STUDY OF HEAVY METAL TOLERANCE, ACCUMULATION AND
RECOVERY IN HYPERACCUMULATOR PLANT SPECIES
By
RENGASAMY BOOMINATHAN
A thesis submitted for the degree of Doctor of Philosophy
School of Biotechnology and Biomolecular Science
The University of New South Wales
June,2002 Dedicated To My Mother TABLE OF CONTENTS
Page
Table of Contents
List of Figures vm
List of Tables 11 L- .. . XXl L~ 6\- Abb'£evfaa c.-J.Ot !$, List of Appendices xxn
Abstract xxm
Acknowledgments XXVI
CHAPTER 1 - INTRODUCTION
1. 1 Heavy Metals In The Environment 1
1. 1. 1 Cadmium 1
1. 1. 2 Nickel 2
1. 2 Phytoremediation 3
1. 3 Phytomining 4
1. 4 Hyperaccumulators 6
1. 5 Mechanisms of Heavy Metal Accumulation and Tolerance 8
1. 5. 1 Compartmentation 9
1. 5. 1. 1 Distribution Between Organs 9
1. 5. 1. 2 Intraorgan and Intracellular Distribution 12
1. 5. 2 Complexation With Organic Acids 14
1. 6 Oxidative Stress 16
1. 6. 1 Superoxide Dismutase 18
1. 6. 2 Catalase 20
1. 6. 3 Ascorbate Peroxidase 21 11
1. 6. 4 Hydrogen Peroxide 23
1. 6. 5 Lipid Peroxidation 24
1. 6. 6 Glutathione 26
1. 6. 6. 1 Effect of Heavy Metal 27
1. 6. 6. 2 Glutathione and Hydrogen Peroxide 28
1. 6. 7 Sulfhydryl Groups 29
1. 7 Phytochelatins 31
1. 8 Hairy Root Cultures 33
1. 9 Aims of This Study 35
CHAPTER 2 - MATERIALS AND METHODS
2. 1 Hairy Root Cultures 36
2. 1. 1 Nickel Hyperaccumulator (A. bertolonii) and Non-hyperaccumulator
(N. tabacum) 38
2. 1. 1. 1 Short-term Ni Uptake by Live and Dead Biomass 38
2. 1. 1. 2 Culture Experiments 38
2. 1. 1. 3 Effect ofH+-ATPase Inhibitor 38
2. 1. 2 Cadmium Hyperaccumulator (T. caerulescens) and Non-hyperaccumulator
(N. tabacum) 39
2. 1. 2. 1 Short-term Cd Uptake by Live and Dead Biomass 39
2. 1. 2. 2 Culture Experiments 39
2. 1. 2. 3 Effect ofH+-ATPase Inhibitor 39
2. 1. 2. 4 Effect of Glutathione Synthesis Inhibitor 39
2. 1. 2. 5 Effect of Free Radical Generators 40
2. 1. 2. 6 Effect of Free Radical Scavenger 40
2.2 Nickel Recovery 41 lll
2.2. 1 Hairy Root Cultures 41
2.2.2 Whole Plant Cultivation 41
2.2.3 Nickel Recovery by Furnace Treatment 42
2. 3 Analytical Procedures 43
2. 3. 1 Biomass Fresh and Dry Weight 43
2.3.2 Heavy Metal Concentrations 43
2.3.3 Distribution of Cd or Ni in Hairy Roots 44
2. 3. 3. 1 Apoplasm and Symplasm 44
2.3.3.2 Microscope Analysis 44
2.3.4 Organic Acids 45
2.3.4. 1 Extraction 45
2.3.4.2 Gel Filtration 46
2.3.4.3 HPLC Measurements 46
2.3.5 Superoxide Dismutase Assay 47
2.3.6 Catalase Assay 47
2.3. 7 Ascorbate Peroxidase Assay 48
2.3. 8 Hydrogen Peroxide Assay 48
2.3.9 Estimation of Malondialdehyde 49
2. 3. 10 Total Glutathione Assay 49
2. 3. 11 Estimation of Free -SH groups on Root Cell Surfaces 50
2.3. 12 Field Emission Scanning Electron Microscope (FESEM) Analysis 51
2. 3. 13 X-ray Diffraction Analysis 51
2.3. 14 Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES)
Analysis 51
2.3. 15 Statistical Analysis 52 lV
CHAPTER 3 - RESULTS
3. 1 Growth of Hairy Roots 53
3. 1. 1 Nickel Hyperaccumulator (A. bertolonii) and Non-hyperaccumulator 53
(N. tabacum)
3. 1. 1. 1 Effect of Ni 53
3. 1. 1. 2 Effect of Ni and H+-ATPase Inhibitor 56
3. 1. 2 Cadmium Hyperaccumulator (T. caerulescens) and Non-hyperaccumulator
(N. tabacum) 57 p• I. 2-• la_ [ 1=tel: o:f. In.4, ol f"·le.d-iu r • , 'p I--\ 3. 1. 2. 1b Effect of Cd and Zn 57
3. 1. 2. 2 Effect of Cd and H+-ATPase Inhibitor 60
3. 1. 2. 3 Effect of Cd and Glutathione Synthesis Inhibitor 62
3. 1. 2. 4 Effect of Cd and Free Radical Generators 63
3. 1. 2. 5 Effect ofCd and Free Radical Scavenger 65
3. 2 Heavy Metal Uptake 66
3. 2. 1 Nickel Accumulation in A. bertolonii and N. tabacum Hairy Roots 66
3. 2. 1. 1 Live and Dead Biomass 66
3. 2. 1. 2 Effect ofH+-ATPase Inhibitor 68
3. 2. 2 Cadmium Accumulation in T. caerulescens and N. tabacum Hairy Roots 69
3. 2. 2. 1 Live and Dead Biomass 69
3.2.2.2 EffectofZn 71
3. 2. 2. 3 Effect ofH+-ATPase Inhibitor 72
3. 2. 2. 4 Effect of Glutathione Synthesis Inhibitor 74
3. 2. 2. 5 Effect of Free Radical Generator 75
3. 2. 2. 6 Effect of Free Radical Scavenger 75
3. 3 Heavy Metal Distribution 77
3. 3. 1 Nickel Distribution in A. bertolonii and N. tabacum Hairy Roots 77 V
3. 3. 1. 1 Effect of Solvent Incubation Time and Agitation 77
3. 3. 1. 2 Effect ofH+-ATPase Inhibitor 78
3. 3. 1. 3 Microscope Analysis 81
3. 3. 2 Cadmium Distribution in T. caerulescens and N tabacum Hairy Roots 84
3. 3. 2. 1 Effect of Solvent Incubation Time and Agitation 84
3.3.2.2 EffectofZn 84
3. 3. 2. 3 Effect ofH+-ATPase Inhibitor 87
3. 3. 2. 4 Effect of Glutathione Synthesis Inhibitor 88
3. 3. 2. 5 Microscope Analysis 90
3. 4 Organic Acids 93
3. 4. 1 Concentration of Organic Acids in A. bertolonii and T. caerulescens Hairy
Roots 93
3.4.2 Association of Ni and Cd with Organic Acids 97
3.5 Oxidative Stress Parameters 102
3. 5. 1 Nickel Hyperaccumulator and Non-hyperaccumulator 102
3. 5. 1. 1 Superoxide Dismutase 102
3. 5. 1. 2 Catalase 104
3. 5. 1. 3 Ascorbate Peroxidase 105
3. 5. 1. 4 Hydrogen Peroxide 107
3. 5. 1. 5 Malondialdehyde 108
3. 5. 1. 6 Sulphydryl Groups on Root Cell Surfaces 110
3.5.2 Cadmium Hyperaccumulator and Non-hyperaccumulator 111
3. 5. 2. 1 Superoxide Dismutase 111
3.5.2.2 Catalase 113
3.5.2.3 Ascorbate Peroxidase 114
3. 5.2.4 Hydrogen Peroxide 116 Vl
3. 5. 2. 5 Malondialdehyde 117
3. 5. 2. 6 Total Glutathione 119
3. 5. 2. 7 Sulphydryl Groups on Root Cell Surfaces 120
3. 6 Nickel Recovery 124
3. 6. 1 Alyssum bertolonii Hairy Roots 124
3. 6. 1. 1 Inductively Coupled Plasma-Atomic Emission Spectrometry
(ICP-AES) Analysis 124
3. 6. 1. 2 Furnace Treatment 125
3. 6. 1. 2a Effect of Time 125
3. 6. 1. 2b Effect oflnitial Concentration of Ni 126
3. 6. 1. 2c Effect of Nitrogen 131
3. 6. 2 Berkheya coddii Plants 134
3. 6. 2. 1 Biomass Dry Weight and Ni Uptake 134
3. 6. 2. 2 ICP-AES Analysis 135
3. 6. 2. 3 Furnace Study 136
CHAPTER 4 - DISCUSSION
4. 1 Growth Analyses 141
4. 2 Nickel Uptake and Distribution 143
4. 3 Cadmium Uptake and Distribution 146
4. 4 Organic Acid Complexation 148
4. 5 Oxidative Stress in Ni-hyperaccumulator and Non-hyperaccumulator 150
4. 6 Oxidative Stress in Cd-hyperaccumulator and Non-hyperaccumulator 154
4. 7 Nickel Recovery 160
4. 7. 1 Alyssum bertolonii Hairy Roots 160
4. 7. 2 Berkheya coddii Plants 161 Vll
CHAPTER 5 - CONCLUSIONS
5. 1 Nickel-hyperaccumulator and Non-hyperaccumulator 164
5. 2 Cadmium-hyperaccumulator and Non-hyperaccumulator 165
5. 3 Nickel Recovery 166
REFERENCES
APPENDIX Vlll
LIST OF FIGURES
Figure 1.1
Possible mechanisms of metal tolerance in plants.
Figure 1.2
Interactions between the reduced and oxidised forms of glutathione and ascorbate in removal ofH2O2.
Figure 3.1
Growth of hairy roots of A. bertolonii without and with 25 ppm Ni.
Figure 3.2
A. bertolonii hairy roots in B5 medium without Ni after 21 days and with 25 ppm Ni after 28 days.
Figure 3.3
Growth of hairy roots of N. tabacum without and with 25 ppm Ni.
Figure 3.4
N tabacum hairy roots in B5 medium without Ni and with 25 ppm Ni after 21 days.
Figure 3.5
Growth of hairy roots of A. bertolonii without Ni and DES with 100 µM DES and 25 ppm
Ni+ 100 µM DES.
Figure 3.6
Growth of hairy roots of N. tabacum without Ni and DES with 100 µM DES and 25 ppm Ni
+ 100 µM DES.
Figure 3.7
Hairy roots of T caerulescens cultured without Cd and with 20 ppm Cd after 21 days.
Figure 3.8
Growth of hairy roots of T caerulescens without Cd and Zn with 20 ppm Cd and 20 ppm
Cd + 33 ppm Zn. lX
Figure 3.9
Growth of hairy roots of N. tabacum without Cd and Zn with 20 ppm Cd and 20 ppm Cd +
33 ppmZn.
Figure 3.10
Hairy roots of N. tabacum cultured without Cd and with 20 ppm Cd after 21 days.
Figure 3.11
Growth of hairy roots of T caerulescens without Cd and DES, with 100 µM DES and 20 ppm Cd + 100 µM DES.
Figure 3.12
Growth of hairy roots of N. tabacum without Cd and DES, with 100 µM DES and 20 ppm
Cd + 100 µM DES.
Figure 3.13
Growth of hairy roots of T caeru/escens without Cd and BSO, with 100 µM BSO and 20 ppm Cd + 100 µM BSO.
Figure 3.14
Growth of hairy roots of N. tabacum without Cd and BSO, with 100 µM BSO and 20 ppm
Cd + 100 µM BSO.
Figure 3.15
Growth of hairy roots of T caerulescens without Cd and CHP, with 33 µM CHP and 20 ppm
Cd + 100 µM CHP.
Figure 3.16
Growth of hairy roots of N. tabacum without Cd and CHP, with 33 µM CHP and 20 ppm Cd
+ 100 µMCHP.
Figure 3.17
Growth of hairy roots of T caerulescens without Cd and MAN, with 50 mM MAN and 20
ppm Cd + 50 mM MAN. X
Figure 3.18
Growth of hairy roots of N. tabacum without Cd and MAN, with 50 mM MAN and 20 ppm
Cd + 50 mM MAN.
Figure 3.19
Ni uptake by live and dead hairy roots of A. bertolonii in short-term experiment with an initial concentration of 25 ppm Ni.
Figure 3.20
Ni uptake by live and dead hairy roots of N. tabacum in short-term experiment with an initial concentration of25 ppm Ni.
Figure 3.21
Effect of 100 µM of the ATPase inhibitor, DES, on Ni uptake by hairy roots of A. bertolonii with an initial concentration of 25 ppm Ni. Hairy roots were grown with DES and without
DES.
Figure 3.22
Effect of 100 µM of the ATPase inhibitor, DES, on Ni uptake by hairy roots of N. tabacum with an initial concentration of 25 ppm Ni. Hairy roots were grown with DES and without
DES.
Figure 3.23
Cd uptake by live and dead hairy roots of T. caerulescens in short-term experiment with an initial concentration of 20 ppm Cd.
Figure 3.24
Cd uptake by live and dead hairy roots of N. tabacum in short-term experiment with an initial concentration of 20 ppm Cd.
Figure 3.25
Effect of 33 ppm Zn on Cd uptake by hairy roots of T. caerulescens with an initial concentration of 20 ppm Cd. Hairy roots were grown with Zn or without Zn. Xl
Figure 3.26
Effect of 33 ppm Zn on Cd uptake by hairy roots of N tabacum with an initial concentration of 20 ppm Cd. Hairy roots were grown with Zn or without Zn.
Figure 3.27
Effect of 100 µM of the ATPase inhibitor, DES, on Cd uptake by hairy roots of T. caerulescens with an initial concentration of 20 ppm Cd. Hairy roots were grown with DES and without DES.
Figure 3.28
Effect of 100 µM of the ATPase inhibitor, DES, on Cd uptake by hairy roots of N tabacum with an initial concentration of 20 ppm Cd. Hairy roots were grown with DES and without
DES.
Figure 3.29
Effect of 100 µM of the GSH synthesis inhibitor, BSO, on Cd uptake by hairy roots of T. caerulescens with an initial concentration of 20 ppm Cd. Hairy roots were grown with BSO and without BSO.
Figure 3.30
Effect of 100 µM of the GSH synthesis inhibitor, BSO~ on Cd uptake by hairy roots of N tabacum with an initial concentration of 20 ppm Cd. Hairy roots were grown with BSO and without BSO.
Figure 3.31
Effect of 33 µM of the free radical generator, CHP, and 50 µM of the free radical scavenger,
MAN, on Cd uptake by hairy roots of T. caerulescens with an initial concentration of 20 ppm Cd. Hairy roots were grown without CHP and MAN, with CHP and MAN. Xll
Figure 3.32
Effect of 33 µM of the free radical generator, CHP, and 50 µM of the free radical scavenger,
MAN, on Cd uptake by hairy roots of N. tabacum with an initial concentration of 20 ppm
Cd. Hairy roots were grown without CHP and MAN, with CHP and MAN.
Figure 3.33
The distribution of Ni between the apoplasm and symplasm fractions of A. bertolonii hairy roots cultured with an initial concentration of 25 ppm Ni.
Figure 3.34
The distribution of Ni between the apoplasm and symplasm fractions of N. tabacum hairy roots cultured with an initial concentration of 25 ppm Ni.
Figure 3.35
The distribution of Ni between the apoplasm and symplasm fractions of A. bertolonii hairy roots cultured with an initial concentration of 25 ppm Ni+ 100 µM DES.
Figure 3.36
The distribution of Ni between the apoplasm and symplasm fractions of N. tabacum hairy roots cultured with an initial concentration of 25 ppm Ni + 100 µM DES.
Figure 3.37
Localisation of Ni using DMG visualised under a light microscope for 21-day-old in A. bertolonii and N. tabacum hairy roots.
Figure 3.38
The distribution of Cd between the apoplasm and symplasm fractions of T caerulescens hairy roots cultured with an initial concentration of 20 ppm Cd.
Figure 3.39
The distribution of Cd between the apoplasm and symplasm fractions of N. tabacum hairy roots cultured with an initial concentration of 20 ppm Cd. Xlll
Figure 3.40
The distribution of Cd between the apoplasm and symplasm fractions of T. caerulescens hairy roots cultured with an initial concentration of 20 ppm Cd + 33 ppm Zn.
Figure 3.41
The distribution of Cd between the apoplasm and symplasm fractions of N tabacum hairy roots cultured with an initial concentration of20 ppm Cd + 33 ppm Zn.
Figure 3.42
The distribution of Cd between the apoplasm and symplasm fractions of T. caerulescens hairy roots cultured with an initial concentration of 20 ppm Cd + 100 µM DES
Figure 3.43
The distribution of Cd between the apoplasm and symplasm fractions of N tabacum hairy roots cultured with an initial concentration of 20 ppm Cd + 100 µM DES
Figure 3.44
The distribution of Cd between the apoplasm and symplasm fractions of T. caerulescens hairy roots cultured with an initial concentration of 20 ppm Cd + 100 µM BSO
Figure 3.45
The distribution of Cd between the apoplasm and symplasm fractions of N tabacum hairy roots cultured with an initial concentration of 20 ppm Cd + 100 µM BSO
Figure 3.46
Localisation of Cd using BTAN-D visualised under a light microscope for 21-day-old T. caerulescens and N tabacum hairy roots.
Figure 3.47
Citric acid content of A. bertolonii hairy roots cultured with and without 25 ppm Ni.
Figure 3.48
Malic acid content of A. bertolonii hairy roots cultured with and without 25 ppm Ni. XIV
Figure 3.49
Malonic acid content of A. bertolonii hairy roots cultured with and without 25 ppm Ni.
Figure 3.50
Citric acid content of T. caerulescens hairy roots cultured with and without 20 ppm Cd.
Figure 3.51
Malic acid content of T. caerulescens hairy roots cultured with and without 20 ppm Cd.
Figure 3.52
Malonic acid content of T. caerulescens hairy roots cultured with and without 20 ppm Cd.
Figure 3.53
The percentages of Ni in aqueous in extract and debris of A. bertolonii hairy roots cultured with 25 ppm Ni.
Figure 3.54
The percentages of Cd in aqueous in extract and debris of T. caerulescens hairy roots cultured with 20 ppm Cd.
Figure 3.55
Typical gel filtration elution profile for an aqueous extract of 14-day-old A. bertolonii hairy roots cultured with 25 ppm Ni.
Figure 3.56
Typical gel filtration elution profiles for an aqueous extract of 14-day-old T. caerulescens hairy roots cultured with 20 ppm Cd.
Figure 3.57
The percentages of Ni associated with organic acids in aqueous extracts of A. bertolonii hairy roots cultured with 25 ppm Ni.
Figure 3.58
The percentages of Cd associated with organic acids in aqueous extracts of T. caerulescens hairy roots cultured with 20 ppm Cd. xv
Figure 3.59
The percentages of total Ni associated with organic acids and in free aqueous form of A.
bertolonii hairy roots cultured with 25 ppm Ni.
Figure 3.60
The percentages of total Cd associated with organic acids m free aqueous form of T
caerulescens hairy roots cultured with 20 ppm Cd.
Figure 3.61
Superoxide dismutase activity in A. bertolonii hairy roots grown without and with Ni at an initial concentration of 25 ppm.
Figure 3.62
Superoxide dismutase activity in N. tabacum hairy roots grown without and with Ni at an initial concentration of 25 ppm.
Figure 3.63
Catalase activity in A. bertolonii hairy roots grown without and with Ni at an initial concentration of 25 ppm.
Figure 3.64
Catalase activity m N. tabacum hairy roots grown without and with Ni at an initial concentration of 25 ppm.
Figure 3.65
Ascorbate peroxidase activity in A. bertolonii hairy roots grown without and with Ni at an
initial concentration of 25 ppm.
Figure 3.66
Ascorbate peroxidase activity in N. tabacum hairy roots grown without and with Ni at an
initial concentration of 25 ppm. XVI
Figure 3.67
Levels of hydrogen peroxide in A. berto/onii hairy roots cultured without and with Ni at an initial concentration of 25 ppm.
Figure 3.68
Levels of hydrogen peroxide in N. tabacum hairy roots cultured without and with Ni at an initial concentration of 25 ppm.
Figure 3.69
Levels of malondialdehyde in A. bertolonii hairy roots cultured without and with Ni at an initial concentration of 25 ppm.
Figure 3.70
Levels of malondialdehyde in N. tabacum hairy roots cultured without and with Ni at an initial concentration of 25 ppm.
Figure 3.71
Levels of cell surface -SH groups in A. bertolonii hairy roots grown without and with Ni at an initial concentration of 25 ppm Ni.
Figure 3.72
Levels of cell surface -SH groups in N. tabacum hairy roots grown without and with Ni at an initial concentration of 25 ppm Ni.
Figure 3.73
Superoxide dismutase activity in T caeru/escens hairy roots grown without and with Cd at an initial concentration of 20 ppm.
Figure 3.74
Superoxide dismutase activity in N. tabacum hairy roots grown without and with Cd at an initial concentration of 20 ppm. XVll
Figure 3.75
Catalase activity in T. caerulescens hairy roots grown without and with Cd at an initial
concentration of 20 ppm.
Figure 3.76
Catalase activity m N. tabacum hairy roots grown without and with Cd at an initial
concentration of 20 ppm.
Figure 3.77
Ascorbate peroxidase activity in T. caerulescens hairy roots grown without and with Cd at an initial concentration of 20 ppm.
Figure 3.78
Ascorbate peroxidase activity in N. tabacum hairy roots grown without and with Cd at an initial concentration of 20 ppm.
Figure 3.79
Level of hydrogen peroxide in T. caerulescens hairy roots grown without BSO + Cd, with
Cd, with BSO and with BSO + Cd. The initial concentration of Cd was 20 ppm and BSO concentration was 100 µM.
Figure 3.80
Level of hydrogen peroxide in N. tabacum hairy roots grown without BSO + Cd, with Cd, with BSO and with BSO + Cd. The initial concentration of Cd was 20 ppm and BSO concentration was 100 µM.
Figure 3.81
Level of malondialdehyde in T. caerulescens hairy roots cultured without and with Cd at an
initial concentration of 20 ppm.
Figure 3.82
Level of malondialdehyde in N. tabacum hairy roots cultured without and with Cd at an
initial concentration of 20 ppm. xvm
Figure 3.83
Level of total glutathione (GSSG + GSH) in T. caerulescens hairy roots grown without BSO
+ Cd, with Cd, with BSO, with BSO + Cd. The initial concentration of Cd was 20 ppm and the BSO concentration was 100 µM.
Figure 3.84
Level of total glutathione (GSSG + GSH) in N. tabacum hairy roots grown without BSO +
Cd, with Cd, with BSO with BSO + Cd. The initial concentration of Cd was 20 ppm and the
BSO concentration was 100 µM.
Figure 3.85
Surface -SH groups in T. caerulescens hairy roots grown without BSO + Cd, with Cd, with
BSO and with BSO + Cd. The initial concentration of Cd was 20 ppm and the BSO concentration was 100 µM.
Figure 3.86
Surface -SH groups in N. tabacum hairy roots grown without BSO + Cd, with Cd, with BSO and with BSO + Cd. The initial concentration of Cd was 20 ppm and the BSO concentration was 100 µM.
Figure 3.87
Typical results for elemental composition of A. bertolonii biomass with and without Ni. The biomass Ni concentration in this sample was 5.5% (w/w).
Figure 3.88
Elemental composition of A. bertolonii hairy roots with initial (0 h) Ni concentration of
5.5% (w/w) and samples with initial Ni concentration of 6.1 % (w/w), after treatment for 3-
17 h in the furnace at 1200°C with air.
Figure 3.89
Metal residue obtained after furnace treatment of A. bertolonii biomass (7. 7% w/w Ni) at
1200°C with air passing through the furnace. XIX
Figure 3.90
Crystal-like structure of metal residue from A. bertolonii biomass (7.7% w/w Ni) after furnace treatment at 1200°C for 17 hours with air passing- through the furnace.
Figure 3.91
Qualitative elemental analysis (under FESEM) ofresidue obtained after furnace treatment of
A. bertolonii biomass containing 7.7% (w/w) Ni at 1200°C for 17 h.
Figure 3.92
Qualitative elemental analysis of alumina tile under FESEM.
Figure 3.93
Qualitative elemental analysis, using FESEM, of residue obtained after furnace treatment of
A. bertolonii biomass containing 1.9% Ni (w/w) for 17 h ·at 1200°C.
Figure 3.94
XRD analysis of metal residue obtained after furnace treatment of A. bertolonii biomass containing 7.7% (w/w) Ni for 17 hat 1200°C with air.
Figure 3.95
Elemental composition of residues after 17 h furnace treatment of A. bertolonii hairy root biomass containing Ni at different initial concentrations.
Figure 3.96
Crystal-like structure observed under FESEM for A. bertolonii hairy roots with 4.4% (w/w)
Ni treated at 1200°C for 5 h with air passing through the furnace.
Figure 3.97
Elemental composition of A. bertolonii biomass containing 4.4% (w/w) Ni after furnace treatment at 1200°C for 17 h with nitrogen and for 5 h with air.
Figure 3.98
Biomass dry weight of B. coddii grown at different initial Ni concentrations in the soil. XX
Figure 3.99
Ni concentration in the biomass of B. coddii.
Figure 3.100
Elemental composition of B. coddii biomass grown without and with Ni.
Figure 3.101
Qualitative elemental analysis, using FESEM, of ash obtained after furnace treatment of B. coddii biomass containing 0.55% (w/w) Ni for 3 hat 1200°C with air.
Figure 3.102
Qualitative elemental analysis, using FESEM, of ash obtained after furnace treatment of B. coddii biomass containing 0.55% (w/w) Ni for 24 hat 1200°C with air.
Figure 3.103
Elemental composition of B. coddii biomass containing 0.55% (w/w) Ni after treatment in the furnace at 1200°C with air for different times.
Figure 3.104
XRD analysis of B. coddii biomass containing 0.55% (w/w) Ni after treatment in the furnace at 1200°c with air for 3 + 6 h.
Figure 3.105
XRD analysis of B. coddii biomass containing 0.55% (w/w) Ni after treatment in the furnace at 1200°c with air for 17 + 24 h. XXl
LIST OF TABLES
Table 1.1 Hyperaccumulators of Ni, Cd, Zn, Pb, Co and Cu from various parts of the
world.
Table 2.1 Composition of Gamborg's B5 medium.
Table 3.1 Effect of treatment duration and agitation on the amounts of Ni recovered from
the cell wall fractions of replicate samples treated with solvents.
Table 3.2 Ni concentrations in the apoplasm and symplasm fractions of hairy roots
averaged over the culture period.
Table 3.3 Effect of treatment duration and agitation on the amounts of Cd recovered from
the cell wall fractions of replicate samples treated with solvents.
Table 3.4 Cd concentrations in the apoplasm and symplasm fractions of hairy roots
averaged over the culture period.
Table 3.5 The effect ofDTNB infiltration time on surface -SH group concentration in T.
caerulescens roots.
Table 3.6 The effect of Cd on -SH analysis.
Table 3.7 Effect of Cd on -SH analysis in extracts of T. caerulescens roots cultured with
Cd.
Table 3.8 Alyssum bertolonii biomass before and after furnace treatment for various
times at 1200°C with air.
Table 3.9 Alyssum bertolonii with various initial Ni concentrations before and after
furnace treatment for 17 hat 1200°C with air.
Table 3.10 B. coddii with 0.55% (w/w) Ni burnt at 1200°C with air passing through the
furnace for different time periods. LIST OF ABBREVIATIONS
AA Ascorbate AAPH 2,2'-azobis (2-amidinopropane) hydrochloride AOS Active oxygen species APX Ascorbate peroxidase ATPase Adenine triphosphatase BSO Buthionine sulfoximine BTAN-D Benzothiazolylazonaphthaol derivatives CAT Catalase CCD Charge coupled device CHP Cumene hydroperoxide DES Diethylstilbestrol DHA Dehydroascorbate DMG Dimethy lglyoximine DTNB 5,5' dithio-(2-nitrobenzoic acid) EDTA Ethylene diethyl tetraacetate FESEM Field emission scanning electron microscope GSH Reduced glutathione GSSG Oxidised glutathione GR Glutathione reductase H202 Hydrogen peroxide HPLC High pressure liquid chromatography ICP-AES Inductively coupled plasma-atomic emission spectrometry MAN D-Mannitol MDA Malondialdehyde NADPH Nicotinamide adenine diphosphate (reduced) PC Phytochelatin SOD Superoxide dismutase TBA Thiobarbituric acid TCA Trichloroacetic acid TNT 2,4,6,-trinitro toluene XRD X-ray diffraction xxn
LIST OF APPENDICES
Appendix 1. Standard curve for Cd determination.
Appendix 2. Standard curve for Ni determination. XXlll
Abstract
Hairy root cultures of the Ni-hyperaccumulator, Alyssum bertolonii, the Cd hyperaccumulator, Thlaspi caerulescens, and the non-hyperaccumulator, Nicotiana tabacum, were used to study growth characteristics, heavy metal uptake and distribution, and the difference between hyperaccumulators and non-hyperaccumulator in terms of their antioxidative responses to heavy metals. Heavy metal complexation with organic acids was investigated for the Ni- and Cd-hyperaccumulators. Techniques were also developed for recovery of Ni from A. bertolonii hairy roots and Berkheya coddii plants using furnace treatment.
Growth of A. bertolonii roots was unaffected by 25 ppm Ni, whereas Ni had a detrimental impact on growth of N. tabacum roots. There was no significant difference in Ni accumulation between the A. bertolonii and N. tabacum hairy roots grown in liquid medium.
About 85-95% of Ni in the roots was associated with the cell symplasm for both species.
Experiments with diethylstilbestrol (DES), a H+ -ATPase inhibitor, revealed that although
DES increased Ni accumulation in both species, Ni transport across the plasma membrane was restricted. In A. bertolonii hairy roots, Ni levels in the cell wall were increased with
DES. After treatment with 25 ppm Ni, about 28% of the total Ni in A. bertolonii roots was associated with citric, malic and malonic acids.
Studies on antioxidative responses showed that, without Ni, superoxide dismutase
(SOD) and catalase (CAT} activities in A. bertolonii hairy roots were significantly greater than in N. tabacum roots by an average factor of about 2.4 and 510, respectively. With 25 ppm Ni, SOD and CAT activities were reduced significantly in A. bertolonii roots. In N. tabacum roots, Ni had a negligible effect on SOD activity but reduced CAT activities to undetectable levels after 5 days of Ni treatment. In contrast, endogenous APX activities in N. tabacum roots were, on average, 1.8 times higher than those in A. bertolonii roots. Upon XXIV
exposure to 25 ppm Ni, APX activity was decreased by an average of 61 % in A. bertolonii
roots and 19% in N. tabacum roots, relative to the corr~sponding control roots without Ni.
Thus, typical antioxidant responses were not observed in either species.
After Ni treatment, H20 2 concentrations were increased by an average of 3.6-fold in
A. bertolonii roots and 8.6-fold in N. tabacum roots. With Ni, a consistent increase in
malondialdehyde (MDA) concentrations in N. tabacum roots suggested that destabilisation
of plasma membranes may have occurred due to lipid peroxidation. Levels of surface -SH
groups in A. bertolonii roots treated with Ni were similar to those in the controls. The
reduction in surface -SH groups in Ni-treated N. tabacum roots is consistent with oxidation of -SH groups being a major toxic effect of H20 2 build up.
Growth analyses showed that T. caerulescens roots were tolerant and N. tabacum roots were not tolerant of 20 ppm Cd. Without Cd, the growth of both species was unaffected by buthionine sulfoximine (BSO), a glutathione synthesis inhibitor, cumene hydroperoxide (CHP), a free radical generator, and D-mannitol (MAN), a free radical scavenger. However, another free radical generator, 2,2 '-azobis(2-amidinopropane) dihydrochloride (AAPH), severely inhibited the growth of T. caerulescens roots.
Studies of metal uptake revealed that dead biomass of T. caerulescens and N. tabacum accumulated 40 to 50% higher concentrations of Cd than the respective live roots.
Zn, BSO, CHP and MAN did not have a significant impact on Cd accumulation in T.
caerulescens roots, whereas Zn and BSO treatments produced a small increase on Cd
accumulation in N. tabacum roots. Metal distribution studies showed that 75-78% of total
Cd in T. caerulescens and N. tabacum roots was localised in the cell apoplasm. Treatment
with DES increased the concentration of Cd in the symplasm of T. caerulescens roots about
6-fold with retention of root viability, and also increased total Cd uptake levels. After
treatment with 20 ppm Cd, about 13% of the total Cd in T. caerulescens roots was associated
with citric, malic and malonic acids. XXV
Without Cd, endogenous SOD and CAT activities in T. caerulescens roots were
about 1.8 and 300 times higher than in N. tabacum roots, respectively. Cd did not have a
significant effect on SOD activity but induced CAT activity in T. caerulescens roots. In
contrast, Cd reduced SOD activities significantly in N. tabacum roots.
Upon exposure to Cd, BSO, and BSO + Cd, H2O2 levels in T. caerulescens roots
were essentially unchanged. However, N. tabacum roots failed to control H2O2
accumulation, which may have resulted in oxidative damage to membranes and proteins and
growth inhibition. The depletion of glutathione levels in T. caerulescens did not affect Cd tolerance or the ability to control H2O2 levels, indicating that glutathione does not play a pivotal role in the Cd-hyperaccumulator. In contrast, glutathione depletion had a significant impact in N. tabacum roots and reduced its tolerance to Cd. A significant increase in surface
-SH groups was observed in T. caerulescens roots upon Cd exposure, whereas the effect of
Cd on surface -SH groups was negligible in N. tabacum roots. Addition of Cd increased
MDA levels in both T. caerulescens and N. tabacum roots, but the concentration of MDA in
Cd-treated N. tabacum roots was about 80% higher than in Cd-treated T. caerulescens.
Nickel recovery from A. bertolonii hairy roots was achieved after furnace treatment.
The metallic residues after furnace treatment of biomass containing above 5.1 % (w/w) Ni
contained about 60% Ni. However, when nitrogen was· passed through the furnace, nickel
oxide formation was inhibited. In Berkheya coddii plants, the maximum Ni accumulation
achieved in the dry biomass was 0.55% (w/w). After furnace treatment, the B. coddii
biomass yielded a sulphur-free 'bio-ore' containing 8.6% Ni.
This study demonstrates that although Ni and Cd are non-redox-active metals, they
induced oxidative stress during metal-accumulation processes m hairy roots.
Hyperaccumulating plant species could be a powerful tool for phytoremediation and
phytomining. XXVI
ACKNOWLEDGMENTS
I wish to express my deep sense of gratitude to Associate Professor Pauline M. Doran for her bright ideas, constructive criticism and great moral support throughout the course of this study.
I would like to thank Mr. Malcolm H. Noble for his assistance with HPLC, to Dr. Tatjana
Nedelkoska for initiating hairy roots, and to Mr. Geoff McDonnell for his assistance with the glass house experiments. I am glad to extent my thanks to Associate Professor Veena
Sahajwalla, and to Mr. N.M. Saha-Chaudhury, School of Material Sciences and Engineering,
UNSW, for their help with the furnace experiments. My thanks also to the members of Lab
125 for their help and co-operation.
Finally, my special thanks to my family members, Dr. D. Navaneetham and Dr. S.
Valliappan for their continuous moral support and encouragement. CHAPTER 1 - INTRODUCTION
1. 1 Heavy Metals In The Environment
Heavy metals from various sources such as fertilizers, sewage sludge amendment, fossil fuel combustion and disposal of industrial waste pollute the environment. Heavy metals such as cadmium (Cd), copper (Cu), chromium (Cr), mercury (Hg), nickel (Ni), zinc (Zn), iron (Fe), etc., are used for various domestic and industrial applications because of their valuable physical and chemical properties. As the world population increases, the consumption of heavy metals also increases markedly. As a result, utilization of large quantities of heavy metal generates substantial amounts of heavy metal pollutants, which are eventually released into the environment. The pollutants use air, water and soil as carriers for transport to plants, animals and human beings. Heavy metal accumulation in living organisms causes severe damage and poses a threat to ecosystems.
1. 1. 1 Cadmium
Among the heavy metals released into the environment, Cd (atomic number 48; atomic weight 112.4) is one of the greatest concerns. Cadmium is discharged into the environment
from mining, smelting and industries producing nickel-cadmium batteries, plastic containing
Cd, pigments, stabilizers, fertilizers, etc., Cadmium is readily accumulated from the
environment by many living organisms and, in some cases, the bioconcentration factors
(metal concentration in dry biomass/initial metal concent~ation in contaminated media) are in
the order of thousands. In plants, Cd accumulates primarily in roots and to a lesser extent in
leaves (Mitchell and Fretz, 1977), whereas, in certain aquatic organisms, Cd accumulates in
the whole body and in soft tissues (Conway, 1978; Riigsgard et al., 1987). Humans normally
absorb Cd into their bodies either by ingestion or inhalation. It has been estimated that most 2 ingested Cd comes from terrestrial foods and to a certain extent from aquatic foods and drinking water (WHO, 1992). The Cd content in terrestrial food arises from atmospheric deposition and uptake of Cd by plants from fertilizers, sewage sludge and manure. In humans, acute Cd poisoning causes nausea, vomiting, diarrhea and severe bronchial and pulmonary irritation. The major hazard to human health from Cd is its chronic accumulation in the kidneys where it can cause dysfunction if the concentration in the kidney cortex exceeds 200 mg/kg fresh weight (Fassett, 1980; Waldron, 1979).
1. 1. 2 Nickel
Nickel (atomic number 28; atomic weight 58.7) is a ubiquitous trace metal that occurs in rocks, soil, water, air and fossil fuels. Ni is released into the environment in various chemical forms by anthropogenic activities such as Ni mining, smelting, and manufacture of stainless and alloy steels. Ni is also discharged into the environment as an air pollutant from fossil fuel combustion and, to a certain extent, incineration of sewage sludge and waste.
