PHYTOCHELATININDUCTION BY TRACE METALS
IN MARINE MICROALGAE
by
Beth A. Ahner
B. S., Civil Engineering
Massachusetts Institute of Technology, 1989
Submitted to the Department of Civil and Environmental Engineering
in Partial Fulfillment of the
Requirements for the Degree of
Doctor of Philosophy
at the
Massachusetts Institute of Technology
September 1994
© 1994 Massachusetts Institute of Technology
All Rights Reserved
Signature of Author ....................
.......................
Department of Civil and Environmental Engineering
Certified by .......................
Professor Frangois M. M. Morel
Thesis Supervisor
Accepted
by ..
.
- . . . --....................
Joseph M. Sussman
Chairman, Departmental Committee on Graduate Studies
- ss
I
b t
9y
Adcr
Ev~'9
gartfr Eno
Co
a,
PHYTOCHELATIN INDUCTION BY TRACE METALS IN MARINE ALGAE
by
BETH A. AHNER
Submitted to the Department of Civil and Environmental Engineering
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy in Civil Engineering
Abstract
Phytochelatins are metal-binding peptides produced enzymatically by higher
plants, fungi and algae in response to many metals, particularly cadmium. Using a very
sensitive HPLC method, I have for the first time quantified phytochelatin concentrations in
laboratory cultures of phytoplankton grown in trace metal defined medium at environmentally relevant metal concentrations and in marine field samples. Phytochelatins are quantified in several marine phytoplankton when exposed to a range of free cadmium ion
concentrations and in Thalassiosira weissflogii, a marine diatom, upon exposure to a
series of trace metals. All the phytoplankton species tested contain phytochelatin even
when there is no added cadmium and elevated phytochelatin concentrations are induced
by cadmium even at very low concentrations. At each cadmium level, the phytochelatin
concentrations measured in most of the species were fairly uniform, and as cadmium
increased, the phytochelatin concentrations also steadily increased as a function of the free
cadmium ion concentration. Within the range of metal concentrations that span those typically found in the marine environment, cadmium, and to a lesser extent copper, are the
most effective inducers of phytochelatins in T.weissflogii; the generality of this result is
confirmed by short term experiments with two other phytoplankton species.
Coastal and open ocean measurements of phytochelatin concentrations (normalized to chlorophyll a) are similar to those measured in laboratory cultures and incubations
of natural seawater samples with added cadmium and copper confirm the induction of the
peptides by these metals. A year long study of phytochelatin concentrations along a
transect from Boston Harbor to Massachusetts Bay reveals decreasing seaward concentrations for most of the year. A comparison of phytochelatin concentrations and copper measurements from several coastal areas confirms the result that phytochelatins are a function
of the free metal rather than total metal. In the Equatorial Pacific, particulate phytochelatin concentrations are similar to those measured in coastal areas.
Given proper calibration and normalization techniques, phytochelatin measurements could become a way to monitor the effects of anthropogenic metal inputs, since
they seem to provide a convenient measure of the metal stress resulting from the complex
mixture of trace metals and chelators in natural waters.
Thesis Supervisor: Dr. Frangois Morel
Title: Professor of Civil and Environmental Engineering
Acknowledgments
I would like to thank everyone who contributed to this thesis either in a scientific
capacity or in some personal way (in most cases both). First I thank my thesis advisor,
Francois Morel, for steadfast guidance throughout my graduate career. In the laboratory as
well as on the squash court he has been a mentor and a friend. I would also like to thank
the other members of my thesis committee who provided fresh new insights throughout
the development of this thesis: Phil Gschwend, Sallie Chisholm, Jim Moffett at Woods
Hole Oceanographic Institute and Neil Price at McGill University. In particular, I want to
thank Neil Price for teaching me nearly all of my laboratory technique and in large part
initiating the topic of my thesis.
I would also like to thank my colleagues at the Parson's Laboratory who helped me
through the years, in particular Jenny Lee and Don Yee. They both made significant contributions to the advancement of this project in the early years and were there for me emotionally and scientifically during the frequent, yet inevitable, ups and downs of my work.
I also owe a great deal of gratitude to my family and to Kirk Sigel, my partner in
life, for the love and patience that has sustained me through the more difficult parts of this
journey. My parents have always been very supportive of my education and without their
help and encouragement I would not be where I am now. Kirk has supported me throughout my graduate education, providing me with computer advise and equipment, but more
importantly providing emotional support upon which I could always depend.
3
Table of Contents
Page
Abstract
........................
Acknowledgments
................
Chapter
1
Introduction.....................
References....................
.................................. 2
.................................. 3
.................................. 9
.. 15
Chapter 2
.. 17
Initial Laboratory and Field Studies.
...................... 18
...............
............... ...................... 18
............... ......................20
............... ......................22
............... ......................28
............... ......................29
Abstract...............
Introduction............
Materials and Methods...
Results and Discussion...
Acknowledgments
.......
References.............
Chapter 3
An Interspecies comparison.
.........................................
31
Abstract...............
......................................... 3 2
Introduction............ .........................................
32
Materials and methods ...
34
.................
.........................................
Results................ .........................................
Discussion.............
Acknowledgments
.......
38
.........................................
47
.........................................50
References............. .........................................
51
Chapter 4
Induction by Various Metals ....
54
Abstract............
................................
55
............
............ ................................
55
Materials and Methods ............
. . . . . . . . . . . . .. . . . . .. . .. . . . . . .56
Results............. ............ ................................
59
Discussion.......... ............ . . . .............................
67
Acknowledgments .... ............ ................................72
References.......... ............ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .7 3
Introduction.........
Chapter 5
Phytochelatins in Coastal Waters
.....................................76
Abstract...................
77
77
Introduction................
4
...
Page
78
Materials and Methods...........
Results........................
86
Discussion.....................
Acknowledgments
...............
References. ....................
..........................
..........................
92
95
..........................
96
Chapter 6
Phytochelatins in the Equatorial Pacific.
...............................97
Abstract............ ..................
...................
98
...................
98
Introduction......... ..................
Materials and Methods ..................
100
...................
100
Results............. ..................
...................
Discussion.......... ..................
108
...................
111
Acknowledgments .... ..................
...................
112
References.......... ..................
Chapter 7
FutureWork.............
.........................................114
References.............
120
Appendix A
Methodological Details of Phytochelatin Analysis.....
5
*000.-·.-.
.121
List of Figures
Page
Chapter
1
Figure 1. Structure of phytochelatin................................
. 11
Chapter 2
Figure 1. Sampling locations in Massachusetts Bay ......................
Figure 2. Phytochelatin concentrations in Thalassiosira weissflogii in
response to cadmium ......................................
Figure 3. Phytochelatin concentrations in transect of Mass. Bay ............
Figure 4. Phytochelatin concentrations over time in addition experiment .....
21
23
25
26
Chapter 3
Figure 1. Typical HPLC chromatograms for phytochelatin analysis .........
Figure 2. Calibration of phytochelatin concentrations ....................
Figure 3. Phytochelatin concentrations in phytoplankton in response to
cadmium
...............................................
Figure 4. Phytoplankton growth rates vs. cadmium in solution .............
Figure 5. Intracellular cadmium vs. concentration in solution ..............
Figure 6. Ratios of phytochelatin to intracellular cadmium ................
37
39
40
41
45
46
Chapter 4
Figure 1. Phytochelatin concentrations in T. weissflogii induced by
manymetals............................................
Figure 2. Short term induction of phytochelatins by many metals ..........
Figure 3. Intracellular metal concentrations in T. weissflogii and the ratios
of phytochelatin to metal..................................
Figure 4. Time evolution of phytochelatin concentrations upon changing
cadmium
exposure.......................................
.60
. 63
. 66
.68
Chapter 5
Figure 1. Phytochelatin concentrations in Mass. Bay plotted as a function of
distance from harbor .....................................
.87
Figure 2. Phytochelatin concentrations (n=2, 3, & 4) in Mass. Bay in
.88
Figure 3. Phytochelatin concentrations in Mass. Bay plotted as a function
of salinity..............................................
.90
Figure 4. Phytochelatin concentrations plotted as a function of copper
October...............................................
concentration. ..........................................
6
.91
Page
Chapter 6
Figure 1. Equatorial Pacific station locations ...........................
Figure 2. Depth profiles of phytochelatin concentrations in the Equatorial
101
Figure 3. Surface nitrate concentrations as a function of latitude............
Figure 4. Depth profiles of nitrate concentrations .......................
Figure 5. Depth profile of phytochelatin concentrations (n= 2, 3 & 4)
at Station 7 .............................................
Figure 6. Phytochelatin measurements after incubations with various metal
104
105
Pacific.................................................102
additions
...............................................
106
107
Appendix A
Figure 1. Example chromatograms...................................
7
123
List of Tables
Page
Chapter 3
Table 1. Marine phytoplankton species surveyed........................
Table 2. Phytochelatin and intracellular metal concentrations in
phytoplankton
............................................
35
42
Chapter 4
Table 1. Speciation of EDTA-complexed metals in Aquil medium ..........
Table 2. Speciation of chloride complexed metals in Aquil medium .........
Table 3. Phytochelatin and intracellular metal concentrations in
T. weissflogii exposed to various metals .......................
Table 4. Phytochelatin concentrations from short term metal addition
57
58
61
experiments
.............................................
64
Table 5. Typical total metal concentrations in coastal waters ..............
70
Chapter 5
Table
Table
Table
Table
1. Sampling dates, times and tidal schedule.......................
2. Boston Harbor- Mass. Bay sampling locations...................
3. Temperature, phytochelatin, and nutrient data for transects.........
4. Phytochelatin and copper measurements from Cape Cod and
Providence Harbor........................................
79
80
81
85
Chapter 7
Table 1. Phytochelatin measurements in marine waters...................
116
Appendix A
Table 1. Amino acid analysis of phytochelatin fractions ..................
8
124
Chapter
1
Introduction
9
Concern over the effect of anthropogenic trace metal inputs into coastal waters has
prompted much scientific investigation into the interaction of these metals with phytoplankton which are the base of the food chain in marine ecosystems. Toxicological studies of all kinds have been done with many metals and a large number of phytoplankton
species. Parameters such as growth and/or assimilation rates (Sunda and Guillard, 1976;
Brand et al., 1986), accumulation factors (Fisher et al., 1984) have been measured to
gauge relative toxicity or availability. These studies have largely established ranges of
metal ion concentrations which are toxic to particular organisms. However, these studies
typically treat the phytoplankton cell as a "black box" and little information about changes
in the internal physiology of the cell has been available.
A first step toward understanding the intracellular mechanisms of response to toxic
metals was the discovery of a specific metal binding peptide-phytochelatin-
first identi-
fied in yeast (Murasugi et al., 1981) and higher plants (Grill et al., 1985; Robinson, 1989;
Rauser, 1990) and then found to be synthesized in a large number of algal species (Gekeler
et al., 1988; Robinson, 1989). Many marine algae have been screened for production of
this peptide and most were found to contain copious amounts when exposed to very high
concentrations of cadmium (Maita and Kawaguchi, 1989; Wikfors et al., 1991). The function of phytochelatin appears to be analogous to that of metallothionein (Grill et al., 1987),
a well studied metal binding protein found in animals. Upon exposure to metals, these
peptides are produced (by gene transcription in the case of metallothionein or an enzymatic reaction in the case of phytochelatin) and they then chelate the intracellular metals
by coordination to the sulfide of the frequent cysteine residues.
Phytochelatin is produced enzymatically by phytochelafin synthase (Grill et al.,
1989) from the precursor glutathione, a tripeptide of glutamate, cysteine and glycine. The
terminal glycine is cleaved and the y-glu-cys is joined to another glutathione to form the
n=2 dimer (Figure 1); additional -glu-cys can be added stepwise to the peptide creating
longer chains (n= n +1). In higher plants the repeating unit has been found to reach n=1l1
(Grill et al., 1985). Phytochelatin synthase has been isolated from cell suspension cultures
of Silene cucubalus and has been fairly well characterized; it is constitutive and requires a
metal for activity. The enzyme activity appears to be self-regulated in that the phytochela-
10
SH
~
~-
H
H
0
1
'I1
C
-N--C
!
N -C
N/C
-
I0
I~~~
COOH
COOH-
-NN-ICOOH
0
I
H
n
n
Phytochelatin
[y-glutamyl-cysteine] n-glycine (n= 2-11)
Figure 1. Structure of phytochelatin (-glutamyl-cysteinyl)n glycine (n=2-11).
11
tin product chelates the metal from the enzyme rendering the enzyme inactive (Loeffler et
al., 1989).
Alternate forms of phytochelatins have been found in a several higher plants which
contain other forms of glutathione; in a legume of the order Fabales homo-phytochelatin
3 -ala] replaces phytochelatin (Grill et al., 1986) and in several species of
[(y-glu-cys)n-P
rice from the family Poaceae, hydroxymethyl-phytochelatin [(Y-glu-cys)n-ser]has been
found in addition to the common phytochelatin peptides (Klapheck et al., 1994). Also
peptides of the structure (y-glu-cys)n (desGly-phytochelatin) have been found in some
yeast (Mehra and Winge, 1988) and plant species (Bernhard and Kagi, 1987), and an additional synthesis mechanism involving the polymerization of y-glu-cys has been described
in vitro (Hayashi et al., 1991).
These numerous laboratory studies lead to the obvious question of whether these
metal binding peptides are produced by plants in the natural environment. Two separate
studies measured phytochelatins in higher plants and stream bed mosses at sites highly
contaminated by mine tailings (Grill et al., 1988; Jackson et al., 1991), but phytochelatin
concentrations were not measurable in the same species outside of the contaminated areas.
Environmental studies have thus been limited by methodological sensitivity.
