I Table of contents

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I

Table of contents

Summary. Different Phytoextraction Techniques of Heavy Metals i

Chapter 1. Introduction 1

1.1 – Toxic heavy metals 1

1.1.1 – Sources, contamination, characteristics and specific effects 1 1.1.2 – Legislation toward heavy metals contamination: the European

and Italian situations

5 1.1.3 – Remediation technologies: an evaluation 10

1.2 – Phytoremediation 13

1.2.1 – Long-term continuous phytoextraction 14 1.2.2 – Chelator-assisted phytoextraction 16 1.2.3 – Exudates role in metal exclusion and hyperaccumulation

phenomena

28 1.2.4 – Genetic engineering of plants: what and how 32 1.3 – Arsenic: a concern among heavy metals 45

1.3.1 – Arsenic: fate and occurrence in different environmental substrates

46

1.3.2 – Arsenic in plants: uptake 52

1.3.3 – Arsenic in plants: toxicity, metabolism and detoxification 57

1.4 – The dual face of copper 62

1.4.1 – Copper: fate and occurrence in soils 63 1.4.2 – Copper in plants: uptake and translocation 64 1.4.3 – Copper in plants: toxicity and detoxification 71 1.5 – Zinc, Cadmium and Lead: other toxic metals in plant system 80 1.5.1 – Zinc: functions, toxicity and detoxification in plants 80 1.5.2 – Cadmium: toxicity and detoxification in plants 82 1.5.3 – Lead: toxicity and detoxification in plants 84

References 86

Outline of the thesis. Different Phytoextraction Techniques of Heavy Metals

119

Chapter 2. Materials and methods 123

2.1 – Phytoextraction experiments 123

2.1.1 – Chemicals 123

2.1.2 – Germination and tolerance index tests 123

2.1.3 – Hydroponic experiments 124

2.1.4 – Speciation modelling 125

2.1.5 – Pot experiments 125

2.1.6 – Sequential extraction of metals 126 2.1.7 – Desorption of metals from soil by chelators 127

2.1.8 – Extractable metals in soil 127

2.1.9 – Determination of EDDS and NTA in soil 128 2.1.10 – Wild species-assisted phytoextraction by B. carinata 128 2.1.11 – Flavonoids, organic and phenolic acids in root exudates 129

2.1.12 – Statistical analysis 130

2.2 – Arsenic 131

2.2.1 – Chemicals 131

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2.2.3 – Root desorption procedure 131

2.2.4 – Time-dependent kinetics of As(III) and As(V) uptake 132 2.2.5 – Concentration-dependent kinetics of As(III) and As(V) uptake 133 2.2.6 – Arsenate and arsenite influxes at different phosphate

concentrations

133 2.2.7 – Measurements of membrane integrity 133 2.2.8 – Arsenic and potassium determination 134

2.3 – Copper 135

2.3.1 – Uptake kinetics 135

2.3.1.1 - Chemicals 135

2.3.1.2 – Plant culture 135

2.3.1.3 – Time-dependent kinetics of Cu(II) uptake 136 2.3.1.4 – Concentration-dependent kinetics of Cu(II) uptake 136 2.3.1.5 – Measurements of membrane integrity 137 2.3.1.6 – Concentration-dependent uptake kinetics of Cu-NTA and

Cu-EDDS complexes

137 2.3.1.7 – Copper and potassium determination 138

2.3.1.8 – Statistical analysis 138

2.3.2 – Copper, NTA and [S,S]-EDDS accumulation in plant tissues 138 2.3.2.1 – Copper, NTA and [S,S]-EDDS determination in plant tissues 138

2.3.3 – Copper transport in xylem sap 140

2.3.3.1 – Chemicals 140

2.3.3.2 – Xylem sap collection 140

2.3.3.3 – Xylem sap analysis 141

2.3.3.4 – Amino acids detection in B. carinata xylem sap 142 2.3.3.5 – Free Cu2+ concentration vs pH titrations 143

2.4 – Genetic engineering of tobacco plants 145

2.4.1 – Plasmid construction 145

2.4.2 – Tobacco transformation procedure 145 2.4.3 – Confirmation of tobacco transformation 147 2.4.4 – RNA isolation, Northern-Blot analysis and RT-PCR 148 2.4.5 – Experimental conditions and design 150

References 152

Chapter 3. Results 155

3.1.1 – Selection of the species 155

3.1.2 – Brassica carinata hydroponical assay 158

3.1.3 – Pot experiments 160

3.1.4 – Extractable metals in time 165

3.1.5 – Biodegradation of EDDS and NTA in soil 166 3.1.6 – Wild species-assisted phytoextraction by B. carinata 166

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3.1.7 – Wild species root exudates 168

3.2 – Arsenic 173

3.2.1 – Time-dependent kinetics of As(III) and As(V) uptake 173 3.2.2 – Concentration-dependent kinetics of As(III) and As(V) uptake 174 3.2.3 – Measurement of membrane integrity 176 3.2.4 – As(III) and As(V) influxes at different phosphate concentrations 176

3.3 – Copper 178

3.3.1 – Time-dependent kinetics of Cu(II) 178 3.3.2 – Concentration-dependent kinetics of Cu(II) 178 3.3.3 – Measurement of membrane integrity 179 3.3.4 – Concentration-dependent kinetics of [S,S]-EDDS-Cu and

NTA-Cu

180 3.3.5 – Long-term copper, EDDS and NTA accumulation in plant

tissues

181

3.3.6 – Xylem sap analysis 185

3.3.7 – Amino acids in B. carinata xylem sap 185

3.3.8 – Cu in xylem sap 188

3.3.9 – Brassica carinata xylem sap and simulated saps for excess of copper

189 3.3.10 – Effect of Cu excess treatments on xylem sap histidine and

proline

192 3.3.11 – Brassica carinata xylem sap and simulated saps for copper

starvation

193

3.4 – AtMT2b-transformed tobacco 195

3.4.1 – As(III) resistance and accumulation tests 195

Chapter 4. Discussion 199

4.1.1 – Chemically-assisted phytoextraction experiments 199 4.1.2 – Wild species-assisted phytoextraction experiments 204 4.1.3 – Genetic engineering approach: AtMT2b-transformed tobacco

plants

208

4.2 - Arsenic 210

4.3 - Copper 214

4.3.1 – Mechanisms of free and complexed copper uptake 214 4.3.2 – Mechanism of copper translocation 221

References 226

Conclusions. Different Phytoextraction Techniques of Heavy Metals 235