Normally, trace levels of Ni ions are required for several biological processes. For example, urease, methyl coenzyme M reductase, hydrogenase and carbon monoxide dehydrogenase have been characterised as Ni-containing enzymes (Thauer et al., 1980; Hausinger, 1987;
Walsh and Orme-Johnson, 1987). However, Ni becomes toxic to biological systems at elevated concentrations. In addition, the biological roles of Ni in plants (Dalton et al., 1988), animals (Nieboer et al., 1988) and microorganisms (Hausinger, 1987) have been revealed.
Studies on the carcinogenicity of Ni in humans have indicated that exposure to sparingly soluble compounds of Ni via the respiratory route results in respiratory cancer, whereas lung cancer is associated with soluble Ni (Hayes, 1997). 3
1. 2 Phytoremediation
Unlike organic compounds that can be mineralized, the remediation of inorganics requires removal or conversion into a biologically inert form. Metal-contaminated sites including soil, water and sediments can be remediated in two different ways: by physiochemical extraction and by bioremediation. Physiochemical methods involve laborious and expensive excavation and land filling strategies. On the other hand, bioremediation is considered a cost-effective, safe, environmentally friendly and more selective process (Crawford and Crawford, 1996).
Generally, bioremediation refers to the use of microorganisms to remove or detoxify contaminants from polluted sites. In recent years, bioremediation has attracted much attention for cleaning up the environment because of its advantages over physiochemical extraction methods. As bioremediation involves the use of lower organisms to remove contaminants, phytoremediation is an emerging technology based on the use of plants to remove or detoxify pollutants from contaminated sites.
Phytoremediation refers to a diverse collection of technologies that use either natural or genetically engineered plants to remediate contaminated environments (Flathman and Lanza,
1998). Phytoremediation comprises various processes, including (Salt et al., 1998):
• phytoextraction: the use of pollutant-accumulating plants to remove metals or organics
from soil by concentrating them in harvestable parts;
• phytodegradation: the use of plants and associated microorganisms to degrade organic
pollutants;
• rhizofiltration: the use of plant roots to absorb and ad~orb pollutants, mainly metals, from
water and aqueous waste streams;
• phytostabilisation: the use of plants to reduce the bioavailablity of pollutants m the
environment; 4
• phytovolatilisation: the use of plants to volatilise pollutants; and
• the use of plants to remove pollutants from air.
An important pathway for human exposure to toxic metals is through inhalation of
suspended soil particulate matter and ingestion of contaminated food via suspended matter
deposits on food plants (Schnoor et al., 1995). Plants provide ground cover and stabilise soil on contaminated sites, thereby reducing wind-blown dl,lsts. Thus, phytoremediation offers the advantage of eliminating secondary air- or water-borne wastes. Increasingly, phytoremediation is being viewed as a cost-effective and user-friendly alternative to traditional methods of environmental clean-up (Boyajian and Carreria, 1997). In addition to low cost, phytoremediation offers a number of performance-based advantages. It provides a permanent solution (i.e., permanently removing the contaminant from the polluted media) and, as an in situ method, it allows remediation to take place without unduly disturbing the contaminated site (Raskin and Ensley, 2000).
Recently, the perspectives and applications of phytoremediation have been discussed in detail by Raskin and Ensley (2000). Nowadays phytoremediation is gaining acceptance by public and regulatory agencies, and may eventually emerge as the preferred method for treating sites contaminated with heavy metals or radionuclides.
1. 3 Phytomining
Phytomining is a developing technology that can be used to remediate metal contaminated
soil or recover metal from soil-surface ores on a commercial basis. In phytomining
processes, plants are grown in metal-containing soil, harvested, removed and burnt to ash. 5
This ash, termed 'bio-ore', can then be treated to recover the metal (Robinson et al., 1997b).
Phytomining has several advantages over conventional mining (Brooks et al., 1998), such as:
• Phytomining has minimal detrimental environmental consequences
• Phytomining can be used where conventional mining is too expensive
• After phytomining, the area is ready for normal crop cultivation
• Bio-ore contains higher concentrations of metal than conventional ore
• Bio-ore needs less storage space
• Bio-ore contains low levels of sulphur and thus does not contribute to acid rain
Several kinds of plant have been identified which are capable of growing in metal-containing
soil and accumulating heavy metals. In 1995, the U.S. Bureau of Mines conducted a
preliminary study of the potential of farming the Ni-hyperaccumulating plant, Streptanthus polygaloides, to recover Ni from soils (Nicks and Chambers, 1995). In addition, Alyssum
bertolonii has been identified as a potential agent for phytoremediation and phytomining of
Ni (Robinson et al., 1997a). It was further suggested that A. bertolonii or other Alyssum
species might be used for phytomining throughout the Mediterranean area including
Anatolia, as well as in Western Australia and the western United States, as these regions
contain large areas of low-grade surface Ni ores. The suitability of Berkheya coddii for
phytomining of Ni from Ni-rich soil has been evaluated by Robinson et al. (1997b).
The use of Alyssum plant species for Ni/Co phytomining has been demonstrated by Chaney
et al. ( 1998). Selected genotypes of Alyssum were cultivated in Ni/Co-rich soil. The plant
materials were harvested by cutting off the plant at soil. level then left to dry. After drying,
the plant materials were collected from the field using normal agricultural practices of
haymaking, incinerated, then reduced to an ash with or without energy recovery. It was 6 suggested that the ash may be treated further by smelting/roasting/sintering at 500-l 500°F to leave a residue metal with few contaminants, before final recovery using conventional metal refining methods such as acid dissolution and electrowining. However, the results from experimental investigation of the recovery of metals from plant biomass have not yet been reported.
1. 4 Hyperaccumulators
The term 'hyperaccumulator' describes a plant with an abnormally high metal accumulation capability. The definition of hyperaccumulators in terms of the metal concentration that can be accumulated in the biomass varies from metal to metal. For example, plant species which contain >1000 µgig Cu, Cr, Pb, Ni or 100 µg/g Cd or 10,000 µgig Zn in their above-ground dry biomass have been termed metal hyperaccumulators (Baker and Brooks, 1989; Reeves et al., 1995). Certain plant species occurring naturally in various parts of the world have been identified as hyperaccumulators. Table 1.1 lists examples of hyperaccumulating species and their biomass metal concentrations.
A major attraction associated with the use of hyperaccumulators in phytoremediation and phytomining is the possibility of employing species that remove large amounts of a particular element from the soil without significant chemical intervention. The important factors involved in the application of hyperaccumulators are the rate of biomass production, coupled with the concentration of metal transferred to the plant organs. Few hyperaccumulators have been studied agronomically, and the yields of plant dry matter that might be achieved under optimum conditions of climate, nutrition and plant density are not known. However, experimental work using Thlaspi caerulescens for remediation of soil containing fairly low levels of contamination from industrial sewage sludge has shown that, 7 at a soil Zn concentration of 444 mg/kg, mature plants can contain 5000-7000 mg Zn/kg dry matter, and that removal of 30 kg/ha is possible with a single crop (Baker et al., 1994).
Similarly, 0.143 kg/ha of Cd could be removed by T. caerulescens from soil containing 13.6 mg/kg Cd, while Ni-hyperaccumulating Alyssum species producing about 23 tonne/ha dry matter could remove 1.34 kg/ha of Ni from soil with a low Ni concentration of 35 mg/kg.
Table 1.1: Hyperaccumulators of Ni, Cd, Zn, Pb, Co, and Cu from various parts of the world (Derived from Raskin and Ensley, 2000).
Species Maximum metal Location concentration (mg/kg dry weight) Berkheya coddii (Ni-hyperaccumulator) 11,600 South Africa
Thlaspi caerulescens (Cd-hyperaccumulator) 2,130 Western and
Central Eroupe
Thlaspi brachypetalum (Zn-hyperaccumulator) 15,300 France
Acer pseudoplatanus (Pb-hyperaccumulator) 1,955 UK
Crotalaria cobalticola (Co-hyperaccumulator) 3,010 Za'ire
Vigna dolomitica (Cu-hyperaccumulator) 3,000 Za'ire
However, the practical utility of many hyperaccumulators for phytoremediation and phytomining may be limited, because many of these species are slow-growing and produce little shoot biomass, thus severely restricting their potential application. Transferring the genes responsible for the hyperaccumulating phenotype to high shoot-biomass-producing non-hyperaccumulator plants has been suggested as a potential avenue for enhancing phytoremediation or phytomining as a viable commercial technology (Brown et al., 1995; 8
CMYdonllL:nS Kramer and ~hardom, 2001 ). In addition, genes encoding metal-binding proteins, such as metalloenzymes, metallothioneins and phytochelatins, metal storage proteins and metal activated enzymes could be introduced into plants to improve their metal accumulation capability and heavy metal tolerance (Raskin, 1996).
Despite the large body of literature available regarding the ecology, evolution, genetics and physiology of plants adapted to grow at high concentrations of heavy metals, the mechanism(s) by which plants cells become tolerant of toxic concentrations of heavy metals are not well understood. As a result, progress towards genetic engineering of non hyperaccumulator plants has been hindered by a lack of understanding of the basic molecular, biochemical and physiological mechanisms involved in heavy metal accumulation. Thus, it is essential to better understand the basic mechanisms of hyperaccumulation and metal tolerance in plants species before genetic modification can be attempted.
1. 5 Mechanisms of Heavy Metal Accumulat_ion and Tolerance
It is believed that there are two basic strategies of metal tolerance in plants: exclusion, whereby plants avoid excess metal uptake; and accumulation and sequestration, whereby plants take up large amounts of metal but are able to use various detoxification strategies to render it harmless. Plants that follows the exclusion strategy cannot be metal hyperaccumulators. Thus, metal-accumulating plant species must have unique characteristics allowing them to accumulate, distribute and sequester heavy metals in their organs.
Once heavy metals are taken up inside plants, tolerance ·to metal could be brought about by distributing it among the plant organs, by intracellular compartmentation and by chelation with organic, inorganic and protein molecules. These strategies are illustrated in Figure 1.1. 9
(i) Metal binding to wall (ii) Reduced transport across cell membrane
High metal
Low metal
Protein complexes (metallothionines & phytochelatins) (v) Metal (iii) Active efflux chelation
Metal Organic complexes
Inorganic complexes (e.g. sulphides)
Cell membrane and wall Metal
(iv) Compartmentalisation
Figure 1.1 Possible mechanisms of metal tolerance irt plants (Derived from Tomsett and Thurman, 1988)
1. 5. 1 Compartmentation
I. 5. I. I Distribution Between Organs
Once metal ions have entered the roots, they can be either stored or exported to the shoots.
Enhancement of metal translocation from roots to shoots is believed to play a vital role in tolerance and hyperaccumulation mechanisms. Metal-accumulating plants translocate remarkably high concentrations of metals from the root-soil interface and sequester these metals in above-ground tissues. Some findings suggest that restriction of root-to-shoot transport of metals could be detrimental to plant tolerance mechanisms. 10
Translocation of Cd between the organs of Cd-hyperaccumulators and non hyperaccumulators has been studied. The distribution of Cd in various plant species has shown that hyperaccumulators tend to accumulate large amounts of Cd in their shoots compared with the roots, whereas the opposite pattern of Cd accumulation was found in non hyperaccumulators. Brown et al. (1994 and 1995) investigated the phytoremediation potential of T. caerulescens in Cd-contaminated media (soil or nutrient solution). The results indicated that T. caerulescens effectively translocated Cd from the contaminated media to the plant shoots. In addition, Arabidopsis halleri was also found to accumulate Cd in the shoots (Kilpper et al., 2000). However, in non-hyperaccumulator plants such as soybean, Cd was strongly retained by the roots (Cataldo et al., 1981 ). Similarly, growth inhibition was found in water hyacinth plants growing with Cd and it was observed that the total content of
Cd in the roots was higher than in the leaves (Fett et al., 1994). Cieslinski et al. (1996) demonstrated that in strawberry plants over 90% of the total Cd taken up during growth in
Cd-treated soil accumulated in the roots regardless of the Cd level in the soil. Studies of Cd accumulation and distribution in sunflower (Helianthus annuus) revealed that Cd was accumulated preferentially in the roots although its concentration increased also in the shoots or leaves (Simon, 1998). However, similar levels of Cd accumulation were found in both root and shoot tissues of Azolla filiculoides plants (Sela et al., 1988).
Translocation of Ni between the roots and shoots has been investigated both in oiler J!Af?/41j: /vi;:JI, :it11½ hyperaccumulators and non-hyperaccumulators. It was found tha1) ~ Nfaccumulated in ahJi t,J(I}; Jnc 1f'rX~' d above-ground-level biomas~ the tolerance capacity of the plant~(Kramer et al.,
1996). Thus, differences in root-to-shoot translocation could be a potential factor in determining tolerance and the accumulation capability of Ni-hyperaccumulators. In a study I I to test the tolerance of A. bertolonii seedlings to Ni (Gabbrielli et al., 1991 ), it was observed that Ni was rapidly carried to and accumulated in the shoots. Similarly, the Ni hyperaccumulator, A. troodii, accumulated about five times more Ni in the aerial parts than in the roots (Homer et al., 1991 a). Kramer et al. (1996) reported that a significant level of
Ni-tolerance was related to higher Ni accumulation in the shoots than in the roots of the Ni hyperaccumulator, A. lesbiacum. However, levels of Ni translocation in the Ni hyperaccumulator T goesingense and the non-hyperaccumulator T arvense were very similar (Kramer et al., 1997).
Some hyperaccumulators have a characteristic of multi-element hyperaccumulation capacity depending on the precise elemental composition of the soil or nutrient solution. For instance,
T caerulescens is considered as both a Cd- and Zn-hyperaccumulator because of its co tolerance of these heavy metals. Experiments have been performed on Zn-hyperaccumulators and non-hyperaccumulators to determine the distribution of Zn between plant organs. The results showed that plant shoots were the maJor accumulation sites for Zn hyperaccumulators, while non-hyperaccumulators stored Zn mainly in the roots. T caerulescens translocated large amounts of Zn from contaminated soil or nutrient solution to the shoots (Brown et al., 1994 and 1995). Similar results for T caerulescens were reported by Frey et al. (2000). Vazquez et al. (1994) studied the sites of Zn accumulation in T caerulescens and found that the leaves accumulated higher average Zn concentrations than the roots. In comparative studies, whereas T caerulescens had much higher concentrations of Zn in the shoots than in the roots, the non-hyperaccumulator, T ochroleucum, accumulated higher concentrations of Zn in the roots than in the shoots (Shen et al., 1997).
Similar studies by Lasat et al. (2000) showed that the Zn-non-hyperaccumulator, T arvense, accumulated about 80% of its total Zn in the roots while 20% was detected in the shoots. In 12
contrast, T. caerulescens accumulated about 60% of total Zn in the shoots with the rest
accumulated in the roots.
I. 5. I. 2 Intraorgan and Intracellular Distribution
Plants have a range of potential mechanisms at the cellular level for detoxification of, and
thus tolerance to, heavy metals. At the subcellular level, the central vacuole appears to be a
suitable storage reservoir for excessively accumulated metals. However, how metals are compartmentalised inside cells is almost completely unknown so far. Some experiments have been performed to gain information about heavy metal distribution at the subcellular
level in both hyperaccumulator and non-hyperaccumulator plant species.
In roots of the Cd-hyperaccumulator, T caerulescens, Cd accumulated mainly in the
apoplast and, to a lesser extent, in vacuoles (Vazquez et al., 1992). However, mesophyll cells
in leaves were determined as a major storage site for Cd in A. halleri, which was recently
discovered to be a Cd-hyperaccumulator (Ktipper et al., 2000). Localisation of Cd in roots of
Zea mays revealed that Cd was detected in the walls of the seive elements and in the middle
lamella, separating the endodermis from the pericycle. These findings suggested that Cd was
principally bound to ion exchange sites on pectic residues in the cell wall (Khan et al., 1984 ).
A previous study by Lindsey and Lineberger (1981) of Phaseolus vulgaris roots also found
that most of the tissue Cd was accumulated in the middle lamella of root tissue. Subcellular
fractionation studies using roots and leaves from seedlings and mature plants of bean showed
that more than half of the tissue Cd occurs in soluble form, the remainder being distributed
nearly equally between the cell wall and organelle fractions (Weigel and Jager, 1980;
Cataldo et al., 1981 ). Localisation of Cd in roots of Agrostis gigantea and Z. mays showed
that Cd was present in the cytoplasm and vacuoles of differentiating and mature cells and in
the nuclei of undifferentiated cells (Rauser and Ackerley, 1987). In addition, Cd 13 accumulation was not detected in the cell walls and epidermal cells. Localisation analyses of
Cd accumulation in tobacco leaves indicated that Cd accumulated predominantly in vacuoles
(Vogeli-Lange and Wagner, 1990).
Studies on localisation of Ni in Ni-hyperaccumulators have shown that the vacuoles of leaves are generally the major storage sites for Ni accumulation, although in some species epidermal cells also retain large amounts of Ni. However, the subcellular distribution of Ni in leaves may vary between hyperaccumulator and non-hyperaccumulator species. It was found that in the Ni-hyperaccumulator, T montanum, Ni in the leaves was localised in the subsidiary cells that surround guard cells but not in guard cells or in other more elongate epidermal cells (Heath et al., 1997). A study was conducted on the leaf epidermis of eight
Ni-hyperaccumulators (Psaras et al., 2000) and the results showed that in all species, Ni was excluded from guard cells and, in species possessing hairy leaves, Ni was excluded from the hairs. However, in some of the hyperaccumulator species, Ni was present in subsidiary cells, at fow levels, while the sites of high accumulation were found in epidermal cells. The form of Ni in leaves of an Ibrian sub-species of the Ni-hyperaccumulator, A. serpyllifolium, was investigated (Brooks et al., 1981) and it was found that Ni was located as a water-soluble polar complex in the vacuoles. Sagner et al. (1998) studied the distribution of Ni in shoot tissue of Sebertia acuminata and found that Ni was located mainly in lacticifers. Subcellular localisation of Ni in leaves of the hyperaccumulator, T goesingense, and the non hyperaccumulator, T arvense (Kramer et al., 2000) showed that the hyperaccumulator accumulated approximately two-fold more Ni in the vacuole than the non-hyperaccumulator, under Ni exposure conditions that were non-toxic to both species. In support of these findings, Gries and Wagner (1998) found that root vacuoles were not a major compartment for Ni accumulation in oat. 14
Localisation of other metals has also been studied in hyperaccumulators. In addition to being divalent cations, Cd and Zn have similar characteristics in terms of their subcellular distribution in plant tissues (Ktipper et al., 2000; Vazquez et al., 1992). Subcellular localisation of Zn in the Zn-hyperaccumulator, T caerulescens, showed that high concentrations of Zn were present in the cell walls of both the epidermal cells and mesophyll cells of leaves, indicating that apoplastic compartmentation might be one of the mechanisms involved in Zn tolerance by T caerulescens (Frey et al., 2000). However, in other work at a solution concentration of 10 µM Zn, T caerulescens roots accumulated equal concentrations of Zn in the apoplast and vacuoles, whereas at 100 µM Zn, higher concentrations of Zn were detected in the vacuoles than in the apoplast (Vazquez· et al., 1994). In a previous study, localisation of Zn in T caerulescens roots showed that Zn accumulated mainly in the apoplast and, to a lesser extent, in vacuoles (Vazquez et al., 1992). Compartmental analysis of Zn in the non-hyperaccumulator, T arvense, demonstrated that Zn was sequestered in the root vacuole, which retarded Zn translocation to the shoots (Lasat et al., 2000).
1. 5. 2 Complexation With Organic Acids
Plants accumulate a variety of metal-complexing substances upon exposure to excessive metal levels, such as organic acids, phytochelatins (PCs), histidine, etc. (Grill et al., 1987;
Kersten et al., 1980; Kramer et al., 1996; Kneer and Zenk, 1992). In normal metabolism, these intracellular complexing substances serve to keep the intracellular availability of essential metals within certain limits. Complexing substances may also act to decrease the availability of non-essential metals. Complexation of metal ions by specific high-affinity ligands reduces the concentration of free metal ions inside the cell, thereby reducing their phytotoxicity. Successful detoxification of heavy metals requires the formation of a stable organometallic complex and permanent storage in a physiologically inert cell compartment. 15
Information about the complexation of Cd with organic acids in plants is limited. However,
the possible involvement of organic acids in Cd detoxification has been discussed; Cataldo et
al. ( 1988) indicated that the in viva complexation pattern of Cd with organic acids differed
from in vitro complexation of Cd in soybean xylem exudates. An in vitro study of Cd
interaction with organic acids in the xylem cell walls of tomato plants showed that Cd
complexation with organic acids could be an important factor for its longitudinal and lateral
movement in the xylem (Senden et al., 1992). Relationships between Cd and organic acids in
tobacco suspension cells were investigated by Kortz et al. ( 1989). The results indicated that
Cd complexation with organic acids may be a principal means for accumulation of Cd in the presence and absence of Cd-binding peptide.
A possible role of organic acids, such as citric, malic and malonic acid, in complexation of
Ni inside plant cells has been investigated in detail. A prospective involvement of malic and malonic acids in Ni complexation was revealed for A. bertolonii by Pelosi et al. (1976). In
addition, Brooks et al. ( 1981) showed that Ni was associated principally with malic and malonic acids, which are present in high concentrations in Ni-hyperaccumulators. However,
in the Ni-hyperaccumulator, Psychotria douarrei, Ni was found as a Ni-malate complex
(Kersten et al., 1980). An organo-metallic complex of Ni was isolated from Ni
hyperaccumulating plants and Ni was found as a negatively charged citratonickelate (11)
complex with Ni(H2O)l+ as the major cationic constituent (Lee et al., 1977). Similarly,
purified extracts of Ni-hyperaccumulating plants from New Caledonia contained Ni as a
citrate complex (Lee et al., 1978). Yang et al. (1997) also found that high Ni accumulation in
shoots of ryegrass was closely related to high xylem transport rates of Ni and the
accumulation of organic acids, citric and malic acid in particular. 16
Complexation of organic acids with other metals hyperaccumulated by plants has also been studied. The role of malic acid has been investigated in various Zn-hyperaccumulators and non-hyperaccumulators and the possibility of Zn detoxification has been considered
(Mathys, 1977; Shen et al., 1997; Tolra et al., 1996). It was suggested that malate may act as a complexing agent for Zn within the plasma of Zn-hyperaccumulators, allowing Zn to be eliminated from the plasma into the vacuole by a special transport mechanism (Mathys,
1977). ~im"i=ffirtfr'":-if · rule" of malate ~rctrelation-=1ias4reerr"""'Jm>P6'§eo~ tcyperaucumN1affi:r··-::E- -caer~c@s.:::(£:hen-~'er-ur.;--1"997; ··Tolra ·er--zrl:, 1996). However, accumulation of malic acid in callus culture of Anthoxanthum odoratum was not seen to have a primary role in the mechanism of Zn-tolerance (Quersh et al., 1986). In contrast, accumulation of citrate in the root sap of Deschampsia caespitosa was strongly correlated with the accumulation of Zn and it was believed that Zn was present mainly as Zn-citrate
(Godbold et al., 1984).
1. 6 Oxidative Stress
In plants, oxidative stress is known to be generated through reactive radicals from a number of environmental factors including light, temperature, salinity, water, mineral deficiency, heavy metals and air pollutants. Reactive oxygen species such as 10 2 (singlet oxygen), 0 2-
(superoxide anion), OH• (hydroxyl radical) and H20 2 (hydrogen peroxide) are formed due to the incomplete reduction of oxygen molecules. Among the three species, OH• is more reactive than the others. However, 0 2- is moderately reactive. Although 0 2 - and H20 2 are not as reactive as OH•, they are sources of OH-, which is highly reactive with membrane lipids (De Vos and Schat, 1991).
An excessive concentration of redox-active heavy metals such as Fe, Cu, Co, etc., might lead to oxidative stress because of the ability of these metals to generate free radicals. When 17 there are free ionic metals, especially Cu and Fe, in a cell, the probability of oxidation by
H20 2 or reduction by 0 2- will be high. The required reduction of the metal can be provided by cellular reductants such as 0 2-, ascorbate or thiols {Girotti, 1985). The highly reactive
OH• radicals are produced in the presence of certain metal ions (e.g. Cu and Fe) in a cell as indicated in the following Fenton reactions (De Vos and Schat, 1991 ) .
...
...
In contrast to redox-active metals, non-redox-active metals are unable to participate in redox cycle reactions that produce highly reactive OH•. For instance, as non-redox-active metals,
Cd and Ni cannot generate free radicals directly in single-electron reactions. However, by inactivating proteins including antioxidant enzymes, or by causing the depletion of low molecular-weight antioxidants, high concentrations of Cd or Ni may disrupt the balance of formation and destruction of active oxygen species_ associated with normal cellular metabolism (Dietz et al., 1999).
Most heavy metals are strong oxidants and may disturb cell metabolism to increase the production of reactive oxygen species, which in turn could enhance the activity of antioxidant enzymes (Gwozdz et al., 1997). One possible mechanism by which elevated concentrations of heavy metals may damage plant tissues is the stimulation of free radical production by imposing oxidative stress (Foyer et al., 1997). For example, redox-active 18 heavy metals, especially Fe and Cu, are thought to play an important role in the onset of lipid peroxidation. These metals catalyse the formation of OH• and are capable of starting the peroxidative process by primary initiation (Halliwell and Gutteridge, 1989; Aust et al.,
1985).
In general, plants have enzymatic and non-enzymatic defense mechanisms against oxidative stress. In non-enzymatic processes, antioxidant substances such as glutathione, ascorbate, and a-tocopherol are involved in the detoxification of destructive free radicals. The enzymatic defense mechanisms include antioxidant enzymes such as superoxide dismutase
(SOD), catalase (CAT) and ascorbate peroxidase (APX). The activity of one or more of these enzymes is generally increased in plants exposed to stressful conditions, and this elevated activity is correlated to increased stress tolerance (Allen, 1995).
1. 6. 1 Superoxide Dismutase
The superoxide radical is an initiator of a chain reaction leading to the production of more toxic oxygen species, which can disrupt the structural and functional integrity of cellular membrane systems in plants. Superoxide radicals are converted into H2O2 by SOD. In eukaryotic cells SOD usually occurs in two forms, viz. CuZnSOD, which is located mainly in the cytoplasm and the chloroplasts, and MnSOD: which is located mainly in the mitochondria. In addition, SOD also exists as FeSOD, which is distributed throughout the cells but is associated particularly with chloroplasts (Bowler et al., 1994). Hydrogen peroxide produced from SOD-catalysed reactions is not extremely reactive but is known to inactivate SOD as well as several Calvin-cycle enzymes (Halliwell and Gutteridge, 1989).
Thus, it is important to limit the level of H2O2 in plant cells. 19
The effects of heavy metal on SOD activities have been studied in various plant species including one report on a hyperaccumulator species. The response of SOD activities to metal stress varies with plant species and with the metal involved. In the Ni-hyperaccumulator, A. argenteum, elevated concentrations of Ni resulted in decreased SOD activity, whereas increasing amounts of Cd coincided with increasing SOD activity (Schickler and Caspi,
1999). However, under similar experimental conditions, the non-hyperaccumulator, A. maritimum, displayed decreased SOD activity at the lower concentration of each metal and significantly higher activity at the highest level. Induction of SOD activity was observed when seedlings of pigeonpea were treated with Ni, while the activity of SOD was unaffected under Ni stress in Z. mays (Rao and Sresty, 2000; Baccouch et al., 2001). Somashekaraiah et al. (1992) reported on the protective role of SOD against the toxicity of Cd to chlorophyll in germinated seedlings of Phaseolus vulgaris. However, Cd ions did not have a significant effect on SOD activities in pea leaf peroxisomes, barley seedlings or maize plants (Romero
Puertas et al., 1999; Patra and Panda, 1998; Logriffoul et al., 1998). Initial induction of SOD activity was detected following the application of Cd to tobacco cells and Scots pine roots
(Piqueras et al., 2000; Schiitzendiibel et al., 2001), while a continuous increase of SOD activity was observed in Cd-treated radish seedlings (Vit6ria et al., 2001). In contrast, a significant decrease in SOD activity was determined in soybean nodules and roots, rice leaves and sunflower seedlings following application of Cd (Balestrase et al., 2001; Chien et al., 2001; Gallego et al., 1996a). In addition to Cd and Ni, the effects of Cu, Fe and Pb on (\f./HJ SOD activities als~ been investigated in various plant species (Navari-Izzo et al. 1998;
Ramadevi and Prasad, 1998; Weckx and Clijsters, 1996; Gallego et al., 1996a; Gwozdz et al., 1997). 20
1. 6. 2 Catalase
Catalase is a heme protein located mainly in the peroxisomes and also in glyoxisomes. It
plays an important role in reducing oxidative stress by catalysing the oxidation of H20 2 in
plants.
CAT 2 H2O + 02
In recent years, experiments have been conducted to determine the effects of heavy metal on
CAT activities in different plant species. The results showed that the CAT response varies
from metal to metal and depends on the metal concentration. The effect of heavy metals on
CAT activities in hyperaccumulator species has not been reported. However, the impact of heavy metals such as Cd, Ni, Cu, etc., has been investigated in non-hyperaccumulator
species.
Vit6ria et al. (2001) correlated the increase of CAT activities with Cd accumulation in radish tissues. In addition, stimulation of CAT activity in the presence of Cd in Scots pine roots, barley seedlings, sunflower plants and potato tuber has been reported (Schtitzendtibel et al.,
2001; Patra and Panda, 1998; Gallego et al., 1996b; Stroinski and Zielezinska, 1997). A
slight increase of CAT activities in pea leafperoxisomes (Romero-Puertas et al., 1999) and a
sharp decrease in tobacco cells (Piqueras et al., 2000) has been detected following the
application of Cd. Although Cd reduced the CAT activities in sunflower leaves and mung
bean seedlings (Gallego et al., 1996a; Somashekaraiah et al., 1992), Cd at a solution
concentration of 50 µM did not induce any change in the CAT activity of soybean nodules
and roots compared with corresponding controls (Balestrasse et al., 2001 ). In seedlings of
mung bean, the activity of CAT was significantly reduced in the presence of Cd compared
with control seedlings (Somasekaraiah et al., 1992). After five days of Cd-treatment, CAT 21 activities were decreased m sunflower plants then increased thereafter (Gallego et al.,
1996b).
Studies on the effect of Ni on seedlings of pigeonpea revealed that CAT activities dropped substantially with increasing concentration of externally supplied Ni ions (Rao and Sresty,
2000), while CAT activity was increased after 24 h of Ni treatment in Z. mays (Baccouch et al., 2001). Similarly, the effect of Cu on CAT activities in Ceratophyllum demersum and oat leaves has been investigated (Ramadevi and Prasad, 1998; Luna et al., 1994) while the effect of Pb on Lupinus luteus roots is reported by Gwozdz et al. ( 1997).
1. 6. 3 Ascorbate Peroxidase
Catalase produces molecular oxygen and water from two_molecules ofH2O2. Since these two molecules must impinge simultaneously at the active site, CAT has a very high maximum velocity, but a very poor affinity for its substrate. In contrast, APX has a high affinity for
H2O2 but requires a reducing substrate, ascorbate. Superoxide dismutase transforms superoxide into H2O2, which oxidises ascorbate by means of APX. The reaction sequence is closed by the reduction of dehydroascorbate (DHA) involving reduced glutathione (GSH), glutathione reductase (GR) and NADPH. The redox cycle of glutathione and ascorbate (AA) is illustrated in Figure 1.2. Ascorbate, the substrate for APX, is typically present at high levels in plant tissues and is involved as a reducing agent in numerous physiological processes, including processes related to regulation of growth, differentiation and plant metabolism. Ascorbate reacts with and destroys many types of free radical, and its principal role in plant cells appears to be as an antioxidant involved in the detoxification ofH2O2.
Ascorbate peroxidase plays an essential protection role in active oxygen scavengmg processes when co-ordinated with SOD activity (Massacci et al., 1995). In the Ni hyperaccumulator, A. argenteum, elevated concentrations of Ni resulted an elevated APX 22 activity, whereas there was no significant change in APX activity m response to Cd
(Schickler and Caspi, 1999). Under similar experimental conditions, the non hyperaccumulator A. maritimum showed elevated levels of APX activity in the presence of
Ni, but the APX activity remained unchanged in response to Cd. Stimulation of APX activity was found in Z. mays roots in the presence of Ni (Baccouch et al., 2001). Roots and leaves of
P. vulgaris and suspension cultures of tobacco cells contained elevated APX activities after exposure to Cd (Chaoui et al., 1997; Piqueras et al., 2000).
B10S YNTH ES IS NAOP+ NADPH
y ~@:>~ 2GSH ,. (~GSSG
DHA AA p+ NADPH MDHA
y DEGRADATION/EXPORT
Figure 1.2 Interactions between the reduced and oxidised forms of glutathione and ascorbate in removal ofH20 2 (Noctor et al., 1998).
In addition, significant enhancement of APX activity was determined in Cd-treated seedlings of P. aureus, pea leaf peroxisomes, seedlings of barley and soybean nodules and roots
(Shaw, 1995; Romero-Puertas et al., 1999; Patra and Panda, 1998; Balestrasse et al., 2001).
It has been found that induction of APX is a time-dependent reaction; stimulation of APX activity was observed in Scots pine roots after 24 h and in sunflower plants after 5 d of Cd- 23 treatment, respectively (Schiltzendtibel et al., 2001; Gallego et al., 1996b). In contrast, Cd reduced the APX activity in sunflower leaves, rice leaves and sunflower leaf discs (Gallego et al., 1996a; Chien et al., 2001; Groppa et al., 2001).
Studies on the effect on APX activity of heavy metals other than Cd and Ni have also been conducted in various plant species. The effect of Cu on APX activity was investigated in P. vulgaris, C. demersum and sunflower leaves (Weckx -and Clijsters 1996; Ramadevi and
Prasad, 1998; Gwozdz et al., 1997; Gallego et al., 1996a). The response of APX to Zn in P. vulgaris, Pb in L. luteus and Fe in sunflower leaves has been reported by several groups
(Chaoui et al., 1997; Gwozdz et al., 1997; Gallego et al., 1996a).
1. 6. 4 Hydrogen Peroxide
Hydrogen peroxide has at least three functions in plant tissues, including wall lignification, protein crosslinking and signal transduction (Low and Merida, 1996). However, the control of H20 2 levels is essential to prevent oxidative damage to membranes and proteins. In relation to heavy metal treatment, the response of H20 2 has been studied in various plant species. The analyses of H20 2 in pea leaf peroxisomes showed a rise of about two-fold in the
H20 2 content in Cd-treated plants (Romero-Puertas et al., 1999), whereas, a 2.5-fold increase in H20 2 in Cd-treated potato tubers has been estimated compared with controls (Stroinski and Zielezinska, 1997). In addition, rapid generation of H20 2 upon Cd exposure in tobacco cell culture and accumulation of H20 2 in Scots pine roots has also been reported (Piqueras et al., 1999; Schiltzendtibel et al., 2001). Although reports are available on the effect of Cu and
Pb on H20 2 levels in different plant species (Weckx and Clijsters 1996; Gwozdz et al.,
1997), there has been little investigation of the effect of Ni on H20 2 concentrations in plants. 24
1. 6. 5 Lipid Peroxidation
The plant plasma membrane is the outer permeability barrier of the plant cell and the first
part of the cell to sense changes in the environment. The plasma membrane lipid
composition can be affected by various kinds of stress. Changes in the composition or
molecular arrangement of membranes might play a role in heavy metal resistance, either by
modifying the permeability of membranes to ions or by altering the activities of membrane
bound enzymes (Verkleij and Schat, 1989). Membrane peroxidation is associated with lipid
gel phase formation, modification of membrane permeability and conformational changes of
membrane-bound proteins (Chia et al., 1984). One of the most damaging processes in cells is the peroxidation of membrane lipids. The lipid peroxidation process as well as some of its by-products may severely affect the functioning of biological membranes and may finally
cause cell death (Kappus, 1985).
Redox-active metals, particularly Fe and Cu, are capable of catalysing the formation of OH• which initiates peroxidative degradation of polyunsaturated fatty acid chains in membrane
lipids. Lipid peroxidation is a chain reaction that can be subdivided into three reaction phases
(De Vos and Schat, 1991):
1. Abstraction of a hydrogen atom from a methylene group of a fatty acid chain (LH) by OH•
OH• + LH ----•
2. In the presence of 0 2, the lipid radical (L•) will rapidly form a lipid peroxy radical
L. + 02 • Loo·
3. The peroxy radical (Loo·) abstracts a hydrogen atom from another fatty acid chain (L'H)
Loo·+ L'H LOOH + L'• 25
The second lipid radical (L'•) can enter phase 2 and the result is a chain of reactions 2 and 3.
This is called the propagation phase. The lipid hydroxides formed during the propagation phase are relatively stable. However, in the presence of Fe or Cu ions they are transformed into more reactive species.
LOOH + Men+ ------,~- Lo· + Me(n+I)+ + OH-
LOOH + Me(n+I)+ -----1~· Loo• + Men+ + H+
Like peroxy radicals (Loo·), alkoxy radicals (LO•) are capable of abstracting hydrogen from unsaturated fatty acids.
The lipid peroxidation process generates several additional products: peroxy radicals may form cyclic endoperoxides, which can hydrolyse to form malondialdehyde (MDA).
Malondialdehyde, a cytotoxic product of lipid peroxidation, is normally considered as the major thiobarbituric acid (TBA) reacting compound. MDA is thought to result from the breakdown of fatty acid species with three or more isolated double bonds, such as linolenic acid and arachidonic acid (Kappus, 1985). The breakdown of linolenic acid produces several aldehydes other than MDA, some of which may react with TBA to form products absorbing at both 532 nm and 450 nm (Kosugi and Kikugawa, 1989). Membrane destabilisation is generally attributed to lipid peroxidation, due to an increased production of toxic oxygen free radicals (Tappel, 1973).
