Investigations into Lead (Pb) Accumulation in Symphytum officinale L.:

329 Pages·2009·5.46 MB·English

Investigations into Lead (Pb) Accumulation in
Symphytum officinale L.:
A Phytoremediation Study

A thesis
submitted in fulfilment
of the requirements for the degree
of
Doctor of Philosophy in Plant Biotechnology
by
Lily Chin

School of Biological Sciences
2007
[© 2007 Lily Chin, all rights reserved]
i
ABSTRACT
Lead (Pb) is the number one heavy metal pollutant in the environment. The high cost
and environmental concerns of conventional remediation technologies has led to an
emerging alternative technology for heavy metal remediation: phytoremediation. This
study was set out to advance Pb phytoremediation by investigating plant-associated
factors (e.g. polyphenol levels, Pb-tannin chelation, and superoxide dismutase activity)
and chemical-based factors (e.g. concentration of Pb, and the type and dosage of
chelating agents in treatments) that may affect Pb accumulation.
Using a hydroponic system, sand-grown Symphytum officinale L. plants were
exposed to nutrient solutions with or without lead nitrate (Pb(NO ) ) and
3 2
ethylenediamine tetraacetic acid (EDTA). Using flame atomic absorption spectroscopy
(to measure Pb content) and bovine serum albumin-protein precipitation (to measure
polyphenol and tannin levels), a significant in vivo correlation between tannin level and
Pb accumulation level was observed in roots of plants exposed to all Pb treatments.
Higher tannin containing-lateral roots accumulated significantly more Pb than lower-
tannin main roots. Transmission electron micrographs of unchelated Pb-treated plants
supported these findings, whilst dialysis-based in vitro Pb-chelation studies with crude
S. officinale root polyphenol extracts did not. The dialysis method was likely to be
subject to fructan interference. A new, more accurate and simple method based on
tannin immobilisation was consequently developed. Results using this method
supported the hydroponic trends. This new method was also verified with purified
tannic acid (from Sigma). Together, these findings demonstrate that S. officinale root
tannins have the ability to chelate Pb. This may be a mechanism to cope with Pb stress
(adaptive tolerance). Despite the typical signs of Pb stress at root level (e.g. root growth
inhibition, and degraded cytoplasms), shoots showed no signs of stress under any Pb
treatments. Most importantly, since this chelation-based tolerance mechanism also
influences the accumulation levels, the phytochemical composition of plants should also
be considered when screening plants for phytoremediation.
The level of Pb accumulated in the shoots depended on the concentration of
Pb(NO ) and presence of chelating agents (EDTA or N-[2 acetamido] iminodiacetic
3 2
acid (ADA)) in the nutrient solution. The highest level of Pb in shoots was between
0.05-0.06% (d.w. on average) using EDTA or ADA, well short of the 1% (d.w.) shoot
accumulation target for Pb phytoextraction. The highest level of Pb in the roots (and of
ii
all measurements) was with unchelated 500 μM Pb(NO ) ; on average 2% (d.w.)
3 2
accumulated in root. Overall, since S. officinale accumulated Pb predominately in the
roots, it is most suited for rhizofiltration and phytostabilisation. Whilst chelating agents
enhanced Pb accumulation in shoots, root levels were unexpectedly reduced compared
to unchelated Pb treatments. The level of Pb translocated did not completely account for
this loss. Minor factors relating to EDTA desorption of roots, EDTA specificity, and
charge repulsion of the PbEDTA complex may account for some of the loss, but the
main cause remains unclear.
In vitro S. officinale cultures were developed and somaclonal variation (involving
Pb pre-treatment of petioles) was used as a tool to further investigate, and attempt to
improve its Pb phytoremediation potential. The shoots and roots of plants produced
from petioles pre-treated with Pb(NO ) appeared more stressed than those without Pb
3 2
pre-treatment. After re-treatment with Pb (Pb(NO ) or PbADA), plants developed from
3 2
most Pb pre-treated petioles appeared to have reduced Pb accumulation and polyphenol
levels, and increased superoxide dismutase activity in roots (although no statistically
significant trends were found). Overall, plants produced from Pb pre-treated petioles in
this study may have less phytoremediation potential.
iv
TABLE OF CONTENTS
Page
ABSTRACT………………………………………………………….…………...…….i
TABLE OF CONTENTS……………………………………………….……….….. iii
LIST OF FIGURES……………………...…………………………………….…... xiv
LIST OF PLATES……………………………………………………….……...….. xix
LIST OF TABLES………………………………………………………………...... x xi
ABBREVIATIONS……………………………………………………………....… x xii
ACKNOWLEDGEMENTS………………………………………………….….... xxiv

