerant plants, such as Arabidopsis trichome (Gutiérrez
et al., 2000) and Sedum alfredii (Sun et al., 2007).
The depletion of GSH level in plants may, therefore, increase the susceptibility of BY2 cells. Similar to our findings, Xiang et al. (2001) reported that Arabidopsis plants with low
GSH level were hypersensitive to cadmium stress due to the limited capacity of these plants
to make PCs, as well as the GSH-deficient mutant cad2-1 was also found to be moresensitive to AsO4- (Li et al., 2006). Therefore, the present investigation suggests that thedecrease of total GSH and GSH by exogenous proline application may increase thesensitivity of BY-2 cells to AsO4-. Therefore, the present investigation suggests that thedecrease of both total GSH and GSH by proline at higher concentration may increase thesensitivity of BY-2 cells to AsO4-.
In this study, compared with control, exogenous proline at 10 mM decreased thearsenate reductase activity at AsO4- stress condition. Our data showed that exogenous
proline at 10 mM did not show any effect on arsenate reductase activity compared with79

arsenate stress (Fig. 4.4). It is reported that the reduction of arsenate to arsenite is the main
aspect of arsenic detoxification in plants (Zhang et al., 2002) which accelerates theconversion of arsenate to arsenite, leading detoxification and sequestration of arsenite.
These results suggest that in the presence of arsenate, exogenous proline did not detoxifyarsenate from BY-2 cells.
Previous studies reported that the antioxidant enzymes (e.g. SOD) are important forplants to defend the oxidative stress caused by arsenate (Cao et al., 2004; Srivastava et al.,2005). In this study, arsenate stress significantly increased the SOD activity compared with
control. In the presence of arsenate, proline at 10 mM decreased the SOD activity comparedwith AsO4- stress (Fig. 4.5). Previous reports indicated that antioxidant enzyme system would
be activated to avoid the oxidative damages effectively. It is also reported that PCs and antioxidative enzymes are considered as an important defense system to detoxify heavy metals
and metalloids (Sneller et al., 1999; Schmöger et al., 2000). Considering the previous reports,
our present study suggests that arsenate stress enhances the damaging effect of BY-2 cellby decreasing the SOD activity in presence of exogenous proline.
In our findings, arsenate decreased the glutathione pool in the presence of proline, andproline did not show increasing activity of arsenate reductase as well as decreased the SOD
activity in the presence of arsenate. Although further investigation is necessary to elucidate
the enhancement of sensitivity to arsenate by proline, there is a possible reason as follows(Fig. 4.15). Arsenate is converted to arsenite by arsenate reductase and arsenite can act as
an inhibitor of pyrroline-5-carboxylate dehydrogenase (P5CDH) (Nirenberg and Jakoby,
1960; Strecker, 1960), which is a member of the superfamily of aldehyde dehydrogenases.
On the other hand, proline is catabolized to cytotoxic glutamate-5-semialdehyde(GSA)/pyrroline-5-carboxylate (P5C) by proline dehydrogenase (ProDH) and then GSA/P5C
is converted to glutamate by P5CDH (Deuschle, 2001). Taken together, BY-2 cells can
produce GSA/P5C due to the catabolism in response to application of proline but cannot
80eliminate the catabolites due to the inhibition of P5CDH under arsenate stress condition.

Glutamate

Pyrroline-5-carboxylate

Pyrroline-5-carboxylate (P5C)