Although non-redox-active metals such as Cd and Ni are unable to participate in redox cycle reactions and produce highly reactive OH-, it has been demonstrated that non-redox-active metals induce changes in lipid peroxidation in various plant species. Although there is no information available on the effect of heavy metals on lipid peroxidation in hyperaccumulators, investigations have been conducted using non-hyperaccumulator 26
species. For instance, Cd has been found to induce lipid peroxidation in sunflower leaf discs,
sunflower plants, P. aureus, P. vulgaris, barley seedlings and tobacco cells (Groppa et al.,
2001; Gallego et al., 1996b; Somashekaraiah et al., 1992; Patra and Panda, 1998; Piqueras et
al., 2000). In contrast, a slight decrease of lipid peroxidation was observed in Cd-treated pea
leaf peroxisomes (Romero-Puertas et al., 1999). In Cd-treated P. vulgaris, the concentration
of MDA was significantly increased compared to the controls, indicating the enhanced lipid
peroxidation (Chaoui et al., 1997). In addition, a study on the effect of Ni in pigeonpea
plants revealed that increasing concentrations of externally supplied Ni increased lipid
peroxidation in both the shoots and the roots (Rao and Sresty, 2000). The enhancement of
lipid peroxidation in Ni-treated Z. mays has been described as a major cause of Ni-induced
oxidative stress (Baccouch et al., 1998 and 2001).
Apart from investigations on the effect of non-redox-active metals on lipid peroxidation,
studies have also been carried out on lipid peroxidation in response to redox-active metals
such as Cu. A significant increase in the level of lipid peroxidation products was observed in the primary leaves of P. vulgaris and C. demersum plants after application of Cu (Weckx and
Clijsters, 1996; Ramadevi and Prasad, 1998). Luna et al. (1994) also found that high concentrations of Cu caused increased rates of lipid peroxidation in oat leaves. It was
indicated that an enhanced production of oxygen free radicals was responsible for peroxidation of the membrane lipids, and the degree of peroxidative damage of cells was
controlled by the potency of the anti oxidative enzyme system.
1. 6. 6 Glutathione
Accumulation of metals m vanous parts of higher plants is often accompanied by an
induction of a variety of cellular changes, directly or indirectly, which contribute to the metal
tolerance capacity of the plant. Glutathione is the most important free thiol that plays a 27
prominent role in defense against free radicals. The reduced form of glutathione (GSH) is a
tripeptide thiol with the formula y-glu-cys-gly. Glutathione is synthesised from its
constituent amino acids in two sequential, A TP-dependent enzymatic reactions catalysed by y-glutamylcysteine synthetase and glutathione synthetase (GS), respectively. GSH exhibits
high chemical reactivity both as a reducing agent and as a nucleophile, participating in the
elimination of reactive oxygen species via thiol-disulphide redox reactions, and in
detoxification of electrophilic metabolites by conjugation reactions, respectively. Isoenzymes of the glutathione S-transferase family are capable of ·catalyzing both above reactions of
GSH, while glutathione reductase (GR) and APX are important in the reduction of oxidised glutathione (GSSG) to GSH, thereby maintaining a high GSH/GSSG ratio (Noctor et al.,
1998; Mauch and Dudler, 1993). In cells where GSH is found, the reduced tripeptide form
exists interchangeably with the oxidised form. While GR uses NADPH to reduce GSSG to
GSH (Figure 1.2), various free radicals and oxidants are able to oxidise GSH to GSSG. GSH reacts with many oxidants such as H20 2, 10 2, 0 2- and OH•_ When cellular thiol compounds
are affected by oxidative stress, the primary oxidative product of GSH is its disulphide,
GSSG (Jocelyn, 1972). Upon oxidative stress, the glutathione redox status may shift to a more oxidised form due to increased GSH oxidation and/or decreased GSSG reduction (De
Vos et al., 1992).
1. 6. 6. 1 Effect ofHeavy Metal
The effect of heavy metal accumulation on GSH and GSH-related compounds has been
investigated in various plant species. Glutathione depletion following Cd exposure has been
observed in cultured cells and roots (Scheller et al., 1987; Rauser et al., 1991; Ruegsegger
and Brunold, 1992; Klapheck et al., 1995; Schneider and Bergmann, 1995). Brassicajuncea
overexpressing GS contained higher concentrations of GSH than wild-type plants and this 28 was also the case after Cd treatment. It has been concluded that in the presence of Cd, the GS enzyme is rate limiting for the biosynthesis of GSH (Zhu et al., 1999a). However, Cd stress led to a decrease in the total glutathione content and an increase in the content of Cd in both tolerant and non-tolerant carrot cells (Kim et al., 2000). The overexpression of y glutamylcysteine synthetase activity and foliar GSH accumulation in transformed poplar allowed greater tissue Cd accumulation but had only a marginal effect on Cd tolerance in poplar. However, glutamate dehydrogenase and GR activities were unchanged by exposure to Cd (Arisi et al., 2000). It was reported that following the application of Ni, the total glutathione content was reduced in seedlings of pigeonpea while a decreased level of GSH and increased proportion of GSSG was estimated in Ni-treated Scots pine seedlings (Rao and
Sresty, 2000; Kukkola et al., 2000).
The effect of Cu and other metals on GSH levels revealed that depletion of GSH levels occurred after metal treatment. In C. demersum treated with a solution concentration of 2 µM
Cu, GSH levels were increased compared with the control. However, 4 µM Cu decreased the
GSH levels, possibly due to greater oxidation in the presence of Cu. This decrease could also be partly due to the limited activity of antioxidant enzymes fostering oxidation (Ramadevi and Prasad, 1998). In addition, an increase in the ratio of GSH:GSSG under oxidative stress has been demonstrated by Emir et al. (1997). Sunflower leaves treated with metal ions (Fe
(II), Cu (II) or Cd (II)), showed a decrease in GSH content.
I. 6. 6. 2 Glutathione and Hydrogen Peroxide
Glutathione reacts rather slowly with H2O2 and direct GSH reduction of H2O2 is not a major route of H2O2 destruction in plants. Smith et al. ( 1984) proposed that under conditions where
H2O2, directly or indirectly, causes oxidation of GSH to GSSG, this regulatory reaction 29 would allow the increased production of GSH during photorespiration. A role of GSH in protection against oxidative stress is the re-reduction of ascorbate in the ascorbate- glutathione cycle (Figure 1.2) (Foyer and Halliwel, 1976; Nakano and Asada, 1980). In the above-mentioned reaction, GSH acts as a recycled intermediate in the reduction of H20 2 using electrons derived from H20 (Foyer, 1997). However, efficient GSH recycling depends on the level of GR in the system. The synthesis of GSH was shown to respond either directly or indirectly to H20 2 in potato tuber (Stroinski and Zielezinska, 1997). The authors showed that the inhibition of CAT by 3-amino-1,2,4-triazol leads to leakage of H20 2 from the peroxisomes and a stimulation of GSH synthesis. Glutathione accumulation was found to compensate for decreases in the capacity of other antioxidants, for example in CAT-deficient mutants and in plants where CAT activity has been reduced by antisense technology (Smith et al., 1984; Smith, 1985; Charnnogpol et al., 1996; Willekens et al., 1997). Srivastava and
Dwivedi (1998) suggested that salicylic-acid-mediated inactivation of CAT and peroxidase in pea seedlings caused a concomitant increase in GR activity along with an increased
GSH:GSSG ratio. They indicated that the increase might protect the cells against oxidative damage by the increased H20 2 level upon salicylic acid treatment.
1. 6. 7 Sultbydryl Groups
Sulphur is an essential element for living organisms. For example, sulphur-bacteria use sulphur as the free element, plants use sulphate or sulphide and higher animals use the cysteine and methionine residue of proteins. Sulphur occurs in cells in three principal forms: sulphide, sulphate and cellular -SH and SS groups. -SH groups, unless masked as in some proteins, are chemically the most active groups in cells. Biologically, the most important -2 property of -SH groups is that they can oxidise. Sulphur has valencies ranging from} to 6 so that several types of oxidation product can be formed. Disulphides are much less active than
-SH groups and function as stable elements of structure in proteins. However, various 30 reagents can cleave disulphides and so convert them back to -SH groups or their derivatives
(Jocelyn, 1972).
Interaction of heavy metals with functional protein -SH groups has been generally proposed as the mechanism of inhibition of several physiological reactions (Sandmann and Boger,
1980). Cd had strong affinity for the essential sulphydryl groups of enzymes or structural proteins in plants and animals (Dabin et al., 1978; Braude et al., 1980). The results of Obata and Umebayashi ( 1993) indicated that the concentration of -SH compounds produced in roots of six plant species was proportional to the degree of Cd incorporation in the plant cells. This may be one mechanism for Cd tolerance in plants. Nussbaum et al. ( 1988) reported that cysteine accumulation increased at a lower level of Cd and decreased at higher concentrations in the roots of Z. mays. Cohen et al. (1998) studied the sulfhydryl-modifying effect of the Fe content in pea seedlings. It was found that low levels of Fe in the seedlings elicited a 50% increase in reduced sulfhydryl groups on the root surface. However, information about the possible involvement of sulfhydryl groups in Ni accumulation is limited.
Zinc may protect sulfhydryl groups in membrane proteins from undergoing oxidative reactions with free radicals and transition metals at the exterior surface of animal cell plasma membranes (Bettger and O'Dell, 1991). However, in higher plants, Zn-deficient barley roots contained lower concentrations of reactive sulfhydryl groups when compared with roots of
Zn-sufficient barley seedlings (Welch and Norvell, 1993). These results suggested that Zn ions may be required to protect reactive sulfhydryl groups in plant root cell exterior surface
0 membrane proteins from oxidative reactions that can be caused by free radicals (02 - or OH ) or transition metals capable of undergoing oxidation-reduction reactions. 31
1. 7 Phytochelatins
Phytochelatins (PCs) are small, cysteine-rich heavy-metal-binding peptides inter-connected
through thiolate coordination. PCs consist of repetitive y-glutamylcysteine units with
carboxyl-terminal glycine, ranging between 5 and 17 amino acids in length. Phytochelatins
may be involved in the accumulation, detoxification and metabolism of metal ions such as
Cd, Zn, Cu, Pb and Hg in plant cells. The y-glutamyl linkages present in these peptides imply
that they are not synthesised via mRNA. The biosynthesis of PCs from the precursor GSH is dt.PePh ~(I j catalysed by the enzyme y-glutamylcysteine ~l _tfanspeptidase, which transfers a y-
glutamylcysteine unit from GSH or PC to an acceptor GSH or PC molecule (Grill et al.,
1989).
Many studies on Cd accumulation in non-hyperaccumulator species have indicated that PC
molecules play an important role in the detoxification of Cd. When sunflower plants (H. annuus) were treated with CdCh, the Cd ion enhanced the content of PCs, which protected the plants against Cd toxicity (Raggio and Moro-Raggio, 1996). It was further observed that
an initial decrease in the GSH content occurred due to an enhancement in PC synthesis.
When non-tolerant Azuki bean (Vigna angularis) cells were treated with Cd, the cells did not
contain PC peptides, unlike tomato (Lycopersicon esculentum) cells that had a substantial
tolerance to Cd (lnouhe et al., 2000). A strong decrease of GSH content due to the toxic
effects of Cd or due to consumption for the synthesis of PCs has been indicated by Gallego
et al. ( 1996b ). When barley seedlings were treated with Cd, it was found that PC levels were
increased (Patra and Panda, 1998). Cd tolerance related to PCs was studied in maize, rye and
wheat seedlings (Wojcik and Tukendorf, 1999). The results showed that in maize, the
amount of PC-glutathione derivatives synthesised was sufficient for binding the total pool of 32 the metal taken up, but the quantity of y-Glu-Cys peptides formed in wheat and rye appeared to be insufficient for detoxification of the metal.
Buthionine sulfoximine (BSO) is a specific inhibitor of y-glutamylcysteine synthetase, the first enzyme of GSH synthesis. The effect of BSO and heavy metals on PC levels has been investigated in several non-hyperaccumulator plant species. BSO caused little or no reduction of growth in tobacco in the absence of Cd, but growth was greatly reduced in cultures exposed to BSO and Cd (Reese and Wagner, 1987). The decreased cell growth was directly correlated with decreased levels of Cd-binding peptide and increased levels of what was thought to be free Cd. When Daucus carota was treated with Cd, it produced PCs; however, no PC synthesis was detected in controls or in the presence of BSO (Toppi et al.,
1999). The inhibition of PC synthesis by BSO was also observed by Grill et al. (1985 and
1987). In contrast, overexpression of y-glutamylcysteine synthetase in Indian mustard increased PC biosynthesis, which in tum enhanced Cd tolerance and accumulation (Zhu et al., 1999b ). A study of the effect of BSO and Cu on sensitive and tolerant Silene cucubalus showed that the induction of oxidative stress by Cu was ~oupled with a lowered GSH content and was significantly increased after pre-treatment of plants with BSO (De Vos et al., 1992).
However, even at low levels of GSH, oxidative stress (either lipid peroxidation or the GSH redox ratio) was not observed with the redox-inactive metal, Cd. These results indicate that
Cu may specifically cause oxidative stress by depletion of the antioxidant GSH due to PC synthesis. It has been reported that Ni does not activate PC synthesis in plants (Guo and
Marschner, 1995; Zenk, 1996; Sagner et al., 1998). 33
1. 8 Hairy Root Cultures
Hairy roots are obtained by transformation of plant tissue with Agrobacterium rhizogenes bacteria. Hairy roots have been used in various fundamental studies of plant biochemistry, physiology and molecular biology and also in agricultural, horticultural and large-scale tissue culture applications. Hairy root cultures have become an experimental tool in plant biology because they:
• can be grown in isolated and controlled conditions
• have high growth rates
• exhibit biochemical and genetic stability
• can be grown in growth-regulator-free media
• can be developed from a variety of plant species
A significant amount of research has been carried out on secondary metabolite production in hairy roots. For example, synthesis of indole alkaloids (Bhadra and Shanks, 1997) and tropane alkaloids (Mahagamasekera and Doran, 1997) in hairy root cultures has been studied, while monoclonal antibody production in hairy roots has been reported by
Wongsamuth and Doran (1997). However, limited infonnation is available about the use of hairy roots in phytoremediation and phytomining studies.
Recently, hairy roots have been used to study heavy metal accumulation in hyperaccumulator plant species (Nedelkoska and Doran, 2000a, 2000b and 2001). It was demonstrated that hairy roots of the Ni-hyperaccumulator species, A. bertolonii, A. tenium and A. troodii, have the capacity to accumulate and tolerate high levels of Ni without the involvement of translocation mechanisms. Similar experiments were conducted using the
Cd-hyperaccumulator T caerulescens demonstrating the capacity of hairy roots of this species to grow and accumulate large amounts of Cd. In non-hyperaccumulators, Cd 34
accumulation has been studied using hairy roots of several species. Cadmium uptake in hairy
root culture of D. carota was studied and it was observed that hairy roots accumulated 0.87 ±
0.1 0 µmoll g fresh weight of Cd without any reduction in fresh weight or total protein
content (Toppi et al., 1999). A comparative study was conducted using hairy root cultures of
Adenophora lobophylla and A. potaninii exposed to Cd (Wu et al., 2001). The results
showed that at a solution concentration of 50 µM Cd, more extensive growth inhibition and
higher Cd accumulation were detected in A. lobophylla than A potaninii. The biochemical
changes upon Cd accumulation in hairy roots have been studied for horseradish (Armoracia
rusticana) and it was found that exposure to Cd induced the synthesis of new groups of SH
containing peptides as well as PCs (Kubota et al., 2000). Cd uptake by cultures of
transformed hairy roots of Solanum nigrum has been investigated (Macek et al., 1994 and
1997). It was found that Cd accumulation was dependent on external metal concentration and treatment time; the cellular level of Cd reached 96.5 µgig to 24,455 µgig of dry weight after
2 h of exposure to solutions with initial Cd concentrations of 0.2 ppm and 2000 ppm, respectively (Macek et al., 1994). In another study, the influence of Cd on the growth,
morphology and physiology aspects of S. nigrum hairy roots was evaluated in the presence
of Cd (Macek et al., 1997). Cd accumulation in hairy roots of sugar beet (Beta vulgaris ),
tobacco (N. tabacum) and morning glory (Calystegia sepium) was studied by Metzger et al.
( 1992). It was found that B. vulgaris hairy roots were highly sensitive to Cd, while those of
N. tabacum and C. sepium were more tolerant. However, Cd accumulation was higher in B.
vulgaris and C. sepium than in N. tabacum. The feasibility of using transformed roots to
estimate the availability of Cd in metal contaminated materials, such as sewage sludges, was
also suggested.
Hairy roots have also been applied in studies to determine the potential of plants for
remediation of xenobiotic pollutants. Mackova et al. ( 1997) demonstrated that high levels of 35
peroxidase activity in hairy root cultures of A. rusticana, S. aviculare, Atropa belladona and
S. nigrum led to the degradation of PCBs (polychlorinated biphenyls) in the growth medium.
The ability of plant species to transform TNT (2,4,6-trinitrotoluene) was also demonstrated
using hairy roots of Catharanthus roseus (Hughes et al., 1997). In addition, TNT metabolites
were isolated and characterized in axenic root cultures of C. roseus (Bhadra et al., 1999). It
was observed that TNT was reduced to monoaminodinitrotoluenes such as 2-amino-2,6-
dinitrotoluene and 4-amino-2,6-dinitrotoluene in the detoxification process carried out by
hairy roots.
1. 9 Aims of This Study
As an experimental tool, hairy roots have several advantages over whole plants (Section 1.8).
In this study, hairy roots of the Ni-hyperaccumulator, A. bertolonii, the Cd-hyperaccumulator
T caerulescens, and the non-hyperaccumulator, N tabacum, will be used to investigate the biochemical changes that take place during heavy metal accumulation, as a function of time
(0--28 days). In addition, processes for recovery of Ni from the biomass of A. bertolonii
hairy roots and Berkheya coddii plants will be studied·. Thus, the main objectives of this
study are:
1. To investigate heavy metal (Cd or Ni) accumulation and distribution in hairy roots of
hyperaccumulator and non-hyperaccumulator plant species
11. To study the interactions of Cd and Ni with organic acids during the accumulation
processes
111. To determine the role of antioxidative systems and oxidative damage associated with
heavy metal accumulation in hairy roots
1v. To investigate the role of GSH in Cd accumulation in hairy roots
v. To study the potential of the Ni-hyperaccumulating plant, B. coddii, for phytomining
ofNi. 36
CHAPTER 2 - MATERIALS AND METHODS
2. 1 Hairy Root Cultures
Hairy roots of Thlaspi caerulescens (Cd hyperaccumulator), Alyssum bertolonii (Ni hyperaccumulator) and Nicotiana tabacum (non-hyperaccumulator) were initiated previously
(Nedelkoska and Doran, 2000 and 2001; Wongsamuth and Doran, 1997). The hairy roots were maintained on solid Gamborg's B5 complex medium (Table 2.1) (Sigma, USA) with
3% (w/v) sucrose (Ajax Chemicals, Australia) and 0.2% (w/v) Phytagel (Sigma). The hairy roots were routinely sub-cultured into liquid B5 medium and used as an inoculum for the experiments after 14 days. The liquid B5 medium contained Gamborg's B5 complex medium powder with 3% (w/v) sucrose and the pH adjusted to 5.8 using 0.1 M KOH. Fifty mL of liquid B5 medium was distributed into 250-mL Erlenmeyer flasks and sterilised for 20 min at 121 °C and 15 psi.
In the experiments with Ni, 25 ppm Ni as NiCh·6H2O was added to the liquid medium and the pH adjusted to 5.8 before sterilisation. In the experiments with Cd, 20 ppm Cd as
Cd(NO3)z·4H2O was added before pH adjustment. In the experiments with Zn, 33 ppm Zn as
ZnSO4·7H2O was added to the liquid medium before pH adjustment.
All experiments were conducted in 250-mL Erlenmeyer flasks containing 50 mL liquid B5 medium. The flasks were inoculated with 1 g fresh weight of hairy roots and placed in a dark 37
Table 2.1 Gamborg's B5 medium (Sigma)
Component Concentration (mg L- 1)
(NH4)zSO4 134.0
H3BO3 3.0
CaC}z 113.24
CoC}z·6H2O 0.025
CuSO4·5H2O 0.025
Na2-EDTA 37.25
FeSO4·7H2O 27.85
MgSO4 122.09
MnSO4·H2O 10.00
Na2MoO4 ·2H2O 0.25
Kl 0.75
KNO3 2500
NaH2PO4 130.5
ZnSO4·7H2O 2.00
Organics
Myo-inositol 100
Nicotinic acid (free acid) 1
Pyridoxine· HCl 1
Thiamine·HCI 10 38
2. 1. 1 Nickel Hyperaccumulator (A. bertolonil) and Non-hyperaccumulator (N.
tabacum)
2. 1. 1. 1 Short-term Ni Uptake by Live and Dead Biomass
Short-term experiments were conducted to investigate Ni uptake over 9 h. Erlenmeyer flasks
containing medium with Ni were inoculated with 1 g fresh weight of live or dead hairy roots.
The dead hairy roots were produced by autoclaving the roots for 20 min at 121 °C. Triplicate
flasks were harvested periodically for measurement of root dry weight, Ni concentration in
the biomass and liquid volume.
2. 1. I. 2 Culture Experiments
Hairy roots were cultured in medium with and without Ni. Triplicate flasks were harvested periodically over a period of 21-28 days for time-course measurements of root fresh weight, root dry weight, Ni concentration in the biomass, liquid volume, superoxide dismutase
(SOD) activity, catalase (CAT) activity, ascorbate peroxidase (APX) activity, hydrogen peroxide (H2O2) concentration, malondialdehyde (MDA) concentration, free -SH
concentration on the root cell surfaces, organic acid content and Ni localisation.
2. J. 1. 3 Effect of II'-ATPase Inhibitor
In some experiments, diethlystilbestrol (DES: Sigma) was added to the medium as an
inhibitor of plasma membrane H+-ATPase. To obtain a medium concentration of 100 µM
DES, 0.0268 g was dissolved in 0.5 mL of 1 M KOH and added to 1 liter of B5 medium with
and without Ni, before the pH was adjusted to 5.8. Triplicate flasks were harvested
periodically over a period of 21-28 days for time-course measurements of root dry weight,
Ni concentration in the biomass and liquid volume. 39
2. 1. 2 Cadmium Hyperaccumulator (T. caerulescens) and Non-hyperaccumulator
(N. tabacum)
2. 1. 2. 1 Short-term Cd Uptake by Live and Dead Biomass
Short-term experiments were conducted to investigate Cd uptake over 9 h. Erlenmeyer flasks containing medium with Cd were inoculated with 1 g fresh weight of live or dead hairy roots. The dead hairy roots were produced by autoclaving the roots for 20 min at 121 °C.
Triplicate flasks were harvested periodically for measurement of root dry weight and Cd concentration in the biomass and liquid volume.
2. 1. 2. 2 Culture Experiments
Hairy roots were cultured in medium with and without Cd. Triplicate flasks were harvested periodically over a period of 21-28 days for time-course measurements of root fresh weight, root dry weight, Cd concentration in the biomass, liquid volume, SOD activity, CAT activity, APX activity, H20 2 concentration, MDA concentration, total glutathione concentration, free -SH concentration on the root cell surfaces, organic acid content and Cd . . ~/2 1, -l)cJ:i c~-ePo-irnh,1(',z were al!!c> Larr/Pd ouf locahsatlon. ,,_/, a1 In some experiments, diethlystilbestrol (DES: Sigma) was added to the medium as an inhibitor of plasma membrane H+-ATPase as described in Section 2.1.1.3. Triplicate flasks were harvested periodically over a period of 21-28 days for time-course measurements of root dry weight, Cd concentration in the biomass and liquid volume. 2. 1. 2. 4 Effect of Glutathione Synthesis Inhibitor A stock solution was prepared by dissolving 0.0446 g of buthionine sulfoximine (BSO: Sigma) in 20 mL of Milli-Q water. The solution was filter-sterilised using 0.2-µm filters 40 (Sartorius, Goettingen, Germany). To achieve a concentration of 100 µM BSO, 0.5 mL of sterile BSO stock solution was added to 50 mL 85 medium with and without Cd. Triplicate flasks were harvested periodically over a period of 21-28 days for time-course measurements of root fresh weight, root dry weight, Cd concentration in the biomass, liquid volume, H20 2 concentration, MDA concentration, total glutathione, and the concentration of free -SH groups on the root cell surfaces. 2. 1. 2. 5 Effect of Free Radical Generators The effect of free radical generators on hairy root growth was studied usmg cumune hydroperoxide (CHP: Sigma). A stock solution was prepared by dissolving 23.8 µL of CHP in 10 mL of Milli-Q water. The solution was filter-sterilised using 0.2-µm filter (Sartorius). One hundred µL of sterile CHP stock solution was added to 50 mL B5 medium with and without Cd, to attain a concentration of 33 µM CHP. Triplicate flasks were harvested periodically over a period of 21-28 days for time-course measurements of root dry weight, Cd concentration in the biomass and liquid volume. In another experiment, growth of T. caeru/escens hairy roots was tested at 2, 4 and 6 mM concentrations of the free radical generator, 2,2' -azobis (2-amidinopropane) hydrochloride (AAPH: Sigma) (Ohlsson et al., 1995). Roots were cultured in the presence of AAPH for 21 days. 2. 1. 2. 6 Effect of Free Radical Scavenger D-mannitol (MAN: Sigma) was used as a free radical scavenger in cultures of hairy roots. A stock solution was prepared by dissolving 0.182 g of MAN in 20 mL Milli-Q water. The solution was filter-sterilised using a 0.2-µm filter (Sartorius). To attain a final concentration 41 of 50 mM MAN, 0.5 mL of sterile MAN stock solution was added to 50 mL B5 medium with and without Cd. Triplicate flasks were harvested periodically over a period of 21-28 days for time-course measurements of root dry weight, Cd concentration in the biomass and liquid volume. 2. 2 Nickel Recovery 2. 2. 1 Hairy Root Cultures Alyssum bertolonii hairy roots were incubated in liquid B5 medium containing different concentrations (100-5000 ppm) of Ni for 9 h to achieve a range of biomass Ni concentrations. The hairy roots were separated from the Ni solutions for measurement of root dry weight before being treated for Ni recovery (Section 2.2.3). 2. 2. 2 Whole Plant Cultivation Berkheya coddii seeds were obtained from Dr. Chris Anderson, Massey University, New Zealand. The seeds were germinated at 25°C in a potting mix (34% (w/w) black soil, 33% (w/w) coco peat and 33% (w/w) coarse propagation sand) under a light intensity of 1500- 2000 lux provided by fluorescent lights (18W; Osram, Australia). After 3-4 weeks, the seedlings were transplanted into 2-litre plastic pots containing potting mix and approximately 20 g of Osmocote Plus (Scotts Australia, Castle Hill, Australia) slow-release fertilizer. Saucers were placed under each of the pots. Two weeks after transplantation, Ni was added as Ni(NO3) 2·6H2O (Sigma-Aldrich) to the potting mix to give concentrations of 0, 1, 2, 3, and 4 mg/g of soil. The Ni salt was buried 1-2 cm into the potting mix and watered in to dissolve. The plants were cultivated for a period of 4 months (April to July 2001) in the glasshouse at the University of New South Wales. The glasshouse temperature was controlled to between 20°C and 25°C during this experiment and the plants were watered on 42 alternate days. After 4 months, the above-ground biomass was harvested for measurement of fresh weight and dry weight. The dry biomass was then treated for Ni recovery. 2. 2. 3 Nickel Recovery by Furnace Treatment This experiment was carried out using A. bertolonii hairy roots and above-ground-level B. coddii plant biomass. The A. bertolonii hairy roots were dried in an oven at 50°C and then ground to a powder using a mortar and pestle. The B. coddii plant biomass was powdered using a grinding mill. The initial Ni concentration in the biomass was measured as described in Section 2.3.2. About 1.0-1.5 g of ground biomass was formed into a pellet, placed on a pre-weighed alumina tile and inserted into a furnace (Furnace Manufacturers, Sydney, Australia). The furnace was fabricated from double skin stainless steel with a welded tubular steel chassis and fan cooler (Mehta and Sahajwalla, 2000). Resistance heating was provided using a SUPER-KANTHAL (a trademark of Kanthal corporation, Bethel, CT, USA) heating element fed with a low voltage-high current power supply to achieve the experimental temperature. The sample was held on a specimen holder, which was pushed to the centre of the hot zone in the furnace using a stainless steel/alumina rod. The furnace was operated at 1200°C with either air or nitrogen passing through. A high quality, high resolution charge coupled device (CCD) camera was used to visualise the processes occurring in the furnace. The output from the camera was channelled to a video cassette recorder and television monitor. The ground biomass was treated in the furnace for different time periods. After removal from the furnace, the residue on the tile was weighed and used for field emission scanning electron microscope (FESEM), X-ray diffraction (XRD) and inductively coupled plasma-atomic emission spectrometry (ICP-AES) analyses. 43 2. 3 Analytical Procedures 2. 3. 1 Biomass Fresh and Dry Weight Harvested hairy roots were filtered under vacuum usin_g Whatman No. filter paper and weighed to obtain the biomass fresh weight. Root dry weight was obtained by drying the filtered roots in an oven (Contherm, Australia) at 50°C to constant dry weight. Harvested above-ground B. coddii plant biomass was weighed directly to obtain the biomass fresh weight, then oven-dried at 50°C to constant dry weight. 2. 3. 2 Heavy Metal Concentrations The analysis of metal concentrations in dry biomass and liquid samples was performed using an atomic absorption spectrophotometer (Varian SpectrAA 200, Australia) in flame mode. The dried biomass was ground using a mortar and pestle and the resulting powder (0.07-0.1 g) was mixed with 4 mL concentrated nitric acid (Merck, Germany) in a glass tube (Hach, Germany) and mixed thoroughly. The tube was incubated for 2 hat 140°C under pressure in a block heater (Grant Instruments, Royston, UK). After complete digestion, the contents were diluted with Milli-Q water and analysed for heavy metal content. The liquid samples were filtered through 0.45-µm filters (Gelman, USA) and analysed directly for heavy metal content. The heavy metal concentrations were estimated using Varian SpectrAA Software, version 1.1. The Cd concentrations were determined usmg a wavelength of 228.8 nm and the Ni concentrations were measured at a wavelength of 352.4 nm. Standard Cd solutions were prepared by dissolving Cd(NO3)2·4H2O in Milli-Q water at concentrations of 1, 2, 3 and 4 ppm for development of a Cd standard curve (Appendix 1). The standard curve for Ni was 44 constructed by dissolving NiCh·6H2O in Milli-Q water at concentrations of I, 2, 3 and 4 ppm (Appendix 2). 2. 3. 3 Distribution of Cd or Ni in Hairy Roots 2. 3. 3. 1 Apoplasm and Symplasm The distribution of heavy metal (Ni or Cd) between the apoplasm and symplasm of hairy roots was measured for both the Cd and Ni-hyperaccumulators and the non hyperaccumulator. The harvested hairy roots were filtered under vacuum using Whatman No. 1 filter paper then immersed in methanol:chloroform (2: 1 v/v) for 3 days (Hart et al., 1992). After 3 days, the roots were filtered and washed with 1.5 L of Milli-Q water. This treatment yields cell wall (apoplasm) preparations of hairy roots while maintaining the structure and morphological characteristics of the intact roots. Cd or Ni levels were measured in roots without solvent treatment and in the cell wall preparations remaining after the treatment. Experiments were conducted to test the sensitivity of the cell wall isolation procedure to the duration of solvent treatment and agitation conditions. A. bertolonii hairy roots incubated with 25 ppm Ni for 14 days were treated with methanol:chloroform for 1, 2 and 3 days before washing with Milli-Q water and analysis of Ni in the cell wall fractions. Before separation from the solvent, some samples were also shaken from 20 min on an orbital shaker operated at 70 rpm. Similar test experiments were conducted using T caerulescens hairy roots incubated with 20 ppm Cd. 2. 3. 3. 2 Microscope Analysis Benzothiazolylazonaphthaol derivatives (BTAN-D) for Cd visualisation were prepared according to the method of Sumi et al. ( 1982). Diazotization was carried out by adding 1.4 g 45 aqueous 3% NaNO2 to a reaction mixture containing 3 g of 2-aminobenzothiazole (Sigma), 15 mL formic acid, 22 mL concentrated H2SO4 and 10 mL Milli-Q water at a temperature below 5°C. The diazonium solution was left in an ice-bath for 30 min with continuous stirring. Three g of P-naphthols (Sigma) in ethanol solution was added slowly to the diazonium solution while maintaining the low temperature. The crude BT AN-D product was washed with methanol twice to remove unreacted P-naphthols and crystallized 3-4 times from dioxane. The staining solution was prepared by dissolving 3 mg of BTAN-D in 1 mL dimethylsulphoxide (Sigma) containing one drop of 0.5 M KOH. After complete solubilisation of BT AN-D, the solution was diluted with 30 mL of Milli-Q water. About 1 g of fresh hairy roots was fixed in acetone for 60 s. Ten ml of staining solution was then added and left overnight at room temperature (22°C). When the roots were examined under a light microscope (Olympus BH-2, Japan) with camera (Olympus OM-2) attachment, Cd complexes were stained blue. Nickel in the roots was visualized by staining with 0.1 M dimethlyglyoximine (DMG: Sigma) (Sagner et al., 1998). An aqueous DMG solution was prepared by dissolving 1.16 g DMG in 0.1 M KOH. About 1 g of fresh hairy roots was fixed in acetone for 60 s. Ten ml of aqueous DMG solution was added and left overnight at room temperature (22°C). Complexation with Ni yielded a scarlet colour that was· observed using a light microscope and attached camera. 2. 3. 4 Organic Acids 2. 3. 4. 1 Extraction Harvested hairy roots were filtered under vacuum using Whatman No. 1 filter paper then frozen at -20°C. The frozen roots were freeze-dried at -50°C until a constant dry weight 46 was obtained. The freeze-dried roots were homogenised with Milli-Q water using a mortar and pestle at room temperature then sonicated for 15 min (Lee et al., 1977; Brooks et al., 1981 ). The samples were centrifuged at 3000 rpm for 15 min and the resultant supernatant used for gel filtration. The cell debris was dried at 50°C for heavy metal analysis. 2. 3. 4. 2 Gel Filtration Sephadex G-10 (Pharmacia) was packed into a column ( 45 x 2.5 cm) and used for organic acid fractionation of hairy root extracts (Lee et al., 1977; Brooks et al., 1981; Kerstein et al., 1980; Sagner et al., 1998). The column was pre-equilibrated and eluted with Milli-Q water at a flow rate of 40 mL h- 1. Fractions of 4 mL were collected. These fractions were filtered through a 0.45-µm filter (Acrodisc, Gelman, USA) then analysed for organic acids and heavy metal. After elution with 200 mL of Milli-Q water, free Cd was eluted with 0.3 M potassium chloride (BDH, England) and free Ni was eluted with 20 mM citric acid (BDH, England). Gel filtrations were performed using triplicate samples from metal-treated and control root cultures. 