The objectives of this study were to determine whether marine phytoplankton
made phytochelatins under conditions of moderate to low metal exposure and to examine
the relationship between metal concentrations and the resulting phytochelatin concentrations. The use of trace metal defined medium enabled me to quantify phytochelatins at
extremely low free metal concentrations and ensured that steady state conditions were
maintained throughout exponential growth of the cells. I also wanted to determine
whether phytochelatin was present in natural marine algal populations and whether these
concentrations were controlled by the concentrations of particular metals in the seawater.
To this end I developed a sensitive analytical HPLC technique (detailed in Chapter 3 and
Appendix A) which employs the sulfide specific fluorescent tag, monobromobimane. The
new method has made possible measurements of phytochelatins in phytoplankton cultured
at very low free metal ion concentrations and in natural assemblages of marine algae from
several field sites.
The higher plant literature has established that at high concentrations many metals,
12
such as Cd, Cu, Pb, Zn, Ni, and Hg, can induce phytochelatin production, but that cadmium is the most effective inducer (Grill et al., 1987). Chapter 2 describes phytochelatin
production by a marine diatom, Thalassiosira weissflogii, in response to variable cadmium
concentrations. Intracellular phytochelatin concentrations are dependent on the concentration of cadmium in the medium and even 30 pM inorganic cadmium induces significant
concentrations above what is measured in cell cultured in medium containing no added
cadmium. Field data from Boston Harbor and Massachusetts Bay are introduced in this
same chapter.
Measurements of particulate phytochelatin in natural seawater samples quantify
the integrated exposure of a wide variety of organisms to a large number metals. Does the
amount of phytochelatin reflect exposure to the concentrations of particular metals? To
answer this question, one needs to know whether individual species produce similar
amounts of phytochelatin (normalized to some parameter such as chlorophyll a) in
response to changing metal concentrations and which metals are the most important
inducers of phytochelatin at free metal concentrations found in the marine environment.
I cultured several marine phytoplankton, with the help of undergraduate research
student Shing Kong, in artificial seawater with defined trace metal concentrations and
quantified the steady state phytochelatin concentrations (Chapter 3). Most species produced increasing amount of phytochelatin with increasing cadmium concentrations and
the relative concentrations synthesized by the various species were similar to each other.
In the following chapter (Chapter 4) I characterize the response of primarily one organism,
Thalassiosira weissflogii, to many different metals. At concentrations typical of the
marine environment only cadmium and copper should directly influence phytochelatin
concentrations. All the other metals tested had no effect at low concentrations and only a
few induced production at concentrations higher than are likely to be encountered in the
natural environment.
Measurements of phytochelatin concentrations have been made in coastal and
open ocean phytoplankton populations. Chapter 5 details an examination of seasonal samples from a transect from Boston Harbor to Massachusetts Bay, and of September samples
from several harbors in coastal southeastern New England. The transects reveal a decreasing seaward trend in phytochelatin concentrations, with some interesting secondary fea-
13
tures probably due to the complexation of metals by organic material. Phytochelatin
concentrations measured in samples from the September sampling of various harbors
demonstrate an inverse correlation with the concentration of copper complexing agents.
Phytochelatin concentrations show no relationship to total copper, but are related to measured free cupric ion concentrations and this relationship is quite similar to that obtained
for laboratory cultures.
Open ocean samples consist of depth profiles from the Equatorial Pacific along a
south to north transect across the Equatorial upwelling. The phytochelatin concentrations
measured in this area are very similar to those measured in the coastal samples. There are
not large differences between profiles in and out of the upwelling zone, but most profiles
have a subsurface maximum in phytochelatin concentrations. Addition of cadmium to
incubation experiments yielded increased concentrations of phytochelatins over controls
at all stations tested, but copper additions did not stimulate production in samples collected outside the upwelling zone, suggesting the presence of biogenic chelating agents in
the water advected from the upwelling zone.
The phytochelatin concentrations measured in these field samples reflect exposure
of a mixed community of phytoplankton to metals, primarily cadmium and copper. It is
likely that phytochelatin measurements could be used in conjunction with other monitoring techniques to evaluate the exposure of phytoplankton to these metals.
14
References
Bernhard, W. R. and H. R. Kigi. 1987. Purification and characterization of atypical cadmium-binding polypeptides from Zea mays. Experientia. 52 (Suppl.): 309-315.
Brand, L., W. G. Sunda and R. R. L. Guillard. 1986. Reduction of marine phytoplankton
reproduction rates by copper and cadmium. J. Exp. Mar. Biol. Ecol. 96: 225-250.
Fisher, N. S., M. Bohd and J.-L. Teyssi6. 1984. Accumulation and toxicity of Cd, Zn, Ag,
and Hg in four marine phytoplankters. Mar. Ecol. Prog. Ser. 18: 201-213.
Gekeler, W., E. Grill, E.-L. Wmnacker and M. H. Zenk. 1988. Algae sequester heavy metals via synthesis of phytochelatin complexes. Arch. Microbiol. 150: 197-202.
Grill, E., W. Gekeler, E.-L. Winnacker and M. H. Zenk. 1986. Homo-phytochelatins are
heavy metal-binding peptides of homo-glutathione containing Fabales. FEBS Lett. 205:
47-50.
Grill, E., S. Loeffler, E.-L. Winnacker and M. H. Zenk. 1989. Phytochelatins, the heavy-
metal-binding peptides of plants, are synthesized from glutathione by a specific yglutamylcysteine dipeptide transpeptidase (phytochelatin synthase). Proc. Natd.Acad. Sci.
U.S.A. 86: 6838-6842.
Grill, E., E.-L. Winnacker and M. H. Zenk. 1985. Phytochelatins: the principal heavymetal complexing peptides of higher plants. Science. 230: 674-676.
Grill, E., E.-L. Wminnacker
and M. H. Zenk. 1987. Phytochelatins, a class of heavy-metalbinding peptides from plants, are functionally analogous to metallothioneins. Proc. Natl.
Acad. Sci. U.S.A. 84: 439-443.
Grill, E., E.-L. Winnacker and M. H. Zenk. 1988. Occurrence of heavy metal binding phytochelatins in plants growing in a mining refuse area. Experientia. 44: 539-540.
Hayashi, Y., C. W. Nakagawa, N. Mutoh, M. Isobe and T. Goto. 1991. Two pathways in
the biosynthesis of cadystins (yEC)nGin the cell-free extract system of the fission yeast.
Biochem. Cell. Biol. 66: 288-295.
Jackson, P. P., N. J. Robinson and B. A. Whitton. 1991. Low molecular weight metal complexes in the fresh water moss Rhynchostegium riparioides exposed to elevated concentrations of Zn, Cu, Cd, and Pb in the laboratory and field. Environ. Exper. Bot. 31: 359-366.
Klapheck, S., W. Fliegner and I. Zimmer. 1994. Hydroxymethyl-phytochelatins
15
[(y-
glutamylcysteine)n-serine] are the metal-induced peptides of the Poaceae. Plant Phys. 104:
1325-1332.
Loeffler, S., A. Hochberger, E. Grill, E.-L. Winnacker and M. H. Zenk. 1989. Termination
of the phytochelatin synthase reaction through sequestration of heavy metals by the reaction product. FEBS Lett. 258: 42-46.
Maita, Y. and S. Kawaguchi. 1989. Amino acid composition of cadmium-binding protein
induced in a marine diatom, Phaeodactylum tricornutum. Bull. Environ. Contain. Toxicol.
43: 394-401.
Mehra, R. K. and D. R. Winge. 1988. Cu(I) binding to the Schizosaccharomycespombe yglutamyl peptides varying in chain lengths. Arch. Biochem. Biophys. 265: 381-389.
Murasugi, A., C. Wada and Y. Hayashi. 1981. Cadmium-binding peptide induced in fission
yeast, Schizosaccharomyces pombe. J. Biochem. 90: 1561-1564.
Rauser, W. E. 1990. Phytochelatins. Annu. Rev. Biochem. 59: 61-86.
Robinson, N. J. 1989. Algal metallothioneins: secondary metabolites and proteins. J. Appl.
Phychol. 1: 5-18.
Robinson, N. J. 1989. Metal-binding polypeptides in plants. p. 195-214. In A. J. Shaw
[eds.], Heavy metal tolerance in plants: Evolutionary aspects. CRC Press.
Sunda, W. G. and R. R. L. Guillard. 1976. Relationship between cupric ion activity and the
toxicity of copper to phytoplankton. J. Mar. Res. 34: 511-529.
Wikfors, G. H., A. Neeman and P. J. Jackson. 1991. Cadmium-binding polypeptides in
microalgal strains with laboratory-induced cadmium tolerance. Mar. Ecol. Prog. Ser. 79:
163-170.
16
Chapter 2
Initial Laboratory and Field Studies
In press: Proceedings of the National Academy of Science
Co-authors:Neil M. Price
Frangois M. M. Morel
17
Abstract
Phytochelatins are small metal-binding polypeptides synthesized by algae in
response to high metal concentrations. Using a very sensitive HPLC method, we have for
the first time quantified phytochelatins from phytoplankton in laboratory cultures at environmentally relevant metal concentrations and in marine field samples. Intracellular concentrations of phytochelatin, in the diatom Thalassiosira weissflogii, exhibit a distinct
dose response with free cadmium ion concentration in the medium mium -
not with total cad-
and are detectable even when [Cd+ 2] is less than 10-12M. In Massachusetts Bay,
phytochelatin levels (normalized to chlorophyll a) in the particulate fraction are similar to
those measured in laboratory cultures exposed to picomolar [Cd+2 ] and exhibit a decreasing seaward trend. Incubations of natural samples with added cadmium confirmed the
induction of these peptides by this metal. Ambient phytochelatin concentrations thus
appear to provide a measure of the metal stress resulting from the complex mixture of
trace metals and chelators in natural waters.
Introduction
First identified in fission yeast (Murasugi et al., 1981), phytochelatins have now
been found to be ubiquitous in plants, algae and many fungi (Grill et al., 1985; Grill et al.,
1987; Robinson, 1989; Rauser, 1990). Like metallothioneins- the primary metal-binding
peptides in animals and prokaryotic organisms- they detoxify intracellular metals by
binding them through a thiolate coordination. Phytochelatins are enzymatic products
(Grill et al., 1989) with the amino acid composition (-Glu -Cys)n-Gly,n= 2-11 (Grill et
al., 1985). The enzyme, phytochelatin synthase, is activated in vitro in the presence of
metals and deactivated when the metals have been bound by the newly synthesized phytochelatins (Grill et al., 1989; Loeffler et al., 1989). Phytochelatin synthase adds the y-GluCys from glutathione, (-Glu -Cys)-Gly, to another glutathione (to make n=2) or to
another phytochelatin chain to increase the repeating unit from n to n+l. Longer chain
18
lengths bind metals more tightly in vitro (Loeffler et al., 1989), but their cellular roles are
otherwise unknown.
On the basis of the laboratory data, plants growing in areas contaminated by metals
are expected to contain phytochelatins. The presence of these peptides may serve as a bioindicator of metal exposure (Robinson, 1989). Indeed two field measurements have documented elevated phytochelatin concentrations in plants growing near mine tailings, areas
of extreme metal pollution (Grill et al., 1988; Jackson et al., 1991).
Extrapolating laboratory data on phytochelatin production by phytoplankton in
culture to natural waters is not straightforward. All laboratory experiments to date have
been performed at very high metal concentrations (20- 1000 pM) (Gekeler et al., 1988;
Wikfors et al., 1991). Such concentrations are 10,000 to 100,000 times greater than the
total dissolved concentrations of metals in surface seawater (e.g. Cd= 2-4 x 10-12M and
Cu= 0.5 x 10-9M (Coale and Bruland, 1988; Bruland, 1992)) including those near sewage
outfalls (Wallace et al., 1988). Even control cultures, to which metals are not deliberately
added, may contain higher metal concentrations than seawater, because of metal impurities in the chemical reagents used to prepare media and in the culture flasks. The problem
is further complicated by the presence of unidentified natural chelating agents in coastal
(Nimmo et al., 1989) and oceanic waters (Coale and Bruland, 1988; Bruland, 1989; Moffett et al., 1990; Bruland, 1992) which may dramatically alter the chemical speciation of
the metal and hence its biologically availability. The biological effects of metals on phytoplankton are known to depend usually on the free rather than on the total metal concentrations (Sunda and Guillard, 1976; Morel and Hering, 1993), but this has not been studied
with regard to phytochelatin induction. Thus the complexity of the metal chemistry in natural waters precludes a simple extrapolation of laboratory data and direct field measurements are needed to demonstrate the existence of phytochelatin in natural phytoplankton
populations.
In this study we use a new analytical measurement technique for phytochelatins to
establish the phytochelatin response of phytoplankton in culture at environmentally relevant cadmium concentrations. We also demonstrate that the response is indeed controlled
by the free rather than the total cadmium. Finally we present data on phytochelatin concentrations in natural samples from Boston Harbor and Massachusetts Bay which exhibit a
19
systematic decreasing seaward trend, away from metal sources in the harbor.
Materials and Methods
For laboratory experiments, the marine diatom Thalassiosira weissflogii, clone
Actin, was cultured in the chemically defined artificial seawater medium Aquil (Price et
al., 1991) at several cadmium concentrations. Medium is treated with Chelex-100 to
remove trace metal impurities and the background total cadmium concentration is estimated to be < 1 nM, equivalent to [Cd+2 ] < 10-13 M. Cadmium concentrations in the
medium were adjusted by equimolar cadmium-EDTAadditions as discussed in Price et al
(1991).The resulting free cadmium ion concentrations were calculated with the thermodynamic equilibrium model MINEQL (Westall et al., 1976). Cells were grown at a constant
20° C with continuous light at 120 BE m 2 s-1 in acid-cleaned polycarbonate bottles.