Arsenite

GSSG
Arsenate reductase

Spontaneous
GSH
Glutamate-5-semialdehyde (GSA)
Arsenate
Proline dehydrogenase (ProDH)
ProlineFigure 4.15 A hypothetical flow-diagram showing the inhibition of proline catabolic processby arsenite. Proline is degraded to glutamate catabolizing by the central enzymes ProDH
and P5CDH. Arsenite inhibits P5CDH activity and hence accumulates P5C/GSA which is
toxic to cells. GSH, reduced glutathione; GSSG, oxidized glutathione.
Plants have evolved various potential mechanisms to protect against the adverse
effects of heavy metal toxicity. Amino acids and their derivatives are also functions as a metalchelator that conferred to plants resistance to toxic levels of metal ions (Manara, 2012). It isreported that the concentrations of amino acid in plants under arsenic chronic stress play avital role. Arsenic toxicity altered the amino acid content and antioxidant activity (Dwivedi etal., 2010). The mechanism of 10 mM proline-enhanced negative effects of arsenate is novelone, which is also important to understand mitigatory roles of proline in BY-2 cells. In thisstudy, we investigated the effects of some stress-related organic molecules on the BY-2 cell
growth both under arsenate stress and non-stress conditions to clarify the proline-enhanced
cell growth reduction by arsenate.
The report suggests that there is an interaction between arginine and proline
metabolism, especially when they are applied exogenously. Arginine is degraded to proline
81and then further catabolized following the metabolic pathway similar to proline (Thompson,1980). In the present study, we found that arginine at 0.5 mM to 10 mM did not affect BY-2
cell growth (Fig. 4.6) but in the presence of AsO4-, like 10 mM proline, 10 mM arginine
inhibited the growth of BY-2 cell and the inhibition is more pronounced than AsO4- stress only
(Fig. 4.7). As exogenously applied arginine showed a similar effect, like proline, on BY-2 cell
growth suggesting that arginine followed the same catabolic pathway like proline and alsoenhances the sensitivity of BY-2 cell growth to arsenate.
Arsenic is non-essential and generally toxic to plants. It is reported that a coordinatedresponse of thiolic ligands and stress-responsive amino acids seems to play a role in arsenic
tolerance in plants to achieve the effective complexation of arsenic by PCs (Tripathi et al.,2013). Here, we investigated the effect of exogenous alanine on BY-2 cell growth both atarsenic stress and non-stress condition. Alanine at 0.5 mM to 10 mM did not affect BY-2 cell
growth (Fig. 4.8). In the present of AsO4-, neither 0.5 mM alanine nor 1 mM alanine affected
BY-2 cell growth but 10 mM alanine significantly ameliorates the AsO4- induced growth
inhibition (Fig. 4.9). Previous reports suggest that alanine help in the regulation of
intracellular pH without causing phytotoxicity (Sousa and Sodek, 2002), and Thakur and Rai(1985) observed that exogenous application of alanine delayed wilting under stressconditions in maize. Alanine, unlike proline, followed separate catabolic pathway and theprevious report indicated that alanine removes the feedback inhibition of proline synthetic
pathway, therefore increased the accumulation of proline during stress condition (Zhang etal., 1995; Hong et al., 2000). It is well known that during stress condition proline accumulationis necessary for the mitigation of stresses. As alanine, does not follow catabolic pathway
similar to proline, arsenate did not show any inhibitory actions. Together, these results
indicated that exogenous alanine mitigates arsenate induced growth inhibition.
It is well known that glutamate is a common biosynthetic precursor of proline (Moat etal., 2003). In the present study, exogenous glutamate at 0.5 mM, 1mM and 10 mM did not82

show any effect on BY-2 cell growth compared with control (Fig.4.10). In the presence ofarsenate, glutamate at 0.5 mM to 10 mM did not improve arsenate induced growth inhibition
of BY-2 cell (Fig. 4.11). These results suggest that glutamate did not mitigate the AsO4induced BY-2 cell growth reduction.
Betaine has been accumulated by plants and studied extensively as a compatible
solute. Exogenous application of betaine increased the tolerance of plants to abiotic stress(Chen and Murata, 2008). However, the protective effect of betaine against arsenate toxicity
in BY-2 cells needs to be investigated. Exogenous betaine at 0.5 mM and 1 mM did not show
any effect on BY-2 cell growth but significantly inhibited by 10 mM (Fig. 4.12). At AsO4- stress,
both 0.5 mM and 1mM betaine did not improve arsenate induced growth inhibition of BY-2
cell (Fig. 4.13). Though exogenous betaine has been shown to mitigate NaCl- or Cd -induced
growth inhibition of tobacco BY-2 cells (Hoque et al., 2007; Islam et al., 2009), our results
suggests that betaine did not mitigate the AsO4- induced cell growth reduction.
In conclusion, exogenous proline plays dynamic roles in BY-2 cells under arsenic
stress, which depends on its level of concentration. In our study, exogenous proline at higher
concentration did not mitigate arsenate stress and further sensitizes the BY-2 cells toarsenate. Though our previous reports have been shown that exogenous proline suppress
cell death and confer tolerance to NaCl stress and Cd stress (Haque et al., 2007, 2008; Banu
et al., 2009; Islam et al., 2009) but exogenous proline at higher concentration did notameliorate arsenate stressed BY-2 cells growth inhibition. The enhancement of stressing
effect by arsenic stress in the presence of higher proline will open new insights for further
studies of stress mitigation in plants.