2. 3. 4. 3 HPLC Measurements Concentrations of citric, malic and malonic acids in the gel-filtered fractions were measured using HPLC (High Pressure Liquid Chromatography, .Waters, USA). The HPLC system consisted of a Waters 510 HPLC pump, a Waters 715 auto-sampler (WISP) and a Waters 480 tunable absorbance detector adjusted to 210 nm. Separation of citric, malic and malonic acids was performed using an Aminex HPX-87H organic acids column (300 x 7.8 mm) protected by an Aminex-85H guard cartridge (BioRad, USA). The column was maintained at a constant temperature of 60°C in an electric column heater. Sulphuric acid at a 47 concentration of 5 mM was used to elute the samples at a flow rate of 0.6 mL min- 1• Maxima-820 software (Dynamic Solutions, Millipore, USA) was used to detect the peaks. 2. 3. 5 Superoxide Dismutase Assay One gram fresh weight of hairy roots was homogenized in cold 50 mM sodium carbonate/sodium bicarbonate buffer (pH 9.8) using a pre-chilled mortar and pestle in an ice bath. The homogenate was centrifuged at 14000 x g for 20 min at 4°C. The resultant supernatant was analysed for superoxide dismutase (SOD) activity according to the method of Misra and Fridovich (1972). The reaction mixture contained 50 mM sodium carbonate/sodium bicarbonate buffer (pH 9.8), 0.2 mM ethylene diethyl tetraacetate (EDTA) and 0.6 mM epinephrine (Sigma) in a total volume of 3 mL (Somashekaraiah et al., 1992). Adrenochrome formation was observed at 470 nm in a dual-beam spectrophotometer. One unit of SOD activity is defined as the amount of enzyme causing 50% inhibition of epinephrine auto-oxidation. 2. 3. 6 Catalase Assay A known weight (1.0-1.5 g) of hairy roots was homogenized in cold 50 mM sodium phosphate/potassium phosphate buffer (pH 7.0) using a pre-chilled mortar and pestle in an ice bath. The extract was centrifuged at 14000 x g for 20 min at 4°C. The resultant supernatant was used for analyses of catalase (CAT) and ascorbate peroxidase (APX). The activity of CAT was determined according to the UV method of Aebi (1974). The reaction mixture contained 800 µL of sodium phosphate/potassium phosphate buffer and 100 µL of root extract. The reaction was started by adding 100 µL of 30 mM H20 2 (Ajax Chemicals). CAT activity was determined from the rate of decomposition of H20 2 at 240 nm and 48 1 1 calculated using an absorbance coefficient for H20 2 of 0.036 mM- cm- • One unit of CAT activity gives a rate of H20 2 decomposition of 1 µmole per minute. 2. 3. 7 Ascorbate Peroxidase Assay Hairy root extract was prepared as described above for the CAT assay (Section 2.3 .6). The activity of APX was measured as described in the literature (Nakano and Asada, 1981; Luna et al., 1994; Ramadevi and Prasad, 1998). The reaction mixture consisted of 675 µL of 50 mM sodium phosphate buffer (pH 7.0) containing 0.2 mM EDTA, 175 µL of 0.5 mM ascorbic acid (Sigma), 50 µL (50 µg) of bovine serum albumin (Sigma) and 50 µL of root extract. The reaction was started by adding 50 µL of 250 mM H20 2• The oxidation of ascorbic acid was followed as a decrease in absorbance at 290 nm and the APX activity was calculated using an absorbance coefficient for ascorbic acid of 2.6 mM- 1cm- 1• One unit of APX activity gives an ascorbic acid oxidation rate of 1 µmole per minute. 2. 3. 8 Hydrogen Peroxide Assay A known quantity (1.0-1.5 g fresh weight) of hairy roots was homogenized with 0.2 g activated charcoal (May and Baker Ltd., Dagenham, England) and 5 mL of 5% (w/v) trichloroacetic acid (TCA: Ajax Chemicals) using a pre-chilled mortar and pestle in an ice bath. The homogenate was filtered through four layers of cheesecloth and centrifuged at 14000 x g for 15 ruin at 4°C. The supernatant was separated and filtered through a 0.45-µm filter (Acrodisc, Gelman) and used for the assay. The assay mixture contained 50 µL of the supernatant, 1.95 mL of 100 mM potassium phosphate buffer (pH 8.4) and 1 mL of colourimetric reagent (1:1 (v/v) of 0.6 mM 4-(2-pyridylazo) resorcinol (disodium salt) (Sigma) and 0.6 mM potassium titanium oxalate (Fluka, Germany)). The reaction mixture was incubated at 45°C in a heating block for 60 min. and the absorbance of the reaction 49 mixture was read at 508 nm. The concentration of H20 2 was determined from the difference in absorption between the samples and blanks, using H20 2 (30% v/v) as a standard. The blank solution contained 50 µL of 5% (w/v) TCA and 1.95 mL of 100 mM potassium phosphate buffer (pH 8.4) (Patterson et al., 1984; Becana et al., 1986). 2. 3. 9 Estimation of Malondialdehyde Lipid peroxidation was determined by the concentration of malondialdehyde (MDA) formed in a reaction mixture (Heath and Packer, 1968; Sreenivasulu et al., 1999). Hairy roots (1.0-1.5 g fresh weight) were homogenized in 5 mL of 0.1 % (w/v) TCA. The homogenate was centrifuged at 10000 x g for 5 min. To 1 mL of the supernatant, 4 mL of 20% (w/v) TCA containing 0.5% (w/v) thiobarbituric acid (Sigma) was added. The solution was heated at 95°C for 30 min in a block heater and then quickly cooled on ice. The mixture was centrifuged at 10000 x g for 15 min and the absorbance was measured at 532 nm. MDA levels were calculated from a 1,1,3,3-tetraethoxypropane (Sigma) standard curve (Manjari and Das, 1998; Kumar and Das, 1993). 2. 3. 10 Total Glutathione Assay A known quantity (1.0-1.5 g fresh weight) of hairy roots was homogenized with 0.5 g sand and 5 mL of 5% (w/v) TCA using a mortar and pestle, The cellular debris and sand were removed after centrifugation at 10000 x g for 10 min at 4 °C. The deproteinised supernatant was extracted 5 times with ether to remove the TCA. The residual ether was evaporated and the resultant solution assayed for total glutathione. The reaction mixture consisted of 0.4 mL of 0.1 M sodium phosphate buffer (pH 7.5) containing 5 mM EDTA, 0.2 mL of 6 mM 5,S'dithio-(2-nitrobenzoic acid) {DTNB: Sigma), 0.2 mL of 1 mM NADPH and the sample extract. The reaction was initiated by adding one unit of yeast glutathione reductase (Sigma) 50 and the change in absorbance was followed at 412 nm (Anderson, 1985, Smith et al., 1984). The concentration of total glutathione was calculated from a glutathione (Sigma) standard curve. 2. 3. 11 Estimation of Free -SH Groups on Root Cell Surfaces Reduced sulfhydryl groups on the root cell surfaces (presumably on the plasma membrane surface and/or within the cell wall (Cohen et al., 1998)) were determined according to the method of Sedlak and Lindsay (1968). One gram of intact roots was submerged in sulfhydryl reaction buffer (0.2 M Tris-HCl and 0.02 M Na-EDTA, pH adjusted to 8.2 with NaOH) and incubated at room temperature for 15 min with shaking at 50 rpm. The reaction was initiated by adding 250 µL of 10 mM DTNB solution then incubated at room temperature (22°C) for 15 min with shaking at 50 rpm. After the 15 min reaction period, the absorbance of the assay solution was measured at 412 nm with a spectrophotometer. The concentration of surface -SH was calculated using a cysteine (Sigma) standard curve. The influence of DTNB infiltration time during the free -SH group analysis was tested using T caerulescens hairy roots. Infiltration times between 15 and 120 min were investigated. In addition, the possibility of Cd interference in the total soluble -SH analysis was also tested using T. caerulescens hairy roots. Approximately 1 g of fresh hairy roots was homogenized in 5 mL of 0.2 M Tris buffer (pH 8.2) containing 0.2 M EDT A using a mortar and pestle. The homogenate was centrifuged at 10000 x g for 10 min at 4°C. To a known volume of the supernatant, 0.1 mL of 10 mM DTNB, 1.9 mL of methanol and 200 µg of Cd was added. The mixture was centrifuged at 10000 x g for 10 min at 4 °C and the absorbance was measured at 410 nm (Sedlak and Lindsay, 1968). A similar experiment was conducted using cysteine (Sigma) instead of root homogenate with and without addition of Cd. 51 2. 3. 12 Field Emission Scanning Electron Microscope (FESEM) Analysis The metal residues on the tiles after furnace treatment (Section 2.2.3) were analysed for elemental composition and morphology using FESEM (S-4500, Hitachi, Japan). All samples were placed onto the surface of a double-sided conductive carbon tape, which was affixed to a copper stub. For each sample, a series of random fields was chosen, and images at a wide range of magnifications were recorded. 2. 3. 13 X-ray Diffraction Analysis A Philips 112 X-ray diffractometer (Philips Analytical, Almelo, The Netherlands) was used to record the intensity of X-rays scattered from the examined samples. Copper Ka radiation (30 Kv, 30 mA) was used as the X-ray source (Lu et al., 2001). Samples were packed into a rectangular cavity in an aluminium holder and scanned iri a step-scan mode (0.05°/step) over an angular range of 10-90° (20). Scattered X-ray intensities were collected for 2.5 s for each step and peaks were identified using the Powder Diffraction File (International Center for Diffraction Data, Pennsylvania, USA, 1995). To provide sufficient sample mass for XRD analysis, some residues after furnace treatment of B. coddii biomass containing 0.55% (w/w) Ni for different times were combined. The residues obtained after 3 and 6 hat 1200°C were mixed together (3 + 6 h); similarly, residues obtained after 17 and 24 h were also mixed together (17 + 24 h) for XRD analysis. 2. 3. 14 Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES) Analysis The elemental compositions of biomass, metal residue and ash were analysed using ICP AES (GBC, Integra-XMP, Melbourne, Australia). The elements measured were B, Ca, Cu, Fe, K, Mg, Mn, Na, Ni, P, S, and Zn. After acid digestion (Section 2.3.2), the samples were 52 diluted with Milli-Q water and subjected to ICP-AES analysis. Elements (such as C, H, N, and 0) that were not measured by ICP-AES are referred to as 'other elements' occurring in the samples. As for the XRD analysis (Section 2.3 .13 ), the residues obtained after 3 and 6 h furnace treatment of B. coddii biomass containing 0.55% (w/w) Ni at 1200°C were mixed together (3 + 6 h); similarly, residues obtained after 17 and 24 h were also mixed together (17 + 24 h) for ICP-AES analysis. 2. 3. 15 Statistical Analysis The error bars represented in the figures in the Results chapter indicate standard errors about the mean calculated from triplicate cultures. 53 CHAPTER 3 - RESULTS 3. 1 Growth of Hairy Roots 3. 1. 1 Nickel Hyperaccumulator (A. bertoloni1) and Non-hyperaccumulator (N. tabacum) 3. 1. 1. 1 Effect of Ni As shown in Figure 3 .1, in the control A. bertolonii hairy root cultures without Ni, the biomass dry weight increased from 0.10 ± 0.01 g to a maximum of 0.50 ± 0.01 g between 0 and 21 days, where ± indicates standard error from triplicate cultures. Similarly, the biomass dry weight of Ni-treated A. bertolonii hairy roots increased from 0.07 ± 0.001 g to 0.50 ± 0.02 g between O and 21 days. These results indicate that the presence of 25 ppm Ni in the growth medium did not affect the growth of A. bertolonii roots as evidence of Ni tolerance. A. bertolonii roots grown without Ni (21-day-old) and with 25 ppm Ni (28-day old) are shown in Figure 3.2. Roots in both cultures appeared healthy with similar colour. 0.7 0.6 --..... '@) 0.5 ·-(1.) ~ 0.4 "r::)c _,Ol) tl) 0.3 tl) ero 0 0.2 ·-o:i 0.1 0 0 5 10 15 20 25 30 Time (days) Figure 3.1 Growth of hairy roots of A. bertolonii: (•) control (0 ppm Ni) and (A) 25 ppm Ni. 54 Figure 3.2 A. bertolonii hairy roots in B5 medium without Ni after 21 days (left) and with 25 ppm Ni after 28 days (right). Figure 3.3 shows results for the biomass dry weight of N. tabacum hairy roots grown with and without Ni in the growth medium. The maximum level of biomass obtained for the control cultures without Ni at 28 days was 0.53 ± 0.03 g dry weight. For Ni-treated N. tabacum the maximum biomass achieved was 0.13 ± 0.02 g at 21 days. The growth of N. tabacum was severely inhibited in the presence of Ni. The maximum level of biomass measured for Ni-treated N. tabacum was 74% less than that achieved by Ni-treated A. bertolonii. This result indicates that N. tabacum was not tolerant of Ni at the concentration te ted. Browning of the biomass was observed in the N. tabacum cultures after 7-10 days. N. tabacum hairy root cultures grown for 21 days without Ni and with 25 ppm Ni are shown in Figure 3.4. Roots grown without Ni appeared healthy, whereas roots grown with 25 ppm Ni showed signs of disintegration with the colour changing to brown. 55 0.7 0.6 ,,-..,...... s:: oo <1) 0.5 3 >-. '- "O 0.4 -.._,00 r./J r./J 0.3 ro E 0 0.2 CQ 0.1 0 0 5 10 15 20 25 30 Time (days) Figure 3.3 Growth of hairy roots of N. tabacum: (• ) control (0 ppm Ni) and (..&. ) 25 ppm Ni. Figure 3.4 N. tabacum hairy roots in B5 medium without Ni (left) and with 25 ppm Ni (right), after 2 1 days. 56 3. I. I. 2 Effect of Ni and II'-A TPase Inhibitor The effect of H+ -A TPase inhibitor on growth of A. bertolonii hairy roots is shown in Figure 3.5. The presence of 100 µM diethylstilbestrol (DES) in the growth medium suppressed the growth of A. bertolonii severely. However, with the addition of DES only, no browning of the biomass was observed during this experiment and the roots remained healthy in appearance. Addition of 25 ppm Ni with .l 00 µM DES did not change the growth of A. bertolonii significantly compared with 100 µM DES only. 0.7 0.6 ,-.._ ..c:- ·v00 0.5 ~ c 0.4 "O 00 '-" rl'.l 0.3 rl'.l ell E 0 0.2 a5 0.1 0 0 5 10 15 20 25 30 Time (days) Figure 3.5 Growth of hairy roots of A. bertolonii: (•) control (without Ni and DES),(,._) 100 µM DES and (•) 25 ppm Ni + 100 µM DES. Similar experiments were conducted using hairy roots of the non-hyperaccumulator, N. tabacum (Figure 3.6). The biomass dry weight analyses showed that DES-treated N. tabacum roots produced stronger growth than the DES:..treated A. bertolonii roots (Figure 3.5). However, addition of Ni and DES together in the growth medium inhibited the growth of N. tabacum severely, reflecting the toxicity of Ni to N. tabacum roots (Figure 3.3). 3. J. 2. 1 a Effect of Initial medium pH The effect of initial medium pH on growth and Cd accumulation in T caerulescens hairy roo1 studied after 14 days of culture. Figure 3 .6a shows that different initial medium pH values die significantly affect growth of Cd-treated roots. Cd accumulation was also not greatly affected initial medium pH. 0.6 1200 0.5 1000 ~ 8 0 c- :.0 '"' ,..c: 0.4 800 11) ] bJ) ,..c: 00 ·v ..... ·-~ ~ .s ~ r::: c 0.3 0 c "O 600 ·.c '1j ..__,bJ) .!::,ro oo Cl) 0 0 4 5 5.8 6 7 Initial medium pH Figure 3.6a Effect of initial medium pH on growth and Cd uptake by hairy roots of T. caerulescens, after 14 days: (D) biomass dry weight and (.A) Cd concentration. 57 0.7 ,,.....,_ 0.6 ..c::+-' 01) ·v 0.5 ~ ~ 0.4 01) en 0.3 -en ac<:l .9 0.2 co 0.1 0 0 5 10 15 20 25 Time (days) Figure 3.6 Growth of hairy roots of N tabacum: (•) control (without Ni and DES),(.&.) 100 µM DES and(•) 25 ppm Ni+ 100 µM DES. 3. 1. 2 Cadmium Hyperaccumulator (T. caerulescens) and Non-hyperaccumulator (N. tabacum) 3.1. 2. IbE/fecto/CdandZn The effect of Cd and Cd + Zn on growth of Cd-hyperaccumulator and non hyperaccumulator hairy roots was monitored for 21-28 days. Figure 3. 7 illustrates the appearance of 21-day-old T. caerulescens hairy roots cultured without Cd and with 20 ppm Cd. Roots in both cultures appeared healthy and there was no difference in colour between the roots grown with and without Cd. The biomass analyses of control cultures grown without Cd showed that the root dry weight increased from 0.08 g ± 0.001 to 0.46 ± 0.001 g between 0 and 28 days (Figure 3.8). The maximum biomass dry weight obtained for Cd-treated T. caerulescens roots was 0.43 ± 0.03 g at 21 days. Thus, the effect of Cd on the growth of T. caerulescens roots was insignificant and the roots were tolerant of 20 ppm Cd. When T. caerulescens roots were grown with 20 ppm Cd + 33 ppm Zn, the maximum biomass dry weight obtained after 21 days culture was 0.60 ± 0.03 g, which is 58 more than 1.3 times the maximum biomass dry weight of the control and Cd-treated hairy roots. However, the overall result indicates that the presence of Zn with Cd had li ttle effect on the growth of T. caerulescens roots. Figure 3.7 Hairy roots of T. caerulescens cultured without Cd (left) and with 20 ppm Cd (ri ght), after 21 days. 0.7 ,--._.... 0.6 .c -0.0~ 3 0.5 >-. ul-, 0.4 01) '-' (/) (/) 0.3 ro E 0 0.2 co 0.1 0 0 5 10 15 20 25 30 Time (days) Figure 3.8 Growth of hairy roots of T. caerulescens: (• ) control (without Cd and Zn), (• ) 20 ppm Cd and(• ) 20 ppm Cd + 33 ppm Zn. 59 The biomass dry weight results for N. tabacum grown with 20 ppm Cd and 20 ppm Cd + 33 ppm Zn are shown in Figure 3.9. No growth was observed for Cd-treated N. tabacum hairy roots during this experiment. The level of biomass dry weight determined for Cd treated hairy roots was 0.11 ± 0.01 g at 28 days which is 4. 7 times less than the biomass dry weight measured in 28-day-old control cultures without heavy metals. This indicates that N. tabacum was not tolerant of 20 ppm Cd. The presence of 33 ppm Zn with 20 ppm Cd in the growth medium slightly increased the growth of N. tabacum roots compared with the roots treated with Cd only. The biomass dry weight of N. tabacum roots treated with Cd + Zn increased from 0.08 ± 0.01 g to 0.20 ± 0.01 g between 0 and 28 days. Figure 3.10 shows the appearance of N. tabacum roots cultured without Cd and with 20 ppm Cd, at 21 days. Roots grown without Cd appeared healthy, whereas roots grown with 20 ppm Cd did not show any growth after inoculation. Browning of the biomass occurred for N. tabacum cultured with 20 ppm Cd after 7-10 days. 0.7 0.6 ~ -01) ·-Q) 0.5 ~ "tjc 0.4 01) '-" ell 0.3 s~ 0 0.2 ·-o::) 0.1 0 0 5 10 15 20 25 30 Time (days) Figure 3.9 Growth of hairy roots of N. tabacum: (•) control (without Cd and Zn), (A) 20 ppm Cd and (•) 20 ppm Cd + 33 ppm Zn. 60 Figure 3.10 Hairy roots of N. tabacum cultured without Cd (left) and with 20 ppm Cd (right), after 21 days. 3. 1. 2. 2 Effect of Cd and W-ATPase Inhibitor When T. caerulescens hairy roots were grown with DES, growth was inhibited severely (Figure 3.11 ). However, browning of the biomass was not observed during this experiment. The biomass dry weight of DES-treated roots increased from 0.07 ± 0.001 g to 0. 11 ± 0.01 g between 0 and 28 days, while the biomass dry weight of roots treated with Cd + DES increased from 0.07 ± 0.001 g to 0.22 ± 0.03 g. This result indicates that the presence of Cd + DES slightly improved the growth of T. caerulescens after 14 days compared with the roots treated with DES only. Figure 3.12 shows the effect of DES on the growth of N. tabacum roots. DES alone in the growth medium had less effect on the growth of N. tabacum compared with A. bertolonii and T. caerulescens (Figures 3.5 and 3. 11 ). However, when the N. tabacum hairy roots were treated with Cd + DES, growth was inhibited significantly. The growth inhibition caused by Cd in this experiment is consistent with the results shown in Figure 3.9. indicating that N. tabacum is not tolerant ofCd. 61 0.7 ,--.._...... c:: 0.6 ·a:;oJ) ~ 0.5 c "O 0.4 oJ) -.._, en en 0.3 a(,;S 0 0.2 O'.l 0.1 0 0 5 10 15 20 25 30 Time (days) Figure 3.11 Growth of hairy roots of T. caerulescens: (•) control (without Cd and DES), (~) 100 µM DES and(•) 20 ppm Cd + 100 µM DES. 0.7 0.6 ...... c:: -oJ) ·a:; 0.5 ~ c 0.4 "O .._,oJ) en en 0.3 "'a 0 0.2 O'.l 0.1 0 0 5 10 15 20 25 Time (days) Figure 3.12 Growth of hairy roots of N. tabacum: (•) control (without Cd and DES), (~) 100 µM DES and(•) 20 ppm Cd + 100 µM DES. 62 3. 1. 2. 3 Effect of Cd and Glutathione Synthesis Inhibitor The effect of the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), on growth of the T caerulescens roots is shown in Figure 3.13. The presence of BSO or Cd + BSO in the growth medium did not affect the growth of T caerulescens roots significantly. Similar experiments were conducted for N tabacum roots. The biomass dry weight analyses showed that the presence of BSO alone did not affect the growth of N tabacum roots compared with the control roots (Figure 3.14). However, when the roots were grown with Cd + BSO, growth was suppressed significantly compared with the control roots, reflecting the toxicity of Cd to N. tabacum. 0.7 0.6 ...... c:: -.....Of.) Q) 0.5 ~ c 0.4 "C ,,_,Of.) Cl) 0.3 Cl) e~ .9 0.2 co 0.1 0 0 5 10 15 20 25 30 Time (days) Figure 3.13 Growth of hairy roots of T caerulescens: (•) control (without Cd and BSO), (.._) 100 µM BSO and(•) 20 ppm Cd + 100 µM BSO. 63 0.7 ,-._...., 0.6 ..c::co ·a:, 0.5 ::: ~ 0.4 co '-" {/) {/) 0.3 c:1:1 E 0 0.2 o5 0.1 0 0 5 10 15 20 25 Time (days) Figure 3.14 Growth of hairy roots of N. tabacum: (•) control (without Cd and BSO), (.6.) 100 µM BSO and(•) 20 ppm Cd + 100 µM BSO. 3. 1. 2. 4 Effect o/Cd and Free Radical Generators The effect of the free radical generator, cumene hydroperoxide (CHP), on the growth of T caerulescens and N. tabacum roots was studied (Figures 3 .15 and 3 .16). The results showed that CHP in the growth medium did not affect the growth of T caerulescens and N. tabacum roots substantially compared with their respective control cultures. However, the presence of CHP with Cd in the medium of N. tabacum roots reduced the growth by 54% at 21 days compared with the corresponding control roots. In contrast, T caerulescens roots were essentially unaffected by Cd + CHP. When T caerulescens roots were treated with another free radical generator, 2,2 ' - azobis(2-amidinopropane) .fhydrochloride (AAPH), growth was inhibited severely compared with the control roots. At 21 days, the biomass dry weight obtained for roots grown with 2, 4 and 6 mM AAPH was 0.12 ± 0.01, 0.09 ± 0.01 and 0.09 ± 0.01 g, respectively, from an inoculation of0.l ± 0.01 g. 64 0.7 ..c::--- 0.6 bfJ ·a3 0.5 ~ c "O 0.4 bfJ '--' VJ 0.3 roVJ E 0 0.2 O'.l 0.1 0 0 5 10 15 20 25 30 Time (days) Figure 3.15 Growth of hairy roots of T caerulescens: (•) control (without Cd and CHP), (.A.) 33 µM CHP and(•) 20 ppm Cd + 33 µM CHP. 0.7 ..... 0.6 ..c::-- -~ (.) 0.5 ~ >, 1-. "O 0.4 OJ) '-" VJ roVJ 0.3 E .2 0.2 O'.l 0.1 0 0 5 10 15 20 25 Titre (days) Figure 3.16 Growth of hairy roots of N. tabacum: (•) control (without Cd and CHP), (.A.) 33 µM CHP and(•) 20 ppm Cd + 33 µM CHP. 65 3. 1. 2. 5 Effect ofCd and Free Radical Scavenger The effect of the free radical scavenger, 0-mannitol (MAN), on the growth of T. caerulescens and N. tabacum roots is shown in Figures 3.17 and 3.18, respectively. Similar biomass dry weights were observed in the control hairy root cultures and in the MAN-treated cultures of both T. caerulescens and N. tabacum. In addition, when the T. caerulescens roots were grown with Cd + MAN, the growth rate did not change relative to that of the control roots. However, the biomass dry weight of N. tabacum roots was significantly reduced in the presence of Cd and MAN compared with the controls. These results indicate that the effect of MAN on the growth of T. caerulescens and N. tabacum roots was insignificant with and without Cd. Q7 Q6 ,-.._ ..c:-co ·a3 QS ~ € Q4 ,__,co ~ ~ Cl:I Q3 E: 0 iii Q2 Ql 0 0 5 10 15 X) 25 Time (days) Figure 3.17 Growth of hairy roots of T. caerulescens: (•) control (without Cd and MAN), (.A.} 50 mM MAN and(•) 20 ppm Cd + 50 mM MAN. 66 0.7 ...... c::-- 0.6 0/} ·-(l) ~ 0.5 c "O 0.4 0/} '-" Vl Vl 0.3 s~ 0 0.2 ·-t:O 0.1 0 0 5 10 15 20 25 Time (days) Figure 3.18 Growth of hairy roots of N. tabacum: (•) control (without Cd and MAN), (.A.) 50 mM MAN and(•) 20 ppm Cd + 50 mM MAN. 3. 2 Heavy Metal Uptake 3. 2. 1 Nickel Accumulation in A. bertolonii and N. tabacum Hairy Roots 3. 2. 1. 1 Live and Dead Biomass Nickel accumulation in live and dead hairy roots of A. berto/onii was measured over 540 min (Figure 3.19). A similar pattern of Ni accumulation was observed for both the live and dead roots. The average Ni concentration accumulated in live hairy roots during the period of 30-420 min was 1860 ± 150 µgig dry weight while the dead hairy roots recorded 1900 ± 190 µgig dry weight over the same period. Figure 3:20 shows Ni accumulation in live and dead N. tabacum hairy roots. After 300 min, the concentration of Ni accumulated in the dead biomass started to decline compared with the live roots. This result suggest that Ni accumulation in the dead biomass was unstable. However, the average Ni concentration accumulated in live hairy roots during the period of 30-420 min was 1260 ± 88 µgig dry 67 weight, while the dead hairy roots recorded 1290 ± 100 µg/g dry weight over the same period. 0 100 200 300 400 500 600 Time (min) Figure 3.19 Ni uptake by live(•) and dead(•) hairy roots of A. bertolonii. The initial Ni concentration was 25 ppm. 1800 ...------, 1600 ,-.._ ~ .E 1400 ..c: 00 .s -~ 1200 § c ·- -o 1000 ~ 00 -El en soo V ::::1.. u '-' 8 ~ 600 ·-z EO 400 E 200 0 ------r------r------..-----1 0 100 200 300 400 500 600 Time (min) Figure 3.20 Ni uptake by live(•) and dead(•) hairy roots of N. tabacum. The initial Ni concentration was 25 ppm. 68 3. 2. 1. 2 Effect of H+-ATPase Inhibitor The effect of DES on Ni uptake by A. bertolonii and N. tabacum hairy roots is shown in Figures 3.21 and 3.22, respectively. Ni uptake by A. bertolonii and N. tabacum hairy roots was consistently higher in the presence of DES. The maximum level of Ni accumulated in DES-treated A. bertolonii roots was 1279 ± 95 µgig dry weight at 21 days, whereas, in roots without DES, the maximum concentration of Ni recorded was 820 ± 88 µg/g dry weight at 10 days. On average over the time-course, the concentration of Ni in the roots was 1.8 times greater with DES than without DES, for both the A. bertolonii and N. tabacum hairy roots. 1600 ..... 1400 Q) ..c:: -01) ..c::..... ·v 1200 .5 ~ 1:::1 0 1000 ..... "Clc ea 01) .b 01) 800 1:::1 ----- Q) :::t (.) .,__, 1:::1 rn 600 0 rn (.) cd ..... a 400 z .....0 .D 200 0 0 5 10 15 20 25 30 Time (days) Figure 3.21 Effect of 100 µM of the ATPase inhibitor, DES, on Ni uptake by hairy roots of A. bertolonii. Hairy roots were grown with DES(•) and without DES(•). The initial Ni concentration was 25 ppm. 69 2500 ,-.._ V ,..c:: ,..c:: -cl) 2000 .Q) -~ ..... ~ ~ 1500 .s "dc ro cl) -b ~ -----Cl} V ::t 1000 (,) '--'en ~ en 0 ro (,) E 0 500 z i5 0 0 5 10 15 20 25 Time (days) Figure 3.22 Effect of 100 µM of the ATPase inhibitor, DES, on Ni uptake by hairy roots of N. tabacum. Hairy roots were grown with DES (•) and without DES (•). The initial Ni concentration was 25 ppm. 3. 2. 2 Cadmium Accumulation in T. caerulescens and N. tabacum Hairy Roots 3. 2. 2. 1 Live and Dead Biomass Cadmium accumulation in live and dead roots of T. caeru[escens and N. tabacum is shown in Figures 3.23 and 3.24, respectively. The dead biomass of T. caerulescens accumulated more Cd than the live roots throughout this experiment. The average concentration of Cd accumulated in the dead roots was 2810 ± 17 µgig dry weight, whereas the average concentration of Cd accumulated in the live roots was 1680 ± 61 µgig dry weight or 40% less than in the dead roots. The average Cd concentration estimated in the dead roots of N. tabacum was 3720 ± 17 µg/g dry weight, which is abo\lt two-fold higher than the average Cd concentration accumulated in the live roots. Thus, both in the hyperaccumulator and non-hyperaccumulator, the dead biomass tended to absorb more Cd than the live roots. 70 3500 ..... 3000 0 ..c: ..c:..... -OS) ·a:; 2500 .5 ~ c::: c .9..... "d 2000 ell .....I-< OS) c::: ----OS) 0 ::1. 1500 (.) '-' c::: C/l 0 C/l (.) ell 1000 "d E u .9 ,.D 500 0 0 100 200 300 400 500 600 Time (min) Figure 3.23 Cd uptake by live (•) and dead (•) hairy roots of T caerulescens. The initial Cd concentration was 20 ppm. 4500 4000 ..... 0 ..c: 3500 ..c: -OS) ..... ·a:; c::: ..... ~ 3000 c::: 0 c ...... "d 2500 ell .b OS) c::: tin 2000 0 ::t (.) '-' c::: C/l 0 C/l 1500 (.) ell "d 8 U .9 1000 ,.D 500 0 0 100 200 300 400 500 600 Time (min) Figure 3.24 Cd uptake by live(•) and dead(•) hairy roots of N. tabacum. The initial Cd concentration was 20 ppm. 71 3. 2. 2. 2 Effect ofZn The effect of Zn on Cd accumulation in T. caerulescens and N. tabacum hairy roots is presented in Figures 3.25 and 3.26, respectively. In T. caerulescens roots treated with Zn + Cd, Cd concentrations were similar over the time-course to those in roots treated with Cd alone. In contrast, the Cd concentration achieved in Zn + Cd-treated N. tabacum roots was 40% higher than in roots without Zn, on average over the time-course. 2500 (l.) ..c:: 2000 ..c:: -.....-OS) (l.) -i::::: ..... ~ i::::: 0 c -~ "O 1500 I-< OS) i::::: OS) -(l.) --:::t (.) i::::: Vl 1000 --Vl 0 (.) cd "O E u 0 :E 500 0 5 10 15 20 25 30 Time (days) Figure 3.25 Effect of 33 ppm Zn on Cd uptake by hairy roots of T. caerulescens. Hairy roots were grown with Zn (•) or without Zn (•). The initial Cd concentration was 20 ppm. 72 4000 .... 3500 (1) ..c:: ..c:: -on .... .Q) 3000 .5 ~ C c 2500 .9 "O ....Cl:! ....I-< on C on 2000 (1) ---:i u '-" C C/l 0 C/l 1500 u Cl:! "O s u 0 1000. ·-..0 500 0 0 5 10 15 20 25 30 Time (days) Figure 3.26 Effect of 33 ppm Zn on Cd uptake by hairy roots of N. tabacum. Hairy roots were grown with Zn (•) or without Zn (•). The initial Cd concentration was 20 ppm. 3. 2. 2. 3 Effect of Ir-ATPase Inhibitor Cd levels in the biomass of T. caerulescens during the first 10 days with DES were, on average, about 2.4 times higher than those without DES and remained higher throughout the remainder of the culture period (Figure 3.27). At 28 days, the concentration of Cd in the roots grown with DES was 3900 ± 220 µg/g dry weight, whereas the roots grown without DES contained 730 ± 220 µg Cd/g dry weight. In contrast, for N. tabacum, DES had no significant effect on the concentration of Cd in the biomass (Figure 3.28). 73 5000 (!) ..c::---- 4000 ..c::- ·v01) .5 ~ C: 0 c 3000 "O ·-('j 01) -I- C: ----01) -(!) ::i. u 2000 C: VJ 0 -VJ u ('j 8 "Ou .s 1000 .D 0 0 5 10 15 20 25 30 Time (days) Figure 3.27 Effect of 100 µM of the ATPase inhibitor, DES, on Cd uptake by hairy roots of T caerulescens. Hairy roots were grown with DES (•) and without DES (•). The initial Cd concentration was 20 ppm. 2500 ..... (!) -- ..c::..... ~ 2000 ·-(!) .5 ~ c 1500 .s.....= 'O ('j I- 01) ..... 'Bo (!) ::i. u= 1000 i:: VJ 0 -VJ u ('j "O 8 u .s 500 .D 0 0 5 10 15 20 25 30 Time (days) Figure 3.28 Effect of 100 µM of the ATPase inhibitor, DES, on Cd uptake by hairy roots of N. tabacum. Hairy roots were grown with DES (•) and without DES (•). The initial Cd concentration was 20 ppm. 74 3. 2. 2. 4 Effect of Glutatltione Synthesis lnltibitor Cd accumulation in T. caerulescens and N. tabacum roots grown with BSO is presented in Figures 3.29 and 3.30, respectively. The presence of BSO in the growth medium did not affect significantly the Cd uptake level in roots of T. caerulescens. On average over the time period, the Cd concentration in the roots grown with BSO was 1520 ± 120 µg/g dry weight, whereas in the roots grown without BSO the Cd concentration was 14 70 ± 180 µg/g dry weight. In contrast, the presence of BSO in the growth medium of N. tabacum increased the Cd accumulation in the biomass by an average of 31 % compared with the roots treated with Cd only (Figure 3.30). 2500 ..... Q) ..c:: ..c::..... -....01) 2000 Q) .s ~ _gi::: ..... t 1500 ~ 01) i::: 01) Q) ---::!. u i::: rn 1000 0 --rn u Cl:! "O E u _g .0 500 0 0 5 10 15 20 25 30 Time (days) Figure 3.29 Effect of 100 µM of the GSH synthesis inhibitor, BSO, on Cd uptake by hairy roots of T. caerulescens. Hairy roots were grown with BSO (•) and without BSO (•). The initial Cd concentration was 20 ppm. 75 5000 ,-..._ Q) ..... 4000 ...c: ...c: ..... on .s 'a> i:: :::: 3000 .8..... ro t: ~ i:: on Q) '-' u 0 0 5 10 15 20 25 30 Time (days) Figure 3.30 Effect of 100 µM of the GSH synthesis inhibitor, BSO, on Cd uptake by hairy roots of N. tabacum. Hairy roots were grown with BSO (•) and without BSO (•). The initial Cd concentration was 20 ppm. 3. 2. 2. 5 Effect of Free Radical Generator The influence of the free radical generator, CHP, on Cd accumulation in T caerulescens hairy roots is presented in Figure 3.31. The presence of CHP in the growth medium of T caerulescens had a negligible effect on Cd accumulation. The effect of CHP on Cd uptake in N. tabacum roots is shown in Figure 3.32. The effect of CHP on Cd accumulation by N. tabacum was also not significant throughout this experiment. 3. 2. 2. 6 Effect of Free Radical Scavenger The effect of the free radical scavenger, MAN, on Cd accumulation in T caerulescens hairy roots is presented in Figure 3.31. The presence of MAN in the growth medium of T caerulescens had a negligible effect on Cd accumulation. The effect of MAN on Cd uptake in N. tabacum roots is shown in Figure 3.32. The presence of MAN in the growth medium of N. tabacum had a negligible effect on overall Cd accumulation. 76 3000 ,.-.._... 2500 (I) ..c on .9 "a:i .s ~ 2000 c::: >, .9... 43 c,:s on b 1500 c::: ----on (I) :::l. u '-" c:: C/l 0 C/l 1000 u c,:s "Cl E u .9 .D 500 0 0 5 10 15 20 25 30 Time (days) Figure 3.31 Effect of 33 µM of the free radical generator, CHP, and 50 µM of the free radical scavenger, MAN, on Cd uptake by hairy roots of T. caerulescens. Hairy roots were grown without CHP and MAN (control) (•), with CHP (+) and with MAN (•). The initial Cd concentration was 20 ppm. 3000 ,_ 2500 (I) ..c... ..c .... ·oon .s ~ c:: 2000 0 c -~ "Cl ....i... on c:: ----on 1500 (I) :::l. u '-" c:: C/l 0 u ~ 1000 "Cl E u 0 .D ·- 500 0 0 5 10 15 20 25 30 Time (days) Figure 3.32 Effect of 33 µM of the free radical generator, CHP, and 50 µM of the free radical scavenger, MAN, on Cd uptake by hairy roots of N. tabacum. Hairy roots were grown without CHP and MAN (control) (•), with CHP (+) and with MAN(•). The initial Cd concentration was 20 ppm. 77 3. 3 Heavy Metal Distribution 3. 3. 1 Nickel Distribution in A. bertolonii and N. tabacum Hairy Roots 3. 3. 1. 1 Effect ofSolvent Incubation Time and Agitation For determination of Ni distribution, hairy root tissues were fractionated by treatment of the samples with organic solvent. The solid remaining after treatment comprised the cell walls and root hairs and is referred as the apoplasm or cell wall fraction. The symplasm or cell contents were released into the solvent. Usually, the solvent was applied for 3 days before analysis of the cell wall fraction for Ni. Because of the possibility of Ni re distribution during solvent treatment, and because the Ni distribution may be affected by agitation, test experiments were conducted with A. bertolonii hairy roots to study the effect of the duration of solvent treatment and agitation conditions on the cell wall fractionation results. As presented in Table 3.1, although the effect of shaking was not significant, Ni levels in the cell wall fraction tended to increase with treatment duration. However, because virtually all the Ni in A. bertolonii was located in the symplasm rather than in the cell walls as described below, the variations in cell wall Ni indicated Table 3.1 are minor and do not affect overall interpretation of the results. Table 3.1: Effect of treatment duration and agitation on the amounts of Ni recovered from the cell wall fractions of replicate samples treated with solvents. ± indicates standard error from triplicate samples. Ni in the cell wall fraction of A. bertolonii (µg) Duration of treatment (days) Without shaking With shaking 1 60 ± 3.7 67 ± 2.0 2 72 ± 0.25 82 ± 3.3 3 91 ± 2.5 92 ± 1.6 78 3. 3. I. 2 Effect of ff-A TPase Inhibitor The distributions of Ni in hairy roots of A. bertolonii and N. tabacum cultured with and without DES are shown in Figures 3.33-3.36. In A. bertolonii grown without DES, an average of 95 ± 2% of Ni in the roots was associated with the symplasm (Figure 3.33), whereas with DES, only 27 ± 3% of Ni in the roots was found in the symplasm (Figure 3.35). The proportion of Ni in the symplasm was also reduced in DES-treated N. tabacum roots to an average of 67 ± 5% (Figure 3.36) compared with 85 ± 5% without DES (Figure 3.34). These reductions in symplasm Ni occurred even though the concentration of Ni in the A. bertolonii and N. tabacum roots was somewhat greater with DES than without (Figures 3.21 and 3.22). 