Growth was monitored daily with a CoulterR Counter, and rates calculated from the
regression lines of log cell concentration vs. time. Late exponential cells (4.0x104 cells/
ml, 500 mls) were harvested by gentle filtration (< 5 psi) onto Whatman GFIF filters and
stored in liquid nitrogen.
Harvested cells were placed directly from storage into 10mM methanesulfonic
acid at 70° C for 2 minutes to denature proteases. This mixture was then homogenized
with a Wheaton teflon pestle and glass grinding tube on ice. The homogenate was then
centrifuged in a Beckman Microfuge ETM for 10 minutes. Supernatant was retained for
derivatization with the fluorescent tag monobromobimane (Newton et al., 1981; Steffens
et al., 1986) and separation with ion-pair reverse phase chromatography. Analyses were
performed on a Beckman HPLC equipped with a Gilson 121 fluorometer and an Alltech
(2.1 x 250mm) C-18 5g reverse-phase column using an acetonitrile gradient (from 18 to
90%) buffered at pH=4.2 by 42mM acetic acid with 0.64 mM of the ion pairing reagent
tetraoctyl ammonium bromide. The system was optimized for maximum sensitivity by
adjusting the particular ion-pair employed, the column diameter and the sample injection
loop size (20011l). The detection limit is -04).3picomoles per injection.
For field experiments, samples of surface seawater (see map of Figure 1 for location) were dispensed into acid-cleaned polycarbonate bottles. The particulate fraction was
20
42 ° 31']
w
42 ° 24']
42 ° 17']
42 ° 10']
71° 04'W
70° 55'W
70 ° 46'W
70° 37'W
70° 28'W
Figure 1. Map of Boston Harbor and Massachusetts Bay. Sampling stations indicated
by darkened circles; arrow shows sampling location for incubation experiment.
21
collected by gentle filtration (< 5 psi) onto 25mm Whatman GF/F filters. Chlorophyll
samples were processed as described by Parsons et al (1984) and were measured on a
Turner Designs fluorometer. Samples for phytochelatin analysis were treated like the laboratory phytoplankton samples, the only difference being the use of an Alltech Adsorbosphere HS C-18 3g (2.1 x 250mm) reverse-phase column.
Incubation experiments were performed on water collected from the University of
Massachusetts Boston pier (arrow on map of Fig. 1). Water was collected into 10- 2 liter
acid cleaned polycarbonate bottles using an acid cleaned teflon line and a battery operated
MasterflexR peristaltic pump. Incubations were done in a 10° C refrigerator with attenuated light. Aliquots were sampled from the duplicate bottles at 6, 18 and 42 hours.
Results and Discussion
Over a range of free cadmium ion concentrations, from no added cadmium to
lnM, phytochelatins exhibited a distinct dose response (Fig. 2). Each of the oligomers
(n= 2, 3, 4) increased systematically with increasing metal concentrations, the dimer being
dominant in all culture extracts. Internal phytochelatin levels varied by two orders of
magnitude over the range employed, while the growth rate of the culture remained practically unchanged. It is notable that even with no added cadmium and at natural (non-polluted) cadmium concentrations, [Cd 2+] < 10-12M (Bruland, 1992), phytochelatin was
present in significant amounts in the cells, as noted previously by Price and Morel (1990).
This suggests the possibility that, besides detoxification, phytochelatins may serve in
intracellular metal homeostasis as hypothesized by several previous researchers (Grill et
al., 1985; Robinson, 1989; Rauser, 1990). In our experiments so far with other metals, we
have found that Cu, Pb and Ni at free concentrations of 10-9 M or less, also induce phytochelatin in Thalassiosira weissflogii, but not to the extent that cadmium does (Chapter 4).
Many studies of the effects of trace metals on phytoplankton have established that
the toxicity of a metal is dependent on its free ion concentration rather than its total concentration (Sunda and Guillard, 1976; Morel and Hering, 1993). This is also true of phytochelatin production as demonstrated by the filled data points included in Figure 2. These
data represent cultures that contained a tenfold lower total cadmium concentration than
22
600
1500
.C
r=-I
S
a)
C)
o
480
To
0
"--
S
-~
1200
J
360
0
0
_P
rc
600
240
c
0
c
C.)
00
.-J
000 ,
120
300
C
None
12.0
11.0
10.0
9.0
pCd= -log[Cd 2+]
Figure 2. Phytochelatin concentrations in Thalassiosira weissflogii (TW) at varying
free cadmium ion concentrations (pCd = -log [Cd+2 ]); the various chain lengths are
labelled (circle), n=2; (upside down triangle), n=3; (square) n=4. Filled symbols correspond to measurements from cultures in which the total cadmium was ten-fold lower
and the EDTA concentration adjusted to obtain the same pCd. Data points representing no added cadmium and pCd = 12.0 cultures were not duplicated. All other points
are the average of duplicates, error bars being within the symbol for several data
points. Right vertical axis is calculated assuming an average of -6x10 ' 12 grams chl a
per cell. Inset shows detail of n=2 and n=3 values for the three lowest cadmium concentrations. Growth rates were 2.3, 2.6, 2.6, 2.6, & 2.3 doublings per day, in order of
increasing pCd's.
23
the others, but achieved the same free cadmium by lowering proportionally the EDTA in
the medium. All other total metals were adjusted accordingly to maintain their free ion
concentrations constant. The resulting phytochelatin levels are identical to those obtained
at higher total cadmium but same free ion concentrations.
To test the production of phytochelatin in natural phytoplankton populations we
collected samples from Massachusetts Bay in February, April and June of 1993 (see map
Figure 1). Concentrations of the phytochelatin dimer from -2 to 25 pmol (g chl a)-1 were
measured in these samples, values similar to those observed in our laboratory cultures. As
might be expected, the highest values were found near the harbor, which receives sewage
and riverine inputs, and decreased as we sampled farther from anthropogenic sources of
metals (Figure 3). While they are fairly noisy, the three sets of field data are consistent
with each other and the decreasing seaward trend is unmistakable.
To demonstrate that the elevated phytochelatin concentrations in the natural phytoplankton populations evince exposure to metals, we incubated a water sample from Boston Harbor, presumably rich in uncharacterized metal-binding organic material, with
added cadmium. Addition of 100 nM CdC12 promoted high, though variable, phytochelatin concentrations in two separate samples (Figure 4). A sample amended with 5 nM
CdC12 remained indistinguishable from the control, thus indicating, by comparison to the
laboratory data, that less than one picomole (10-12moles) of the cadmium remained
uncomplexed. Addition of 100 tM EDTA resulted in a small but perceptible decrease in
the algal phytochelatin concentrations (from 1.5 to 0.8 gmol (g chl a)-l).
Overall, our laboratory and field data exhibit remarkable consistency and support
the notion that phytochelatins may be a good quantitative indicator of metal exposure.
The similarity between the values obtained in cultures and in natural samples is almost
surprising in view of the crudeness of the normalization to chlorophyll a concentrations.
The agreement among the three sets of field data shows that the gradient in intracellular
phytochelatin concentrations maybe a permanent oceanographic feature in Massachusetts
Bay, presumably reflecting a gradient in metal availability. Clearly one expects the position of peak concentrations to be dependent on the tides - which dominate the currents
and the mixing regime in the bay and were similar for the three sampling episodes.
Because different organisms may respond differently to various metals, it is not possible at
24
25
February 1993
T
o
0
f
3
20
40
April 1993
E
I
I2
i
s
~S.1 ~if
10
20
A
#G
is
M
A.
4M
I
30
40
June 1993
0
i
*#
8
I,S
:..S
no
0
1C
c
20
30
40
Km from Black Falcon Pier (Inner Boston Harbor)
Figure 3. Phytochelatin concentrations normalized to chlorophyll a in particulate samples collected on February 23, April 6 and June 22, 1993 from a seaward transect of
Massachusetts Bay plotted as a function of distance from inner Boston Harbor; data
points are the average of duplicates and where not indicated the error bars are within
the symbol. See Figure 1 for actual location of stations. Points at zero are below the
detection limit and the open circle indicates a sample (the duplicate of which was
below detection) in which a low phytochelatin measurement was divided by a very
low chlorophyll a value.
25
0
54
48
42
36
I
I
I
I
I
I
v
V
+ 100nM Cd
30 f1J,
l_
E
0
/'
/
, /
'V."
10.0
I
V
-cc'
7.5
.v
I'0
5.0
X~~
V
2.5
'9DA
8
+
0.0
!
I
0
10
20
30
EDTA~~~~C
40
50
Time (hours)
Figure 4. Phytochelatin concentrations over time in amended natural seawater samples. Open circles, initial; filled circles, 5nM CdC12 ; inverted triangles, lOOnMCdC12 ;
inverted filled triangles, controls; open squares, lOO1MEDTA. Incubation experiments were performed on water collected from the U. Mass. Boston pier (arrow on
map, Figure 1) on May 12, 1993.
26
this time to say what metals were responsible for the observed phytochelatin gradient
Nonetheless the similarity in the profiles over the seasons (winter to early summer) when
the flora was dominated by different species (Townsend et al., 1990) supports the notion
that the phytochelatin response is sufficiently general to provide an overall measure of
metal exposure, possibly of exposure to a particular metal such as cadmium.
Our measurements of phytochelatin concentrations in phytoplankton cultures at
background trace metal concentrations provide a basis to speculate on the extent to which
these algal chelators may be a significant source of dissolved complexing agents in seawater. For example, if we consider an open ocean chlorophyll a concentration of 0.1 Rg per
liter, a phytochelatin concentration of -2 grmol(g chl a)-1 , and a turnover time of the phytoplankton of -1 day (assuming all of the phytochelatin to be released to the medium) we
can calculate an extracellular turnover time of about 100 days to maintain a concentration
of 20 pM in the water column. Phytochelatins, which are subject to oxidation of the thiols
and breakdown by proteases, would thus seem unlikely to constitute a major fraction of
the mysterious chelators measured by several researchers (Coale and Bruland, 1988; Bruland, 1989; Moffett et al., 1990; Bruland, 1992).
While much additional laboratory and field work at relevant metal concentrations
remains needs to be done to elucidate their exact cause, variations in phytochelatin concentration are measurable in field samples and in phytoplankton at environmentally relevant metal concentrations. Thus phytochelatins appear to provide a measure of the short
term response of the algae to metal stress as imparted by the complex mixture of trace
metals and chelating agents in their external milieu. More importantly, perhaps, the study
of phytochelatin production in situ provides a mechanistic insight into the physiological
response of the phytoplankton and may allow us to causally link increases in metal concentrations to their biological and ecological consequences.
27
Acknowledgments
We thank R. Mason, J. Gawel, J. Lee and P. Gschwend for reviewing this manuscript and the Massachusetts Water Resource Authority for providing logistical support.
Financial support for this work was provided by grants from the National Science Foundation, the Office of Naval Research, the Environmental Protection Agency, the National
Oceanic and Atmospheric Administration, and the Massachusetts Water Resource Authority.
28
References
Bruland, K. W. 1989. Complexation of zinc by natural organic ligands in the central North
Pacific. Limnol. Oceanogr. 34: 269-285.
Bruland, K. W. 1992. Complexation of cadmium by natural organic ligands in the Central
North Pacific. Limnol. Oceanogr. 37: 1008-1017.
Coale, K. H. and K. W. Bruland. 1988. Copper complexation in the Northeast Pacific.
Limnol. Oceanogr. 33: 1084-1101.
Gekeler, W., E. Grill, E.-L. Winnacker and M. H. Zenk. 1988. Algae sequester heavy metals via synthesis of phytochelatin complexes. Arch. Microbiol. 150: 197-202.
Grill, E., S. Loeffler, E.-L. Winmnacker
and M. H. Zenk. 1989. Phytochelatins, the heavymetal-binding peptides of plants, are synthesized from glutathione by a specific yglutamylcysteine dipeptide transpeptidase (phytochelatin synthase). Proc. Natl. Acad. Sci.
U.S.A. 86: 6838-6842.
Grill, E., E.-L. Winnacker and M. H. Zenk. 1985. Phytochelatins: the principal heavymetal complexing peptides of higher plants. Science. 230: 674-676.
Grill, E., E.-L. Winnacker and M. H. Zenk. 1987. Phytochelatins, a class of heavy-metalbinding peptides from plants, are functionally analogous to metallothioneins. Proc. Natl.
Acad. Sci. U.S.A. 84: 439-443.
Grill, E., E.-L. Winnacker and M. H. Zenk. 1988. Occurrence of heavy metal binding phytochelatins in plants growing in a mining refuse area. Experientia. 44: 539-540.
Jackson, P. P., N. J. Robinson and B. A. Whitton. 1991. Low molecular weight metal complexes in the fresh water moss Rhynchostegium riparioides exposed to elevated concentrations of Zn, Cu, Cd, and Pb in the laboratory and field. Environ. Exper. Bot. 31: 359-366.
Loeffler, S., A. Hochberger, E. Grill, E.-L. Winnacker and M. H. Zenk. 1989. Termination
of the phytochelatin synthase reaction through sequestration of heavy metals by the reaction product. FEBS Lett. 258: 42-46.
Moffett, J. W., R. G. Zika and L. E. Brand. 1990. Distribution and potential sources and
sinks of copper chelators in the Sargasso Sea. Deep-Sea Res. 37: 27-36.
Morel, F. M. M. and J. G. Hering. 1993. Principles and Applications of Aquatic Chemistry.
John Wiley and Sons Inc.
29
Murasugi, A., C. Wada and Y Hayashi. 1981. Cadmium-binding peptide induced in fission
yeast, Schizosaccharomyces pombe. J. Biochem. 90: 1561-1564.