CHAPTER 1: INTRODUCTION
1 HEAVY METALS..............................................................................................1
1.1 Definitions................................................................................................1
1.2 Sources of heavy metal pollution..............................................................2
2 LEAD..................................................................................................................2
2.1 Lead pollution...........................................................................................2
2.2 Effect of lead on human health..................................................................3
2.3 Effect of lead on plant growth...................................................................3
3 HEAVY METAL REMEDIATION TECHNOLOGIES..................................5
3.1 Conventional remediation technologies.....................................................5
3.2 Alternative remediation technologies (I) – Micro-organisms.....................6
3.3 Alternative remediation technologies (II) - Phytoremediation...................7
3.3.1 Origin of phytoremediation concept..............................................7
3.3.2 Phytoremediation: definition and classes.......................................7
3.3.3 ‘Pros and cons’ of phytoremediation..............................................8
3.3.4 Ideal attributes of phytoextraction plants.......................................9
3.3.5 Plants used for lead phytoextraction............................................10
iv
4 HEAVY METAL UPTAKE BY PLANTS......................................................11
4.1 Factors influencing heavy metal bioavailability.......................................11
4.1.1 Physical and biological factors....................................................12
4.1.2 Chemical factors (I): soil chemistry.............................................12
4.1.3 Chemical factors (II): chelating agents.........................................12
4.2 Heavy metal uptake, transport and accumulation.....................................13
4.2.1 Metal uptake and transport..........................................................13
4.2.2 Movement route and accumulation of lead in plant tissues...........15
5 HEAVY METAL STRESS AND TOLERANCE IN PLANTS......................16
5.1 Positive effects of reactive oxygen species (ROS)...................................17
5.2 Negative effects of ROS and the antioxidative defence system................17
5.3 Lead and oxidative stress........................................................................18
6 HEAVY METAL TOLERANCE MECHANISMS........................................18
7 MECHANISM 1 (PART I): CONTROLLING METAL-ION LEVELS.......19
7.1 Tissue isolation.......................................................................................19
7.2 Cellular isolation.....................................................................................19
7.3 Detoxification by ligand chelation...........................................................20
8 MECHANISM 1 (PART II): METAL-POLYPHENOL CHELATION........21
8.1 Polyphenols: class and structure..............................................................21
8.1.1 Classes of polyphenols................................................................21
8.1.2 Tannins.......................................................................................22
8.2 General roles of plant polyphenols..........................................................22
8.3 Location of polyphenols in plants............................................................24
8.3.1 Polyphenols at cellular level........................................................24
8.3.2 Polyphenols at tissue level...........................................................25
8.4 Heavy metal tolerance: chelation to polyphenols?...................................26
9 MECHANISM 2: THE ANTIOXIDATIVE DEFENCE SYSTEM (ADS)....27
9.1 Superoxide dismutase (SOD)..................................................................27
9.2 Role of SOD in heavy metal tolerance....................................................27
10 TISSUE CULTURE AND PHYTOREMEDIATION.....................................28
Chapter 2 - Materials and methods 67v
11 MUTANT STUDIES AND PHYTOREMEDIATION………..……...………2 9
11.1 Somaclonal variation approach………………………………………... 29
11.1.1 Type and advantages…………………………………………... 29
11.1.2 Induced somaclonal variation…………………………………..3 0
11.1.3 Developing heavy metal tolerant plants………………...…….. 31
11.2 Genetic modification approach…………………………………………3 1
11.2.1 Transgenic plants……………………………………………….3 2
11.2.2 Risks associated with genetic engineering…………………….. 33
11.3 Chemical mutagenesis and Arabidopsis thaliana………………...…… 34
12 SYMPHYTUM OFFICINALE L. (COMFREY)…………………………….. 35
13 AIM AND OBJECTIVES………………………………………………...…... 35