83

SUMMARY
The toxic metalloid arsenic is widely distributed in soil environment and causes physiologicaland structural disorders in plants. The reduction of crop yield by arsenic stress has beenrecognized as a threat to the sustainable food production. Arsenic occurs predominantly as
inorganic forms such as arsenate (AsO4-) and arsenite (AsO3-). Plants take up arsenic mainly
as arsenate. Proline is accumulated as a compatible solute in plants under various stressconditions. Exogenous proline scavenges free radicles, improves plant metabolism andstimulates plant growth under stress conditions. However, in some cases, exogenous proline
showed its toxicity to plants and caused programmed cell death. It is reported that proline
ameliorates heavy-metal toxicity in plants. However, the role of proline in arsenate-stressed
BY-2 cells remains unclear. In this study, I investigated the effects of exogenous proline on
tobacco BY-2 cells cultured under AsO4- stress and found that proline depending on its
concentrations plays dynamic roles in BY-2 cells such as mitigation of arsenate stress in
response to lower concentrations of proline (e.g., 0.05 mM), whereas sensitization of BY-2
cells to arsenate in response to higher proline treatment (e.g., 10 mM).
The effects of exogenous proline, exogenous arsenate, and the co-treatment ofarsenate and proline on BY-2 cells growth, cell death, and cell number. Here, AsO4significantly inhibited the growth of BY-2 cells at 60 µM but not at either 40 µM or 50 µM.
Proline at 0.05 mM to 10 mM did not affect the cell growth but delayed it at 20 mM. Therefore,
for mitigating the arsenic stress, I examined the effects of a wide range (0.05, 0.1, 0.5, 1,
and 10 mM) of exogenous proline on the inhibition of cell growth by 60 µM arsenate andfound that proline at 0.05 mM alleviated the arsenate-induced cell growth inhibition, but
surprisingly accelerated the growth inhibition at 1 mM and 10 mM. Proline at 0.05 mM and
0.1 mM significantly decreased the number of Evans Blue stained cells but 10 mM proline
boosted the number of stained cells. Proline at 0.05 mM and 0.1 mM increased the total
84

number of cells, whereas 10 mM proline decreased the total number of cells. These results
indicate that the effects of 0.05 mM proline on arsenate-stressed BY-2 cells reversed with
the increase of proline concentration to 10 mM, and also suggests that the lower
concentration of proline mitigates arsenate stress, whereas the higher concentration of
proline sensitizes BY-2 cells to arsenate. To insight into the issue that 10-mM proline
enhances the sensitivity of BY-2 cells to arsenate, I further investigated the effects of 10 mM
proline on BY-2 cells treated with 40 µM arsenate and found that 40 µM arsenate did notinhibit cell growth in the absence of proline but inhibits it in the presence of proline,
suggesting that application of 10 mM proline enhances the adverse effects of arsenate.
Together, these results suggest that proline plays two distinct roles in BY-2 cells in thepresence of arsenate.
The molecular mechanisms of the lower proline in BY-2 cells under arsenate stresswere described. The mechanisms of how proline mitigates stress in plants are not fully
understood but appear to involve its chemical properties and effects on redox systems suchas the glutathione pool. Glutathione is the major source of non-protein thiols in plant cells
and functions as a key component of the antioxidant network. The increased level ofglutathione pool is generally regarded as a protective response against oxidative stress. The
reduced glutathione content is associated with the protection to oxidative stress in plants. In
the presence of AsO4-, 10 mM proline decreased the total glutathione content compared withboth control and arsenate but not 0.05 mM proline. Furthermore, 0.05 mM proline decreased
the reduced glutathione and increased the oxidized glutathione contents compared witharsenate stress, whereas 10 mM proline decreased both reduced and oxidized glutathionelevels. These results suggest that during mitigation process proline maintains glutathione
homeostasis, whereas, during sensitization of BY-2 cells to arsenate, the glutathione pool isdecreased.
Arsenate is readily reduced to arsenite through arsenate reductase using reduced85

glutathione as a reductant. Here, at AsO4- stress condition, compared with control, 0.05 mM
proline did not show any effects on arsenate reductase activity but 10 mM proline decreased
it. Compared with arsenate stress, 0.05 mM proline significantly increased the arsenate
reductase activity, but 10 mM proline did not. These results suggest that proline mitigatesarsenate stress by increasing the arsenate reductase activity which accelerates theconversion of arsenate to arsenite, leading detoxification and sequestration of arsenite. The
activity of antioxidant enzymes is important for stress mitigation. It is also reported that the
SOD activity is decreased at AsO4- stress condition in Glycine max (Chandrakar et al., 2016).
It is reported that the SOD activity is increased at AsO4- stress condition. Here, proline at
0.05 mM and 0.1 mM did not show any effect on the SOD activity in the presence of arsenate.
On the reverse, exogenous proline at 10 mM decreased the activity of SOD compared with
AsO4- stress only.
The above data indicate that 10 mM proline inversely regulates all the parameters
those were positively regulated by 0.05 mM proline, and enhances stressing effects. The
mechanism of 0.05 mM proline-induced stress mitigation is consistent with the previous
reports. However, the mechanism of 10 mM proline-enhanced negative effects of arsenate
is novel one, which is also important to understand mitigatory roles of proline in BY-2 cells.
Although further investigation is necessary to elucidate the enhancement of toxicity ofarsenate by proline, there is a possible reason for the enhancement as follows. Arsenate isconverted to arsenite by arsenate reductase and arsenite can act as an inhibitor of pyrroline5-carboxylate dehydrogenase (P5CDH), which is a member of the superfamily of aldehyde
dehydrogenases. On the other hand, proline is catabolized to cytotoxic glutamate-5semialdehyde (GSA)/pyrroline-5-carboxylate (P5C) by proline dehydrogenase (ProDH) andthen GSA/P5C is converted to glutamate by P5CDH. Together, BY-2 cells can produce
GSA/P5C due to the catabolism in response to application of proline but cannot eliminate
the catabolites due to the inhibition of P5CDH under arsenate stress condition.
86