Newton, G. L., R. Dorian and R. C. Fahey. 1981.Analysis of biological thiols: Derivatization with monobromobimane and separation by reverse-phase high-performance liquid
chromatography. Anal. Biochem. 114: 383-387.
Nimmo, M., C. M. G. van den Berg and J. Brown. 1989. The chemical speciation of dissolved nickel, copper, vanadium, and iron in Liverpool Bay, Irish Sea. Estuarine Coastal
Shelf Science. 29: 57-74.
Parsons, T. R., Y. Maita and C. M. Lalli. 1984. A Manual of Chemical and Biological
Methods for Seawater Analysis. Pergamon Press.
Price, N. M., and others. 1991. Preparation and chemistry of the artificial algal culture
medium Aquil. Biolog. Oceanogr. 6: 443-461.
Price, N. M. and F. M. M. Morel. 1990. Cadmium and cobalt substitution for zinc in a
marine diatom. Nature. 344: 658-660.
Rauser, W. E. 1990. Phytochelatins. Annu. Rev. Biochem. 59: 61-86.
Robinson, N. J. 1989. Algal metallothioneins: secondary metabolites and proteins. J. Appl.
Phychol. 1: 5-18.
Steffens, J. C., D. F. Hunt and B. G. Williams. 1986. Accumulation of non-protein metal-
binding polypeptides (y-glutamyl-cysteinyl)n-glycinein selected cadmium-resistant
tomato cells. J. Biol. Chem. 261: 13879-13882.
Sunda, W. G. and R. R. L. Guillard. 1976. Relationship between cupric ion activity and the
toxicity of copper to phytoplankton. J. Mar. Res. 34: 511-529.
Townsend, D. W., and others. 1990. Seasonality of oceanographic conditions in Massachusetts Bay, Technical Report No. 83. Bigelow Laboratory for Ocean Sciences, West
Boothbay Harbor, Maine.
Wallace, G. T., J. H. Waugh and K. A. Gamrner.1988. Metal distribution in a major urban
estuary (Boston Harbor) impacted by ocean disposal. p. 67-78. In D. A. Wolfe and T. P.
O'Connor eds.], Urban wastes in coastal marine environments. Robert E. Kreiger Publishing Co.
Westall, J. C., J. L. Zachary and F. M. M. Morel. 1976. MINEQL: A Computer Program
for the Calculation of Chemical Equilibrium Composition of Aqueous Systems. R. M. Parsons Lab., Mass. Inst. Technol.
Wikfors, G. H., A. Neeman and P. J. Jackson. 1991. Cadmium-binding polypeptides in
microalgal strains with laboratory-induced cadmium tolerance. Mar. Ecol. Prog. Ser. 79:
163-170.
30
Chapter 3
An Interspecies comparison
Submitted to: Limnology and Oceanography
Co-authors: Shing Kong
Francois M. M. Morel
31
Abstract
Phytochelatins are metal-binding peptides produced enzymatically by higher
plants, fungi and algae in response to many metals, particularly cadmium. We have studied phytochelatin production in several marine phytoplankton exposed to a range of free
cadmium ion concentrations. As a result of increased analytical resolution, we have found
that all the species contain phytochelatin even when there is no added cadmium and that
elevated phytochelatin concentrations are induced by cadmium even at very low and environmentally relevant concentrations (as low as 10-12M free ion concentration). In some
but not all species intracellular cadmium and phytochelatin concentrations are maintained
at a fixed stoichiometric ratio at high cadmium concentrations. Phytochelatin production
and accumulation appear to be regulated in a manner that varies among phytoplankton
species.
Introduction
First identified in fission yeast (Murasugi et al., 1981), phytochelatins have been
shown to be the major intracellular metal binding peptides of plants, algae and some fungi.
While the presence of phytochelatins in phytoplankton exposed to very high metal concentrations is well established, little is known regarding the physiological control of algal
phytochelatins particularly at realistic environmental metal concentrations. Recently we
have measured phytochelatins in the ambient algal population in Massachusetts Bay and
observed a decreasing trend from the Inner Boston Harbor to Massachusetts Bay (Chapter
2 & 5). In this paper we examine the production of phytochelatins in cultures of several
species of marine phytoplankton with varying dissolved cadmium concentrations. In the
next chapter, we study the phytochelatin response of (chiefly) one organism-Thalassiosira weissflogii -
exposed to a variety of metals.
Phytochelatins are polypeptides with the amino acid structure ( -glu-cys)n-gly
where n ranges from 2 to 11 (Grill et al., 1985). The gamma linkage of the glutamate and
32
cysteine residues involves the carboxyl of the functional chain of glutamate and the amino
group of the cysteine. Phytochelatin production is enhanced when organisms are exposed
to high concentrations of a wide range of metals (Grill et al., 1985). An enzyme, phytochelatin synthase, isolated from a higher plant, catalyzes phytochelatin formation in vitro
(Grill et al., 1989). The enzyme cleaves the terminal glycine from one glutathione (-glucys)-gly and joins the -glu-cys to the amino terminus of another glutathione to form the
n=2 oligomer, the chain can be lengthened stepwise by the joining of additional -glu-cys
moieties to the phytochelatin peptide chain. Phytochelatin synthase requires a metal ion
for activity, and several metals have been shown, at various concentrations, to restore
activity to the apoenzyme in vitro (Grill et al., 1989); cadmium was found to be the most
effective at restoring the activity of the enzyme. The enzymatically produced phytochelatin, (or artificial chelating agents such as EDTA) can turn the enzyme off by chelating the
metal (Loeffier et al., 1989).
Phytochelatins are thought to be the functional analog of metallothioneins- the
primary metal-binding protein in animal and prokaryotic cells. Like metallothionein, they
chelate metals through coordination with the reduced sulfur in cysteine. Reports of sulfur
to metal ratios in the isolated complexes range from 2 to 4. Strasdeit et al (1991) used
EXAFS spectroscopy on phytochelatin complexes isolated from a tobacco plant and demonstrated that each cadmium ion was coordinated by four cysteine sulfurs. Thus if there
are primarily n=2 or n=3 oligomers, more than one peptide must chelate a single metal
ion. Acid labile sulfide, as well as sulfite, has been found to be associated with some
metal-phytochelatins complexes and to increase stability of the metal-peptide cluster
(Murasugi et al., 1983; Steffens et al., 1986; Weber et al., 1987). It has been suggested
that phytochelatin may serve as a temporary carrier of reduced sulfur in the sulfur incorporation pathway (Steffens et al., 1986).
Previous studies with algal cultures have established that phytoplankton produce
phytochelatins when exposed to high metal concentrations. Using analytical refinements,
which permit us to measure very low concentrations of phytochelatins, we examine here
the response of eight species of marine phytoplankton, covering most major taxa, when
exposed to several cadmium concentrations over the range encountered in seawater.
33
Materials and methods
Culturing conditions and medium - All algal species (Table 1) were obtained from
the Culture Collection of Marine Phytoplankton, Bigelow Laboratories for Ocean Sciences. The chemically defined artificial seawater medium AQUIL (Price et al., 1991) was
employed throughout the study. Cadmium concentrations in the medium were varied by a
separate addition of the Cd-EDTA complex. The resulting free metal ion concentrations
(pMe= -log Me+ 2 ) were calculated with the thermodynamic equilibrium model MINEQL
(Westall et al., 1976). In all experiments, total metal and EDTA concentrations were
adjusted to maintain the pMe's of all the other metals in solution at their normal AQUIL
values. Cells were grown at a constant temperature of 20 ° C with continuous light at 120
gE m 2 s 1 in acid-cleaned polycarbonate bottles. Cell density was monitored daily with a
CoulterR Counter or with in vivo fluorescence on a Turner Designs filter fluorometer, and
rates calculated from the regression lines of log cell concentration vs. time or log fluorescence vs. time. Cellular volumes were also measured with the CoulterR Counter. Late
exponential cells were harvested by gentle filtration (with vacuum pressures < 5 psi) onto
Whatman GF/F filters and stored in liquid nitrogen. Small volumes were also collected
for chl a analysis and were treated as outlined in Parsons et al (1984).
Cell preparation and derivatization- Cells and filter were placed directly from liquid nitrogen into 70 ° C acid (10mM methanesulfonic acid) for two minutes to denature
large proteins and enzymes which may break-down phytochelatin in vitro. The mixture
was then homogenized on ice with a tissue grinder and a Wheaton teflon pestle and glass
grinding tube. The homogenate was then transferred to Eppendorf centrifuge tubes and
centrifuged for 10 minutes in a Beckman Microfuge ET M to pellet filter and cell fragments. The supernatant was retained for reaction with the fluorescent tag.
Phytochelatin concentrations were quantified via a sulfur specific fluorescent
tag- monobromobimane (mBrB) (Molecular Probes). Reaction conditions were largely
modelled after the method of Newton et al (1981) with several modifications. The supernatant, prepared as described above, was reacted with excess dithiothreitol (DTT) for 10
minutes to reduce disulfides formed while preparing the cell homogenate. The mixture
was then reacted with the monobromobimane as described (Newton et al., 1981); after 10
34
7""
0
I-
-4
"t
\
o
Ca
I-
c
*N
\N
o-
C1
U)
U)
\0
o
o
Cs4 9
9~
C
N
'
M
o~
en
_
U)
0
I
,}
IDO
in
M
tn
tn
U)
U
I=
00
u;
%6
en
in
0
I"
91
U)
0
U U
.
Q
0
M
U
M
-
U
:F
u
91
0;
M
tn
M
M
00
Z
C1
a
U
U
U
U5
U
U)
u
U)
C4
=
0
C
U
U
>-
U)
U,
o
0U
0©
._
U,
C
U)
U,
0
*-
u
v?
*.
4-.
4 .
_-C
u
U)
U)
4-
C
C.
C4 J
o
C
I-
V.
-0
C
6
V 4-C
U~~~~
--.
En U
m
to
U,
. V U)0
U)
C
U,
0
4h
-
0
Z
rz
o
6. .
I
.e
U)
z
U)
U)
'Z
IIt
.%
-
I)
6t
t
Z
-Z.
R
Q
I,
4
35
Z
to
E.,
lqll
minutes excess cysteine was added to react the remaining mBrB which elutes near the n=2
oligomer. The cys-mBrB peak is the first large off-scale peak in Figure la. The sample
was then acidified with 1M acetic acid to stabilize the reaction mixture.
HPLC Chromatography- Chromatography was performed on a Beckman HPLC
equipped with a Gilson 121 filter fluorometer (310-410 nM excitation, 475-650 nM emission). An Alltech Adsorbosphere HS C-18 3g reverse-phase column (2.1 x 250mm) was
employed with an acetonitrile gradient (from 18 to 90%) buffered at pH = 4.2 by 42 mM
acetic acid with 0.64 mM of the ion pairing reagent tetraoctyl ammonium bromide. Phytochelatins have previously been quantified with monobromobimane (Steffens et al., 1986;
Kneer et al., 1992). Our chromatographic method uses an ionpair to separate phytochelatin from interferences. Monobromobimane reacts non-specifically with many nucleophiles (including water), as well as with any sulfide containing cellular constituents or
added reducing agent DfT creating many interfering reagent peaks (Newton et al., 1981).
The carboxyl groups of the phytochelatin are charged at the pH of the running buffer and
thus interact with the positively charged ammonium ion of the ionpair, separating the phytochelatin from most of the reagent peaks and the large peaks of the DTT that elute chiefly
from 0 to 25 minutes (Figure la).
Typical chromatograms for a blank (reagents only), a synthetic n=2 standard, and a
cell extract from 50-80 minutes are shown in Figure lb. The n=2 oligomer elutes roughly
at 53 minutes, the n=3 at 70 minutes and the n=4 at 73 minutes. The standard mixture has
some impurities, thus small stray peaks occur such as the one in between the two reagent
peaks present in the blank. The elution of the n=2 oligomer and the two reagent peaks
occurs slightly earlier in the cell extract chromatogram; this is due to the large amount of
organic material present in the cell extract sample. Coelution of the synthesized standards
(n= 2, 3 &4) with the phytochelatin peaks in the cell extract, as well as amino acid analysis
of collected peak fractions confirmed the identity of the peaks as phytochelatins. Amino
acid analysis (acid hydrolysis followed by phenylisothiocyanate pre-column derivatization, data shown in Appendix A) and synthesis of the phytochelatin standards were performed at the MIT Biopolymers Laboratory.
Calibration of peak area to concentration was done with the synthetic standard
n=2; a linear relationship holds for a wide range of concentrations (from 1 to 400 pico-
36
A.
1.0
U
4 ton
_
\
1 UU
0.8
80 .*
.-
U
0
0.6
60
40
0.4
c
C.)
a
0
a'-
20
C
0.2
20
0.0
00
L
O_
('
0
20
40
60
80
Time (minutes)
B.
0.6
0.4
0.2
0.0
U
0.6
0
1!
1
0.4
0.2
11
0.0
0.6
0.4
0.2
0.0
50
60
70
80
Time(minutes)
Figure 1. A. A chromatogram of a blank (reagents only). Fluorescence (left y-axis) is
plotted vs. time from 0 to 80 minutes. The percentage acetonitrile (right y-axis) is
plotted as a dotted line over time. B. Chromatogram of fluorescence vs. time from 50
to 80 minutes; top is blank, middle is n=2 standard, and bottom is a T. weissflogii cell
extract. Phytochelatin oligomers are marked by arrows.
37
moles) (Figure 2). The n= 3 and 4 peak areas were calibrated with the n=2 calibration
curve as there is a linear increase in fluorescence with increasing number of tags per molecule. Utilizing a 200gl injection loop with this small bore column gives us a detection
limit of -0.3 pmol.