100 ..... z 80 ~...... 0 4-c 0 60 ~ 0.0 .....ro .::: 40 ~ 2 ~ ii.. 20 0 0.4 1 3 5 7 10 14 21 28 Time (days) Figure 3.33 The distribution of Ni between the apoplasm (D) and symplasm (•) fractions of A. bertolonii. The roots were cultured with 25 ppm Ni. 79 100 z 80 ~ 2- '- 0 60 (l) Of) ro c:: -(l) 40 I-,u (l) 0... 20 0 0.4 1 3 5 7 10 14 21 Time (days) Figure 3.34 The distribution of Ni between the apoplasm (0) and symplasm <•) fractions of N. tabacum. The roots were cultured with 25 ppm Ni. 100 z 80 ~ -.....0 '- 0 60 (l) Of) .....ro c:: 40 (l) ~ (l) A. 20 0 0.4 1 3 5 7 10 14 21 28 Time (days) Figure 3.35 The distribution of Ni between the apoplasm (0) and symplasm (•) fractions of A. bertolonii. The roots were cultured with 25 ppm Ni + 100 µM DES. 80 100 z 80 '"@...... 0 4-< 0 60 V bI) .....ro s:: V 40 (.) I,., V 0... 20 0 0.4 1 3 5 7 10 14 21 Time (days) Figure 3.36 The distribution of Ni between the apoplasm (0) and symplasm <•) fractions of N. tabacum. The roots were cultured with 25 ppm Ni+ 100 µM DES. As shown in Table 3.2, the concentration of Ni in the symplasm of A. bertolonii was reduced, on average, to 43% of the value without DES. At the same time, the average concentration of Ni in the apoplasm of A. bertolonii was more than 25 times higher as a result of DES treatment. Transport of Ni from the apoplasm to the symplasm was therefore significantly inhibited in DES-treated A. bertolonii roots. When the higher Ni content of the N. tabacum roots with DES (Figure 3.22) is taken into account, Ni concentrations in both the apoplasm and symplasm of N. tabacum were greater with DES than without (Table 3.2). The greater increase occurred in the apoplasm, where the average concentration of Ni was over 6.6 times higher with DES. The concentration of Ni in the symplasm also increased to a value 46% higher than that without DES. 81 Table 3.2: Ni concentrations m the apoplasm and symplasm fractions of hairy roots averaged over the culture period. ± indicates standard error. Average Ni concentration in the Average Ni concentration m the apoplasm fraction symplasm fraction Plant species (µg/g whole dry weight) -(µgig whole dry weight) Without DES With DES Without DES With DES A. bertolonii 30 ± 15 760 ± 56 650 ± 74 280 ± 37 N. tabacum 74 ± 40 490 ± 66 740 ± 43 1080 ± 150 3. 3. 1. 3 Microscope Analysis The results for Ni localisation using metal-specific staining are presented in Figure 3.37 for both A. bertolonii and N. tabacum. In 21-day-old A. bertolonii roots, Ni was accumulated along the length of the roots and at the tips, although there was a slight reduction in Ni intensity behind the root apex near the meristem. The control roots stained with DMG but without Ni did not show any colour development (Figures 3.37a and 3.37c). Similar results were obtained for 21-day-old N. tabacum roots stained with DMG, except that the root tips were free of Ni (Figures 3.37e-3.37h). 82 Figure 3.37 Localisation of Ni using DMG visualised under a light microscope for 21-day old A. bertolonii and N tabacum hairy roots (a) A. bertolonii hairy root without Ni, stained with DMG (control); (b) A. bertolonii hairy root with Ni, stained with DMG; (c) A. bertolonii hairy root tip without Ni, stained with DMG ( control); ( d) A. bertolonii hairy root tip with Ni, stained with DMG; (e) N tabacum hairy root without Ni, stained with DMG (control); (f) N. tabacum hairy root with Ni, stained with DMG; (g) N. tabacum hairy root tip without Ni, stained with DMG (control); (h) N. tabacum hairy root tip with Ni, stained with DMG. The bars in the bottom-centre of each photograph represent 200 µm. 83 (a) (b) (c) (d) ... c-1 ~ _· ... - • . ,,., . ., '. . - ... .. '.. ' ' (e) (f) (g) (h) 84 3. 3. 2 Cadmium Distribution in T. caerulescens and N. tabacum Hairy Roots 3. 3. 2. 1 Effect ofSolvent Incubation Time and Agitation As described in Section 3.3.1.1, test experiments were also conducted with T caerulescens hairy roots to study the effect of the duration of solvent treatment and agitation conditions on the cell wall fractionation results. Results presented in Table 3.3 indicates that neither the duration of the treatment nor shaking of the samples affected the amount of Cd recovered from the cell wall fractions of T caerulescens. These data also demonstrate the reproducibility of the results from solvent treatment. Table 3.3: Effect of treatment duration and agitation on the amounts of Cd recovered from the cell wall fractions of replicate samples treated with solvents. ± indicates standard error from triplicate samples. Cd in the cell wall fraction of T caerulescens (µg) Duration of treatment (days) Without shaking With shaking 1 370 ± 13 374 ± 19 2 366 ± 30 376 ± 5.9 3 357 ± 3.6 348 :;i: 12 3.3.2.2 Effecto/Zn The results for Cd distribution between the apoplasm and symplasm of T caerulescens and N. tabacum hairy roots are illustrated in Figures 3.38-3.41. Most of the Cd was retained in the cell walls. An average of 78 ± 4% of Cd in the T caerulescens hairy roots (Figure 3.38) and 75 ± 5% of Cd in the N. tabacum hairy roots (Figure 3.39) was found in the apoplasm fraction during incubation with 20 ppm Cd. The distribution of Cd did not change significantly when the root were cultured with 33 ppm Zn as well as 20 ppm Cd: on 85 average, 80 ± 4% Cd in T caerulescens (Figure 3.40) and 88 ± 3% of Cd in N. tabacum (Figure 3 .41) was found in the cell walls in the presence of Zn. 100 "c:i u 80 '@..... 0 ...... 60 0 0 oJj .....ell 40 s::: 0 e0 20 0.-. 0 0.4 1 3 5 7 lO 14 21 28 Time (days) Figure 3.38 The distribution of Cd between the apoplasm (D) and symplasm (•) fractions of T caerulescens. The roots were cultured with 20 ppm Cd. 100 "c:i u 80 -.....ell .....0 ..... 60 0 0 oJj .....ell 40 s::: 0 e0 0.-. 20 0 0.4 1 3 5 7 10 14 21 Time (days) Figure 3.39 The distribution of Cd between the apoplasm (D) and symplasm <•) fractions of N. tabacum. The roots were cultured with 20 ppm Cd. 86 100 "du cii 80 .....0 (.,.... 0 (I) 60 b1) ..... "'i::: (I) 40 (.) I- (I) 0... 20 0 0.4 1 3 5 7 10 14 21 28 Time (days) Figure 3.40 The distribution of Cd between the apoplasm (0) and symplasm (•) fractions of T caerulescens. The roots were cultured with 20 ppm Cd + 33 ppm Zn. 100 "d u 80 cii...... 0 (.,.... 0 60 (I) b1) ..... "'i::: (I) 40 ~ (I) 0... 20 0 0.4 1 3 5 7 10 14 21 Time (days) Figure 3.41 The distribution of Cd between the apoplasm (0) and symplasm (•) fractions of N. tabacum. The roots were cultured with 20 ppm Cd + 33 ppm Zn. 87 3. 3. 2. 3 Effect of Ir-A TPase Inhibitor The effect of DES on the Cd distribution in T caerulescens and N. tabacum hairy roots is presented in Figures 3.42 and 3.43. On average, 51 ± 5% of the Cd in the T caerulescens hairy roots and 88 ± 2% of the Cd in the N. tabacum hairy roots were found in the cell wall fractions with DES. The percentage of apoplastic Cd in T caerulescens was lower than without DES (Figure 3.38). Taking into account the greater Cd content in the biomass with DES (Figure 3.27), the average concentration of Cd in the cell walls of T caerulescens was similar with and without DES (Table 3.4), but Cd levels in the symplasm increased markedly by a factor of over 600%. For N. tabacum, the Cd distribution and Cd concentrations in the biomass fractions were similar with and without DES (Figures 3.39 and 3.43 and Table 3.4). 100 u"O 80 -.....~ .....0 c..., 0 Q) 60 01) ~ § e 40 Q) ~ 20 0 0.4 1 3 5 7 10 14 21 28 Time (days)" Figure 3.42 The distribution of Cd between the apoplasm (0) and symplasm C•) fractions of T caerulescens. The roots were cultured with 20 ppm Cd + 100 µM DES. 88 100 "O u 80 ~...... 0 '- 0 60 Q) on .....ro c:: 40 Q) (.) 1- Q) 0... 20 0 0.4 1 3 5 7 10 14 21 Time (days) Figure 3.43 The distribution of Cd between the apoplasm (D) and symplasm (a) fractions of N tabacum. The roots were cultured with 20 ppm Cd + 100 µM DES. Table 3.4: Cd concentrations in the apoplasm and symplasm fractions of hairy roots averaged over the culture period. ± indicates standard error from triplicate samples. Average Cd concentration in the Average Cd concentration in the apoplasm fraction symplasm fraction Plant species (µgig whole dry weight) (µgig whole dry weight) Without DES With DES Without DES With DES T caerulescens 1100 ± 190 1610±210 280 ± 80 1700 ± 280 N. tabacum 1500± 150 1470 ± 35 450 ± 140 220 ± 44 3. 3. 2. 4 Effect of Glutathione Synthesis Inhibitor · The effect of BSO on the Cd distribution between the symplasm and apoplasm of T caerulescens and N tabacum hairy roots is shown in Figures 3.44 and 3.45. For both species, the presence of BSO in the growth medium did not affect the Cd distribution significantly compared with the roots treated with Cd alone. T caerulescens grown with BSO accumulated 85 ± 4% of the total Cd in the apoplasm on average over the time period 89 (Figure 3.44), compared with 78 ± 4% without BSO (Figure 3.38). Similar results were obtained for N. tabacum; that is, on average, 80 ± 5% of the total Cd was accumulated in the apoplasm of roots grown with BSO (Figure 3.45) and 75 ± 5% without BSO (Figure 3.39). 100 "O u 80 ~...., ....,0 4-< 60 · 0 Q) I:)[) ....,cd 40 s::::: eQ) Q) P-, 20 0 0.4 1 3 5 7 10 14 21 28 Time (days) Figure 3.44 The distribution of Cd between the apoplasm (D) and syrnplasm <•) fractions of T. caerulescens. The roots were cultured with 20 ppm Cd + 100 µM BSO. 100 "Ou 80 -cd 0 - 60 4-<- 0 Q) ....,~ 40 s::::: Q) eQ) P-, 20. 0 0.4 1 3 5 7 10 14 21 Time (days) Figure 3.45 The distribution of Cd between the apoplasm (D) and syrnplasm C•) fractions of N. tabacum. The roots were cultured with 20 ppm Cd + 100 µM BSO. 90 3. 3. 2. 5 Microscope Analysis The results from Cd localisation using a Cd-specific stain (BT AN-D) visualised under a light microscope are shown in Figure 3.46. Although Cd was distributed throughout the length of the T caerulescens hairy roots (Figure 3 .46b ), Cd accumulated particularly at the root tips (Figure 3 .46d). N. tabacum exposed to Cd became dark brown in colour after 21 days. N. tabacum root hairs stained strongly for Cd (Figure 3.46f); however, in contrast to T caerulescens, the root tips of N. tabacum were essentially Cd-free (Figure 3.46g). 91 Figure 3.46 Localisation of Cd using BT AN-D visualised under a light microscope for 21-day-old T. caerulescens and N. tabacum hairy roots. (a) T. caerulescens hairy root without Cd, stained with BTAN-D (control); (b) T. caerulescens hairy root with Cd, stained with BTAN-D; (c) T. caerulescens hairy root tip without Cd, stained with BTAN-D (control); (d) T. caerulescens hairy root tip with Cd, stained with BTAN-D; (e) N. tabacum hairy root without Cd, stained with BTAN-D (control); (f) N. tabacum hairy root with Cd, stained with BT AN-D; (g) N. tabacum hairy root tip with Cd, stained with BTAN-D. The bars in the bottom-centre of each photograph represent 200 µm. · 92 ,. • (a) (b) (c) (d) (e) (f) (g) 93 3. 4 Organic Acids 3. 4. 1 Concentrations of Organic Acids in A. bertolonii and T. caerulesce11s Hairy Roots The concentrations of citric, malic and malonic acids in aqueous extracts of A. bertolonii hairy roots cultured with and without Ni are shown in Figures 3.47-3.49. The presence of Ni did not induce accumulation of any of the three organic acids; on the contrary, citric acid levels were somewhat lower in the Ni-treated biomass than in the control cultures after 7 days (Figure 3.47). On a molar basis, malic acid was the most abundant of the three acids. Over the entire culture period, the average molar ratio of citric : malic : malonic acid without Ni was 1.0 : 1.9 ± 0.1 : 1.4 ± 0.3. With Ni, the ratio was essentially the same at 1.0 : 2.1 ± 0.2 : 1.4 ± 0.2. The sum of the concentrations of citric, malic and malonic acids in A. bertolonii was, on average, 432 ± 65 µmol/g dry weight without Ni and 388 ± 46 µmol/g dry weight with Ni. 250 i:: 0 200 ·-ea ..c:---+-' ~ 01) +-' i:: (!) (!) ·- (.) ~ 150 i:: 0 (.) ~ -c, 01) (.) ;:::::. 100 ·-ro 0 (.) s . i:: ::1. +-' u -- 50 0 0 5 10 15 20 25 30 Time (days) Figure 3.47 Citric acid content of A. bertolonii hairy roots cultured with (•) and without (•) 25 ppm Ni. 94 350 300 c:: .9...... ro ..c:: .....H -OJ} 250 c:: ·o3 (1) u ~ c:: 200 0 c u "O "O OJ} 150 ·u ---- ro 0a -~ ::t 100 "c; '-' :E 50 0 0 5 10 15 20 25 30 Time (days) Figure 3.48 Malic acid content of A. bertolonii hairy roots cultured with (•) and without (•) 25 ppm Ni. 250 c:: .....0 ..... 200 ~ ..c:: .!:l -OJ} c:: (1) ·o3 u s:: ~ 150 0u c "O "O ·g OJ} ---- u 0 100 ·a a::t 0 '-' ro -:E 50 0 0 5 10 15 20 25 30 Time (days) Figure 3.49 Malonic acid content of A. bertolonii hairy roots cultured with (•) and without ( •) 25 ppm Ni .. 95 The concentrations of organic acids in aqueous extracts of T. caerulescens hairy roots grown with and without Cd are shown in Figures 3.50-3.52. Although the levels of each acid varied during the cultures, there was no significant difference between the Cd-treated roots and the controls. On a molar basis, citric acid was the least abundant of the three organic acids. Averaged over the entire culture period, the molar ratio of citric : malic : malonic acid in T. caerulescens was 1.0 : 3.3 ± 0.4 : 3.2 ± 0.9 without Cd and 1.0 : 2.9 ± 0.5 : 2.4 ± 0.5 with Cd, indicating that Cd treatment did not substantially alter the balance of these acids. The average total concentration of citric, malic and malonic acids in T. caerulescens was 421 ± 53 µmol/g dry weight without Cd and 431 ± 41 µmol/g dry weight with Cd. 180 160 s::::: 140 .....0 ...... c:-- .....~ .....bi) 120 6 0 (.) ~ s::::: 100 0 (.) ~ 80 .....~ bi) (.) ;::::. Cd 0 60 (.) 8 ·i:: ..... ::I. 40 C - 20 0 0 5 10 15 20 25 30 Time (days) Figure 3.50 Citric acid content of T. caerulescens hairy roots cultured with (•) and without(•) 20 ppm Cd. 96 450 400 0= ...... 350 ~ ..c--- .....1--< ·vo1} 300 =Q) (.) ~ 250 =0 c (.) "O 200 "O o1} ·c:5 ;:::. ro 0 150 E -~ :::t 100 -ro '--' ::E 50 0 0 5 10 15 20 25 30 Time (days) Figure 3.51 Malic acid content of T. caerulescens hairy roots cultured with (•) and without ( •) 20 ppm Cd. 300 0= 250 ...... ro ..... b ..c--- .....o1} 200 =Q) Q) (.) ~ =0 (.) c 150 "O "O -~ o1} (.) --0 100 ·a -E 0 :::t ro '--' -::E 50 0 0 5 10 15 20 25 30 Time (days) Figure 3.52 Malonic acid content of T. caerulescens hairy roots cultured with (•) and without(•) 20 ppm Cd. 97 3. 4. 2 Association of Ni and Cd with Organic Acids On average over the culture period, 36 ± 2% of total Ni in the A. bertolonii roots (Figure 3.53) and 35 ± 3% of total Cd in the T. caerulescens roots (Figure 3.54) was in water soluble form and could be recovered by aqueous extraction of the biomass. The water insoluble form of Ni or Cd was determined from the debris obtained after aqueous extraction of the hairy roots. 100 90 z 80 cil..... 70 .....0 ...... 60 0 Typical gel filtration elution profiles for extracts of A. bertolonii and T. caerulescens are shown in Figures 3.55 and 3.56. Citric, malic and malonic acids were eluted with 80-140 mL water; citric acid eluted first and malonic acid last. As indicated in Figure 3.55, Ni was eluted in two stages. After elution of the organic acids with 200 mL water, free aqueous Ni was eluted using 20 mM citric acid. The amount of free Ni was always much lower than that found in the organic acid fractions. 98 100 90 u"O 80 '@..... 70 .....0 <+-, 60 0 II) 50 01) .....ro 40 i::::: II) ...u 30 II) 0... 20 10 0 3 5 7 14 21 28 Time (days) Figure 3.54 The percentages of Cd in aqueous extract (0) and debris C•) of T caerulescens hairy roots cultured with 20 ppm Cd. 5 --E 0. 0. 4 z-- "O i::::: ro 3 01) --E ·u"O-- 2 ro u -~ 1 ...01) 0 0 1 8 15 22 29 36 43 50 57 64 71 78 85 92 99 Fraction ( 4 ml) Figure 3.55 Typical gel filtration elution profile for an aqueous extract of 14-day-old A. bertolonii hairy roots cultured with 25 ppm Ni. Citric acid ( + ), malic acid ( • ), malonic acid (0), Ni (•). 99 As shown in Figure 3.56, Cd was also eluted in two stages. In the first stage, Cd was collected with the organic acid fractions by elution with water; in the second stage Cd was eluted alone by passing 0.3 M KC! through the column after 200 mL of water. A substantial quantity of unassociated Cd was obtained from the T caerulescens extracts in the second-stage elution. 1 8 15 22 29 36 43 50 57 64 71 78 85 92 Fraction (4 mL) Figure 3.56 Typical gel filtration elution profile for an aqueous extract of 14-day-old T caerulescens hairy roots cultured with 20 ppm Cd. Citric acid ( + ), malic acid ( • ), malonic acid (0), Ni (•). The percentages of Ni and Cd associated with organic acids in the aqueous extracts of A. bertolonii and T caerulescens are shown in Figures 3.57 and 3.58. The percentage of Ni associated with organic acids in the A. bertolonii extracts was consistently high at 80-90%, except at the end of the culture period (Figure 3.57). Taking into account the percentage of total Ni in the biomass that was found in the aqueous extracts for each sample, an average of 7.5 ± 2% of the total Ni in A. bertolonii occurred in free aqueous form, while 28 ± 2% was complexed with organic acids (Figure 3.59). The percentage of acid-associated Cd in 100 the T caerulescens extracts varied between 18% and 63% (Figure 3.58); however, most of the Cd was not complexed with organic acids most of the time. Taking into account the percentage of total Cd in the biomass that was found in the aqueous extracts for each sample, an average of 21 ± 2% of the total Cd in T caerulescens hairy roots was in free aqueous form and 13 ± 3% was associated with organic acids (Figure 3.60). 100 90 80 ..... u ro z 1--< 70 c...... 0 >< V V 60 co en ro ;:I ..... 0 50 C V V u ;:I 1--< CT' 40 V ro A.. .s 30 20 10 0 3 5 7 14 21 28 Time (days) Figure 3.57 The percentages of Ni associated with organic acids in aqueous extracts of A. bertolonii hairy roots cultured with 25 ppm Ni. (D) % Ni associated with organic acids and (•) % free Ni. 101 100 90 ...., 80 "O (.) ell u ....,i... 70 4-, :> Figure 3.58 The percentages of Cd associated with organic acids in aqueous extracts of T caerulescens hairy roots cultured with 20 ppm Cd. (D) % Cd associated with organic acids and (•) % free Cd. 100 90 80 --z ctj.... 70 ....0 60 4-, 0 V 50 OJ) ....ell s::: 40 V (.) i... 30 V p... 20 10 0 3 5 7 14 21 28 Time (days) Figure 3.59 The percentages of total Ni associated with organic acids (D) and in free aqueous form <•) in A. bertolonii hairy roots cultured with 25 ppm Ni. 102 100 90 u"O 80 '"@..... 70 .....0 60 '-+-< 0 (1) 50 en .....ell 40 t::: (1) u 30 i... (1) 0... 20 10 0 3 5 7 14 21 Time (days) Figure 3.60 The percentages of total Cd associated with organic acids (D) and in free aqueous form (•) in T caerulescens hairy roots cultured with 20 ppm Cd. 3. 5 Oxidative Stress Parameters 3. 5. 1 Nickel Hyperaccumulator and Non-hyperaccumulator 3. 5. 1. 1 Superoxide Dismutase Activities of superoxide dismutase (SOD) in A. bertolonii and N. tabacum hairy roots were estimated with and without Ni in the growth medium over a period of 21-28 days. The SOD activity was reduced by 30% in Ni-treated A. bertolonii roots compared with the control roots, on average over the time period (Figure 3.61). Figure 3.62 shows the activities of SOD estimated in N. tabacum hairy roots cultured with and without Ni. The presence of Ni did not alter the SOD levels compared with the corresponding control roots. On average over the time course, the SOD activity without Ni was 205 ± 32 Units/g fresh weight and 213 ± 14 Units/g fresh weight with Ni. However, without Ni, the endogenous 103 SOD activity in A. bertolonii hairy roots was significantly greater than in N. tabacum, by an average factor of about 2.4. 900 .:::>-. 800 > ...... 700 u ,.i::: (,;l -- b1} 11) ·;; CJl 600 .....(,;l ~ ~ ,.i::: CJl 500 E 11) CJl <.t:: :.a b1} 400 11) ....___ :'2 .....CJl 300 ;,( ·a 0 i.. ::J 11) '-' 200 0. ~ er:, 100 0 0 5 10 15 20 25 30 Time (days) Figure 3.61 Superoxide dismutase activity in A. bertolonii hairy roots grown without (•) and with (•) Ni at an initial concentration of 25 ppm. 900 .....>-, ·;; 800 ...... ·-u --,.i::: 700 (,;l b1} 11) ·;; CJl 600 .....(,;l ~ ~ ,.i::: CJl 500 E 11) CJl <.t:: :.a b1} 400 11) ....___ :'2 .....CJl 300 ;,( ·a 0 i.. ::J 11) '-' 200 0. ~ er:, 100 0 0 5 10 15 20 25 30 Time (days) Figure 3.62 Superoxide dismutase activity in N. tabacum hairy roots grown without(•) and with (•) Ni at an initial concentration of 25 ppm. 104 3. 5. 1. 2 Catalase The levels of catalase (CAT) activity in A. bertolonii hairy roots were measured in time course experiments with and without Ni present in the growth medium (Figure 3.63). Between 1 and 14 days, the Ni-treated A. bertolonii hairy roots produced less CAT activity compared with the control roots. On average over the culture period, the CAT activity was decreased by 38% in A. bertolonii hairy roots cultured with Ni compared with the hairy roots cultured without Ni. The results for CAT activity in N. tabacum hairy roots (Figure 3.64) show that N. tabacum produced significantly lower levels of CAT activity compared with A. bertolonii hairy roots. The CAT activity determined in A. bertolonii hairy roots cultured without Ni was 513 ± 109 times higher than the activity determined in N. tabacum roots cultured without Ni, on average over the time-course. However, Ni further reduced the CAT activity in N. tabacum roots compared with the control roots. 100 ..... 80 ..c:: >, -1:)1) ..... ll.) > ·- ·-..... ~ 60 ·-0 ..c:: ro ell ll.) ll.) ellro ti:: 40 ro 1:)1) ..... ell -ro ---..... u 8 20 '-" 0 0 5 10 15 20 25 30 Time (days) Figure 3.63 Catalase activity in A. bertolonii hairy roots grown without (•) and with(•) Ni at an initial concentration of 25 ppm. 105 0 5 10 15 20 25 30 Time (days) Figure 3.64 Catalase activity in N. tabacum hairy roots grown without (•) and with(•) Ni at an initial concentration of 25 ppm. 3. 5. 1. 3 Ascorbate Peroxidase Hydrogen peroxide is decomposed in plant cells by ascorbate peroxidase (APX) using ascorbate as substrate. Thus, APX activities were measured in A. bertolonii and N. tabacum hairy roots with and without Ni present in the growth medium. Ni reduced the APX activity in A. bertolonii hairy roots significantly compared with the control roots (Figure 3.65). The maximum level of APX activity was 0.4 ± 0.04 Units/g fresh weight without Ni, whereas in Ni-treated hairy roots, the maximum level was 0.16 ± 0.01 Units/g fresh weight. Figure 3.66 shows the levels of APX activity determined in N. tabacum hairy roots cultured with and without Ni. Without Ni, N. tabacum roots produced 0.55 ± 0.04 Units/g fresh weight while A. bertolonii roots produced 0.34 ± 0.04 Units/g fresh weight of APX activity, on average over the time course. Thus, without Ni APX activities in N. tabacum hairy roots were, on average, 1.8 times those in A. bertolonii hairy roots (Figures 3.65 and 3.66). Exposure to Ni reduced APX activity in both species. This effect was more 106 pronounced in A. bertolonii, which suffered an average reduction in APX activity of 61 % due to Ni treatment (Figure 3.65). In comparison, Ni decreased APX activities in N. tabacum hairy roots by an average of 19% (Figure 3.6~)- The maximum APX activity in Ni-treated N. tabacum roots was 2.9 times that in Ni-treated A. bertolonii. 0.7 ------0.6 0.1 • • 0------~------1------ 0 5 10 15 20 25 30 Time (days) Figure 3.65 Ascorbate peroxidase activity in A. bertolonii hairy roots grown without(•) and with (•) Ni at an initial concentration of 25 ppm. 0 5 10 15 20 25 30 Time (days) Figure 3.66 Ascorbate peroxidase activity in N. tabacum hairy roots grown without (•) and with (•) Ni at an initial concentration of 25 ppm. 107 3. 5. 1. 4 Hydrogen Peroxide Hydrogen peroxide (H 20 2) is a reactive oxygen species and also an indicator of oxidative stress. The concentration of H20 2 accumulated in hairy roots of A. bertolonii and N. tabacum was measured with and without Ni (Figures 3.67 and 3.68). The overall results show that Ni-treated A. bertolonii and N. tabacum roots produced higher levels of H20 2 than the corresponding control roots. H20 2 levels in Ni-treated A. bertolonii hairy roots were, on average, 3.6-fold higher than without Ni (Figure 3.67). The average increase in H20 2 concentration in N. tabacum was substantially higher at 8.6-fold, following a large 5.5-fold increase in H20 2 levels at the beginning of the culture period (Figure 3.68). The maximum H20 2 concentration in Ni-treated N. tabacum hairy roots was 1.6 times that in Ni-treated A. bertolonii. 0 5 10 15 20 25 30 Time (days) Figure 3.67 Levels of hydrogen peroxide in A. bertolonii hairy roots grown without(•) and with (•) Ni at an initial concentration of 25 ppm. 108 2000 1000 0 •• • • • • 0 5 10 15 20 25 Time (days) Figure 3.68 Levels of hydrogen peroxide in N. tabacum hairy roots grown without (•) and with (•) Ni at an initial concentration of 25 ppm. 3. 5. 1. 5 Malondialdehyde Lipid peroxidation levels in the hairy roots were determined by measunng the concentration of malondialdehyde (MDA) produced. MDA levels were similar in roots of A. berto/onii cultured with and without Ni (Figure 3.69), indicating that the effect of Ni on lipid peroxidation was small. On average over the time period, the concentration of MDA determined in roots cultured with Ni was 12 ± 1.5 nmole/g fresh weight, compared with 15 ± 1.5 nmole/g fresh weight without Ni. Figure 3.70 shows the concentration of MDA in N. tabacum roots grown with and without Ni. The results indicate that Ni in the medium increased lipid peroxidation in N. tabacum roots by about 30% compared with the control roots, on average over the time period. The maximum concentration of MDA was 12 ± 1.0 nmole/g fresh weight in roots grown without Ni and 17 ± 1.4 nmole/g fresh weight with Ni. 109 25 :::: .::2 ~ 20 ...... 1-, ...... :::: ...c:-- V 01) u :::: ·v 0 ~ 15 u ...c: V en "0 V >-. t.l:: ...c: 01) 10 V "Cl ------~ cii 0 ~:::: § 5 0 ,__, cii ~ 0 0 5 10 15 20 25 30 Time (days) Figure 3.69 Levels of malondialdehyde in A. bertolonii hairy roots grown without ( •) and with (•) Ni at an initial concentration of 25 ppm. 0 5 10 15 20 25 30 Time (days) Figure 3.70 Levels of malondialdehyde in N. tabacum hairy roots grown without(•) and with (•) Ni at an initial concentration of 25 ppm. 1 I 0 3. 5. 1. 6 Sulphydryl Groups on Root Cell Surfaces The levels of sulphydryl (-SH) groups on root cell surfaces were estimated in A. bertolonii and N. tabacum hairy roots cultured with and without Ni in the growth medium. Similar levels of -SH groups were found for A. bertolonii with and without Ni (Figure 3.71). On average over the time-course, the concentration of -SH groups determined in Ni-treated roots was 293 ± 60 nmole/g fresh weight, compared with 303 ± 51 nmole/g fresh weight in the control roots. Figure 3. 72 shows the concentration of surface -SH groups determined in the N. tabacum hairy roots. In contrast with A. bertolonii, Ni in the growth medium reduced the concentration of surface -SH groups by about 45% in N. tabacum compared with the control roots. Levels of surface -SH in the N. tabacum roots remained relatively constant with culture time. 600 .:: .s..., 500 ('j ..., ...,I-< --..c: .:: O[) Q) u .Q) 400 .:: 0 ::: u ..c: ::r: ~ 300 en cJ::::: I O[) Q) u Q) 200 ~ 0 I-< - ;:::S C/l § '-" 100 -~ u 0 0 5 10 15 20 25 30 Time (days) Figure 3. 71 Levels of cell surface -SH groups in A. bertolonii hairy roots grown without (•) and with(•) Ni at an initial concentration of 25 ppm. 11 1 600 c:: .8.... 500 Cl:! ...... I- ..i::::-- c:: ~ Q) u ·a3 c:: ::: 400 0 ..i:::: u Vl ::r: Q) CZl tb 300 I ~ Q) (.) ----~ ~ 0 200 I- ;:::$ E Vl c:: v -- 100 u 0 0 5 10 15 20 25 30 Time (days) Figure 3.72 Levels of cell surface -SH groups in N tabacum hairy roots grown without ( •) and with (•) Ni at an initial concentration of 25 ppm. 3. 5. 2 Cadmium Hyperaccumulator and Non-hyperaccumulator 3. 5. 2. 1 Superoxide Dismutase Superoxide dismutase (SOD) activities were measured in T caerulescens and N tabacum hairy roots in time-course experiments over a period of 21-28 days. SOD activity decreased with culture time in T. caerulescens and N tabacum roots grown with and without Cd (Figures 3.73 and 3.74). There was no significant difference in the SOD activities in T caerulescens roots cultured with and without Cd. Without Cd, the endogenous SOD activity in T caerulescens (Figure 3.73) was about 1.8 times that in N tabacum (Figure 3.74). In contrast with T caerulescens, SOD activities in N tabacum were 40-90% lower in Cd-treated roots compared with the controls. 112 700 >-. ·;- 600 ,-... ·--g ..c::- 00 500 V . ii; C/l .....ro :3: ;::1 ..c:: 400 C/l a V C/l :.a <.l::: 300 V 00 "O ---C/l -~ ·a- 200 0 I-< ;:::i V '-" 0.. ;::1 100 r:./1 0 0 5 10 15 20 25 30 Time (days) Figure 3.73 Superoxide dismutase activity in T. caerulescens hairy roots grown without (•) and with(•) Cd at an initial concentration of 20 ppm. 700 c 600 :~ ,-... (.) -ro ..c::-on 500 V ';jj roC/l ::: ;::1 ..c:: 400 - C/l a V C/l <.l::: ·-"O 300 V on "O ---C/l ·->< -~ 200 8 ·-;:J V ,._, g. 100 r:./1 0 0 5 10 15 20 25 30 Time (days) Figure 3.74 Superoxide dismutase activity in N tabacum hairy roots grown without(•) and with(•) Cd at an initial concentration of 20 ppm. 113 3. 5. 2. 2 Catalase The effect of Cd on catalase (CAT) activities in T caerulescens and N. tabacum hairy roots was monitored over a culture period of 21-28 days. Endogenous CAT activities in T caerulescens roots (Figure 3.75) were, on average, over 300 times greater than in N. tabacum (Figure 3.76), and were induced after 5 days of Cd treatment. Although short term CAT induction may have occurred in the N. tabacum roots during the first 3 days of Cd exposure, this response was not sustained. 120 -..... 100 ...... >-. ~ ;> Q.} ...... ~ 80 ..c:: ~ en Q.} Q.} en t,b 60 cd cd 0.0 -..... en cd .....----..... 40 u i::::: ;::J -- 20 0 0 5 10 15 20 25 30 Time (days) Figure 3.75 Catalase activity in T. caerulescens hairy roots grown without(•) and with (•) Cd at an initial concentration of 20 ppm. 114 0 5 10 15 20 25 30 Time (days) Figure 3.76 Catalase activity in N. tabacum hairy roots grown without(•) and with(•) Cd at an initial concentration of 20 ppm. 3. 5. 2. 3 Ascorbate Peroxidase Measurement of ascorbate peroxidase (APX) activities m T caerulescens showed that endogenous APX activities in T caerulescens (Figure 3. 77) were roughly half those in N. tabacum (Figure 3. 78). However, on average over th_e time-course, the APX activities measured in Cd-treated T caerulescens roots were similar to the control roots. The maximum APX activity in N. tabacum roots grown without Cd was 0.62 ± 0.01 Units/g fresh weight at 5 days, whereas 0.43 ± 0.06 Units/g fresh weight was measured as the maximum activity in 21-day-old Cd-treated T caerulescens roots. The effect of Cd on APX activities in N. tabacum roots was negligible. 115 0.7 .....;>-. 0.6 :E..... ,-...... (.) ..c: ro oJ) 0.5 (I) tl) ·v ro ~ ....."O ..c: 0.4 tl) >< (I) .....0 (I) r.t: 0.. oJ) 0.3 .....(I) ----.....tl) ro ·a 0.2 ,f ~ 0 '-' (.) Figure 3. 77 Ascorbate peroxidase activity in T caerulescens hairy roots grown without (•) and with(•) Cd at an initial concentration of 20 ppm. 0.7 ------. 0.6 0.5 0.4 0.3 0.2 0.1 0 ------,-----,.------,-----,.------1 0 5 10 15 20 25 30 Time (days) Figure 3.78 Ascorbate peroxidase activity in N tabacum hairy roots grown without (•) and with (•) Cd at an initial concentration of 20 ppm. 116 3. 5. 2. 4 Hydrogen Peroxide The concentration of H20 2 accumulated in T caerulescens and N. tabacum hairy roots was measured under various experimental conditions (Figures 3.79 and 3.80). H2O2 concentrations in T caerulescens hairy roots were similar with and without Cd (Figure 3.79). BSO by itself and BSO + Cd also had a negligible effect on H2O2 levels in T caerulescens compared with the control cultures. In N. tabacum roots (Figure 3.80), H2O2 concentrations without Cd were approximately constant and similar to those in T caerulescens. However, with Cd, levels of H2O2 in the N. tabacum roots were up to 5.1 times higher than those measured without Cd. Treatment of N. tabacum roots with BSO only also increased H2O2 levels by an average factor of about 2.5 compared with the control cultures without BSO. Exposure of N. tabacum roots to BSO + Cd increased the accumulation of H2O2 even further, especially during the early stage of the culture. § 2400 -~ -. 2100 ... .E g -~ 1800 8 ~ 1500 (I) ...c:: "O en .>< ~ 1200 0 00 ----- g_~ ~ 900 i::: 0 ~ § 600 0 '-" 300 -a>-. ::c: 0 ------~---- 0 5 10 15 20 25 30 Time (days) Figure 3.79 Level of hydrogen peroxide in T caerulescens hairy roots grown without BSO + Cd (control)(•), with Cd (•), with BSO (0) and with BSO + Cd (D). The initial concentration of Cd was 20 ppm and the BSO concentration was 100 µM. 117 2400 2100 1800 1500 1200 900 600 300 • • • • 0 -+------.------...------~----...------1 0 5 10 15 20 25 Time (days) Figure 3.80 Level of hydrogen peroxide in N. tabacum hairy roots grown without BSO + Cd (control) (•), with Cd (•), with BSO (0) and with BSO + Cd (0). The initial concentration of Cd was 20 ppm and the BSO concentration was 100 µM. 3. 5. 2. 5 Malondialdehyde Lipid peroxidation levels in T. caerulescens and N. tabacum hairy roots were estimated by measuring the concentration of MDA produced in the roots. During this study, the concentration of MDA estimated in Cd-treated N. tabacum roots. was higher than the concentration estimated in Cd-treated T. caerulescens roots. The presence of Cd in the culture medium increased the concentration of MDA in both T. caerulescens (Figure 3.81) and N. tabacum (Figure 3.82). In 21-day-old T. caerulescens roots grown with Cd, the MDA level of 17 ± 1.3 nmol/g fresh weight was 6.6 times higher than the concentration measured in the corresponding control roots. Cd increased lipid peroxidation by 72% in T. caerulescens and by 46% in N. tabacum roots on average over the time-course compared with the control roots. Thus, Cd accumulation induced lipid peroxidation both in the hyperaccumulator and non-hyperaccumulator roots. 118 i::: 0 27 -~ Z" 24 ...... c: 8 -~ 21 8 ! 18 15 ~>. cl::~ ~ 0/j 12 :9 Q) -~ 0 9 -g § 0 -- 6 ~ :::E 3 0 5 10 15 20 25 30 Time (days) Figure 3.81 Level of malondialdehyde in T. caeru/escens hairy roots without (•) and with (•) Cd at an initial concentration of 20 ppm. 0 5 10 15 20 25 30 Time (days) Figure 3.82 Level of malondialdehyde in N. tabacum hairy roots without(•) and with (•) Cd at an initial concentration of 20 ppm. 119 3. 5. 2. 6 Total Glutathione The total glutathione (GSH + GSSG) levels in T. caerulescens and N. tabacum roots are shown in Figures 3.83 and 3.84. The presence of Cd and BSO in the culture medium of T. caerulescens roots reduced the total glutathione levels ·relative to the control roots, in the order of BSO + Cd > BSO > Cd. Similar results were obtained for N. tabacum roots cultured with Cd, BSO and BSO + Cd. However, the concentration of total glutathione determined under normal growth conditions (without Cd or BSO) as 149 ± 9 nmole/g fresh weight for T. caerulescens roots and 42 ± 3.1 nmole/g fresh weight for N. tabacum roots, on average over the time-course. This indicates that T. caerulescens roots produced comparatively high levels of endogenous glutathione. 200 150 100 50 o------.------.,------1 0 5 10 15 20 25 30 Time (days) Figure 3.83 Level of total glutathione (GSSG + GSH) in T. caerulescens hairy roots grown without BSO + Cd (control)(•), with Cd (•), with BSO (0) and with BSO + Cd (0). The initial concentration of Cd was 20 ppm and the BSO concentration was 100 µM. 120 250 .9= .... 