I investigated the effects of some stress-related organic molecules on the BY-2 cell
growth both under arsenate-stress and non-stress conditions to compare the results withproline effects, as well as to clarify the proline-enhanced cell growth reduction by arsenate.
It has been reported that arginine and proline are synthesized independently but notdegraded independently. There is a considerable interconnection between their catabolic
pathways when applied exogenously. Arginine is degraded to proline and then further
catabolized following the metabolic pathway similar to proline. Here, I found that 10 mM
arginine did not affect BY-2 cell growth, but in the presence of arsenate, like 10 mM proline,
10 mM arginine enhanced the cell growth reduction by arsenate. As arginine follows proline
degradation pathway, so the effects of arsenate in BY-2 cells in the presence of proline orarginine also same. This result also strengthens the arsenite-induced P5CDH inhibitory
mechanism of negative effects by arsenate. Alanine, unlike proline, follows separate
catabolic pathway and alanine inhibits arginine degradation process. Here, exogenous
alanine did not affect BY-2 cell growth at 0.5 mM to 10 mM. In the presence of AsO4-, neither
0.5 mM nor 1 mM alanine affected the cell growth but 10 mM alanine significantly recovered
the AsO4--induced growth inhibition. These results suggest that exogenous alanine mitigatesarsenate stress. As alanine, does not follow catabolic pathway similar to proline, arsenate
did not show any inhibitory actions. Betaine is a compatible solute that accumulates in plants
and mitigates stress. Here, I found that exogenous betaine at 0.5 mM and 1 mM did notaffect BY-2 cell growth and did not improve arsenate-induced cell growth reduction. Unlike
proline effects, the arsenate in the presence of betaine did not enhance the cell growth
reduction, suggesting that arsenate, particularly in the presence of proline, increased thesensitivity of BY-2 cells.

87CONCLUSIONS
Proline plays dual roles in BY-2 cells under arsenate stress, which depends on its level ofconcentration. The lower concentration of proline mitigates arsenate stress in BY-2 cells but
the higher concentration of proline sensitizes that cells to arsenate.
-

Proline at 0.05 mM mitigated AsO -induced growth inhibition of BY-2 cells by
4decreasing reduced GSH and increasing oxidized GSH. Proline at 0.05 mM increased thearsenate reductase activity that utilized reduced GSH as a reductant, which leads to theconversion of arsenate to arsenite and detoxification processes. Thus, proline mitigatesarsenate stress in BY-2 cells.
Proline at 10 mM decreased the total GSH, reduced GSH and oxidized GSH as well
as did not increase the arsenate reductase activity which is responsible for the conversionof arsenate to arsenite. Moreover, 10 mM proline decreased the antioxidant enzyme such
SOD activity.

88

ACKNOWLEDGEMENTS
It is the blessings of the creator who enable and keep me in good health to carry out myresearch and writing dissertation to complete my doctoral course.
I would like to express my cordial gratitude and sincere appreciation to my researchsupervisor Dr. Yoshiyuki Murata, Professor, Faculty of Agriculture, Okayama University,
Japan for your kind and scholastic guidance throughout the doctoral course.
I also express my sincere thanks to Dr. Yoshimasa Nakamura for your valuable discussion
and suggestions throughout the course of this study. It is always enlightening andenjoyable to talk with you.
It is my immense pleasure to acknowledge Dr. Shintaro Munemasa for his help and valuableadvice during the research period.
Thank goes to Toshiyuki Nakamura for your suggestions regarding scientific meetings anduse of laboratory equipment.
Thank goes to Professor Yoshinobu Kimura, Faculty of Agriculture for his valuable comments.
I would like to express my heartiest appreciation and indebtedness to Md. Yeasin Prodhan
for being the best companion, continuous cooperation and for encouraging me
throughout the study period. I am also grateful to my beloved son Md. Nahin Prodhan
for his special sacrifice.
Thank goes to Anna Yonezawa for her cordial help during the study period. 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. Abdul Majid and Mst. Rowsan Ara.

89

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