Cadmium quotas- Cells were cultured as described above with the addition of carrier free 109Cdto the growth medium. The cells went through at least 6 doublings in the
presence of the tracer. Near the end of exponential growth, cells were collected onto
25mm 0.2- 3pgm(depending on cell size) Poretics polycarbonate membrane filters with
gentle filtration, incubated 15 minutes on the filter with lmM diethylenetriaminepentaacetic acid (DTPA) dissolved in seawater to remove surface bound 109Cd,and then rinsed
with three aliquots of filtered seawater (Lee et al., submitted). The cadmium quotas for
the coccolithophores include cadmium incorporated into the coccolith. The isotope
remaining on the filter was counted by liquid scintillation in a Beckman LS 1801. A calculated specific activity was used to determine the metal quotas of the cells at the various
pCd's. Radiolabelled cells were fixed with Lugol's Solution (Parsons et al., 1984) and
counted microscopically with a Fuchs-Rosenthal haemacytometer.
Results
Eight species of eucaryotic algae were cultured at several cadmium concentrations
to quantify intracellular phytochelatin concentrations (Figure 3). The range of free cadmium ion concentrations was chosen to encompass values typical of the marine environment, from pCd=13 measured in the open ocean by Bruland (1992), to pCd= 9
corresponding to a total inorganic cadmium concentration of 10 nM, well above maximum
coastal cadmium concentrations (Wallace et al., 1988; Flegal and Safiudo-Wilhelmy,
1993) and high enough to inhibit the growth rates of some organisms (Figure 4).
All species examined produced measurable quantities of phytochelatin at all cadmium concentrations. Our data unequivocally demonstrate the presence of phytochelatins
even at the lowest free metal concentrations. Phytochelatin concentrations are given normalized to chlorophyll a (
-glu-cys= 2(n=2) + 3(n=3)+ 4(n=4), EC values; Table 2) to
allow for comparison between species and with field data. At no added cadmium
38
700
600
500
ea
2!
cc
400
300
200
100
A
200
100
300
400
Concentration (picomoles)
Figure 2. Calibration curve of n=2, concentration (0- 400 pmol) vs. peak area. Insert is
an enlargement the standard curve at very low concentrations from 0.5 to 5 picomoles.
Line represents a best fit regression through the data points.
39
,uu
on
Om
1500
480
1200
225
360
900 go
150
240
600
120
300
.en
1.9U
T. ocetnica
90
60
.
75
-ZZ
-A-
au
30
0
0
0
g~~~~~
O
___
IOU
1500
625
1250
500
1000
375
750
D. tertioUecta.
585
215
390
0
a
250
a
75
500
0
125
0
I
_,
_
.-
250
o
a
_
0
0
_
0
9~~~~~~~~~
]......
0M
@1
225
WUU
a0
0
P. ca.reae
0
.
_
195
188
-
450
225
150
150O
300
75
150
113
75
38
0
_
a
_
O
_
f~~~l^
*_ ^
1.U
;U.u
ee ~~~~~~~~~~
qg.un
tz ^
O0u.U
G;
P. lduheri
B. pyrgmaea
37.5
11.3
37.5
25.0
7.5
25.0
48.8
32.5
I
12.5
0.0
_._-
3.8
i
.
n
i
_._
12.5
n
16.3
vw.
.
13.0 12.0 11.0 10.0 9.0
.
.
.
I
.
,
0
v.w
13.0 12.0 11.0 10.0 9.0
pCd= -log [Cd+2]
Figure 3. Phytochelatin concentrations in the different phytoplankton species at steady
state growth as a function of free cadmium ion concentration. The left y-axis is in
units of gmol g chl a and the right y-axis is in units of amol celll; note the difference
in scale for each of the graphs. Open circle plot the n=2 oligomer, open triangles, n3;
and open squares, n4.
40
....
X
m
l
lZ
I
I
E
1.0
0.5
T. ueissflogi
0.0
I
j
N
I
I
l
T. ocecZm'c
!
I
1 I
I
I
;lI
NI
1.0
0.5
W
03
0.0
N
D. terttollecta
.~~\
.
.
I
T. nazWcLato
,I
I
!
il
!
i
I
Z
T
Ii
.
I
1.0
0.5
E. humzei
P. crterae
0.0
!
.I
.l .
l
B
-
1.0
0.5
0.0
P. luthe7-i
I
pCd-13
12
11
H. pygrnaea
I
I
10
9
pCd,3
pod-13
12
12
11
11
10
0t
9
9
pCd= -log[Cd+2]
Figure 4. Relative phytoplankton growth rates at the different free cadmium ion concentrations. Maximum growth rates listed in Table 1.
41
00
0
0
C
atr,
..
0
ocu
CE
-
0\
1!
0Cl4
0
u
0~~
Cu
."I
'
0
0
00
i-.
0U
0)
0
o
0
0
C)
o
Cu
-
:R
0
Cl4
.05
0._
8
.
uii
U
N~
7i
C)
0
Cu
II
.t+
ClII
Cu
-C
00
.
_.
1:~~~~I.
lV
°
0o
-1:
CO
: 0
Sc
U
0
tr)
.*c o
0
-o
Cl4
0
0
CN:0
II
Qu
Cu
Cu
:c
. 0~
oCZ
UI
*0lf
M :
-
,.
r-
r0
.
N
.
C-)
C>
E-.
Cl
*
N
-"
o-
1)
oo,
en
C)
O0
oo
N
.
_4
_l
It
II
"t
s
oo .
:0
:1
II
I
_ ,
'
00
u
tD
\C
N
.
.1
N
't
.
I
0
00o:
Ct
V.
-II
e
00
N~
u
oo
UI
0,_0
-
E
Cu
00
0
d
uC
00~ .Y
_Cu)
Il
I
I11
-
0,
_ct'
"C
N
I
-
:.0xo
'.0
o
Cl4
ir
if
_
-
0c
:
: 't
._
en
Cl
I~
E
o-t-C-t~
~-
00
Cu-C
oD
C
.Co
Cu=
o -
u
3 u a U.) la
3Z5 3 a
U a L)
d 3U
Cu .0
Cu a
°2
U s
o
;b
Cw C
C
IIr
1.
Cl 1.
I
C o
11§
g
*i 4--o
Q?
b
o4
04r
Q
42
(labelled as pCd=13) the range of phytochelatin produced is fairly tight, from 4- 40 gmol
(g chl a)-1 (Table 2). For T. weissflogii, the value of EC in parentheses (E y-glu-cys= 34
amol cell'), is on the same order as metal quotas measured in this species (50 and 30 amol
cell-1 for Zn and Co respectively; Morel et al., 1991).
The chlorophyll a per cell (Table 1) used to normalize the phytochelatin data is the
average of cultures grown at various pCd's and constant light. The actual chlorophyll a
per cell was variable, but the variation was never more than ±50% and there was no clear
pattern of change with respect to cadmium concentration in the medium.
All species, except for the dinoflagellate, exhibited increasing concentrations of
phytochelatin with increasing free cadmium (Figure 3). In most species a measurable
response occurred at a very low free cadmium concentrations, much lower than concentrations that affected growth rate, and one that is likely to be found in seawater. Thus phytochelatin production is probably an important physiological response in the field (Chapter
2). P. lutheri, a prymnesiophyte, only increases phytochelatin production when the cadmium concentrations reach pCd=10. Only the dinoflagellate H. pygmaea maintains a constant phytochelatin concentration over the whole range of pCd's, suggesting that an
alternative detoxification mechanism for cadmium may be employed by this organism.
Within the variability measured among species there do not appear to be trends
according to phylogenetic groupings. Of the two diatoms that were assayed, T. weissflogii
contains from 13-2400 gmol y-glu-cys (g chl a)-1 whereas only -34-410 gmol total yglu-cys (g chl a)-1 were measured in T. oceanica. The two coccolithophores, E. huxleyii
and P. carterae, also produce very different amounts, over the range from pCd=13 to 10:
~40-900 gmol y-glu-cys (g chl a)-land -3-270 gmol y-glu-cys(g chl a)-1 respectively.
The same is true of the two chlorophytes investigated. Moreover, there do not appear to
be systematic differences in either phytochelatin concentrations or the onset of the phytochelatin induction between coastal and oceanic species: T. weissflogii and P. carterae are
coastal isolates whereas T. oceanica and E. huxleyii are pelagic.
Most species tested produce markedly more of the dimer (n=2) than of the other
oligomers, although ratios between the oligomers depend on the cadmium exposure. The
chlorophyte D. tertiollecta, like higher plants, produces significantly more of the longer
chain length phytochelatins (n=3 and n=4) at the three highest cadmium concentrations.
43
In the prymnesiophyte E. huxleyii the trimer and dimer are at similar concentrations and
both respond dramatically to increased cadmium. Clearly the synthesis of the longer chain
oligomers is regulated in different ways by different phytoplankton species.
To compare the amount of phytochelatin produced in the cells to the amount of
intracellular cadmium, we measured the cellular cadmium concentrations for all the species at all pCd's (Figure 5). All species exhibited increasing cellular cadmium concentrations with increasing free cadmium in the medium. The normalization to grams of
chlorophyll a per cell (intracellular cadmium on a per cell basis is listed in Table 2) separates the species into two groupings- four species accumulate about ten times more cadmium than the other four. The two oceanic species, T. oceanica and E. huxleyii fall
together into the group containing more intracellular cadmium. For all the species, the
increase in intracellular cadmium was considerably less than that in the extracellular free
cadmium concentrations, the maximum being a factor of 103for P. lutheri and the minimum a factor of 10 for T. weissflogii over a range of 104 in Cd+ 2 .
The ratios of intracellular phytochelatin to cadmium concentrations are plotted for
each pCd in Figure 6 ((y-glu-cys) per Cd mol molr, values from Table 2). The dotted
line indicates the 2:1 ratio, the maximum capacity of phytochelatin to chelate the cadmium
in a bidentate complex. Four of the eight species, T. oceanica, T. maculata, E. huxleyii,
and P. lutheri, display a similar pattern of phytochelatin production normalized to intracellular cadmium. At the lowest concentration of cadmium (pCd=13), all species contain
more than enough phytochelatin to complex intracellular cadmium and it is likely that
other metals, such as Zn or Co, are binding a significant fraction of the total phytochelatin.
With increasing cadmium, each of these four species appears to regulate phytochelatin
production to follow internal cadmium: the ratios approach constant values near the 2:1
line. The data for the other four species suggest a more complicated relationship between
phytochelatin and internal cadmium. Only the dinoflagellate has ratios that drop significantly below the 2:1 line, once again suggesting that this organism has an alternative
mechanism for dealing with intracellular cadmium. T. weissflogii exhibits an increasing
ratio of phytochelatin to internal cadmium with increasing cadmium concentrations. The
ratio also increases for P. carterae, but levels off and eventually decreases at the highest
cadmium concentration when growth is significantly affected. Conversely D. tertiollecta
44
10 3
10 2
101
.0
0
P-4
100
10 - 1
0- 2
10-3
13
12
11
10
9
pCd= -log [Cd+2]
Figure 5. Log intracellular cadmium concentration (normalized to chl a cell- l ) plotted
vs. free cadmium ion concentration. Average cellular concentrations of chl a cell 1 for
each species is given in Table 1. Error bars are the average of two duplicates; where
not shown error bars are within symbol.
45
10
T tuefr.
T. ce
ca
12
101
~~~~~~~~~~.
.GUt
10
le
I,
I
0
E
101
IOe
G)
0
E
lo'
D. trtioUcta
.~~~~~~~
...
T. m*uvcuoa
,,,,
_._
0
-E
0
U
P
e
10
id'
id-,
to'
'.1
P. a ,
I,0
10
0,
loe
. uzl ii
,
P.
cajrte
H:.
hu:=leyii~
10
lo"
i0d
lo'
'
lo
-
10
i'pCd-13
12
11
10
9
pCd-13
12
11
10
9
pCd= -log[Cd+2]
Figure 6. Log of the ratio of phytochelatin ( y-glu-cys= 2(n=2) +3(n=3) +4(n=4)) to
intracellular cadmium plotted vs. free cadmium ion concentration. The dotted line
represents the 2:1 line. Error bars are the average of two duplicates; where not shown
error bars are within symbol.
46
displays a steadily decreasing ratio for the whole range of cadmium concentrations while
there is no effect on growth rate.
Discussion
The use of a chemically defined and metal-buffered medium has allowed us to
evaluate the relationship between metal exposure and phytochelatin production in marine
phytoplankton in steady exponential growth. Phytochelatin production, for most species
in this study, is an important intracellular process, likely the primary mechanism for
detoxification once cadmium enters the cell. We have demonstrated, as a result of
increased resolution and methodological sensitivity, that all the species contain phytochelatin even when there is no added cadmium and that phytochelatin production in phytoplankton is induced by cadmium even at very low and environmentally relevant
concentrations. In all but one organism, phytochelatin production is a function of the free
cadmium in the medium. Comparison of internal cadmium and phytochelatin concentrations has revealed that the regulation of phytochelatin synthesis and polymerization varies
widely among phytoplankton species and is not simply a function of intracellular cadmium.
All previous studies of phytochelatin production in phytoplankton have been done
at high metal concentrations in unbuffered systems under non-steady state conditions
(upon Cd addition to the culture medium, intracellular cadmium concentrations first
increase as cadmium is taken up by the cells and then decrease as the cell biomass
increases). Comparisons are thus difficult; still it is instructive to compare our maximum
concentrations to these other studies. Gekeler et al (1988) exposed many algal species to
20gLmCd(N0 3 ) 2 and measured values of -5-50 gmol y-glu-cys g-1 protein. A rough estimate of -40 g protein per g chl a, yields concentrations (200-2000 gmol (g chl a)-1' that
are very similar to those measured at the highest pCd in this study - 80-2800 Pmol (g chl
a)-1 (excluding H. pygmaea-Table 2). It is interesting that a completely different set of
phytoplankton (including both marine and freshwater species) yields nearly the same
range of concentrations.