200 ~ -:c bJl II)= (.) ·a; 0= ~ 150 (.) ..c: rf) II) II) 0= c.t:: ~.... cIJ 100 ro ---II) ....::l -0 - E bJl = 50 ~.... '-' 0 t""' 0 0 5 10 15 20 25 Time (days) Figure 3.84 Level of total glutathione (GSSG + GSH) in N. tabacum hairy roots grown without BSO + Cd (control)(•), with Cd (•), with BSO (0) and with BSO + Cd (0). The initial concentration of Cd was 20 ppm and the BSO concentration was 100 µM. 3. 5. 2. 7 Sulphydryl Groups on Root Cell Surfaces The effect of 5,5 'dithio-(2-nitrobenzoic acid) (DTNB) infiltration time on the analysis of surface -SH group concentration in T. caerulescens hairy roots is presented in Table 3.5. The results indicate that the infiltration time did not have a significant effect on the results for surface -SH concentration. Similarly, the potential interference of Cd in the -SH analysis was tested using cysteine (Table 3.6) and extracts of T. caerulescens roots (Table 3. 7). The results show that addition of Cd to root extracts before -SH analysis had no effect on the measurement, indicating that the DTNB reaction is not influenced by the presence of Cd per se, and that any differences in -SH concentration do not reflect differences in root Cd content. 121 Table 3.5: The effect of DTNB infiltration time on surface -SH group concentration in T. caerulescens roots. ± indicates standard error from triplicate samples. Incubation time Concentration of surface -SH (min) groups (nmole/g fresh weight) 15 123.0 ± 11.0 20 109.5 ± 7.2 60 99.3 ± 6.7 90 84.6 ± 2.6 120 121.2 ± 19.3 Table 3.6: Effect of Cd on -SH analysis. Cysteine (0.01 M) Absorbance Absorbance (µL) without Cd with 200 µg Cd 40 0.188 0.173 60 0.258 0.248 80 0.320 0.324 100 0.392 0.399 120 0.447 0.452 150 0.507 0.510 122 Table 3.7: Effect of Cd on -SH analysis in extracts of T caerulescens roots cultured without Cd. Extract (µL) Absorbance without Absorbance with 200 µg Cd Cd 100 0.070 0.067 200 0.127 0.127 300 0.180 0.182 400 0.230 0.232 The concentration of -SH groups on the cell surfaces in T caerulescens hairy roots was measured using roots cultured without and with Cd, BSO and Cd + BSO. The results show that the presence of Cd in the culture medium increased the concentration of surface -SH groups by more than 50% on average over the time period, compared with the control roots (Figure 3.85). However, BSO eliminated completely the stimulatory effect of Cd on surface -SH groups in T caerulescens. Similar experiments were conducted for N. tabacum and it was found that the effects of Cd, BSO and BSO + Cd on the concentration of surface -SH groups were insignificant, compared with the control (Figure 3.86). In contrast with the results for T caerulescens, BSO did not change surface -SH levels in N. tabacum, with or without Cd. 123 1000 i:: 0 900 ...... c:: ---01) 800 ~ .Q) i:: Q) 700 u ~ i:: ..c:: 0 VJ "600 u Q) :r:: r.!:: 500 r/J 01) 400 Q)·~ 0 u ~.... § 300 ;:::s '-" r/J 200 100 0 0 5 10 15 20 25 30 Time (days) Figure 3.85 Surface -SH groups in T. caerulescens hairy roots grown without BSO + Cd (control) (•), with Cd (•), with BSO (0) and with BSO + Cd (D). The initial concentration of Cd was 20 ppm and the BSO concentration was 100 µM. 1000 ------, 900 800 700 600 500 400 300 200 ! 100 0 -+-----.------.-----""T'"""-----r------t 0 5 10 15 20 25 Time (days) Figure 3.86 Surface -SH groups in N. tabacum hairy roots grown without BSO + Cd (control) (•), with Cd (•), with BSO (0) and \\'.ith BSO + Cd (D). The initial concentration of Cd was 20 ppm and the BSO concentration was 100 µM. 124 3. 6 Nickel Recovery 3. 6. l Alyssum bertolonii Hairy Roots 3. 6. I. I Inductively Coupled Plasma-Atomic Emission Spectrometry (JCP-AES) Analysis The results from ICP-AES analysis of A. bertolonii hairy root biomass with and without Ni are shown in Figure 3.87. The results presented in Figure 3.87 are for roots with a particular Ni concentration of 5.5% (w/w) but are typical of A. bertolonii biomass with other Ni contents. Without Ni, K accounted for about 9% of the total mass, whereas in biomass with Ni the concentration of K was about 5%. The other elements measured in this experiment (B, Ca, Cu, Fe, Mg, Mn, Na, Ni, P, S, and Zn) were present at concentrations below 1% (w/w). In A. bertolonii roots with and without Ni, other elements not measured by ICP-AES, such as C, H, 0 and N, accounted for about 90% of the biomass. 100 en en 90 C<:l E 80 0 :E 70 ea..... 60 .....0 s::::: 50 ·-(l) 40 l:)J) .....C<:l 30 s::::: (l) ~ 20 (l) p.... 10 0 ;:::s p.... s::::: u z z N Elements Figure 3.87 Typical results for elemental composition of A. bertolonii biomass with (•) and without (D) Ni. The biomass Ni concentration in this sample was 5.5% (w/w). 125 3. 6. 1. 2 Furnace Treatment 3. 6. 1. 2a Effect of Time Ground biomass of A. bertolonii containing 6.1 % (w/w) Ni was treated at l 200°C for 3-17 h with air passing through the furnace. The results showed that similar weight losses of 93- 94% were observed between 3 and 17 h (Table 3.8), indicating that treatment time after 3 h did not make any difference in terms of weight loss for A. bertolonii hairy roots. At the end of the experiments, metal residue was found on the tiles recovered from the furnace for all samples. The elemental composition of the residues obtained after furnace treatment of A. bertolonii hairy roots for various times is presented in Figure 3.88. Biomass before treatment in the furnace (0 h), contained about 5.5% Ni, 5% K and 0.5% P. Other elements such as C, H, 0 and N not determined by ICP-AES accounted for above. 80% of the biomass. After furnace treatment of A. bertolonii hairy roots containing 6.1 % Ni (w/w) for 3-17 h, the metal residues contained above 80% Ni. The residues obtained after 3 and 6 h furnace treatment contained above 5% P; residues after 9 and 17 h furnace treatment contained about 1.7% P. However, in all the samples the other major elements measured below ·1 % of the total mass. Table 3.8: Alyssum bertolonii biomass before and after furnace treatment for various times at 1200°C with air. Time (h) Initial Residue (g) % weight loss biomass (g) 3 1.6 0.10 93.6 6 1.5 0.10 93.6 9 1.5 0.10 . 93.5 17 1.6 0.11 93.5 126 120 JOO VJ VJ ('3 E 80 Elements Figure 3.88 Elemental composition of A. bertolonii hairy roots with initial (0 h) Ni concentration of 5.5% (w/w) and samples with initial Ni concentration of 6.1 % (w/w), after treatment for 3-17 h in the furnace at 1200°C with air; 0 h (a ), 3 h (0 ), 6 h (D ), 9 h ( ) and 17 h c• ). 3. 6. 1. 2b Effect of Initial Concentration of Ni Ground biomass of A. bertolonii hairy roots contai ning various Ni concentrations (0.4-7.7% (w/w)) was burnt at 1200°C for 17 h with air passing through the furnace. It was observed that with decreasing Ni concentration, the percentage weight loss increased (Table 3.9). This indicates that high levels of Ni in the biomass reduced the percentage weight loss. The metallic residue after furnace treatment of A. bertolonii hai ry roots with 7.7% (w/w) Ni is shown in Figure 3.89. 127 Table 3.9: Alyssum bertolonii biomass with various initial Ni concentrations before and after furnace treatment for 17 h at l 200°C with air. Initial Initial Ni Residue (g) % weight loss biomass concentration (g) % (w/w) l.9 7.7 0.22 88.7 1.5 5.9 0.10 93.1 1.4 5.1 0.09 96.8 1.4 4.6 0.07 94.8 1.4 3.0 0.05 96.5 1.3 1.9 0.03 97.6 1.4 0.4 0.02 98.3 Figure 3.89 Metal residue obtained after furnace treatment of A. bertolonii biomass (7.7% w/w Ni) at I 200°C for 17 h with air passing through the furnace. 128 Field emission scanning electron microscopy (FESEM) analysis showed that the elimination of elements such as C, H, 0 and N and crystal formation of metal during the burning process increased with initial Ni concentration in the biomass. The residue obtained from the biomass containing 7.7% (w/w) Ni showed a crystal-like structure under FESEM (Figure 3.90) and the qualitative elemental analyses showed that the residue contained Ni, Al and 0 (Figure 3.91 ). The Al and, to a certain extent, the O content of the residue was associated with the background (tile) composition (Figure 3.92). The residue from furnace treatment of biomass with a lower Ni concentration of 1.9% (w/w) contained a greater number of impurities such as Mg, Ca, Fe and Pas well as Ni, Al and O (Figure 3.93). Figure 3.90 Crystal-like structure of metal residue from A. bertolonii biomass (7.7% w/w Ni) after furnace treatment at I 200°C for I 7 hours with air passing through the furnace. 129 cps 25 Ni 20 15 0 Ni f Energy (keV) Figure 3.91 Qualitative elemental analysis (under FESEM) of residue obtained after furnace treatment of A. bertolonii biomass containing 7.7% (w/w) Ni at 1200°C for 17 h. cps indicates cycles per second. cps 0 8 6 4 2 2 4 e Energy (keV) Figure 3.92 Qualitative elemental analysis of alumina tile under FESEM. cps indicates cycles per second. 130 cps .----;,p 'I I 0 ,11 Energy (keV) Figure 3.93 Qualitative elemental analysis, using FESEM, of residue obtained after furnace treatment of A. bertolonii biomass containing 1.9% Ni (w/w) for 17 hat 1200°C. cps indicates cycles per second. XRD analysis ofresidue obtained from the biomass with 7.7% (w/w) Ni showed that most of the peaks detected correspond to the characteristics of NiO (Figure 3.94). C 0 u n t 1200 Figure 3.94 XRD analysis of metal residue obtained after furnace treatment of A. bertolonii biomass containing 7.7% (w/w) Ni for 17 hat 1200°C with air. Degrees 2-Theta is the scanning angle. The marked ( 0) peaks indicate Ni 0. 131 The effect of the initial concentration of Ni on the results from 17 h furnace treatment of A. bertolonii hairy roots was studied for Ni concentrations between 7.7% and 0.4% (w/w). At the end of the furnace treatments, metal residue was obtained for all the samples. These samples were analysed for elemental composition using ICP-AES and the results are shown in Figure 3.95. The percentage of Ni in the residues decreased with decreasing initial Ni concentration. The Ni concentration in the residue obtained from the biomass containing 7. 7% (w/w) Ni was 42 times higher than that in the residue obtained from the biomass containing 0.4% (w/w) Ni. However, the concentration of 'other elements' (elements not determined by ICP-AES analysis, such as C, H 0, and N) increased with decreasing initial concentration of Ni in the biomass. For instance, the levels of 'other elements' was 30% of the total biomass with an initial concentration of 7.7% (w/w) Ni, whereas the levels measured for the biomass with an initial concentration of 0.4% (w/w) Ni was about 97%. Apart from Ni, the levels of P and, in some samples, K accounted for between 1 and 4% of the total elemental composition. Most of the elements indicated in Figure 3.96 contributed below 1% of the total composition. 3. 6. I. 2c Effect ofNitrogen A. bertolonii biomass with 4.4% (w/w) Ni was treated at 1200°C for 17 h with nitrogen gas passing through the furnace. At the end of this experiment, instead of a metal residue, a substantial amount of black powder was found on the tile corresponding to a weight loss of about 88%. A similar experiment was conducted for A. bertolonii biomass with 4.4% (w/w) Ni with air passing through the furnace for 5 h. At the end of this experiment, a metal residue was found on the tile corresponding to a 93% weight loss. When this residue was examined under FESEM, a crystal-like structure was found (Figure 3.96). 132 120 -.------, 100 - V, V, e 0 ~ e Figure 3.95 Elemental composition of residues after I 7 h furnace treatment of A. bertolonii hairy root biomass containing Ni at different initial concentrations (w/w) of: 0.4% (D ), 1.9% c• ), 3.0% (D ), 4.6% ( ), 5.1 % c• ), 5.9% (D ), 7.7% (D ). Figure 3.96 Crystal-like structure observed under FESEM for A. bertolonii hairy roots with 4.4% (w/w) Ni treated at 1200°C for 5 h with ai r passing through the furnace. 133 The results from elemental analysis of samples treated under nitrogen and air are presented in Figure 3.97. The loss of 'other elements' was significantly inhibited under the nitrogen atmosphere compared with the sample treated with air passing through the furnace. The level of 'other elements' measured in the sample treated with nitrogen was about 3.5 times higher than in the sample treated with air. Ni contributed about 78% of the total mass of the residue obtained under air compared with only 45% in the sample treated under nitrogen. However, similar levels of P were measured for samples treated under nitrogen and air. 90 80 - C/l C/l cd 70 a . ~ 60 +-' 0 +-' 50 . '- - 0 Q) - OJ) 40 cd +-' s::: . Q) 30 (.) I-< Q) 20 A,. 10 - ra 0 I I I I I I I I I I I I zcd z r./) Elements Figure 3.97 Elemental composition of A. bertolonii biomass containing 4.4% (w/w) Ni after furnace treatment at 1200°C for 17 h with nitrogen (D) and for 5 h with air(•). 134 3. 6. 2 Berkheya coddii Plants 3. 6. 2. 1 Biomass Dry Weight and Ni Uptake The above-ground biomass dry weight analyses showed that the maximum level of growth of B. coddii plants, 68 ± 7 g biomass dry weight, occurred at an initial Ni concentration of 1 mg/g soil (Figure 3.98). The level of biomass production was 3. 7 times higher than for the corresponding control plants. The growth of Ni-treated plants decreased with increasing initial Ni concentration in the soil above 1 mg/g. The Ni accumulation analyses showed that the concentration of Ni in the biomass increased with increasing initial Ni concentration in the soil (Figure 3.99). The maximum concentration of Ni obtained was about 5500 µg/g dry weight or 0.55% (w/w). 80 70 ,-.., 01) '-" 60 ...c::~ -~01) 50 ~ 40 "Oc Cl) Cl) 30 sro 0 20 ·-o::i 10 0 0 1 2 3 4 5 Initial concentration of Ni (mg/g soil) Figure 3.98 Biomass dry weight of B. coddii grown at different initial Ni concentrations in the soil. The error bars represent standard errors from five replicates. 135 6000 5000 (l) --..c:: -CJ) ..c:: . a3 - 4000 .5 ~ c:: .s -0c ro CJ) 3000 -I-...... __ c:: on -(l) :i u .._,, c:: rn 2000 0 rn u ro E z .s 1000 .0 0 0 1 2 3 4 5 Initial concentration of Ni in the soil (mg/g soil) Figure 3.99 Ni concentration in the biomass of B. coddii. The error bars represent standard errors from five replicates. 3. 6. 2. 2 ICP-AES Analysis Elemental analysis of B. coddii biomass grown with and without Ni was performed using ICP-AES (Figure 3.100). Approximately 90% of the total elemental composition was accounted for by 'other elements' not measured by ICP-AES, such as C, H, 0 and N, both with and without Ni. The K content measured in the biomass of B. coddii without Ni was 1.8 times higher than the K content with Ni. Similar levels of Ca were measured in both Ni treated and control B. coddii biomass. However, in B. coddii grown with Ni, the P levels were reduced by 2.5 times compared with plants grown without Ni. 136 100 90 - - '/J 80 - '/Jro s 70 - 0 :.0 60 ~...... 0 ...... so 0 Q) Oil 40 .....ro i::: Q) ....(.) 30 Q) 0... 20 10 - 0 ~ rl. -, ~ G <.,~ + ~~ ~~ ~'?> ~ ~ 'V~ ~,, -+' o;;s; Elements Figure 3.100 Elemental composition of B. coddii biomass grown without (D) and with 3. 6. 2. 3 Furnace Study Ground biomass of B. coddii containing 0.55% (w/w) Ni was burnt for different times at 1200°C with air passing through the furnace. The duration of the furnace treatment did not affect the weight loss of the samples (Table 3.10), and all treatments left ash on the tile. The ash obtained after 3 h and 24 h of furnace treatment was analysed under FESEM for qualitative elemental composition (Figures 3.101 and 3.102). The results showed that both samples had similar elemental composition with significant levels of Ca, except that there was no sulphur in the ash of the 24 h sample. 137 Table 3.10: B. coddii with 0.55% (w/w) Ni burnt at I 200°C with air passing through the furnace for different time periods. Time (h) Initial biomass dry Residue (g) % of weight weight (g) loss 3 2.13 0.11 94.7 6 2.09 0.11 94.6 9 2.16 0.1 I 94.7 17 2.22 0.12 94.7 24 2.22 0.11 94.9 2 4 6 8 10 Energy (ke\l) Figure 3.101 Qualitative elemental analysis, using FESEM, of ash obtained after furnace treatment of B. coddii biomass containing 0.55% (w/w) Ni for 3 hat 1200°C with air. cps indicates cycles per second. 138 2 4 6 8 Energy (ke\/) Figure 3.102 Qualitative elemental analysis, using FESEM, of ash obtained after furnace treatment of B. coddii biomass containing 0.55% (w/w) Ni for 24 h at I 200°C with air. cps indicates cycles per second. The results from elemental analysis of B. coddii biomass containing 0.55% (w/w) Ni after furnace treatment for various times are represented in Figure 3.103. The residues obtained after furnace treatment at 1200°C for 3 and 6 h were mixed together (3 + 6); similarly, residues obtained after 17 and 24 h were also mixed together (17 + 24) before ICP-AES analysis. In biomass before furnace treatment (0 h), the level of 'other elements' not measured by ICP-AES was about 90% of the total mass. In the furnace-treated samples, the 'other elements' accounted for about 40% of the total mass independent of the duration of the treatment. In addition, Ca and Ni were concentrated by up to 17-fold in furnace-treated samples compared with the 0 h sample that was not treated in the furnace. Similarly, levels of P were about 19 times higher in furnace-treated samples than in the biomass of B. coddii with Ni not treated in the furnace. 139 100 90 80 Figure 3.103 Elemental composition of B. coddii biomass containing 0.55% (w/w) Ni after treatment in the furnace at 1200°c with air for different times: 0 h (D ), 3 + 6 h C• ), 9 h ( ), 17 + 24 h (D ). Ash obtained after furnace treatment of B. coddii biomass containing 0.55% (w/w) Ni at 1200°C with air for 3 + 6 h and 17 + 24 h was examined under XRD. Major peaks identified fo r the 3 + 6 h sample were CaO, NiO and Ca-Ni complex (Figure 3.104). Peaks recorded fo r the 17 + 24 h sample correspond to Ca 0 , Ni 0 , Ca-Ni complex and CaO4 (Figure 3.105). These results indicate that the ash produced by furnace treatment contained mainl y Ca and Ni as major elements. 140 4000 C 0 u n a t 3000 C 2000 ,-,,.-. -.-.-. -I ..,...,....-,-.,..,...,..,...,...... ,...,..,..,...,..,._ -,.,--,-,--.,...,...---- 0__.,______, -. -1-' .,.., 1 30 40 ~ ~ ro oo ~ 100 Degrees 2-Theta Figure 3.104 XRD analysis of B. coddii biomass· containing 0.55% (w/w) Ni after treatment in the furnace at 1200°C with air for 3 + 6 h. Peaks are identified as (a) Cao, (b) NiO and (c) Ca-Ni complex 4000·--,------, C 0 u n a 3000 b C C 2000 1000 d -~~J o,_.______...,.....,.._,...... ,..,..,1..,....,..,.....,..,.....,..,..,,..,..,.....,...,.....,..,..,.,..,..,.....,..,.....,.,..l"'T'"I"'!""!"'!'.....,-:,-:-, -:-1 __,, ___. 30 40 50 60 70 80 90 100 Degrees 2-Theta Figure 3.105 XRD analysis of B. coddii biomass containing 0.55% (w/w) Ni after treatment in the furnace at 1200°C with air for 17 + 24 h. Peaks are identified as (a) CaO, (b) NiO, (c) Ca-Ni complex and (d) CaO4• 141 CHAPTER 4 - DISCUSSION 4. 1 Growth Analyses Plants growing in a heavy metal polluted environment are adapted to survive using one of two strategies: 'avoidance' by which the plant is protected externally from the influence of stress, and 'tolerance', by which the plant survives the -effects of internal stress caused by elevated metal concentrations (Baker, 1987). Plant species devoid of both these strategies are unable to withstand or grow against the metal toxins and become hypersensitive to heavy metals. In this work, the biomass dry weight analyses revealed the extent of Ni tolerance in A. bertolonii and N. tabacum hairy roots (Figures 3.1 and 3.3), and Cd tolerance in T. caerulescens and N. tabacum hairy roots (Figures 3.8 and 3.9). Alyssum bertolonii roots were tolerant of 25 ppm (426 µM) Ni, whereas the same concentration of Ni was extremely toxic to N. tabacum · roots. The results for A. bertolonii and N. tabacum roots are in accordance with previous studies performed in this laboratory (Nedelkoska and Doran, 2001). It has been reported that growth inhibition in Ni-non hyperaccumulators is due mainly to the suppression of mitotic activity and energy requiring cellular processes in the cells (Espen et al., 1997; Gabbrielli et al., 1990). Similarly, the Cd-hyperaccumulator, T. caerulescens, did not show any reduction in its growth at 20 ppm (178 µM) Cd compared with the growth of roots without Cd. However, at 20 ppm Cd, N. tabacum hairy roots suffered severe growth inhibition compared with the control roots. Although N. tabacum hairy roots accumulated similar levels of heavy metals compared with A. bertolonii and T. caerulescens hairy roots, it failed to survive the metal concentrations. It is apparent that N. tabacum roots were unable to utilise either of the two 142 survival strategies and are not tolerant of 25 ppm Ni or 20 ppm Cd. Similar results have been reported previously for Cd-non-hyperaccumulators, including N tabacum hairy roots, cauliflower plants, azuki bean cells, and pea plants (Nedelkoska and Doran, 2000; Hasegawa et al., 1997; Inouhe et al., 2000; Dixit et al., 2001; Sandalio et al., 2001). In this study, both the hyperaccumulators and non-hyperaccumulator accumulated heavy metals but the hyperaccumulators most likely employed internal detoxification mechanism(s) to overcome metal toxicity. The causes of the toxicity of the heavy metals to plant roots are unclear. However, it has been suggested that the limited elongation of roots under the influence of Cd can result from inhibited mitosis, decreased synthesis of the components of cell walls, damaged Golgi apparatus or changes in the metabolism of polysaccharides in the root caps (Punz and Sieghardt, 1993). The synthetic estrogen, diethylstilbestrol (DES), is known mainly as an inhibitor of plasma membrane H+-ATPase (Michelet and Boutry, 1995). The plasma membrane is the outer permeability barrier of plant cells and one of the first parts of the cell to sense changes in the environment. Proton-pumping plasma membrane ATPase (H+-ATPase) is responsible for generating the pH and electrochemical potential difference across the membrane that drives secondary transport of a range of nutrients into the cells. Influx of sucrose and hexoses such as glucose and fructose occurs via symporters dependent on the availability of H+ ions outside of the plasma membrane (Bush, 1993; Rausch, 1991). Active uptake of other compounds such as amino acids, peptides and anions is coupled to the proton gradient through operation of specific transmembrane ·carriers (Logan et al., 1997). The electrical potential difference generated by H+-ATPase is also used to drive cation uptake through ion channels. However, other transporters, including for heavy metals, are directly energised by ATP rather than the transmembrane H+ difference (Williams et al., 2000; 143 Palmgren and Harper, 1999), and are thus uncoupled from the activity of H+ -ATPase. As well as playing a key role in transport, H+-ATPase is also a principal regulator of intracellular pH (Palmgren, 1998). Inhibition of H+-ATPase by DES collapses the proton gradient maintained by this enzyme, thus deactivating a variety of secondary transport processes. Acidification of the cytoplasm and / or alkalinisation of the apoplasm can also be expected to occur. The severely reduced growth of DES-treated A. bertolonii (Figure 3.5) and T. caerulescens (Figure 3.11) hairy roots is consistent with the prevention of nutrient uptake following inhibition of H+ - ATPase activity. The reason for the relatively small effect of DES on growth of N. tabacum roots (Figure 3.6) is unclear; in these roots, either the concentration of DES was insufficient to completely inhibit H+-ATPase, or alternative transporters directly energised by ATP were available to provide nutrients. In T. caerulescens and N. tabacum roots, the glutathione synthesis inhibitor, buthionine sulfoximine (BSO), reduced glutathione levels several-fold (Figures 3.83 and 3.84) but the growth of hairy roots was not affected (Figures 3.13 and 3.14). This shows that glutathione did not have a direct role in the growth of hairy roots under usual culture conditions. In T. caerulescens hairy roots, Cd-detoxification was also independent of the levels of glutathione or glutathione-related compounds (Figure 3.84). 4. 2 Nickel Uptake and Distribution In 9-h experiments, dead and live hairy roots of A. bertolonii accumulated similar levels of Ni, indicating that short-term accumulation occurred by simple adsorption due, for example, to the ionic concentration difference between the solid and liquid phases (Figures 144 3.19 and 3.20). Initially, the dead N tabacum hairy roots tended to accumulate more Ni compared with the live roots, but after 5 h, the Ni concentration in the dead biomass started to decline. This suggest that the accumulated Ni was not permanently associated with any kind of cell components and diffused back into the liquid medium. In the long-term experiments, Ni concentrations in A. bertolonii were between 380 and 820 µg/g dry weight and in N tabacum hairy roots between 650 and 930 µgig d: wei&ht (Figures 3.+1 an?. . ,t.f'2, a,fuve-rrou,J. j,,on,.~s 3.22). These values are slightly lower than the threshold of 1000 µg/g dry weig~defined for Ni hyperaccumulation (Baker et al., 1994). However, in previous work with higher concentrations of Ni in the medium, hairy roots of A. bertolonii were shown to accumulate > 1000 µgig dry weight of Ni while retaining the ability to grow (Nedelkoska and Doran, 2001). In the liquid medium culture system employed in this study, metal concentrations in the biomass do not distinguish hyperaccumulator and non-hyperaccumulator phenotypes, as biosorption of Ni readily occurs in plant tissues submerged in Ni solutions (Nedelkoska and Doran, 2001). Instead, the ability of A. bertolonii hairy roots to tolerate high Ni concentrations while maintaining growth distinguish this species from N tabacum (Figure 3.1 ). In contrast, roots of the Ni-hyperaccumulator, A. troodii, accumulated higher concentrations of Ni than the non-hyperaccumulator, Aurinia saxatilis, in a pot trial study (Homer et al., 1991a). Although high levels of Ni accumulation have been reported for the Ni-hyperaccumulators, T. montanum, Dichapetalum gelonioides and Hybanthus species (Boyd and Martens, 1998; Homer et al., 1991b; Brooks et al., 1977), the initial concentration of Ni used in the above studies was much higher than the initial concentration of Ni used in this study. Thus, initial Ni cpncentration also plays a role in Ni accumulation in hyperaccumulating plant species. 145 In contrast with Cd, most of the Ni in A. bertolonii and N. tabacum hairy roots was released with the symplasm during solvent treatment (Figures 3.33 and 3.34). Exclusion of Ni from the interior of A. bertolonii cells therefore appears to play a minor role in Ni tolerance and detoxification. These results differ from those reported for leaves of the Ni hyperaccumulator, T. goesingense, in which 67-73% of the Ni absorbed during hydroponic culture was located in the apoplasm or cell walls (Kramer et al., 2000). Cell wall binding was of more importance to N. tabacum than A. bertolonii hairy roots during the first 5 days of Ni treatment; however, exclusion of Ni from the interior of the cell was unsuccessful in N. tabacum in the longer term (Figure 3.34). Ni was distributed along the length of the A. bertolonii and N. tabacum roots except for the N. tabacum tips (Figure 3.37). Exclusion of heavy metal from the root tips may be a protective response in N. tabacum aimed at avoiding damage to the meristem. In whole plants capable of hyperaccumulating Ni, high concentrations of Ni have been found in the pholem sap (Sagner et al., 1998) and in the xylem for translocation to the shoots (Kramer et al., 1996). The lack of particular concentration of Ni in the vascular structures of A. bertolonii may reflect the absence of a transpiration steam in hairy root cultures and the lack of a mechanism for root-shoot translocation of metals. DES increased the concentration of Ni in A. bertolonii and N. tabacum roots (Figures 3.21 and 3.22). However, the proportion of Ni associated with the cell walls of A. bertolonii was significantly enhanced, causing a substantial increase of Ni concentration in the apoplasm and decrease in the symplasm (Figures 3.33 and 3.3_5). Treatment with DES appears, therefore, to restrict the entry of Ni into the symplasm of A. bertolonii. These results may reflect a coupling between the proton gradient generated by H+-ATPase and transport of Ni across the plasma membrane; alternatively, Ni transport itself may have been inhibited by 146 DES. However, as DES is likely to have affected the activity of other proteins and processes in the cells, this interpretation remains tentative. 4. 3 Cadmium Uptake and Distribution In the short-term experiments, the dead biomass of T caerulescens and N. tabacum tended to accumulate higher concentrations of Cd than the corresponding live roots (Figures 3 .23 and 3.24), which indicates that dead biomass may have more binding sites after autoclaving than the live roots. This result is in accordance with the results obtained for dead microbial biomass (Santa-Maria and Cogliatti, 1998; Volesky and May-Philips, 1994). In the long-term experiments, T caerulescens and N. tabacum accumulated maximum Cd concentrations of 2121 and 2038 µgig dry weight C~, resp. ectively (Figure~ .,;.rz._ a/;,Jt(-- t_,cJu,rcf. /;,om.r4-t 3.25 and 3.26). These values are well above the threshold level (100 µgig dry weight)A defined for Cd-hyperaccumulators (Baker and Brooks, 1989; Reeves et al., 1995). However, the hyperaccumulator was distinguished from the non-hyperaccumulator by growing with high tissue concentrations of Cd. In contrast, the Cd-non-hyperaccumulator, water hyacinth, accumulated several-fold higher concentrations of Cd than the defined threshold concentration but it failed to survive at that concentration (Zhu et al., 1999c). Treatment with DES significantly altered the uptake and distribution of Cd in T caerulescens hairy roots (Figures 3.27 and 3.42). With DES, concentrations of Cd in the biomass were 2-3 times those in roots without DES. Previously, DES was found to increase Cd uptake in T caerulescens hairy roots during the first 9 hours of exposure (Nedelkoska and Doran, 2000). This was attributed to a possible enhancement in biosorption due to H+-ATPase inhibition increasing the negative charge and pH outside the plasma membrane. However, in the present work, as well as an increase in Cd 147 concentration in the roots, there was a shift in the distribution of Cd away from the cell walls into the symplasm and a substantial increase in Cd levels inside the cells. These results suggest that the membrane transporter responsible for Cd uptake into the symplasm of T caerulescens is not coupled to the proton gradient generated by plasma membrane H+-ATPase. It also suggests that the transporters involved in Cd uptake and/or intracellular detoxification are not directly inhibited by DES, which can affect the activity of transport proteins other than plasma membrane H+ -ATPase, including at the plant vacuole (Al Awqati, 1986). Although treatment with DES shut down T caerulescens growth (Figure 3.11) and, presumably, many other cell functions through the effect on nutrient uptake, the roots continued to accumulate high concentrations of Cd while protecting the cells from metal damage. As treatment with DES and inhibition of H+ -ATPase significantly reduces the ATP content of plant cells (Balke and Hodges, 1979), survival of the roots is testament to the resilience of the mechanisms of metal tolerance and hyperaccumulation in T caerulescens, and demonstrates the ability of this species to channel limited resources into metal detoxification under stress conditions. Zn and BSO did not have a significant effect on Cd accumulation in T caerulescens roots (Figures 3.25 and 3.29), whereas Cd accumulation was increased in N tabacum roots with Zn and BSO (Figure 3.26 and 3.30). This may be due to the lack of resistance to biochemical mechanisms in dead N tabacum roots. However, no significant effect on Cd accumulation was observed in T caerulescens and N tabacum roots when the roots were cultured with Cd + CHP and Cd + MAN, respectively (Figures 3.31 and 3.32). Most of the Cd in T caerulescens and N tabacum hairy roots remained with the cell wall after dissolution of the cell contents (Figures 3.38 and 3.39). This is consistent with 148 previous reports that Cd is stored mainly in the apoplast of T. caerulescens roots (Vazquez et al., 1992). The results also indicate that Cd detoxification in T. caerulescens roots occurs predominantly in the cell wall region. Although exclusion from the symplasm may play a role in Cd tolerance, Cd was toxic to N tabacum hairy roots even though 75% of the metal remained at the cell walls. The results for Cd localisation were not affected by co- '2-n-- incubation with Zn and BSO. De is considered to pass rapidly across the plasma membrane of T. caerulescens roots (Lasat et al., 1996). The delay in Cd uptake into the symplasm described previously for T. caerulescens hairy roots (Nedelkoska and Doran, 2000) was not observed in the present work. Nevertheless, symplasm Cd accounted for less than 36% of the total Cd in T. caerulescens for the entire duration of the culture (Figure 3.38) . • 4.4 Organic Acid Complexation Without heavy metal added to the cultures, the average total concentrations of citric, malic and malonic acids in A. bertolonii (Figures 3.47-3.49) and T. caerulescens (Figures 3.50- 3.52) hairy roots were 432 ± 65 and 421 ± 53 µmol/g dry weight, respectively. These are relatively high values; the concentration of the same organic acids in most crop species is within the range 10-90 µmol/g dry weight (calculated from Clark, 1969). This result indicates that high levels of organic acids are a constitutive property of T. caerulescens and A. bertolonii hairy roots. Whole plants of T. caerulescens also contain high endogenous levels of organic acids, particularly malic acid (Tolra et al., 1996; Shen et al., 1997). Complexation between organic acids and hea~y metals has been implicated for metal detoxification, transport and storage in several hyperaccumulator species (Brooks et al., 1981; Kersten et al., 1980; Kramer et al., 2000; Lee et al., 1978; Pelosi et al., 1976; Sagner et al., 1998; Salt et al., 1999; Tolra et al., 1996; Zhao et al., 2000). In the present 149 study, 28 ± 2% of the total Ni in A. bertolonii (Figure 3.59) and 13 ± 3% of the total Cd in T caerulescens (Figure 3.60) hairy roots were associated with organic acids. Organic acid complexation was therefore of greater importance in accumulation of Ni than Cd; however, in both species, most of the Cd or Ni was not present as water-soluble complexes with organic acids. The percentage of total Ni in water-soluble form in A. bertolonii hairy roots (Figure 3.53) was considerably lower than the value of 80% reported by Pelosi et al. (1976) for A. bertolonii leaves, and towards the lower end of the range of 32-77% for leaves of other Ni hyperaccumulators (Brooks et al., 1981; Homer et al., 1991a). This suggests that polar metal complexes, such as those with organic acids, play a smaller role in Ni accumulation by A. bertolonii hairy roots than in whole plants. Previous work has demonstrated that the capacity of Alyssum hairy roots for Ni hyperaccumulation is not as great as whole plants (Nedelkoska and Doran, 2001). Neither citric, malic or malonic acid accumulated to higher levels in the T caerulescens or A. bertolonii hairy roots after addition of Cd or Ni to the cultures. This is consistent with the organic acids already being present in substantial molar excess compared with the amounts of metal taken up by the roots (26:1 for Cd and 37:1 for Ni). Significant additional acid synthesis was therefore not required for metal complexation. A similar lack of response of organic acid levels to Ni has been observed for the Ni hyperaccumulator, A. lesbiacum (Kramer et al., 1996). In contrast, positive correlations have been found between external Zn and organic acid concentrations in the roots of hyperaccumulator plants (Zhao et al., 2000). This may reflect in part the much higher concentrations of Zn taken up by hyperaccumulators compared with Cd and Ni, and the correspondingly lower organic acid to metal ratios. 