CHAPTER 2: MATERIALS AND METHODS

OBJECTIVE 1
1 PLANT PROPAGATION................................................................................38
1.1 Root stock............................................................................................38
1.2 Vegetative plant propagation................................................................38
2 POLYPHENOL STUDIES...............................................................................39
2.1 Histochemical studies...........................................................................39
2.1.1 Detection of polyphenols..........................................................39
2.1.2 Class of tannins.........................................................................39
2.2 Factors affecting polyphenol analysis...................................................39
2.3 Polyphenol and tannin extraction..........................................................40
2.3.1 Sample preparation...................................................................40
2.3.2 Extraction methods...................................................................40
2.4 Polyphenol quantification.....................................................................40
2.4.1 Folin-Ciocalteu assay for polyphenols......................................42
2.4.2 BSA-protein precipitation assay for polyphenols.......................42
2.4.3 Folin-Ciocalteu assay for tannins..............................................42
2.4.4 BSA-protein precipitation assay for tannins..............................43
3 LEAD ACCUMULATION STUDIES (I)........................................................43
Chapter 2 - Materials and methods 6v8i
3.1 Lead effects..........................................................................................43
3.2 Lead accumulation – hydroponic experiments......................................43
3.2.1 Lead treatment in hydroponic nutrient solution.........................43
3.2.2 Preparation of plant material for lead analysis...........................44
3.2.3 Measurement of lead level in samples.......................................46
4 TRANSMISSION ELECTRON MICROSCOPY...........................................46
4.1 Preparation of ultra-thin sections..........................................................46
4.1.1 Fixation....................................................................................46
4.1.2 Post-fixation.............................................................................47
4.1.3 Dehydration..............................................................................47
4.1.4 Infiltration................................................................................47
4.1.5 Embedding...............................................................................47
4.1.6 Sectioning.................................................................................47
4.2 Microscopy..........................................................................................47
5 IN VITRO LEAD CHELATION TO POLYPHENOLS.................................48
5.1 Established protocol: Lavid’s dialysis method......................................48
5.2 Developing a new method: PVPP-immobilised tannin method.............48
5.2.1 Plant material............................................................................49
5.2.2 Polyphenol extraction and assay...............................................49
5.2.3 Preparation of immobilised root tannins....................................49
5.2.4 Lead chelation to immobilised root tannins...............................51
5.3 Lead chelation to immobilised lateral root tannins................................51
5.4 Lead chelation to immobilised purified tannic acid...............................52

OBJECTIVE 2
6 LEAD ACCUMULATION STUDIES (II).......................................................52
6.1 Effect of EDTA level on lead accumulation..........................................53
6.2 Effect of 500 m M Pb(NO ) level on lead accumulation........................53
3 2
7 LEAD UPTAKE STUDIES (III): EDTA STUDIES........................................53
7.1 EDTA pre-treatment studies.................................................................54
7.1.1 Root segment test......................................................................54
Chapter 2 - Materials and methods v6i9i
7.1.2 Whole plant test........................................................................54
7.2 Lead accumulation: alternative chelators (ADA)..................................55
8 DEVELOPING SYMPHYTUM OFFICINALE IN TISSUE CULTURE.......55
8.1 Explant selection..................................................................................55
8.2 Sterilisation regime..............................................................................56
8.3 Media development..............................................................................56
8.3.1 Callus induction media (CIM)...................................................56
8.3.2 Callus growth media (CGM).....................................................57
8.3.3 Root and whole plant induction media (RIM, WPIM)...............57
8.3.4 Propagation of Symphytum officinale plant lines.......................58