At steady state, phytochelatin concentrations are both a function of synthesis and
47
dilution by cell division. Slower growth rates will thus result in higher cellular phytochelatin concentrations. Even when growth rate is decreased, the production rate is still a
function of the trace metal concentration.
The primary function of phytochelatin is thought to be metal detoxification. This
is supported by the binding properties of the peptides, by their induction upon metal exposure and by the control of phytochelatin synthase by metal concentrations. Our steady
state data provide a further quantitative confirmation of the detoxifying role of phytochelatins in phytoplankton. In four of the species we studied, the increase in intracellular cadmium concentration upon cadmium exposure is exactly matched by an increase in
phytochelatin concentration, such that the ratio remains about constant around two y-glucys per cadmium (Figure 6). These algae thus possess a finely tuned control mechanism to
synthesize the exact concentration of phytochelatin necessary to bind intracellular cadmium. It is however likely that a significant fraction of the cadmium is associated with
other cellular components, such as the membrane (Price and Morel, 1990; Reinfelder and
Fisher, 1994). The ratio of cadmium associated with phytochelatins may actually be
closer to the results of Strasdeit et al (1991) who measured four y-glu-cys per cadmium.
It is unlikely that phytochelatin production is the only mechanism of detoxification
in eucaryotic phytoplankton (Robinson, 1989; Wikfdors et al., 1991). Wikfors et al (1991)
tested cadmium-tolerant phytoplankton and reported that at very high concentrations of
cadmium (100-1000 AM) at least one species, Isochrysis galbana, was producing a metalbinding protein, rich in cysteine, that was not phytochelatin. In our study, one organism is
clearly not utilizing phytochelatin production to detoxify cadmium: H. pygmaea does not
increase production of phytochelatin when exposed to greater cadmium concentrations
and the highest internal cadmium concentrations actually exceed those of phytochelatins.
Previous studies have established that some bacteria (Higham et al., 1984), including
many cyanobacteria (Olafson et al., 1979), produce a metallothionein-like protein to complex metals intracellularly. Fungi can make phytochelatins (Murasugi et al., 1981) or metallothionein-like proteins (Lerch, 1980) or both (Mehra et al., 1988). Metallothionein
genes have also been identified in some higher plants (de Miranda et al., 1990; Evans et
al., 1990) and reports of metallothionein-like complexes in plants have been numerous. It
is thus possible that H. pygmaea (or some bacterial symbiont; Dodge, 1973) is synthesiz-
48
ing metallothionein or a metallothionein-like protein instead of phytochelatin.
A comparison of our laboratory data to concentrations of phytochelatin measured
in the field (Chapter 2) reveals that natural populations produce amounts of phytochelatin
that are similar to those measured in cells with low cadmium concentrations. We measured dimer concentrations ranging from 20 pmol (g chl a)-lin Boston Harbor to 2 gmol
(g chl a)-1 in Massachusetts Bay. These values are very similar to those measured in the
pCd 13 toll range. Clearly the variability among species does not allow us to estimate
precisely the free cadmium concentrations in Boston Harbor, even if we assume that cadmium is the principal inducer of phytochelatin in the field. Nonetheless the very low phytochelatin concentrations in Massachusetts Bay are comparable (or even lower than) those
obtained in the laboratory at the lowest free metal concentration (pCd 13) and those in
Boston Harbor are similar to those induced in cultures exposed to low free cadmium (pCd
12- 11). It is thus clear that the cadmium in Boston Harbor and Massachusetts Bay whose
total concentration is in the range 1 pM to 5 nM (Wallace et al., 1988) must be almost
entirely complexed. To attribute the observed phytochelatin gradient to a particular metal
or set of metals requires more information on phytochelatin induction by other metals in
marine algae (Chapter 4).
Since its discovery in the early 80's, many have speculated that phytochelatins
may have functions other than detoxification. Evidence that phytochelatins may act as a
buffer for metals in non-stressed cells was given by Grill et al (1988); they reported that
phytochelatin was induced in tobacco cells when cells were transferred from their growth
medium to fresh standard medium. As the zinc and copper were taken up by the cells
from the fresh medium, phytochelatin production initially increased; as the cell mass further increased and the metals were incorporated into enzymes, the phytochelatins (per volume of culture) then decreased. It is possible that metal concentrations (ZnT= 37 gM and
CUT=0.1 gM) in such "standard" medium may have simply stimulated an initial detoxification response. Perhaps more convincing are in vitro studies that have shown that phytochelatin-metal complexes restore activity to metal-requiring apoenzymes (Thumann et al.,
1991), implying that the same may occur in vivo. Our data showing the presence of phytochelatins in all phytoplankton cells at very low free metal concentrations also implies a
role other than detoxification. The similarities between basal phytochelatin concentrations
49
and those of trace metals under steady state conditions, provide strong support to the idea
that phytochelatins play a fundamental role in metal homeostasis. Such a role may be
potentially important in marine phytoplankton which must grow under conditions of
exceedingly low ambient metal concentrations.
Acknowledgments
A special thanks to Shing Kong, an undergraduate participating in the MIT UPOP
program, who helped to culture some of the phytoplankton and run some of the HPLC
samples. Special thanks to Lynn Roberts, a former student of the Parson's Laboratory,
who suggested integrating a critical component of my HPLC method.
This work was funded by NSF, ONR, EPA, Sea Grant and the MWRA.
50
References
Bruland, K. W. 1992. Complexation of cadmium by natural organic ligands in the Central
North Pacific. Limnol. Oceanogr. 37: 1008-1017.
de Miranda, J. R., M. A. Thomas, D. A. Thurman and A. B. Tomsett. 1990. Metallothion-
ein genes from the flowering plant Mimulus guttatus. FEBS. 260: 277-280.
Dodge, J. D. 1973. The fine structure of algal cells. Academic Press.
Evans, I. M., L. N. Gatehouse, J. A. Gatehouse, N. J. Robinson and R. R. D. Croy. 1990. A
gene from pea (Pisum sativum L.) with homology to metallothionein genes. FEBS Lett.
262: 29-32.
RFlegal,A. R. and S. A. Safiudo-Wilhelmy. 1993. Comparable levels of trace metal contamination in two semi-enclosed embayments: San Diego Bay and South San Francisco Bay.
Environ. Sci. Tech. 27: 1934-1936.
Gekeler, W., E. Grill, E.-L. Winnacker and M. H. Zenk. 1988. Algae sequester heavy metals via synthesis of phytochelatin complexes. Arch. Microbiol. 150: 197-202.
Grill, E., S. Loeffler, E.-L. Winnacker and M. H. Zenk. 1989. Phytochelatins, the heavymetal-binding peptides of plants, are synthesized from glutathione by a specific yglutamylcysteine dipeptide transpeptidase (phytochelatin synthase). Proc. Natl. Acad. Sci.
U.S.A. 86: 6838-6842.
Grill, E., J. Thumann, E.-L. Winnacker and M. H. Zenk. 1988. Induction of heavy-metal
binding phytochelatins by inoculation of cell cultures in standard media. Plant Cell
Reports. 7: 375-378.
Grill, E., E.-L. Winnacker and M. H. Zenk. 1985. Phytochelatins: the principal heavymetal complexing peptides of higher plants. Science. 230: 674-676.
Higham, D. P., P. J. Sadler and M. D. Scawen. 1984. Cadmium-resistant Pseudomonas
putida synthesizes novel cadmium proteins. Science. 225: 1043-1046.
Kneer, R., T. M. Kutchan, A. Hochberger and M. H. Zenk. 1992. Saccharomyces cerevi-
siae and Neurospora crassa contain heavy metal sequestering phytochelatin. Arch.
Microbiol. 157: 305-310.
Lee, J. G., S. B. Roberts and F. M. M. Morel. submitted. Cadmium: a nutrient for the
marine diatom Thalassiosira weissflogii. Limnol. Oceanogr.
51
Lerch, K. 1980. Copper metallothionein, a copper-binding protein from Neurospora
crassa. Nature. 284: 368-370.
Loeffler, S., A. Hochberger, E. Grill, E.-L. Winnacker and M. H. Zenk. 1989. Termination
of the phytochelatin synthase reaction through sequestration of heavy metals by the reaction product. FEBS Lett. 258: 42-46.
Mehra, R. K., E. B. Tarbet, W. R. Gray and D. R. Winge. 1988. Metal-specific synthesis of
two metallothioneins and Ty-glutamylpeptides in Candida glabrata. Proc. Natl. Acad. Sci.
85: 8815-8819.
Morel, F. M. M., R. J. M. Hudson and N. M. Price. 1991. Limitation of productivity by
trace metals in the sea. Limnol. Oceanogr. 36: 1742-1755.
Murasugi, A., C. Wada and Y.Hayashi. 1981. Cadmium-binding peptide induced in fission
yeast, Schizosaccharomyces pombe. J. Biochem. 90: 1561-1564.
Murasugi, A., C. Wada and Y. Hayashi. 1983. Occurrence of acid-labile sulfide in cadmium-binding peptide from fission yeast. J. Biochem. 93: 661-664.
Newton, G. L., R. Dorian and R. C. Fahey. 1981. Analysis of biological thiols: Derivatization with monobromobimane and separation by reverse-phase high-performance liquid
chromatography. Anal. Biochem. 114: 383-387.
Olafson, R. W., K. Abel and R. G. Sim. 1979. Prokaryotic metallothionein: Preliminary
characterization of a blue-green alga heavy metal-binding protein. Biochem. Biophys.
Res. Commun. 89: 36-43.
Parsons, T. R., Y. Maita and C. M. Lalli. 1984. A Manual of Chemical and Biological
Methods for Seawater Analysis. Pergamon Press.
Price, N. M., and others. 1991. Preparation and chemistry of the artificial algal culture
medium Aquil. Biolog. Oceanogr. 6: 443-461.
Price, N. M. and F. M. M. Morel. 1990. Cadmium and cobalt substitution for zinc in a
marine diatom. Nature. 344: 658-660.
Reinfelder, J. R. and N. S. Fisher. 1994. The assimilation of elements ingested by marine
planktonic bivalve larvae. Limnol. Oceanogr. 39: 12-20.
Robinson, N. J. 1989. Algal metallothioneins: secondary metabolites and proteins. J. Appl.
Phychol. 1: 5-18.
Steffens, J. C., D. F. Hunt and B. G. Williams. 1986. Accumulation of non-protein metalbinding polypeptides (y-glutamyl-cysteinyl)n-glycinein selected cadmium-resistant
tomato cells. J. Biol. Chem. 261: 13879-13882.
Strasdeit, H., A.-K. Duhme, R. Kneer, M. H. Zenk, C. Hermes and H.-F. Nolting. 1991.
52
Evidence for discrete Cd(SCys)4 units in cadmium phytochelatin complexes from EXAFS
spectroscopy. J. Chem. Soc., Chem. Commun. 1129-1130.
Thumann, J., E. Grill, E.-L. Wmnacker and M. H. Zenk. 1991. Reactivation of metalrequiring apoenzymes by phytochelatin-metal complexes. FEBS Lett. 284: 66-69.
Wallace, G. T., J. H. Waugh and K. A. Garner. 1988. Metal distribution in a major urban
estuary (Boston Harbor) impacted by ocean disposal. p. 67-78. In D. A. Wolfe and T. P.
O'Connor eds.], Urban wastes in coastal marine environments. Robert E. Kreiger Publishing Co.
Weber, D. N., C. F. Shaw and D. H. Petering. 1987. Euglena gracilis cadmium-binding
protein-II contains sulfide ion. J. Biol. Chem. 262: 6962-6964.
Westall, J. C., J. L. Zachary and F. M. M. Morel. 1976. MINEQL: A Computer Program
for the Calculation of Chemical Equilibrium Composition of Aqueous Systems. R. M. Parsons Lab., Mass. Inst. Technol.
Wikfors, G. H., A. Neeman and P. J. Jackson. 1991. Cadmium-binding polypeptides in
microalgal strains with laboratory-induced cadmium tolerance. Mar. Ecol. Prog. Ser. 79:
163-170.
53
Chapter 4
Induction by Various Metals
Submitted to: Limnology and Oceanography
Co-author: Francois M. M. Morel
54
Abstract
Phytochelatin has been quantified in Thalassiosira weissflogii, a marine diatom,
upon exposure to a series of trace metals (Cd, Pb, Ni, Cu, Zn, Co, Ag, and Hg) at concentrations which would likely be found in the marine environment. Within the range of concentrations relevant to natural waters, cadmium, and to a lesser extent copper, are the most
effective inducers of phytochelatins. The generality of this result was confirmed by short
term experiments with two other phytoplankton species. Quantification of intracellular
cadmium, nickel, and zinc shows that phytochelatin production does not follow a simple
stoichiometric relationship to the metal quotas. The rapid formation of phytochelatin in T.
weissflogii upon cadmium exposure, and the fast elimination when metal exposure is alleviated, reveal a dynamic pool of phytochelatin which is tightly controlled by the cell.
Introduction
Many trace metals have been shown to induce phytochelatin production in plants
(Grill et al., 1987), although the concentration necessary to stimulate the response, as well
as the magnitude of the response, depend on the particular metal. Cadmium has been
found to be the most effective inducer of phytochelatin, yet it is believed that production
of this peptide is a general metal detoxification system (Grill et al., 1985) see reviews by
(Robinson, 1989; Rauser, 1990; Steffens, 1990).