150 4. 5 Oxidative Stress in Ni-hyperaccumulator and Non hyperaccumulator Plant species undergo oxidative stress in various circumstances, including in the presence of heavy metals. Heavy metals, especially redox-active-metals, are strong oxidants and are known to produce free radicals or reactive substances by their oxidation and reduction reactions. These free radicals can enhance or suppress the activity of antioxidant enzymes in a cell system. Generally, plants have enzymatic and non-enzymatic defense mechanisms against in situ free radical products. Among the enzymatic defenses, CAT, SOD and APX play a vital role to eliminate the reactive substances. The contrasting effects of 25 ppm Ni on growth of A. bertolonii and N. tabacum hairy roots (Figure 3 .1) demonstrate that A. bertolonii roots possess superior mechanisms for protection against Ni toxicity. There were substantial differences in antioxidative enzyme activities between the two species. SOD activity was greater in A. berto/onii roots than in N. tabacum, with or without Ni (Figures 3.61 and 3.62). However, Ni reduced SOD activity in the hyperaccumulator while it had a negligible effect on SOD activity in the non-hyperaccumulator. These results for N. tabacum are in accordance with the results obtained for Zea mays seedlings (Baccouch et al., 2001). In contrast, an induction of SOD activity was observed in another non-hyperaccumulator, pigeonpea seedlings, upon exposure to Ni (Rao and Sresty, 2000). CAT activities in Ni-treated A. bertolonii were more th_an two orders of magnitude higher than in Ni-treated N. tabacum, reflecting significantly greater endogenous CAT levels in A. berto/onii (Figures 3.63 and 3.64); however Ni reduced CAT activities in both species. The CAT results obtained for N. tabacum in the present study are consistent with the results 151 reported by Rao and Sresty (2000) for pigeonpea seedlings. However, the above results for N. tabacum are not consistent with the results obtained for Z. mays (Baccouch et al., 2001). In contrast, APX was more active in both the control and Ni-treated N. tabacum roots (Figure 3.66) than in the A. bertolonii roots (Figure 3.65). However, following the application of Ni, APX activity reduced in both species, more significantly in A. bertolonii roots. In contrast, Ni stimulated APX activity in the non-hyperaccumulator species Z. mays (Baccouch et al., 2001). Because H2O2 levels were similar in both A. bertolonii and N. tabacum roots without Ni treatment (Figures 3.67 and 3.68), the results for the control cultures suggest that A. bertolonii roots rely more on CAT, while N. tabacum relies more on APX and the ascorbate/glutathione cycle, to control H2O2 levels. Therefore, although A. bertolonii suffered a significant reduction in APX activity with Ni (Figure 3.61), H2O2 concentrations were lower in the hyperaccumulator species than in the Ni-treated N. tabacum roots (Figures 3.67 and 3.68). Unlike peroxidases such as APX, CAT has the advantage of not requiring an additional source of reducing power for reaction. Presumably, the antioxidative metabolism relied upon by N. tabacum in the absence of significant endogenous CAT activity was detrimentally affected or overstretched in the presence of Ni, resulting in almost immediate 5.5-fold surge in H2O2 concentration (Figure 3.68) and persistent elevated levels of lipid peroxidation (Figure 3.70). H2O2 levels in A. bertolonii roots also increased with Ni (Figure 3.67), but to a lesser extent than in N. tabacum. Higher endogenous CAT activities in A. bertolonii may therefore give this species an advantage relative to N. tabacum for combating Ni-induced oxidative stress. In A. bertolonii, exposure to Ni caused a reduction in the activity of all three antioxidative enzymes measured: SOD, CAT and APX (Figures 3.61, 3.63 and 3.65). This is contrary to 152 the typical antioxidative response to metal-induced stress, whereby the activities of enzymes capable of removing free radicals and active oxygen species are induced. Similar results were found with N. tabacum, except that SOD activities were essentially the same with and without Ni. Significant differences in antioxidative response patterns therefore did not occur between the Ni-hyperaccumulator and non-hyperaccumulator. It has been reported that decreasing CAT and APX activities in Cu-treated oat leaves and Pb-treated Lupinus luteus roots may be an indication that metal ions induced toxic oxygen species (Luna et al., 1994; Gwozdz et al., 1997). However, it was suggested that an induction of APX activity occurred because of Cu-induced oxidative stress in Phaseolus vulgaris and Ceratophyllum demersum (Weckx and Clijsters, 1996; Ramadevi and Prasad, 1998). The reduced activities of SOD, CAT and APX in Ni-treated A. bertolonii roots may be the result of enzyme deactivation, either from direct interaction with Ni ions or active oxygen species (Dat et al., 2000; Dietz et al., 1999), or as the result of enzyme modulation by stress-related effector molecules (Dat et al., 2000; Foyer et al., 1997; Takahashi et al., 1997). It is unlikely that the activity of these enzymes declined because of a lower requirement for antioxidative metabolism, as H2O2 levels were significantly increased in the presence of Ni in both A. bertolonii and N. tabacum hairy roots. However, A. bertolonii was much more tolerant of this condition so that growth remained unaffected. The higher H2O2 levels in the Ni-treated N. tabacum roots could be responsible for oxidative damage to proteins, DNA and other macromolecules required for growth. The reduction in reactive surface -SH groups in Ni-treated N. tabacum roots (Figure 3.72) is consistent with oxidation of -SH groups being a major toxic effect of H2O2 (Dat et al., 2000). In contrast, the maintenance of control levels of surface -SH in A. bertolonii (Figure 3.71) reflects the greater tolerance by the hyperaccumulator species of its accumulated H2O2 levels. In 153 contrast to A. bertolonii roots, Ni slightly increased lipid peroxidation levels in N. tabacum roots (Figure 3.70) which is consistent with results reported for the seedlings of the Ni non-hyperaccumulator, Triticum aestivum (Pandolfini et al., 1992). Similar results were also reported for the Cu-non-hyperaccumulators, C. demersum and P. vulgaris, in response to Cu (Ramadevi and Prasad, 1998; Weckx and Clijsters, 1996). The only other published observations on antioxidativ~ metabolism in hyperaccumulator plants were reported by Schickler and Caspi (1999). Using hydroponic culture of the Ni hyperaccumulator, A. argentenum, these authors analysed SOD and APX activities in the aerial parts of the plant after 14 days of exposure to relatively low Ni concentrations of up to 100 µM. As in the current study, lower SOD activities were observed with Ni treatment relative to the control cultures without Ni. However, in contrast to the present results for A. berto/onii, APX levels in A. agenteum were either unchanged or increased depending on the external Ni concentration. Schickler and Caspi (1999) interpreted their results to suggest that Ni tolerance mechanisms in A. argenteum involved either removing active oxygen species or preventing them from forming, thus reducing the need for antioxidative enzyme activity. The present results showing a substantial increase in H2O2 levels in Ni treated A. bertolonii roots do not support this interpretation. Instead, it appears that strong oxidants such as H2O2 can build up in A. berto/onii without deleterious consequences for growth, indicating a tolerance of active oxygen species independent of the antioxidative enzymes studied. This could occur, for example, if the reactions generating free radicals and other damaging molecules were better compartmentalised in A. bertolonii than in non hyperaccumulators, thus isolating any strong oxidants from sensitive biochemical processes in the cell. This hypothesis applies more appropriately to membrane- 154 impermeable oxygen species such as the superoxide radical (Alscher et al., 1997) than to H20 2, which diffuses freely across membranes (Foyer et al., 1997). 4. 6 Oxidative Stress in Cd-hyperaccumulator and Non hyperaccumulator A major objective in studies of hyperaccumulators is to identify the mechanisms that confer metal tolerance to these species. This work demonstrates that roots of the Cd hyperaccumulator, T caerulescens, are equipped with superior antioxidative defenses compared with the non-hyperaccumulator, N. tabacum. The results show that endogenous SOD activities were higher in the hyperaccumulator than in the non-hyperaccumulator (Figures 3.73 and 3.74). Cd did not have a significant affect on SOD activity in T caerulescens but it reduced SOD activity significantly in N. tabacum roots. In contrast, results have been reported for non-hyperaccumulators indicating that Cd does not have a significant effect on SOD activity (Romero-Puertas et al., 1999; Patra and Panda, 1998; Logriffoul et al., 1998). In addition, an initial induction and continuous increase of SOD activity has been observed for non-hyperaccumulators following the application of Cd (Piqueras et al., 2000; Schiltzendilbel et al., 2001; Vit6ria et al., 2001). However, in this study the results for SOD activity obtained for the non-hyperaccumulator are similar to the results reported for soybean nodules and roots, rice leaves and sunflower seedlings (Balestrase et al., 2001; Chien et al., 2001; Gallego et al., 1996a). A substantially (over 300-fold) higher endogenous CAT activity was estimated in T caerulescens roots compared with N. tabacum roots (Figures 3.75 and 3.76). In T caerulescens, CAT activity was enhanced after 5 days of Cd treatment (Figure 3.75) and T caerulescens was also successful in exerting tight control over H20 2 levels (Figures 3.79). 155 The high endogenous CAT activity in T caerulescens (Figure 3.75), the large increase in CAT activity with Cd treatment (Figure 3.75), and the lack of enhancement of APX activity with Cd (Figure 3.77) suggest that T caerulescens relies more on CAT than APX and the ascorbate-glutathione cycle for elimination of H20 2. Although other reports are not available for the effect of Cd on CAT activity in hyperaccumulators, several groups have investigated the effect of Cd on CAT activity in non-hyperaccumulators. In contrast with the result for N. tabacum roots, Cd stimulated . CAT activity in several non hyperaccumulators (Schutzendiibel et al., 2001; Patra and Panda, 1998; Gallego et al., 1996; Stroinski and Zielezinska, 1997). However, the results for N. tabacum are in accordance with the results reported for sunflower leaves and mung bean seedlings (Gallego et al., 1996a; Somashekaraiah et al., 1992). A dose dependent induction of CAT activity by Cd was reported for seedlings of Hordeum vulgare (Patra and Panda, 1998). Unlike peroxidases such as APX, CAT has the advantage of not requiring an additional source of reducing power. CAT activity is often lower in plant roots and stems than in the leaves (Havir et al., 1996; Shaw, 1995), reflecting the presence of fewer chloroplasts and peroxisomes in nonfoliar tissues and the importance of photosynthesis as a major source of active oxygen species (AOS). CAT deficiency in N. tabacum has been found to confer no physiological disadvantage as long as the light intensity remains low (Willekens et al., 1997). Consistent with this, low endogenous CAT activity in the dark-cultured N. tabacum roots studied in this work did not prevent rapid root growth in Cd-free medium (Figure 3.9). However, dark-grown T caerulescens roots maintained very high levels of endogenous CAT activity in the absence of AOS · generation from photosynthesis, suggesting that T caerulescens roots are prepared for prolific AOS generation associated with other biochemical processes. As roots are the principal entry point for Cd in plants, 156 and as AOS build-up in the form of H20 2 is a common response to Cd in non hyperaccumulator species (Romero-Puertas et al., 1999; Schutzendubel et al., 2001; Stroinski and Zielezinska, 1997), the availability of high constitutive CAT activity in the roots of Cd-hyperaccumulators represents a logical and effective defense strategy. Although the APX activity estimated in T. caerulescens was roughly half that in N. tabacum, Cd did not have a significant effect on the activity of APX in either species. The results obtained in the present study for N. tabacum roots contradicts those showing enhanced APX activity after Cd treatment in Cd-non-hyperaccumulators such as the roots and leaves of P. vulgaris, tobacco cells, P. aureus, pea leaf peroxisomes, seedlings of barley, and soybean nodules and roots (Chaoui et al., 1997; Piqueras et al., 2000; Shaw, 1995; Romero-Puertas et al., 1999; Patra and Panda, 1998; Balestrasse et al., 2001). According to previous reports, Cd enhances H20 2 levels in non-hyperaccumulators (Romero-Puertas et al., 1999; Stroinski and Zielezin.ska, 1997; Piqueras et al., 1999; Schi.itzendiibel et al., 2001). In this study, Cd elicited an increase in H20 2 of up to 5-fold in N. tabacum relative to the control cultures (Figure 3.80). Although H20 2 takes part in several important processes in plant cells, such as cell wall lignification, protein crosslinking, and signal transduction (Low and Merida, 1996), control of H20 2 build-up is essential to prevent oxidative damage to members and proteins. The ability of T. caerulescens roots to keep H202 concentrations in check represents an important feature of the hyperaccumulator phenotype. The ascorbate-glutathione cycle is considered the principal scheme for H20 2 destruction in plants (Noctor et al., 1998), and reliance on it by N. tabacum was evident in the low levels of CAT activity (Figure 3.76) and higher APX activities in N. tabacum compared with T. caerulescens (Figures 3.77 and 3.78). As the 157 APX route for removing H2O2 depends on glutathione. H2O2 levels increased significantly in N tabacum roots when glutathione levels were depressed relative to the control cultures by BSO (Figure 3.80). In contrast with the scenario for N tabacum, Cd tolerance in T. caerulescens roots was largely independent of glutathione levels and, by implication, APX activity, as evidenced by the maintenance of growth (Figure 3.8) and steady H2O2 concentrations (Figure 3.79) in cultures with very low glutathione contents (Figure 3.83) after treatment with BSO + Cd. Although, almost total glutathion depletion occurred following the application of Cd in both T. caerulescens (Figure 3.83) and N tabacum roots (Figure 3.84), the effect was more pronounced in the hyperaccumulator than in the non-hyperaccumulator. However, the results obtained for N tabacum roots are consistent with other reports available for non hyperaccumulators (Scheller et al., 1987; Rauser et al., 1991; Ruegsegger and Brunold, 1992; Klapheck et al., 1995; Schneider and Bergmann, 1995). In this study, the inhibition of glutathione synthesis by BSO caused elevated levels of H2O2 both in the hyperaccumulator and non-hyperaccumulator (Figures 3.79 and 3.80). It has been proposed that increased production of H2O2 may be due to Cd-induced oxidative stress in peroxisomes, where glycolate oxidase catalyses a reaction to produce H2O2 (Romero Puertas et al., 1999). Cd tolerance in the presence of low glutathione levels has been reported previously for roots of other metal-tolerant species (De Vos et al., 1992). By implication, as glutathione is the substrate for phytochelatin synthesis (Zenk, 1996), the retention of Cd tolerance in T. caerulescens roots treated with BSO + Cd suggests that phytochelatins are also not essential for the hyperaccumulator phenotype. This result is consistent with a recent report by Ebbs et al. (2002), which concluded that phytochelatins do not play a significant role in Cd tolerance in T. caerulescens. 158 Lipid peroxidation was increased in both T. caerulescens and N tabacum roots following the application of Cd (Figures 3.81 and 3.82). Increased lipid peroxidation might be an indication of increased production of free radicals (Aust et al., 1985; Halliwell and Gutteridge, 1989). However, the products formed during peroxidation reactions depend on the lipid composition of the membranes as well as on the availability of metal compounds capable of transforming lipid hydroperoxides (De Vos and Schat, 1991). Although reports on lipid peroxidation are limited for Cd-hyperaccumulators, several studies have revealed that Cd induces lipid peroxidation in non-hyperaccumulator species (Groppa et al., 2001; Gallego et al., 1996; Somashekaraiah et al., 1992; Patra and Panda, 1998; Piqueras et al., 2000). The reported results are similar to those found here for N tabacum roots. Apart from Cd, accumulation of Cu significantly increased the level of MDA in C. demersum (Ramadevi and Prasad 1998) and P. vulgaris (Weckx and Clijsters, 1996). Deficiency of antioxidant enzyme activities may result in the enhancement of free-radical-mediated lipid peroxidation (Manohar and Balasubramanian, 1986). Ouariti et al. (1997) proposed that the loss of membrane lipids in tomato plants treated with either Cd or Cu may be related to an enhanced rate of catabolism. In the present study, Cd accumulation induced lipid peroxidation both in the hyperaccumulator and non-hyperaccumulator. However, the levels ofMDA produced in the non-hyperaccumulator were higher than in the hyperaccumulator. In the non-hyperaccumulator, the decreased levels of CAT, SOD and APX would result in elevated levels of oxidative stress and this would, in turn, result in higher levels of lipid peroxidation. The changes in the composition or molecular arrangements of membranes might play a role in heavy metal resistance, either by modifying the permeability of membranes to ions or by altering the activities of membrane-bound enzymes (Verkleij and 159 Schat, 1989). Membrane peroxidation is associated with lipid gel phase formation, modification of membrane permeability and conformational changes of membrane-bound proteins (Chia et al., 1984). Exposure to Cd elicited a burst of surface reactive thiols in T. caerulescens roots (Figure 3.85), but not in N tabacum (Figure 3.86). An increase in reactive -SH groups at the cell walls may be a mechanism for immobilising and detoxifying Cd in the apoplasm, thus protecting the cells from Cd influx. The strong modulating effect of BSO on this response (Figure 3.85) suggests that the -SH burst in T. caerulescens was glutathione-dependent. Nevertheless, increased surface thiol concentrations were not essential for Cd tolerance in T. caerulescens, as normal root growth and H2O2 levels were maintained during treatment with BSO + Cd (Figures 3.13 and 3.79), even though surface -SH levels were low (Figure 3.85) and the glutathione pool was severely depleted (Figure 3.83). In yeast, Fe deficiency resulted in higher levels of reduced sulfhydryl groups at the exofacial plasma membrane surface, as well as higher concentrations of GSH within the cell (Lesuisse and Labbe, 1992). A significant increase in reduced sulfhydryl groups on the root surface of pea seedlings grown without Fe suggested that some type of sulfhydryl modification of plasma membrane components may occur under Fe deficiency and may partially account for the enhanced Cd influx in Fe-deficient plants (Cohen et al., 1998). Welch and Norvell (1993) suggested that Zn may be required to protect reactive sulfhydryl groups in the membrane proteins of root cell exterior surfaces from oxidative reactions. Cd toxicity to plants and animals may be the result of the strong affinity of Cd to essential -SH groups of enzymes or structural proteins (Dabin et al., 1978, Braude et al,, 1980). In this study, Cd induced free -SH groups on the root surfaces of T. caerulescens, whereas BSO did not do so. This may be due to the interaction of Cd with -SH groups in the root surface, the capacity of Cd 160 to induce -SH groups, and also because free active Cd might cause degradation of surface proteins. The free radical generator, cumene hydroperoxide (CHP), and the free radical scavenger, O-mannitol (MAN), added to the growth medium did not have a significant impact on the growth of T caerulescens and N. tabacum (Figures 3.15-3.18). With CHP, the reason could be that the concentration of CHP used in this experiment was not enough to produce high levels of free radicals, or that, after a certain time, CHP may have become unstable in the growth medium. However, when T caerulescens hairy roots were treated with another free radical generator, 2,2 '-azobis(2-amidinoporpane) dihydrochloride (AAPH), growth was severely inhibited, indicating that T caerulescens roots cannot withstand the over production of active oxygen species. 4. 7 Nickel Recovery 4. 7. 1 Alyssum bertolonii Hairy Roots Elemental analysis of A. bertolonii hairy roots revealed that elements such as C, H, 0 and N accounted for more than 80% of the mass of both the Ni-treated and non-Ni-treated hairy roots (Figure 3.87). However, Ni accumulation reduced K levels compared with the hairy roots that were not exposed to Ni. Furnace treatment time had little effect on the elemental composition of the residues from A. bertolonii biomass containing Ni. However, prolonged furnace treatment increased the percentage of Ni by eliminating some unwanted elements in the residue compared with the 3 h sample (Figure 3.88). In addition, it was observed that nickel oxide formation may depend on the initial concentration of Ni in the biomass (Figure 3.90). At high initial Ni concentrations, the proportion of 'other elements' remaining after furnace treatment decreased, indicating the possibility of obtaining Ni with 161 fewer contaminants (Figure 3.96). The furnace treatment of biomass containing Ni under inert conditions (N2) revealed that oxidation was the major process involved in metal concentration. The XRD analysis showed that Ni in the residue of A. bertolonii biomass with 7.7% (w/w) Ni was present mainly in the form ofNiO (Figure 3.95). 4. 7. 2 Berkheya coddii Plants The biomass dry weight analyses indicated that B. coddii may require a minimum level of Ni for healthy growth (Figure 3.99). It was observed that during the cultivation, plants that were not grown with Ni underwent frequent insect attack. Although increasing the soil Ni concentration reduced plant growth, there was no significant difference observed between the biomass dry weight of plants grown with 4 mg of Ni per g of soil and that of plants grown without Ni. However, the Ni concentration in the biomass increased with the initial concentration of Ni in the soil. In this study, the highest level of Ni accumulation achieved was 0.55% (w/w) in the dry matter of above-ground-level biomass at an initial Ni concentration of 4 mg/g soil. A similar study with Alyssum species showed that the dried leaves accumulated 1% (w/w) Ni (Morrison et al., 1980), which is 1.8 times higher than the level obtained for B. coddii in the present study. However, the initial concentration of Ni supplied in the present study was 2.5 times lower than the initial concentration of Ni supplied to the Alyssum species. Thus, a higher initial Ni concentration in the soil might increase the level of Ni accumulation in B. coddii. Elemental analysis of B. coddii biomass with and without Ni indicated that Ni accumulation reduced the levels of K and P compared with the control plants (Figure 162 3.101). Elemental analysis of B. coddii ash with Ni after furnace treatment showed that the ash contained about 35% (w/w) Ca (Figure 3.104). A similar result was also obtained for dried biomass of Hybanthus jloribundus collected from various regions of Western Australia: Ni accumulated with Ca at a ratio of about 1: 1.6 (Farago et al., 1977). This result also indicates that high levels of Ca uptake might have inhibited Ni uptake in B. coddii to certain extent, as previous studies have revealed that high levels of Ca in soil may inhibit Ni accumulation in Alyssum species. Furthermore, it was reported that maintaining low levels of Ca in soil enhanced the uptake of Ni by Alyssum species (Chaney et al., 1998 and 1999), and similar findings were reported for B. coddii plants (Robinson et al., 1999). In addition, Baker et al. (2000) recently reported that extremely low or high levels of Ca may inhibit Ni uptake in Ni-hyperaccumulators. Nicks and Chambers (1995) reported that when dried Streptanthus polygaloides containing 0.36% (w/w) Ni was incinerated, it yielded ash containing 8% Ni with a 22-fold decrease in the biomass. In the present study, dried biomass of B. coddii with 0.55% (w/w) Ni yielded ash containing 8.6% Ni after furnace treatment, with a 20-fold decrease in the biomass. In contrast to the above results, in the present work it was found that A. bertolonii biomass containing 5.1-7.7% (w/w) Ni yielded residues with about 60% Ni after furnace treatment, which is 3.75 times higher than the level of Ni determined in the ash of H. caledonius (Kelly et al., 1995). Furnace treatment of B. coddii biomass with Ni resulted in a concentration of Ni along with elements such as Ca, Mg and P. Comparatively, the levels of Ca were higher than the other major elements. Thus, Ca might have inhibited nickel oxide formation and the elimination of 'other elements' in B. coddii biomass during the furnace treatment. Previously, Robinson et al. (1997a) proposed a model for phytomining of Ni using A. bertolonii that involved harvesting the crops after 12 months and burning 163 them to produce a sulphur-free bio-ore with about 11 % Ni. This study has demonstrated that harvesting B. coddii plants after 4-5 months and treating them in a furnace yields a sulphur-free bio-ore containing 8.6% Ni. 164 CHAPTER 5 - CONCLUSIONS 5. 1 Nickel-Hyperaccumulator and Non-hyperaccumulator • A. bertolonii hairy roots were tolerant of a solution concentration of 25 ppm Ni, and growth was unaffected. However, N. tabacum roots suffered severe growth inhibition at 25 ppm Ni. • Application of DES severely retarded the growth of A. bertolonii hairy roots, and increased the uptake of Ni in A. bertolonii and N. tabacum roots. • Almost all the Ni accumulated in A. bertolonii and N. tabacum roots was located in the symplasm. • In A. bertolonii hairy roots, DES restricted Ni uptake across the plasma membrane and increased Ni levels in the cell walls. • Hairy roots of A. bertolonii contained high constitutive levels of citric, malic and malonic acids and 28% of the Ni accumulated in the roots was associated with these organic acids. • The activities of the antioxidative enzymes SOD, CAT and APX were not enhanced after exposure to 25 ppm Ni in either A. bertolonii or N. tabacum roots. • Compared with N. tabacum roots, A. bertolonii roots may be protected from oxidative damage by higher endogenous activities of CAT and SOD. However, H20 2 levels increased significantly with Ni in both species, the greater increase occurring in N. tabacum. • The results of this work suggest that A. bertolonii possesses detoxification mechanisms that allow unimpeded growth even in the presence of elevated concentrations of active oxygen species. 165 5. 2 Cadmium-Hyperaccumulator and Non-hyperaccumulator • T. caerulescens hairy roots were tolerant of a solution concentration of 20 ppm Cd and growth was unaffected. However, N. tabacum roots suffered severe growth inhibition at 20ppmCd. • Under normal culture conditions, the effects on growth of Zn, BSO, CHP and MAN were negligible for both T. caerulescens and N. tabacum roots. • Treatment with DES severely inhibited the growth of T. caerulescens roots. DES also increased the uptake of Cd in T. caerulescens roots but not in N. tabacum roots. • Under usual culture conditions, Cd was found mostly with the cell walls of T. caerulescens and N. tabacum hairy roots. • Treatment with DES caused a substantial increase in Cd accumulation in the symplasm of T. caerulescens without affecting root viability, thus indicating that the mechanisms of Cd tolerance and hyperaccumulation in this species are not affected by disruption of transmembrane proton gradients. • T. caerulescens hairy roots contained high constitutive levels of citric, malic and malonic acids. About 13% of the Cd accumulated in the roots was associated with these organic acids. • In contrast to N. tabacum, T. caerulescens roots exhibited a robust capacity for tight regulation of H202 levels during exposure to Cd. This ability was associated with a more than 300-fold greater constitutive CAT activity in T. caerulescens than in N. tabacum, and a further increase in CAT activity with Cd treatment. • Depletion of the glutathione pool by BSO did not affect Cd tolerance or the ability of T. caerulescens to control H202 concentrations, indicating that glutathione does not play a pivotal role in Cd hyperaccumulation. 166 • This work indicates that antioxidative capacity is an important factor in the response of T caerulescens roots to Cd. The results suggest that overexpression of CAT and/or SOD in conjunction with other molecular approaches may be a key component to improving Cd tolerance in non-hyperaccumulator plants. 5. 3 Nickel Recovery • Metal residues containing about 60% Ni were obtained from A. bertolonii hairy roots after furnace treatment. • This study demonstrated a method to produce sulphur-free Ni 'bio-ore' from B. coddii plants. • The sulphur-free Ni 'bio-ore' of B. coddii contained 8.6% Ni (w/w) and about 35% Ca (w/w) as major elements. • Further research is required to isolate Ni from the 'bio-ore' of B. coddii. • This work emphasises the feasibility of applying Ni-hyperaccumulating plant species in the field for phytoremediation and phytomining. 167 REFERENCES Aebi, H.E. (1983) Catalases. In: H.U. Bergmeyer, ed, Methods ofEnzymatic analysis, 3rd eds, Vol III. Verlag Chemie, Weinheim, p. 273-286. Al-Awqati, Q. (1986) Proton-translocating ATPase. Annual Review in Cell Biology, 2, 179 -199. Allen, R.D. (1995) Dissection of oxidative stress tolerance using transgenic plants. Plant Physiology, 107, 1049-1054. Alscher, R.G., J.L. Donahue, C.L. Cramer (1997) Reactive oxygen species and antioxidants : relationships in green cells. Physiologia Plantarum, 100, 224-233. Anderson, C.W.N., R.R. Brooks, A. Chiarucci, C.J. LaCoste, M. Leblanc, B.H. Robinson, R. Simcock, RB. Stewart (1999) Phytomining for nickel, thallium and gold. Journal of Geochemical Exploration, 67, 407-415. Arisi, A.C.M, B. Mocquot, A. Lagriffoul, M. Mench, C.H. Foyer, L. Jouanin (2000) Response to cadmium in leaves of transformed poplars overexpressing y-glutamucysteine synthetase. Physiologia Plantarum, 109, 143-149. Aust, S.D., L.A. Morehouse, C.E. Thomas (1985) Role of metals in oxygen radical reactions. Journal ofFree Radical Biology and Medicine, 1, 3-25. Baccouch, S., A. Chaoui, E. El Ferjani (1998) Nickel-induced oxidative damage and antioxidant responses in Zea mays shoots. Plant Physiology and Biochemistry, 36, 689-694. Baccouch, S., A. Chaoui, E. El Ferjani (2001) Nickel toxicity induces oxidative damage in Zea mays roots. Journal ofPlant Nutrition, 24, 1085-1097. Baker, A.J.M. (1987) Metal tolerance. New Phytologist, 106, 93-111. Baker, A.J.M., Brooks, R.R. (1989) Terrestrial higher plants which hyperaccumulate metallic elements-a review of their distribution, ecology and phytochemistry. 1 Arzt1,po11> ri,£-. {IC/85J :z>;f, :,1:n0J,orz _ 61;; c-/.1.. t (("' IA ,1,·1 ; and ~/;, { ,.(f:...: J;si,,/ f,dR M O, °;_':fl al .Jfr;,~.J- Mefl'°dJ rf ().,,--nt-1l11Y!OP'Jt{, //:J / 5+!3- ..5 ~ t;;I /} I u rJ 168 Biorecovery, 1, 81-126. Baker, A.J.M., S.P. McGrath, C.M.D. Sidoli, R.D. Reeves (1994) The possibility of in situ heavy metal decontamination of polluted soils using crops of metal accumulating plants. Resources, Conservation and Recycling, 11, 41-49. Baker, A.J.M., Y-M. Li, A.J. Scott, R.L. Chaney (2000) Recovering metals from soil. GB and US Patent No. WO0028093. Balestrasse, K.B., L. Gartley, S.M. Gallego, M.L. Tomaro (2001) Responses of antioxidant defence system in soybean nodules and roots subjected to cadmium stress. Australian Journal ofPlant Physiology, 28, 497-504. Balke, N.E., T.K. Hodges (1979) Comparison ofreductions in adenosine triphosphate content, plasma membrane-associated adenosine triphosphatase activity, and potassium absorption in oat roots by diethylstilbestrol. Plant Physiology, 63, 53- 56. Becana, M., P. Aparicio-Tejo, J.J. Irigoyen, M. Sanchez-Diaz (1986) Some enzymes of hydrogen peroxide metabolism in leaves and root nodules of Medicago sativa. Plant Physiology, 82, 1169-1171. Bettger, W.J., B.L. O'Dell (1991) A critical physiological role of zinc in the structure and function ofbiomembranes. Life Sciences, 28, 1425-1438. Bhadra, R., J.V. Shanks (1997) Transient studies ofnutri~nt uptake, growth and indole alkaloid accumulation in heterotrophic cultures of hairy roots of Catharanthus roseus. Biotechnology and Bioengineering, 55, 527-534. Bhadra, R., D.G. Wayment, J.B. Hughes, J.V. Shanks (1999) Confirmation of conjugation processes during TNT metabolism by axenic plant roots. Environmental Science and Technology, 33, 446-452. Bielawski., W., K.W. Joy (1986) Reduced and oxidised glutathione and glutathione reductase in tissue of Pisum sativum. Planta, 169, 267-272. Ba1;-f/l!) Y/~11)· V',) u,,J). ll;t:1w11- {__!__CJCJo)_ t;,~ocJM-, .· _ar1t:L. 5(-(CJ (,l7v r-r1dei hli '~ ar1,;f ln 02 ,1 C-1,,vUt/y&_. o/ I l:llN-lcc-Lu;1J1 1;i,1/,//tt't /J-/?eL/2.on-7 5r?ieJ i.Jl- Ju/./.eve_d n1 Bowler, C., W.V. Camp, M.Van Montagu, D. Inze (1994) Superoxide dismutase in plants. Critical Reviews in Plants Sciences, 13, 199-218. Boyajian, G.E., L.H. Carreira (1997) Phytoremediation: a clean transition from laboratory to marketplace? Nature Biotechnology, 15, 127-128. Boyd, R.S., S.N. Martens (1998) Nickel hyperaccumulation by Thlaspi montanum Var. montanum (Brassicaceae): A constitutive trait. American Journal ofBotany, 85, 259-265. Braude, G.L., A.M. Nash, W.J. Wolf, R.L. Carr, R.L. Chaney (1980) Cadmium and lead content ofsoybean products. Journal ofFood Science, 45, 1187-1189. Brooks, R.R., J. Lee, R.D. Reeves, T. Jaffre (1977) Detection ofnickeliferous rocks by analysis ofherbarium specimens of indicator plants. Journal of Geochemical Exploration, 1, 49-57. Brooks, R.R., R.S. Morrison, R.D. Reeves, T.R. Dudley, Y. Akman (1979) Hyperaccumulation of nickel by Alyssum linnaeus (Cruciferae). Proceedings the ofRoyal Society ofLondon. B., 203, 387-403. Brooks, R.R., S. Shaw, A.A. Marfil (1981) The chemical form and physiological function of nickel in some Iberian Alyssum species. Physiologia Plantarum, 51, 167-170. Brooks, R.R., M.F. Chambers, L.J. Nicks, B.H. Robinson (1998) Phytomining. Trends in Plant Science, 3, 359-362. Brown, S.L., R.L. Chaney, J.S. Angel, A.J.M. Baker (1994) Phytoremediation potential of Thlaspi caerulescens and bladder campion for zinc and cadmium contaminated soil. Journal ofEnvironmental Quality, 23, 1151-1157. Brown, S.L., R.L. Chaney, J.S. Angel, A.J.M. Baker (1995) Zinc and cadmium uptake by hyperaccumulator Thlaspi caerulescens grown in nutrient solution. Soil Science Society ofAmerican Journal, 59, 125-133. Bush, D.R. (1993) Proton-coupled sugar and amino acid transporters in plants. Annual 170 Review ofPlant Physiology and Plant Molecular Biology, 44, 513-542. Cataldo, D.M., T.R. Garland, R.E. Wildung (1981) Cadmium distribution and chemical fate in soybean plants. Plant Physiology, 68, 835-839. Catalado, D.M., K.M. McFadden, T.R. Garland, R.E. Wildung (1988) Organic constituents and complexation of Nickel (II), Iron (111), Cadmium (11) and Plutonium (IV) in soybean xylem exduates. Plant Physiology, 86, 734-739. Chamnongpol, S., H. Willekens, C. Langebartels, M. Van Montagu, D. Inze, W. Van Camp (1996) Transgenic tobacco with a reduced catalase activity develops necrotic lesions and induces pathogenesis-related expression under high light. The Plant Journal, 10, 491-503. Chaney, R.L., J.S. Angel, A.J.M. Baker, Y.M. Li (1998) Method for phytomining of nickel, cobalt and other metals from soil. US Patent No. 5,711,784. Chaney, R.L., J.S. Angel, A.J.M. Baker, Y.M. Li (1999) Method for phytomining of nickel, cobalt and other metals from soil. US Patent No. 5,944,872. Chaoui, A., S. Mazhoudi, M.H. Ghorbal, E.E. Ferjani (1997) Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in bean (Phaseolus vulgaris L.). Plant Science, 127, 139-147. Chia, L.S., C.I. Mayfield, J.E. Thompson (1984) Stimulated acid rain induces lipid peroxidation and membrane damage in foliage. Plant, Cell and Environment, 7, 333- 338. Chien, H-F., J-W. Wang, C.C. Lin, C.H. Kao (2001) Cadmium toxicity of rice leaves is mediated through lipid peroxidation. Plant Growth Regulation, 33, 205-213. Cieslinski, G., G.H. Neilsen, E.J. Hogue (1996) Effect of soil cadmium application and pH on growth and cadmium accumulation in roots, leaves and fruit of strawberry plants (Fragaria x ananassa Duch.). Plant and Soil, 180, 267-276. Clark, R.B. (1969) Organic acids from leaves of several crop plants by gas chromatography. 