OBJECTIVE 3
9 IN VITRO LEAD-TREATED PLANTS..........................................................58
9.1 Whole plants pre-treated with lead.......................................................58
9.1.1 Pre-treatment # 1: 0, 500 mM PbEDTA, pH 4.5........................59
9.1.2 Pre-treatment # 2: 0, 500 mM PbEDTA, pH 7.0........................59
9.1.3 Pre-treatment # 3: 0, 500 mM PbEDTA and PbADA, pH 7.0.....60
9.2 Shoots from sand-grown plants pre-treated with lead............................60
9.3 Petioles from in vitro shoots pre-treated with lead................................60
9.3.1 Pre-treatment # 4: 0 to 500 m M Pb(NO ) , pH 4.5.....................60
3 2
9.3.2 Pre-treatment # 5: 0, 500 mM Pb(NO ) and PbADA, pH 4.5....60
3 2
9.4 Propagation of plants pre-treated with lead...........................................61
9.5 Re-treatment regime.............................................................................61
10 INDICATOR TESTS FOR POTENTIAL MUTANTS...................................62
10.1 Histochemical localisation of superoxide anions...................................63
10.1.1 Leaf discs...............................................................................63
10.1.2 Petiole sections.......................................................................64
10.1.3 Roots of intact whole plants....................................................64
10.2 Superoxide dismutase (SOD) activity...................................................64
10.2.1 Extraction...............................................................................64
10.2.2 Superoxide dismutase assay....................................................65
viii

10.2.3 Total protein assay………………………………………………66
11 STATISTICAL ANALYSIS……………………………………………….….. 66

CHAPTER 2: RESULTS AND DISCUSSION

OBJECTIVE 1
1 PLANT PROPAGATION...................................................................................67
2 POLYPHENOL STUDIES.................................................................................67
2.1 Histochemical detection of polyphenols.................................................67
2.2 Class of tannins.......................................................................................68
2.3 Factors affecting polyphenol analysis.....................................................68
2.3.1 Tannin recovery after freeze-drying............................................71
2.3.2 Number of extraction rounds.......................................................71
2.3.3 Extraction solvent........................................................................71
2.3.4 Type of quantitative assay...........................................................75
2.3.5 Extraction method and time........................................................76
2.3.6 Overall methods chosen..............................................................80
2.4 Distribution of polyphenol and tannins in Symphytum officinale...........80
3 LEAD ACCUMULATION STUDIES (I)..........................................................83
3.1 Ashing temperature.................................................................................83
3.2 Lead effect on plants: visual observations..............................................83
4 ROLE OF POLYPHENOLS IN LEAD ACCUMULATION..........................85
4.1 Hydroponic conditions............................................................................85
4.2 Pb uptake and accumulation...................................................................86
4.3 Effect of lead treatments on polyphenol and tannin levels.....................89
4.4 Transmission electron microscopy (TEM).............................................92
5 CORRELATION BETWEEN TANNIN AND LEAD ACCUMULATION 113
5.1 Other possible factors for lead accumlation trends...............................113
5.1.1 Root volume..............................................................................113
ix

5.1.2 Root age.....................................................................................113
6 IN VITRO LEAD CHELATION TO TANNINS............................................114
6.1 Lavid’s dialysis method (I)...................................................................114
6.1.1 Established protocol..................................................................114
6.1.2 Modified protocol......................................................................115
6.1.3 Fructan interference (I)..............................................................121
6.1.4 Fructan interference (II): Direct experimental evidence...........122
6.2 New appproach: Lead chelation to immobilised tannins......................124
6.2.1 Approach to experimental design..............................................124
6.2.2 In vitro lead chelation by lateral and main root tannins............125
6.2.3 Linear in vitro chelation............................................................133
6.3 Lead chelation to immobilised tannins: general points........................133
6.4 Validating immobilised tannin method: purified tannins.....................136
6.4.1 In vitro lead chelation: purified tannic acid...............................136
6.4.2 In vitro lead chelation: fructan-spiked purified tannic acid......139
6.4.3 In vitro chelation observations..................................................143

OBJECTIVE 2
7 LEAD ACCUMULATION STUDIES (II)......................................................145
7.1 Effect of increasing EDTA concentration on lead accumulation.........145
7.2 Effect of 500 μM Pb(NO ) exposure level.........................................147
3 2
8 WHY DOES EDTA REDUCE LEAD ACCUMULATION IN ROOTS?....150
8.1 EDTA pre-treated root segments..........................................................151
8.1.1 Effect of EDTA pre-treatment on lead accumulation level.......151
8.1.2 Effect of EDTA pre-treatment on root appearance...................151
8.2 EDTA pre-treated whole plants............................................................155
8.2.1 Effect of EDTA pre-treatment on lead accumulation level.......155
8.2.2 Effect of EDTA pre-treatment on root appearance...................155