Our goal is to elucidate the factors that control phytochelatin production by phytoplankton. In the previous chapter, we investigated phytochelatin production in response
to cadmium by several phytoplankton species. In this study we examine the response of
Thalassiosira weissflogii to a variety of metals (Cd, Pb, Cu, Ni, Zn, Co, Ag and Hg), all of
which have been found to stimulate phytochelatin production in higher plants. As in our
experiments with cadmium, we tested free metal concentrations that would be encountered in natural seawater in order to evaluate which metals may stimulate this response in
natural populations of algae. We performed short term assays with two other phytoplank-
55
ton species to compare the patterns of the phytochelatin response to various metals.
Finally to assess how changing environmental conditions might be reflected by changes in
cellular concentrations of phytochelatin, we examined the kinetics of phytochelatin production and elimination upon changes in metal exposure.
Materials and Methods
Cell preparation and HPLC Chromatography- T. weissflogii (Actin) was cultured
in Aquil (Price et al., 1991) as detailed in the previous chapter, but with the addition of different metals. These metals were added in addition to the normal trace metals in the Aquil
seawater medium containing either 10 or 100 pM ethylenediaminetetraacetic (EDTA).
Total metal additions (MeT) were made as Me-EDTA complexes to achieve the various
free ion (Me + n ) and total inorganic ion (Me') concentrations (Tables 1& 2). See previous
chapter for methods outlining cell sample preparation and HPLC chromatographic methods.
Metal quota experiments- Cells were cultured as described in the previous paper
with the addition of carrier free radiotracers to the growth medium. Medium was allowed
to equilibrate with the tracer to ensure that it was proportionately distributed between the
free ion and EDTA complex. This was particularly important for nickel which required
one week of equilibration before adding the cells. Near the end of exponential growth,
cells were collected onto 25mm 3gm Poretics polycarbonate membrane filters with gentle
filtration, incubated 15 minutes on the filter with lmM diethylenetriaminepentaacetic acid
(DTPA) dissolved in seawater to remove surface bound
109Cd and
65Zn, and then rinsed
with three aliquots of filtered seawater (Lee et al., submitted). Nickel (63 Ni) containing
cells were incubated first for 3 minutes with a lmM 8-hydroxyquinoline-5-sulfonate seawater solution and then rinsed with filtered seawater (Price and Morel, 1991). Isotope
remaining in the cells on the filter was counted by liquid scintillation in a Beckman LS
1801. A calculated specific activity was then used to determine the metal quotas of the
cells at the various pMe. Radiolabelled cells were spiked with Lugol's Solution (Parsons
et al., 1984) and counted microscopically with a haemacytometer.
Short term metal exposures- Three phytoplankton species, T. weissflogii , Tetra-
56
0 : 3
6-
.
H
H
U
H
0
Z
d
0+
la
.0
N
+-9
-
i<
H
-o
N
~0
1:
U
~oC
u
102
-Ch
I
+
w
.
-
0
.0" .004
+
+
'N.
II II
*0.4
Q
0-
+
1-o
. _
-
;;
C40
I-
8
N
4.)
.
10
0V
U
V:L
+
4.
.C
3
0
+I
0
XS ^
.0S
4IIl&
2.4
+4
_.
U
K4
0
Q
4-
N-
Z
CJ
+
iC
K<
'E.
v
0
Z
K
N
9I
'9
0
K
o
:I
I
Q
'9
_O
0
II
i
oCN
0
K
0
'I
0
K
0
K
._
l
K
:
o0
xC
0_O
R
IF
C5
m
,e
K
-C
4)
1y
:C
0
0
K
'C
C;
i
V
E
-o
K
K
K
K
1;
I?
I _
V
?
C
I
ICll
o
!
O
K
UX
C~
<1
C
0
\'C
Cr
U
C)
v.
~L~
.0
4-
4) i
v
_E
I
I.)
I
I
4.)
0
U)
N
57
IL)
I
X
i
Ci
H
0
0
F.
_
w
L)
v
I
0
to
O0
to =,
*.E
u
-
N
2
ap
_
(N
I
r-
C
I-
E
t~
._
I-
CNy
2
AS
do
U41
'S
m
0
E
N
-o
I-
0
C;
E
.
0
0
V
l-
C)
C
_C;
f
r_ cl
.0
_
_
58
AS
(Ni
selmis maculata
r(TTM),
and Emiliana huxleyii (BT6) (obtained from the Culture Collec-
tion of Marine Phytoplankton, Bigelow Laboratories), were tested for a short term
response to high additions of cadmium, lead, copper and zinc (pMe=9). Duplicate 500 ml
cultures were grown in standard Aquil medium to late exponential, subdivided into four
aliquots and spiked with the different metals added as EDTA complexes at concentrations
necessary to achieve free ion concentrations of 1 nM (pMe=9). Cells were incubated 22
hours and harvested the next day onto 25 umm
Whatman GF/F filters and analyzed for
intracellular phytochelatin as detailed in the previous paper.
Time series experiment- To determine rates of intracellular accumulation and degradation of phytochelatin in T. weissflogii, time course experiments were conducted. In
the first experiment duplicate 500 ml cultures were grown to -3.0 X104 cells/ ml in
medium containing no cadmium, at which time cadmium was added as an EDTA complex
to achieve an equilibrium concentration of pCd=10. The bottles were sampled (in aliquots
of 25- 50 mls) at various times from t=0 to t36 hours. In the second experiment, when T.
weissflogii cells, cultured at a pCd=10, reached late exponential growth phase they were
filtered out onto 47 mm 3gm membrane filters, rinsed with filtered seawater and then
resuspended in fresh Aquil containing no added cadmium with normal EDTA (100pM)
and other trace metals. The duplicate bottles were sampled over time from t=0 to t=39
hours.
Results
The concentration of intracellular phytochelatin in T. weissfiogii increased upon
addition of most metals tested (Figure 1, Table 3). Cadmium stimulated production of
phytochelatins to the greatest extent, concentrations reaching 1500 amole (n=2) cell at
pCd =9, whereas Pb, Cu, and Ni induced much smaller amounts of phytochelatin at the
same free ion concentrations: 220, 50 and 25 amole (n=2) cell 1 respectively. As shown in
the previous chapter, growth rates for T. weissflogii were relatively unaffected by these
cadmium concentrations, but growth rates were reduced at the highest concentrations of
Pb, Hg, Ni and Cu. High lead and mercury concentrations reduced growth by a factor of
almost two, yet intracellular phytochelatin concentrations were relatively low compared to
59
800
SO1500
Cd
480
360
240
,
U
U
L. .4.
X
I
120
0
NONE 12.0
11.0 10.0
100
80
200
900
60
150O
600
40
100
300
20
50
0
0
9.0
none 12.0
pCd- -log [Cd+2]
_
100
250
Pb
1200
11.0 10.0
pPb.- -log
9.0
Pb+2J
_
250
Cu
80
Ni
200
60
150
40
100
m
24
16
C
it
0
20
to
tO
AQUL C
0
8
50
* i
12.0 11.0
13.8
pCu -g
10.0
9.0
so
80
40
20o 0
0
0
_
u
none 12.0 11.0
pN,- -g
[Cu+2]
10.0
9.0
o
,E
Zn
12
AQUIL U
e
40
16
30
12
0
Co
40
30
20
8
4
10
4
10
0
0
0
0
none 12.0 11.0
pZn- -g
10.0
9.0
AQUILCo
12.0
none
Zn+2]
e
[N;+2]
0
16
I.
11.0
0
9)
0
0
ON
.
20
10.0 9.0
pC---log [Co+2]
16
50
40
Ag
120
Hg
40
12
30
8
20
90
30
-0
13.9
13.2
12.9
12.2
60
20
10
10
0
0
30
0
none 23.4 23.1
11.9
pg- -log [Ag+]
22.7
22.4
pH9- -og [H(4+2]
Figure 1. Phytochelatin concentrations in T. weissflogii induced individually by cadmium, lead, copper, nickel, zinc, cobalt, silver, and mercury plotted against the log of
the free metal ion concentration. Standard Aquil concentration of all other metals are
present in medium. Open circles are concentrations of the n=2 oligomer, open triangles, n=3; and open squares, n=4. Error bars represent the average of measurements
from two separate cultures; samples without error bars were not duplicated. The left
y-axis is in units of ,umol (g chl a)-1 and the right axis is in units of amol celll(10
18
mole cell-l); note the different scales in each graph. The conversion from one unit to
the other is based on an average concentration of 2.6 pmol chl a cell-1 in T. weissflogii
(see Table 1, Chapter 3). The standard Aquil concentrations (optimal culture condi-
tions) of copper, zinc, and cobalt are marked with arrows.
60
c4
4)
E
1
00
0.
ca
00
V'1c~)
CD
=4C
_t
-
II
1)
m
"C
0 0en
~00
"It
tn
'I
4) Cl
071
Cl4
P.
0C)
t
Cl4
N
Cl4
E
cO
oCo
C.)
C
4II
0
11N
'I
0.
0
u-N
00
00
Cl
(",
£0
Cl
Cl4
.-
CO5
t
CS
o.,
oo
O
I-
4) 0n
_.
11
0II
0.
0.
CI"C-
11 Cl
0
Cl
CO
.X *-
.
"t
V)
C
CO
PCO
0..
o
4+ 44
CK
4e
-4
RS
-
4\0
4l
C
4) Cl
0
It
Cl
Cl
N
-.
COE
4)
X4.)
+:
QC
=
-~
Cl
CO
~
COCO
.z
4)
o
0.
_
CO .s
"t
~~~~1
0.
+F~~~~
'_O
4+ 4
IU
C.
+
U
*i+1 +D
4
4+
4-
0
4+-
UUU
;C
O
n~'
<
_
4) E
CO o
4-
L4
U~
E
X,
o
C)-E
C.)
4)
t
>oC
E .g=
0._O
COO
a
CO_
XC..)
CO
C
.COQ
OC,,
..0
C.)
o
tn
N
t
-e
.E
CO
u
o.
C,Zu
CO
CO
61
00.
CO
.0
U
CO
C.)
v-
* CO4+*
those induced by cadmium. The same was true for copper and nickel; growth was inhibited at the highest concentrations, yet intracellular phytochelatin barely rose above background levels.
High zinc and cobalt concentrations had no effect on phytochelatin concentrations
or on growth rates up to a pMe=9. Free concentrations of Zn and Co above these levels,
which would rarely be encountered in natural seawater (>10 nM), did induce some phytochelatin production in T. weissflogii (data not shown). At the lowest free zinc ion concentration (pZn=12), zinc is a limiting nutrient for T. weissflogii and the growth rate was
decreased. The corresponding decrease in the n=2 phytochelatin oligomer at this zinc
concentration suggests that sufficient zinc stimulates some phytochelatin production and
phytochelatins may serve as a buffer for zinc. The addition of Ag did not stimulate phytochelatin production or depress growth rates in the range tested. All of these metals have
been shown to induce phytochelatins in higher plants, but at much higher concentrations
than those employed in this study (Grill et al 1987).
To determine whether the response of T. weissflogii to the various metals is typical
of marine algae, we performed short incubations (22 hours) of two other species, E. huxleyii, a prymnesiophyte, and T. maculata, a chlorophyte, with relatively high additions of
Cd, Cu, Zn, and Pb (Figure 2.) The concentrations of phytochelatins induced in these short
term experiments were fairly similar to those measured at steady state in T. weissflogii
(Table 4), and the exceptional ability of cadmium to stimulate phytochelatin synthesis was
not limited to T. weissflogii. For all species, the greatest phytochelatin induction was
obtained with cadmium, followed by lead and copper, and little induction was seen with
zinc. The response of T.maculata to these metals was very similar to that of T. weissflogii,
except that there was relatively more phytochelatin production in response to zinc and less
in response to lead. The pattern of phytochelatin production in E. huxleyii was also much
like that of T. weissflogii, with the exception of the large response to copper. Almost as
much phytochelatin was synthesized in response to copper as to cadmium (Table 4); the
concentration of n=2 actually exceeded the concentration made in response to cadmium.
Production of phytochelatin by T. weissflogii in short term incubations with copper
and lead were slightly greater than at steady state. Over four times as much phytochelatin
was synthesized in response to copper. This difference may be due alternate detoxification
62
800
2000
600
1500
400
1000
200
500
a
0
~P
1
.
76
1U
aculta
T.
Q
hO
0
0
n-4~~~~~~~~~~
T w
60
[i_ =2-E
120
En R.3
so
a
60
I0
45
o15
3
30
30
A
;L V r~
L~~~rLFOA
v
1
v
16
120
90
12
60
8
30
4
n
a
Cd
Cu
Zn
Pb
Figure 2. Short term induction of phytochelatins in T. weissflogii, T. maculata, and E.
huxleyii upon addition of cadmium, copper, zinc, and lead. Metals were added as
EDTA complexes to achieve free metal ion concentrations of lnM. Cells were incubated for 22 hours with the individual metals and then assayed for phytochelatin production. Units of y-axis are as labeled in Figure 1; note the different scales in each
graph. Error bars represent the average of measurements from two separate incubations.
63
Table 4. Phytochelatin concentrations (E -glu-cys gmol (g chl a)-1 ) from short term metal
additions experiments and steady state cadmium exposure concentrations. Individual
oligomer concentrations are plotted in Figure 2.
T. weissflogii
T. maculata
E. huxleyii
Cd
Cu
Zn
Pb
t
3600
170
10
330
t
2400
45
13
190
t
170
44
57
12
t
1800
t
550
340
8.6
40
t 4000
t Short term incubation phytochelatin concentrations.
tSteady state phytochelatin concentrations from Chapter 3.