171 Crop Science, 9, 341-343. Cohen, C.K., T.C. Fox, D.F. Garvin, L.V. Kochain (1998) The role of iron-deficiency stress responses in stimulating heavy metal transport in plants. Plant Physiology, 116, 1063-1072. Conway, H.C. (1978) Sorption of arsenic and cadmium and their effects on growth, micronutrient utilization and photosynthetic pigment composition of Asterionella formosa. Journal ofFish Research Board Canada, 35, 286-294. Crawford, R.L., D.L. Crawford (1996) Bioremediation: Principles and Applications, Cambridge University Press, New York, USA. Dabin, 0., E. Marafante, J.M. Mousny, C. Myttenaere (1978) Absorption, distribution and binding of cadmium and zinc in irrigated rice pl~ts. Plant and Soil, 50, 329-341. Dalton, D.A., S.A. Russell, H.J. Evans (1988) Nickel as a micronutrient element for plants. Bio/actors, 1, 11-16. Dat, J., S. Vandenabeele, E. Vranova, M. Van Montagu, D. Inze, F. Breusegem (2000) Dual action of the active oxygen species during plant stress responses. Cellular and Molecular Life Sciences, 57, 779-795. Davis, M.A., R.S. Boyd (2000) Dynamics of Ni-based defence and organic defences in the Ni hyperaccumulator, Streptanthus polygaloides (Brassicaceae). New Phytologist, 146, 211-217. De Vos, C.H.R., H. Schat (1991) Free radicals and heavy metal tolerance. In: J. Rozema, J.A.C. Verkleij, eds, Ecological Responses to Environmental Stress, Kluwar Academic, Dordrecht, The Netherlands, p. 22-30 De Vos, C.H.R., M.J. Vonk, R. Vooijs, H. Schat (1992) Glutathione depletion due to copper induced phytochelatin synthesis causes oxidative stress in Silene cucubalus. Plant Physiology, 98, 853-858. De Vos, C.H.R., W.M. Ten Bookum, R. Vooijs, H. Schat, L.J. De Kok (1993) Effect of 172 copper on fatty acid composition and peroxidation of lipids in the roots of copper tolerant and sensitive Silene cucubalus. Plant Physiology and Biochemistry, 80, 409- 415. Dietz, K.J., M. Baier, U. Kramer (1999) Free radicals and reactive oxygen species as mediators of heavy metal toxicity in plants. In: M.N.V. Prasad and J. Hagemeyer, eds, Heavy Metal Stress in Plants, Springer, Berlin, Germany, p. 73-97. Dixit, V., V. Pandey R. Shyam (2001) Differential antioxidative responses to cadmium in roots and leaves of pea (Pisum sativum L. cv. Azad). Journal of Experimental Botany, 52, 1101-1109. Ebbs, S., I. Lau, B. Ahner, L. Kochian (2002) Phytochelatin synthesis is not responsible for Cd tolerance in the Zn/Cd hyperaccumulator T_hlaspi caerulescens (J. & C. Persl). Planta, 214, 635-640. Emri, T., I. Pocsi, A. Szentirmai (1997) Glutathione metabolism and protection against oxidative stress caused by peroxides in Penicillium chrysogenum. Free Radical Biology and Medicine, 23, 809-814. Espen, L., L. Pirovano, S.M. Cocucci (1997) Effects of Ni2+ during the early phases of radish (Raphnus sativus) seed germination. Environmental and Experimental Botany, 38, 187-197. Farago, M.E., A.J. Clark, M.J. Pitt (1977) Plants which accumulate metals. Part I. The metal content of three Australian plants growing over mineralised sites. Inorganica Chimica Acta, 24, 53-56. Farrell, K., L. Lu, V. Sahajwalla, V. Harris (1998) The atomic scale structural features of some coals and chars. 8th Australian Coal Science Conference (Proceedings), The Institute of Energy Australia, Sydney, Australia. p 1-6. Fassett, D.W. (1980) Cadmium. In: H.A. Waldron, ed, Metals in the Environment, Academic Press, London, UK, p. 61-110. 173 Fett, J.P., J. Cambraia, M.A. Oliva, C.P. Jordao (1994) Cadmium uptake and growth inhibition in water hyacinths: Effects of nutrient solution factors. Journal of Plant Nutrition, 17, 1205-1217. Flathman, P.E., G.R. Lanza (1998) Phytoremediation: Current views on an emerging green technology. Journal ofSoil Contamination, 7, 415-432. Foyer, C.H., B. Halliwell (1976) The presence of glutathione and glutathione reductase in chloroplasts: a proposed role in ascorbic acid metabolism. Planta, 133, 21-25. Foyer, C.H (1997) Oxygen metabolism and electron transport in photosynthesis. In: J. Scandalios, ed, Oxidative Stress and the Molecular Biology of Antioxidant Defenses, Cold Spring Harbor Laboratory Press, New York, USA, p. 587-621. Foyer, C.H., H. Lopez-Delgado, J.F. Dat, I.M. Scott (199-7) Hydrogen peroxide and glutathione associated mechanisms of acclimatory stress tolerance and signaling. Physiologia Plantarum, 100, 241-254. Frey, B., C. Keller, K. Zierold, R. Schulin (2000) Distribution of Zn in functionally different leaf epidermal cells of the hyperaccumulator Thlaspi caerulescens. Plant, Cell and Environment, 23, 675-687. Gabbrielli, R., C. Mattioni, 0. Vergnano (1991) Accumulation mechanisms and heavy metal tolerance of a nickel hyperaccumulator. Journal ofPlant Nutrition, 14, 1067-1080. Gabbrielli, R., R. Pandolfini, 0. Vergnano, M.R. Palandri (1991) Comparison of two serpentine species with different nickel tolerance strategies. Plant and Soil, 122, 271- 277. Gallego, S.M., M.P. Benavides, M.L. Tomaro (1996a) Effect of heavy metal ion excess on sunflower leaves: evidence for involvement of oxidative stress. Plant Science, 121, 151-159. Gallego, S.M., M.P. Benavides, M.L. Tomaro (1996b) Oxidative damage caused by 174 cadmium chloride in sunflower (Helianthus annuus L) plants. International Journal ofExperimental Botany, 58, 41-52. Girotti, AW. (1985) Mechanisms oflipid peroxidation. Journal ofFree Radical Biology and Medicine, 1, 87-95. Godbold, D.L., W.J. Horst, J.C. Collins, D.A. Thurman, H. Marcchner (1984) Accumulation of zinc and organic acids in roots of zinc tolerant and non-tolerant ecotypes of Deschampsia caespitosa. Journal ofPlant Physiology, 116, 59-69. Gries, G.E., G.J. Wagner (1998) Association of nickel versus transport of cadmium and calcium in tonoplast vesicles of oat roots. Planta, 204, 390-396 Grill, E., E.L. Winnacker, M.H. Zenk (1985) Phytochelatins: the principal heavy- metal complexing peptides of higher plants. Science, 230, 674-676. Grill, E., E.L. Winnacker, M.H. Zenk (1987) Phytochelatins, a class of heavy-metal-binding peptides from plants, are functionally analogous to metallothioneins. Proceedings of the National Academy of Science, USA, 84, 439-443. Grill, E., S. Loffler, E.L. Winnacker, M.H. Zenk (1989) Phytochelatins, the heavy-metal -binding of peptides of plants, are synthesized from glutathione by a specific y glutamylcysteine dipeptidyl transpeptidase (phytochelatin synthase). Proceedings of the National Academy of Science, USA, 86, 6838-6842. Groppa, M.D., M.L. Tomaro, M.P. Benavides (2001) Polyamines as protectors against cadmium or copper-induced oxidative damage in sunflower leaf discs. Plant Science, 161, 481-488. Guo, Y. T ., H. Marschner ( 1995) Uptake, distribution, and binding of cadmium and nickel in different plant species. Journal ofPlant Nutrition, 18, 2691-2706. Gwozdz, E.A., R. Przymusinski, R. Rucinska, J. Deckert (1997) Plant cell responses to heavy metals: molecular and physiological aspects. Acta Physiologia Plantarum, 19, 459-465. 175 Halliwell, B., J.M.C. Gutteridge (1989) Free Radicals in Biology and Medicine. Clarendon Press, Oxford, UK. Hart, J.J., J.M. DiThomaso, D.L. Linscott, L.V. Kochian (1992) Characterization of the transport and cellular compartmentation of paraquat in root of intact maize seedlings. Pesticide Biochemistry and Physiology, 43, 212-222. Hasegawa, I., E.Terada, M. Sunairi, H. Wakita, F. Shinmachi, A. Noguchi, M. Nakajima, J. Yazaki (1997) Genetic improvement of heavy metal tolerance in plants by transfer of the yeast metalliothionein gene (CUPI). Plant and Soil, 196, 277-281. Hausinger, R.P. (1987) Nickel utilization by microorganisms. Microbiology Review, 51, 22- 42. Havir, E.A., L.F. Brisson, I. Zelitch (1996) Distribution of catalase isoforms in Nicotiana tabacum. Phytochemistry, 41, 699-702. Hayes, R.B (1997) The carcinogenicity of metals in humans. Cancer Causes and Control, 8, 371-385. Heath, R.L., L. Packer (1968) Photoperoxidation in isolated chloroplasts. I.Kinetics of peroxidation stoichiometry of fatty acid peroxidation. Archives of Biochemistry and Biophysics, 125, 189-198. Heath, S.M., D. Southworth, J.A. D'allura (1997) Localization of nickel in epidermal subsidiary cells of leaves of Thlaspi montanum var. siskiyouense (Brassicaceae) using energy-dispersive X-ray microanalysis. International Journal of Plant Science, 158, 184-188. Homer, F.A., R.S. Morrison, R.R. Brooks, J. Clemers, R.D. Revees (1991a) Comparative studies of nickel, cobalt and copper uptake by some nickel hyperaccumulators of the genus Alyssum. Plant and Soil, 138, 195-205. Homer, F.A., R.D. Reeves, R.R. Brooks, A.J.M. Baker (1991b) Characterization of the 176 nickel-rich extract from the nickel hyperaccumulator Dichapetalum gelonioides. Phytochemistry, 30, 2141-2145. Hughes, J.B., J. Shanks, M. Vanderford, J. Lauritzen, R. Bhadra (1997) Transformation of TNT by aquatic plants and plant tissue cultures. Environmental Science and Technology, 31, 266-271. Inouhe, M., R. Ito, S. Ito, N. Sasada, H. Tohoyama, M. Joho (2000) Azuki bean cells are hypersensitive to cadmium and do not synthesis phytochelatins. Plant Physiology, 123, 1029-1036. Jocelyn, P.C. (1972) Biochemistry ofthe -SH Group. Academic Press, London, UK, p. 63-136. Kappus, H (1985) Lipid peroxidation: mechanisms, analysis, enzymology and biological relevance. In: H. Sies, ed, Oxidative Stress, Academic Press, London, UK, p. 273- 310. Kelly, P.C., R.R. Brooks, S. Dilli, T. Jaffre (1975) Preliminary observation on the ecology and plant chemistry of some nickel-accumulating plants from New Caledonia. Proceedings ofthe Royal Society ofLondon. B., 189, 69-80. Kersten, N.J., R.R. Brooks, R.D. Reeves, T. Jaffre (1980) Nature of nickel complexes in Psychotria douarrei and other nickel accumulating plants. Phytochemsitry, 19, 1963-1965. Khan, D.H., J.G. Duckett, B. Frankland, J.B. Kirkham (1984) An X-ray microanalytical study of the distribution of cadmium in roots of Zea mays L. Journal of Plant Physiology, 115, 19-28. Klapheck, S., S. Schlunz, L. Bergmann (1995) Synthesis ofphytochelatins and homophytochelatins in Pisum sativum L. Plant Physiology, 107, 515-521. Kneer, R., M.H. Zenk (1992) Phytochelatins protect plant enzymes from heavy metal poisoning. Phytochemistry, 31, 2663-2667. 177 Kim, Y.O., F. Takeuchi, M. Hara, K. Toru (2000) Characterization of cadmium-tolerant carrot cells in response to cadmium stress. Soil Science and Plant Nutrition, 46, 807- 814. Kortz, R.M., B.P. Evangelou, G.J. Wagner (1989) Relationships between cadmium, zinc, Cd-peptide and organic acid in tobacco suspension cells. Plant Physiology, 91, 780- 787. Kosugi, H., K. Kikugawa (1989) Potential thiobarbituric acid-reactive substances in peroxidised lipids. Journal ofFree Radical Biology and Medicine, 7, 205-207. Kramer, U., A.N. Chardonnens (2001) The use of transgenic plants in the bioremediation of soils contaminated with trace elements. Applied Microbiology and Biotechnology, 55, 661-672. Kramer, U., I.J. Pickering, R.C. Prince, I. Raskin, D.E. Salt (2000) Subcellular localisation and speciation of nickel in hyperaccumulator and non-accumulator Thlaspi species. Plant Physiology, 112, 1343-1353. Kramer, U., J.D. Cotter-Howells, J.M. Charnock, A.J.M. Baker, J.A.C. Smith (1996) Free histidine as metal chelator in plants that accumulate nickel. Nature, 379, 635-638. Kramer, U., R.D. Smith, W.W. Wenzel, I. Raskin, D.E. Salt (1997) The role of metal transport and tolerance in nickel hyperaccumulation by Thlaspi goesingense Halacsy. Plant Pysiology, 115, 1641-1650. Kubota, H., S. Kyoko, T. Yamada, T. Maitani (2000) Phytochelatin homologs induced in hairy roots of horseradish. Phytochemistry, 53, 239-245. Kukkola, E., P. Rautio, S. Huttunen (2000) Stress indications in copper- and nickel- exposed Scots pine seedlings. Environmental and Experimental Botany, 43, 197-210. Kumar, K.V., U.N. Das (1993) Are free radicals involved in the pathobiology of human essential hypertension? Free Radical Research and Communications, 19, 59-66. Kupper, H., E. Lombi, F-J. Zhao, S.P. McGrath (2000) Cellular compartmentation of 178 cadmium and zinc in relation to other elements in the hyperaccumulator Arabidopsis halleri. Planta, 212, 75-84. Lagriffoul, A., B. Mocquot, M. Mench, J. Vangronsveld (1998) Cadmium toxicity effects on growth, mineral and chlorophyll contents, and activities of stress related enzymes in young maize plants (Zea mays L.) Plant and Soil, 200, 241-250. Lasat, M.M., A.J.M. Baker, L.V. Kochian (1996) Physiological characterization of root Zn2+ absorption and translocation to shoots in Zn hyperaccumulator and nonaccumulator species of Thlaspi. Plant Physiology, 112, 1715-1722. Lasat, M.M., N.S. Pence, F.G. David, S.D. Ebbs, L.V. Kochian (2000) Molecular physiology of zinc transport in the Zn hyperaccumulator Thlaspi caerulescens. Journal of Experimental Botany, 51, 71-79. Lee, J., R.D. Revees, R.R. Brooks, T. Jaffre (1977) Isolation and identification of a citrato complex of nickel from nickel accumulating plants. Phytochemistry, 16, 1503-1505. Lee, J., R.D. Revees, R.R. Brooks, T. Jaffre (1978) The relation between nickel and citric acid some nickel accumulating plants. Phytochemistry, 17, 1033-1035. Lessuisse, E., P. Labbe (1992) Iron reduction and trans-plasma membrane transfer in the yeast Saccharomyces cerevisiae. Plant Physiology, 100, 769-777. Lindsey, P.A., R.D. Lineberger (1981) Toxicity, cadmium accumulation, and ultrastructural alterations induced by exposure of Phaseolus seedlings to cadmium. Horticultural Science, 16, 434. Logan, H., M. Basset, A.A. Very, H. Sentenac (1997) Plasma membrane transport systems in higher plants: from black boxes to molecular physiology. Physiologia Plantarum, 100, 1-15. Low, P.S., J.R. Merida (1996) The oxidative burst in plant defense: function and signal transduction. Physiologia Plantarum, 96, 533-542. Lu, L., V. Sahajwalla, C. Kong, D. Harris (2001) Quantitative X-ray diffraction analysis and 179 its application to various coals. Carbon, 39, 1821-1833. Luna, C.M., A.C. Gonzalez, V.S. Trippi (1994) Oxidation damage caused by an excess of copper in oat leaves. Plant Cell Physiology, 35, 11-15. Mackova, M., T. Macek, P. Kucerova, J. Burkard, J. Pazlarova, K. Demnerova (1997) Degradation of polychlorinated biphenyls by hairy root culture of Solanum nigrum. Biotechnology Letters, 19, 787-790. Macek, T.E., P. Kotrba, M. Suchova, F. Skacel, K. Demnerova, T. Ruml (1994) Accumulation of cadmium by hairy root cultures of Solanum nigrum. Biotechnology Letters, 16, 621-624. Macek, T.E., P. Kotrba, T. Ruml, M. Suchova, F. Skacel, M. Mackova (1997) Accumulation of cadmium ions by hairy root cultures. In: P.M. Doran, ed, Hairy roots: Culture and Applications. Harwood Academic, Amsterdam, The Netherlands, p. 133-138. Mahagamasekera, M.G.P ., P .M. Doran (1997) Intergeneric co-culture of genetically transformed organs for the production of scopolamine. Phytochemistry, 47, 17-25. Mathys, W. (1977) The role of malate, oxalate and mustard oil glucosides in the evolution of zinc resistance in herbage plants. Physiologia Plantarum, 40, 130-136. Manjari, V., U.N. Das (1998) Oxidant stress, anti-oxidants, nitric oxide and essential fatty acids in peptic ulcer disease. Prostaglandins, Leu!wtrienes and Essential Fatty Acids, 59, 401-406. Manohar, M., K.A. Balasubramanian (1986) Antioxidant enzymes in gastrointestinal tract. Indian Journal ofBiochemistry and Biophysics, 23, 274-278. Massacci A., M.A. Lannelli, F. Pietrini, F. Loreto (1995) The effect of growth at low temperature on photosynthetic characteristics and mechanisms of photoprotection of maize leaves. Journal of Experimental Botany, 46, 119-127. May, M.J., T. Vemoux, R. Sanchez-Fernandez, M.Van Montagu, D. Inze (1998) Evidence Mcrft;)L S· ''\_.' · rD, ~cC,(_ C\910_) 4y0v,Jlh_ 'cf __ f /chl &ll {' .,lfor,10C~ , 711 , , ,· ·,.');·. , -.1 , Cc 1{tfi' 1 , al- CoJ1/rolftl p!I /4 velJ, Ulna?/ c, ':__, (,/.,l'·- c.',> i I fad{)'~- ·~~-I r'A I 0£ A - /;z.7CJ, ~-O(JYJ·/r. ( · ' /U;· ...·1/ ,;;;>-,--, ~ J ... t- . ,, C 180 for posttranscriptional activation of y-glutamylcysteine synthetase during plant stress responses. Proceedings ofthe National Academy-of Science, USA, 95, 12049-12054. Mauch, F., R. Dudler (1993) Differential induction of distinct glutathione S-transferases of wheat by xenobiotics and by pathogen attack. Plant Physiology, 102, 1193-1201. Mazhoudi, S., A. Chaouri, M.H. Ghorbal, E.E. Ferjani (1997) Response of antioxidant enzymes to excess copper in tomato (Lycopersicon esculentum, Mill.). Plant Science, 127, 129-137. Mehta, A.S., V. Sahajwalla (2000) Influence of composition of slag and carbonaceous materials on the wettability at the slag/carbon interface during pulverised coal injection in a blast furnace. Scandinavian Journal ofMetallurgy, 29, 17-29. Mejare, M., L. Bulow (2001) Metal-binding proteins and peptides in bioremediation and phytoremediation of heavy metals. Trends in Biotechnology, 19, 67-73. Metzger, L., I. Fouchault, C. Glad, R. Prost, D. Tepfer (1992) Estimation of cadmium availability using transformed roots. Plant and Soil, 143, 249-257. Meuwly, P., P. Thibault, A.E. Schwan, W.E. Rauser (1995) Three families ofthiol peptides are induced by cadmium in maize. Plant Journal, 7, 391-400. Michelet, B., M. Boutry (1995) The plasma membrane H+-ATPase. Plant Physiology, 108, 1-6. Misra, H.P., I. Fridovich (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. Journal of Biological Chemistry, 247, 3170-3175. Mitchel, C.D., T.A. Fretz (1977) Cadmium and zinc toxicity in white pine, red maple and Norway spruce. Journal ofAmerican Society ofHorticultural Science, 102, 81-84. Moffat, A.S. (1995) Plants proving their worth in toxic metal clean up. Science, 269, 302- 303. Monk, L.S., K.V. Fagerstedt, R.M.M. Crawford (1989) Oxygen toxicity and superoxide 181 dismutase as an antioxidant in physiological stress. Physiologia Plantarum, 76, 456- 459. Morrison, R.S., R.R. Brooks, R.D. Reeves (1980) Nickel uptake by Alyssum species. Plant Science Letters, 17, 451-457. Nakano, Y., K. Asada (1980) Spinach chloroplasts scavenge hydrogen peroxide on illumination. Plant, Cell and Physiology, 21, 1295-1307. Nakano, Y., K. Asada (1981) Hydrogen peroxide scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant, Cell and Physiology, 22, 867-880. Navari-Izzo, F., M.F. Quartacci, C. Pinzino, F.D. Vecchia, C.L.M. Sgherri (1998) Thylakoid-bound and stromal antioxidative enzymes in wheat treated with excess copper. Physiologia Plantarum, 104, 630-638. Nedelkoska T.V., P.M. Doran (2000a) Hyperaccumulation of cadmium by hairy roots of Thlaspi caerulescens. Biotechnology and Bioengineering, 67, 607-615. Nedelkoska T.V., P.M. Doran (2000b) Characteristics of heavy metal uptake by plant species with potential for phytoremediation and phytomining. Minerals Engineering, 13, 549-561. Nedelkoska T.V., P.M. Doran (2001) Hyperaccumulation of nickel by hairy roots of Alyssum species: comparison with whole regenerated plants. Biotechnology Progress, 17, 752-759. Nicks, L.J., M.F. Chambers (1995) Farming for metals. Mining Environmental Management, 15-18. Nieboer, E., RT. Tom, W.E. Sanford (1988) Nickel metabolism in man and animals. In: H. Sigel and A. Sigel, eds, Metal Ions in Biological Systems, Vol. 23. Marcel Dekker, New York, USA, p. 91-121. Noctor, G., A.C.M. Arisi, L. Jouanin, K.J. Kunert, H. Rennenberg, C.H. Foyer (1998) 182 Glutathione: biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants. Journal ofExperimental Biology, 49, 623-647. Nussbaum, S., D. Schmutz, C. Burnold (1988) Regulation of assimilatory sulphate reduction by cadmium in Zea mays L. Plant Physiology, 88, 1407-1410. Obata, H., M. Umebayashi (1993) Production of SH compounds in higher plants of different tolerance to Cd. Plant and Soil, 155/156, 533-536. Ohlsson, A.B., T. Berglund, P. Komlos, J. Rydstrom (1995) Plant defense metabolism is increased by the free radical-generating compound AAPH. Free Radical Biology and Medicine, 19, 319-327. Ouariti, 0., N. Boussama, M. Zarrouk, A. Cherif, M.H. qhorbal (1997) Cadmium and copper induced changes in tomato membrane lipids. Phytochemistry, 45, 1343-1350. Palmgren, M.G. (1998) Proton gradients and plant growth: role of the plasma membrane H+ ATPase. Advances in Botanical Research, 28, 1-70. Palmgren, M.G., J.F. Harper (1999) Pumping with plant P-type ATPases. Journal of Experimental Botany, 50, 883-893. Pandolfini, T., R. Gabbrielli, C. Comparini (1992) Nickel toxicity and peroxidase activity in ~ seedlings of Triticum aestivum L. Plant Cell and Environment, 15, 719-725. Patra, J., M. Lenka, B.B. Panda (1994) Tolerance and co-tolerance of the grass Chloris barbata Sw. to mercury, cadmium and zinc. New Phytologist, 128, 165-171. Patra, J., B.B. Panda (1998) A comparison of biochemical response to oxidative and metal stress in seedlings of barley, Hordeum vulgare L. Environmental Pollution, 101, 99- 105. Patterson, B.D., E.A. MacRae, I.B. Ferguson (1984) Estimation of hydrogen peroxide in plant extracts using titanium (IV). Analytical Biochemistry, 139, 487-492. Pelosi, P., R. Fiorentini, C. Galoppini (1976) On the nature of nickel compounds in Alyssum bertolonii Desv-II. Agricultural and Biological Chemistry, 40, 1641-1642. 183 Piqueras, A., E. Olmos, J.R. Martinez-Solano, E. Hellin (1999) Cd-induced oxidative burst in tobacco BY2 cells: Time course, subcellular location and antioxidant response. Free Radical Research, 31, S33-38. Psaras, G.K., T.H. Constantindis, B. Cotsopouls, Y. Manetas (2000) Relative abundance of Ni in the leaf epidermis of eight hyperaccumulators: Evidence that the metal is excluded from both guard cells and trichomes. Annals ofBotany, 86, 73-78. Punz, W.F., H. Sieghardt (1993) The response ofroots of herbaceous plant species to heavy metals. Environmental Experimental Botany, 33, 85-98. Qureshi, J.A., K. Hardwick, H.A. Collin (1986) Malic acid production in callus cultures of zinc and lead tolerant and non-tolerant Anthoxanthum odoratum. Journal ofPlant Physiology, 122, 477-479. Raggio, M., N. Moro-Raggio (1996) Oxidative damage caused by cadmium chloride in sunflower (Helianthus annuus L.) plants. Experimental Botany, 58, 41-52. Ramadevi, S., M.N.V. Parasad (1998) Copper toxicity in Ceratophyllum demersum L. (Coontail), a free floating macrophyte: Response of antioxidant enzymes and antioxidants. Plant Sceince, 138, 157-165. Rao, K.V.M., T.V.S. Sresty (2000) Antioxidant parameters in the seedlings of pigeon pea (Cajanus cajan (L.) Millspaugh) in response to Zn and Ni stresses. Plant Science, 157, 113-128. Raskin, I. (1996) Plant genetic engineering may help with environment cleanup. Proceedings ofthe National Academy ofScience, USA, 93, 3164-3166. Raskin, I., B.D. Ensley (2000) Phytoremediation of Toxic Metals: Using Plants to Clean up the Environment. John Wiley, New York, USA. Rausch, T. (1991) The hexose transporters at the plasma membrane and the tonoplast of higher plants. Physio/ogia Plantarum, 82, 134-142. Rauser, W.E., C.A. Ackerley (1987) Localisation of cadmium in granules within 184 differentiating and mature root cells. Canadian Journal ofBotany, 65, 643-646. Rauser, W.E., R. Schupp, H. Rennenberg (1991) Cysteine, y-glutamylcysteine and glutathione levels in maize seedlings. Plant Physiology, 91, 128-134. Reese, R.N., G.J. Wagner (1987) Effects ofbuthionine sulfoximine on Cd-binding peptide levels in suspension-cultured tobacco cells treated with Cd, Zn, or Cu. Plant Physiology, 84, 574-577. Reeves, R.D., A.J.M. Baker, R.R. Brooks (1995) Abnormal accumulation of trace metals by plants. Mining and Environment Management, 3, 4-8. Riisgard, H.U., E. Bjomestad, F. Mohlenberg (1987) Accumulation of cadmium in the mussel Mytilus edulis: kinetics and importance of uptake via food and sea water. Marine Biology, 96, 349-353. Robinson, B.H., R.R. Brooks, B.E. Clothier (1999) Soil amendments affecting nickel and cobalt uptake by Berkheya coddii: Potential use for phytomining and phytoextraction. Annals ofBotany, 84, 689-694. Robinson, B.H., A. Chiarucci, R.R. Brooks, D. Petit, J.H._ Kirkman, P.E.H. Gregg, V. De Dominicis (1997a) The nickel hyperaccumulator plant Alyssum bertolonii as a potential agent for phytoremediation and phytomining of nickel. Journal of Geochemical Exploration, 59, 75-86. Robinson, B.H., R.R. Brooks, A.W. Howes, J.H. Kirkman P.E.H. Gregg (1997b) The potential of the high-biomass nickel hyperaccumulator Berkheya coddii for phytoremediation and phytomining. Journal of Geochemical Exploration, 60, 115- 126. Romero-Puertas, M.C., I. McCarthy, L.M. Sandalio, J.M. Palma, F.J. Corpas, M.Gomez, L.A. Del Rio (1999) Cadmium toxicity and oxidative metabolism of pea leaf peroxisomes. Free Radical Research, 31, S25-3 l. Ruegsegger, A., C. Brunold (1992) Effect of cadmium on y-glutamylcysteine synthesis in 185 maize seedlings. Plant Physiology, 99, 428-433. Sagner, S., R. Kneer, G. Wanner, J.P. Cosson, B.D. Neumann, M.H. Zenk (1998) Hyperaccumulation, complexation and distribution of nickel in Sebertia accuminata. Phytochemistry, 47, 339-347. Salt, D.E., R.D. Smith, I. Raskin (1998) Phytoremediation. Annual Reviews ofPlant Physiology and Plant Molecular Biology, 49, 643-668. Salt, D.E., R.C. Prince, A.J.M. Baker, I. Raskin, I.J. Pickering (1999) Zinc ligands in the metal hyperaccumulator Thlaspi caerulescens as determined using X-ray absorption spectroscopy. Environmental Science and Technology, 33, 713-717. Sandalio L.M., H.C. Dalurzo, M. Gomez, M.C. Romero-Puertas, L.A. Del Rio (2001) Cadmium-induced changes in the growth and oxidative metabolism of pea plants. Journal ofExperimental Botany, 52, 2115-2126. Sandmann, G., P. Boger (1980) Copper-mediated lipid peroxidation processes in photosynthetic membranes. Plant Physiology, 66, 797-800. Santa-Maria, G.E., D.H. Cogliatti (1998) The regulation of zinc uptake in wheat plants. Plant Science, 137, 1-12. Scheller, H.V., B. Huang, E. Hatch, P.B. Goldsbrough (1987) Phytochelatin synthesis and glutathione levels in response to heavy metals in tomato cells. Plant Physiology, 85, 1031-1035. Schickler, H., H. Caspi (1999) Response of antioxidative enzymes to nickel and cadmium stress in hyperaccumulator plants of the genus Alyssum. Physiologia Plantarum, 105, 39-44. Schneider, S., L. Bergmann (1995) Regulation of glutathione synthesis in suspension cultures of parsley and tobacco. Botanica Acta, 108, 34-40. Schnoor, J.J., L.A. Licht, S.C. McCutcheon, N.L. Wolfe, L.H. Carreira (1995) Phytoremediation of organic and nutrient contaminants. Environmental Science and 186 Technology, 29, 318-323. Schiltzendilbel, A., P. Schwanz, T. Teichmann, K. Gross, R. Langenfeld-Heyser, D.L. Godbold, A. Polle (2001) Cadmium-induced changes in antioxidative systems, hydrogen peroxide content, and differentiation in Scots pine roots. Plant Physiology, 127, 887-898. Sedlak, J., R.H. Lindsay (1968) Estimation of total, protein-bound, and nonprotein sulfhydryl groups in tissue with Ellman's reagent. Analytical Biochemistry, 25, 192- 205. Sela, M., E. Tetor, E. Fritz, A. Huttermann (1988) Localization and toxic effects of cadmium, copper and uranium in Azolla. Plant Physiology, 88, 30-36 Senden, M.H.M.N., F.J.M. Van Passen, A.J.G.M. Vander Meer, H. Th. Wolterbeek (1992) Cadmium-citric acid-xylem cell wall interactions in tomato plants. Plant, Cell and Environment, 15, 71-79. Shaw, B.P. (1995) Effect of mercury and cadmium on the activities of antioxidative enzymes in the seedlings of Phaseolus aureus. Biologica Plantarum, 37, 587-596. Shen, Z.G., F.J. Zhao, S.P. McGrath (1997) Uptake and transport of zinc in the hyperaccumulator Thlaspi caerulescens and the non-hyperaccumulator Thlaspi ochroleucum. Plant, Cell and Environment, 20, 898-906. Smith, I.K., AC. Kendall, A.J. Keys, J.C. Turner, P.J. Lea (1984) Increased levels of glutathione in a catalase-deficient mutant of barley (Hordeum vulgare L.). Plant Science Letters, 37, 29-33. Smith, I.K., A.C. Kendall, A.J. Keys, J.C. Turner, P.J. Lea (1985) The regulation of the biosynthesis of glutathione in leaves of barely (f!ordeum vulgare L.). Plant Science, 41, 11-17. Simon, L. ( 1998) Cadmium accumulation and distribution in sunflower plant. Journal of Plant Nutrition, 21, 341-352. 187 Somashekaraiah, B.V., K. Padmaja, A.R.K. Prasad (1992) Phytotoxicity of cadmium ions on germinating seedlings ofmung bean (Phaseolus vulgaris): Involvement oflipid peroxides in chlorophyll degradation. Physiologia Plantarum, 85, 85-89. Sreenivasulu, N., S. Ramanjulu, K. Ramachandra-Kini, H.S. Prakash, H. Shekar-Shetty, H.S. Savithiry, C. Sudhakar (1999) Total peroxidase activity and peroxidase isoforms as modified by salt stress in two cultivars of fox-tail millet with differential salt tolerance. Plant Science, 141, 1-9. Srivastava, M.K, U.N. Dwivedi (1998) Salicylic acid modulates glutathione metabolism in pea seedlings. Journal ofPlant Physiology, 153, 409-414. Stroinski, A., M. Zielezinska (1997) Cadmium effect on hydrogen peroxide, glutathione and phtochelatins levels in potato tuber. Acta Physiologia Plantarum, 19, 127-135. Sumi, Y., T. Muraki, T. Suzuki (1982) Histochemical staining of cadmium with benzothiazolylazonaphthol derivatives. Histochemistry, 73, 481-486. Takahashi, H., Z. Chen, H. Du, Y. Liu, D.F. Klessig (1997) Development of necrosis and activation of disease resistance in transgenic tobacco plants with severely reduced catalase levels. Plant Journal, 11, 993-1005. Tappel. A.L. (1973) Lipid peroxidation damage to cell components. Federation Proceedings, 32, 1870-1874. Thauer, R.K., G. Diekert, P. Schonheit (1980) Biological roles of nickel. Trends in Biochemical Science, 11, 304-306. Tolra, R.P., C. Poschenrieder, J. Barcelo (1996) Zinc hyperaccumulation in Thlaspi caerulescens. 2. Influence on organic acids. Journal ofPlant Nutrition, 19, 1541- 1550. Tomsett, A.B., D.A. Thurman (1988) Molecular biology of metal tolerances of plants. Plant, Cell and Environment, 11, 383-394. Toppi, L.S.D, M. Lambardi, N. Pecchioni, L. Pazzagli, M. Durante, R. Gabbrielli (1999) 188 Effects of cadmium stress on hairy roots of Daucus carota. Journal of Plant Physiology, 154, 385-391. Vazquez, M.D., J. Barcelo, Ch. Poschenrieder, J. Madico, P. Hatton, A.J.M. Baker, G.H. Cope (1992) Localization of zinc and cadmium in Thlaspi caerulescens (Brassicaceae ), a metallophyte that can hyperaccumulate both metals. Journal of Plant Physiology, 140, 350-355. Vazquez, M.D., C. Poschenrleder, J. Barcelo, A.J.M. Baker, P. Hatton, G.H. Cope (1994) Compartmentation of zinc in roots and leaves of the zinc hyperaccumulator Thlaspi caerulescens J & C Presl. Botonica Acta, 107, 243-250. Verkleij, J.A.C., H. Schat (1989) Mechanism of metal tolerance in higher plants. In: A.J Shaw, ed, Heavy Metal Tolerance in Plants: Evolutionary Aspects. CRC Press Boca Raton, USA, p. 179-194. Vitoria, A.P., P.J. Lea, R.A. Azevedo (2001) Antioxidant responses to cadmium in radish tissues. Phytochemistry, 57, 701-710. Vogeli-Lange, R., G.J. Wagner (1990) Subcellular localization of cadmium and cadmium binding peptides in tobacco leaves. Plant Physiology, 92, 1086-1093. Volesky, B. H.A. May-Phillips (1994) Biosorption of heavy metals by Saccharomyces cerevisiae. Applied Microbiology and Biotechnology, 42, 797-806. Waldron, H.A (1979) Lecture Notes on Occupational Medicine, 2nd eds, Blackwell Scientific Publications, Melbourne, Australia, p. 27-31. Walsh, C.T., W.H. Orme-Jhonson (1987) Nickel enzymes. Biochemistry, 26, 4901-4906. Weckx, J., H. Clijsters (1996) Oxidative damage and defense mechanisms in primary leaves of Phaseolus vulgaris as a result of root assimilation of toxic amounts of copper. Physiologia Plantarum, 96, 506-512. Weigel, H.J., H.J. Jager (1980) Subcellular distribution and chemical form of cadmium in bean plants. Plant Physiology, 65, 480-482. 189 Welch, R.M., W.A. Norvell (1993) Growth and nutrient uptake by barely (Hordeum vulgare L. cv Herta): studies using an N-(2-hydroxyethyl) ethylenedinitrilotriacetic acid buffered nutrient solution technique. II. Role of zinc in the uptake and root leakage of mineral nutrients. Plant Physiology, 101, 627-631. WHO, (1992) Cadmium. Environmental Health Criteria, 134, 36-45. Willekens, H., S. Chamnongpol, M. Davey, M. Schraudner, C. Langebartels, M. Van Montagu, D. Inze, W. Van Camp (1997) Catalase is a sink for H2O2 and 1s indispensable for stress defence in C3 plants. EMBO Journal, 16, 4806-4816. Williams, L.E., J.K. Pittman, J.L. Hall (2000) Emerging mechanisms for heavy metal transport in plants. Biochimica et Biophysica Acta, 1465, 104-126. Wojcik, M., A. Tukendorf (1999) Cd-tolerance of maize, rye and wheat seedlings. Acta Physiologia Plantarum, 21, 99-107. Wongsamuth, R., P.M. Doran (1997) Production of monoclonal antibodies by tobacco hairy roots. Biotechnology and Bioengineering, 54, 401-415. Wu, S., Y. Zu, M. Wu (2001) Cadmium response of the hairy root culture of the endangered species Adenophora lobophylla. Plant Science, 160, 551-562. Yang, X.E., V.C. Baligar, J.C. Foster, D.C. Martens (1997) Accumulation and transport of nickel in relation to organic acids in ryegrass and maize grown with different nickel levels. Plant and Soil, 196, 271-276. Yang, X., V.C. Baligar, D.C. Martens, R.B. Clark (1996) Plant tolerance to nickel toxicity. 1. Influx, transport, and accumulation of nickel in four species. Journal of Plant Nutrition, 19, 73-85. Zenk, M.H. (1996) Heavy metal detoxification in higher plants-a review. Gene, 179, 21-30. Zhao, F.J., Z.G. Shen, S.P. McGrath (1998) Solubility of zinc and interactions between zinc and phosphorus in the hyperaccumulator Thlaspi caerulescens. Plant, Cell and Environment, 21, 108-114. 190 Zhao, F.J., E. Lombi, T. Breedon, S.P. McGrath (2000) Zinc hyperaccumulation and cellular distribution inArabidopsis halleri. Plant, Cell and Environment, 23, 507-514. Zhu, Y.L., E.A.H. Pilon-Smits, L. Jouanin, T. Norman (1999a) Overexpression of glutathione synthetase in Indian mustard enhances cadmium accumulation and tolerance. Plant Physiology, 119, 73-79. Zhu, Y.L., E.A.H. Pilon-Smits, A.S. Tarun, S.U. Weber, L. Jouanin, N. Terry (1999b) Cadmium tolerance and accumulation in Indian mustard is enhanced by overexpressing y-glutamylcysteine synthetase. Plant Physiology, 121, 1169-1177. Zhu, Y.L., A.M. Zayed, J-H. Qian, M. de Souza, N. Terry (1999c) Phytoaccumulation of trace elements by wetlands plants: II. Water hyacinth. Journal of Environmental Quality, 28, 339-344. 191 APPENDICES APPENDIX 1. Standard curve for Cd determination. Abs New Rational - Cal. Set 1 0 .27 Calibrated 0.20 0.10 0.00~,----...... ::~---~~-----~ 0.0 .0 Cd ppm APPENDIX 2. Standard curve for Ni determination. Abs New Rational - C~l. Set 1 0 .10 Calibrated 0.05 0.00~~------=---=------~""'7" 0.0 2.0 4. Ni ppm