64
strategies given time to adapt to high concentrations of copper under steady state conditions. For both T. maculata and E. huxleyii, the short term response to cadmium was much
less than that achieved at steady state concentrations (Table 4). These species may not
have the reserves of glutathione necessary to produce large amounts of phytochelatin or
cadmium may not be taken up as fast as in T. weissflogii.
Intracellular zinc and nickel was measured in T. weissflogii at the various free
metal concentrations to determine whether phytochelatin production was a function of
either of the metal quotas. These data are plotted in Figure 3 along with the intracellular
cadmium quotas from Chapter 3. As previously described, intracellular cadmium concentrations changed very little over the free cadmium ion concentrations, remaining nearly
constant at 13 amol cell-' at pCd 12 & 11, increasing to only 20 and 50 amol cell 1 at pCd
10 & 9 respectively. Steady state phytochelatin concentrations were not simply a function
of the intracellular cadmium. Phytochelatin pools exceeded cadmium on a mole:mole
ratio by 100times at the highest cadmium concentration. Zinc quotas varied 100 fold over
the range of concentrations tested yet phytochelatin was constant, except at the lowest zinc
concentration. The ratio of y-glu-cys to Zn decreased as a result of the increasing Zn
quota. Total inorganic concentrations of zinc in the medium are much lower than those of
cadmium at the same free metal ion concentration (Cd'= 30 pM vs. Zn'= lpM at the free
ion concentration of 10-12M). Thus the difference in metal quotas at the lowest free ion
concentration may reflect limitation of zinc uptake by diffusion (Hudson and Morel,
1993).
Intracellular nickel quotas ranged from 0.3 to 300 amol cell' from pNi's of 12 to 9
and were a function of free nickel concentrations although the medium contained nitrate
and the cells should have had no nickel requirement (Price and Morel, 1991). Phytochelatin concentrations did not follow increasing nickel quotas, even when nickel reached levels high enough to inhibit growth. Ratios of y-glu-cys to Ni decreased as nickel
concentrations increased because the intracellular nickel increased much more than phytochelatins. There was a slight increase in phytochelatin at the highest concentration, but
not enough to complex the measured nickel quotas (ratio well below the 2:1 line).
The kinetics of phytochelatin production by T. weissflogii, in response to cadmium
additions were also investigated in short term incubation experiments. Upon exposure to
65
·
!
0
a
.P
1
I
I
a.
I
N
.0
-I
0
N
l
-I
:
I1
'
I
l
o
I
aoonb
p / UAD-n-L aqD
(I-i1ie
0
0
Mouwo)sojonb wnwpo
I1
I
I
I
o.
4
CD
0
N
q
i
N
a.
!
qN;
A
II
'I
omonb
uz / Inln-/-
SORoDj
(l- , o #1owo)
o0wo)
sOionb
ouZ
(t--o
solonb
Oug
ii
I
oc°
,.
Z
Z
oa
,:&1
_i'
p
0
a.
I
I
%o
0
,
a
,
.
'.I
a
T
o
a
(-.w sowo)sono
a0fb !N / sA*-ni6B- soaow
oN
(t-lmo WlOLD)so~,on) ID)ION
Figure 3. Intracellular nickel, zinc, and cadmium concentrations in T. weissflogii (bottom) and the ratio of the total y-glu-cys from phytochelatin (I y-glu-cys= 2x(n=2) +
3x(n=3) + 4x(n=4)) to these metal quotas (mole mole 'l ) at the various metal concentrations (top).
66
cadmium (added as an EDTA complex to achieve a pCd =10), we observed immediate
phytochelatin production -within one hour the intracellular values changed from 5 to 180
amole (n=2) cell 1 (Figure 4, a). After 5 hours, the cells had exceeded steady state concentrations (for pCd=10) of ~800 amol (n=2) cell-1 by about 25%. The n=3 oligomer also
increased immediately and overshot the steady state concentration. This result reflects the
rapid enzymatic production of phytochelatin from the large pool of glutathione in the cells
(measured to be approximately 1.5 fmole cell ' ) as has been demonstrated for higher
plants and yeast (Grill et al., 1987; Robinson et al., 1988).
We also investigated the disappearance of phytochelatin upon resuspending the
cells in medium containing no added cadmium (and 100M EDTA). Phytochelatin per
cell decreased rapidly; values changed from -700 amol (n=2) cell-1 to half that in three
hours (Figure 4, b). After 20 hours, concentrations had reached steady state concentrations for cell cultured in medium containing no added cadmium. The insert in Figure Sb
plots the decrease in particulate phytochelatin per liter to demonstrate that the decrease in
cellular phytochelatin concentration was not simply the result of dilution caused by cell
division. These results imply either enzymatic degradation or export of the phytochelatin
from the cell, or some combination of both. This phenomenon is being investigated further by following simultaneously the intracellular concentration of cadmium and phytochelatin.
Discussion
Cadmium is the most effective inducer of phytochelatin production in the marine
diatom T. weissflogii in the range of trace metal concentrations that are likely to be important in natural seawater. Other metals, particularly copper, lead, and mercury, induce
small amounts of phytochelatin within the range of concentrations examined, whereas
some metals, such as zinc, cobalt, and silver, have no effect at all. Limited testing of other
metals besides cadmium on phytochelatin induction in T. maculata and E. huxleyii, supports the finding that cadmium is the most important inducer of phytochelatins. Elevated
intracellular metal concentrations of nickel and zinc in T. weissflogii do not significantly
increase the phytochelatin pool, even when nickel concentrations begin to affect growth.
67
A.
1200
U
1000
_
800
_
.
I
.
I
.
I
,
I
I
0o
0
0
n= 2
1
600
_
400
_-
200
_-
,4
cl
o
-7_
0V
ao
0
I
0
i
0
5
I
i
I
_
10
15
20
25
30
Time after Cd addition (hours)
B.
1000
0
0
0
o0V.
800
I-
v
600
400
oa
0To
0eJ
.
200
0
0
10
20
30
40
50
Time after resuspension (hours)
Figure 4. Timeevolution of phytochelatin concentrations upon changing cadmium
exposure. Open circles represent the n=2 oligomer and open triangles n=3. A. Phytochelatin production in T. weissflogii after addition of cadmium (pCd=10). B. Decrease
in phytochelatin concentrations upon resuspension of cells (cultured at pCd=10) in
Aquil medium containing no added cadmium (100.M EDTA). Insert in B. shows the
decrease in total particulate phytochelatin per liter as a function of time.
68
The rapid accumulation of phytochelatin in T. weissflogii upon exposure to cadmium is
consistent with the enzymatic synthesis found in higher plants. We also observed a rapid
decrease in phytochelatin concentration when cadmium exposure is alleviated, suggesting
that the intracellular phytochelatin pool is tightly controlled.
Other studies of phytochelatin production in phytoplankton have been generally
limited to induction by cadmium, with the exception of two studies. Gekeler et al (1988)
reported stimulation of phytochelatin production in two chlorophytes by a handful of other
metals (Pb, Zn, Ag, Cu, and Hg). In another study Howe and Merchant (1992) examined
the response of Chlamydomonas reinhardtii to additions of micromolar concentrations of
Cd, Hg and Ag, and as in our study, found that phytochelatin was induced to the greatest
extent by cadmium, even at these extremely high concentrations.
A comparison of the total inorganic concentrations of metals that induced phytochelatin at the various pMe's in laboratory experiments to those typical of marine environments (Table 5) supports the notion that, in the field, cadmium should be the most
important inducer of phytochelatins with copper possibly becoming important in some situations. Lead begins to induce phytochelatin at pPb=10 (Pb'= 23 nM), but total concentrations of lead in the field rarely reach 5 nM, and are typically an order of magnitude
lower. Zinc, nickel, cobalt and may exceed the concentrations tested here, but the lack of
significant phytochelatin induction at pMe= 9 compared to that of cadmium, as well as the
strong organic complexation of these metals in natural seawater (Bruland, 1989; Nimmo
et al., 1989; Zhang et al., 1990) will limit the ability of these metals to induce phytochelatins in the environment. Typical silver and mercury concentrations never reach the highest
concentrations tested in this report (Flegal and Saiudo-Wilhelmy, 1993; Mason et al.,
1993). Total concentrations of cadmium in polluted coastal waters may sometimes be as
high as 5 nM. In the absence of organic complexation, this inorganic cadmium concentration would correspond roughly to a pCd of 10. In many species, phytochelatin concentrations are modulated by cadmium even down to pCd=12 (Cd'= 30 pM), typical of
concentrations that are measured in seawater. Total copper concentrations in the field can
exceed those tested in this study, and given the relatively large response of T. weissflogii
and E. huxleyii to short term exposure to copper, it is possible that copper may be important in some field situations.
69
I)
00
-
o1
9
oo
CI
0
00
8
IC2
CD~
0
0
Q
C
Co
CD
Lr4
0
t
0CD
o
F @
C's
Ca
S-Iq
-4
0
_4
E
\o
_
m 0
00
I
.
0
o)
I.
C
C
E .0
En
N
C0
o
LI
I
I.ON
W)
0
-
C
0rX
*So
04)
0X
-
,
ONi '
'N
EUo
cU o
ON I
Cl
o
t
'.
-
_
C7
-e
.°
0
=o -o
C .0 C.
C
C.
C
0C~
C
N
'N
C:t~
C4
0
Eet
u
0
C.
C.)
M
00
1-4
c
-
-
C-
C
E
'-
C
C.)
ie
4)
Ct
.H
.0
,
I
0s
D
a
g
0
I
o
I
o
I.N
0
.0
O
er
C
04
O
s:
.24)
:.0r
o
4c
Y - -v
0o0 0oN
'C
4)
0S
-
C
0
C
c4)
'
14
0
;=
0
=
zr
C.
C.)s
~
Ct
CZ
c t
70
N
'O
Unlike what we observed in many species in response to cadmium, there appears
to be no stoichiometry between phytochelatin and other intracellular metal concentrations.
This may have to do with the ability of the particular metal to effectively activate the phytochelatin synthase. In vitro, Grill et al (1989) observed that, at a given concentration,
cadmium most effectively activated the synthase, whereas Ag and Pb restored 58% and
43% of the activity respectively, and Zn and Hg less than 35%. At the same time, different
metals partition between the membrane and the soluble fraction of the cell in different proportions. For example in Isocrysis galbana, Reinfelder et al (1994) reported that 80% of
the silver (AgT= 17 nM) is membrane bound (pelletted at 750 g for 5 min.), and that
depending on cell growth rate, membrane bound zinc can range from 11 to 61% of the
total. Thus the extent of activation of the phytochelatin synthase enzyme upon exposure
to a metal (and the resulting phytochelatin concentrations) will depend not only on the cellular metal concentration but also on the nature of the particular metal and its partitioning
within the cell.
There is a slight increase in phytochelatin production with increasing copper, but
at the highest copper concentration production seems to level off. This may reflect an
inability to synthesize more or a decrease in cell size under copper stress, but it may also
be due to another detoxification or resistance mechanism being employed under steady
state at high copper concentrations. It has recently been discovered that some higher
plants also have a metallothionein-like gene (de Miranda et al., 1990; Evans et al., 1990).
de Miranda et al (1990) measured increased mRNA transcripts of the metallothionein-like
gene in the copper tolerant plant Mimulus guttalus exposed to copper. The short term
incubations of T. weissflogii with copper overshot the steady state concentration by almost
a factor of ten. This suggests that on the short term phytochelatin may be employed as a
detoxification mechanism for copper, but over time the mechanism may shift to a more
stable, gene regulated mechanism akin to metallothionein production.
The induction of high concentrations of phytochelatins by copper in E. huxleyii
may simply reflect the variability in response among various species exposed to different
metals. But this response may in fact be unique to this species which is uniquely able to
grow at high copper concentrations. Of all the prymnesiophytes, the strains of E. huxleyii
have been observed by Brand et al (1986) to be the most tolerant to copper and cadmium.
71
While its growth is reduced at elevated copper, the organism is able to maintain this
decreased growth up to very high copper concentrations.
Our observation of rapid phytochelatin production upon exposure to cadmium is in
accord with previous studies (Grill et al., 1987; Robinson et al., 1988). The relatively
large intracellular pool of glutathione is converted rapidly into phytochelatin by an enzymatic pathway (Grill et al., 1989). Our observation of the rapid loss of phytochelatin from
the cell upon elimination of cadmium from the medium was less expected. The phytochelatins are either rapidly degraded back into glutathione or amino acids or exported. We
can rule out the export of the cadmium-phytochelatin complex since accumulated cadmium has been shown to remain in the cells upon removal of metal exposure (Harrison
and Morel, 1983). Clearly the dynamics of cellular phytochelatin upon changes in environmental conditions deserve further study. It appears that phytochelatins concentrations
may be carefully controlled by a balance of synthesis and elimination.
Overall it is clear that phytochelatins are important intracellular constituents which
are always present in all of the eucaryotic marine phytoplankton examined in the previous
chapter and the intracellular pool of these peptides is tightly controlled in response to inorganic metal concentrations at levels that are relevant to marine ecosystems. Cadmium,
and to a lesser extent copper, appear to be the metals most likely to induce high phytochelatin concentrations in polluted waters. Thus measurements of phytochelatin in the field,
such as those reported in Chapters 2, 5 & 6, may reflect exposure to cadmium and copper.
Acknowledgments
This work has been funded by grants from NSF, ONR, EPA, MIT Sea Grant, and
the MWRA. Special thanks to Robert Mason who provided cells for the mercury experiments.
72
References
Brand, L., W. G. Sunda and R. R. L. Guillard. 1986. Reduction of marine phytoplankton
reproduction rates by copper and cadmium. J. Exp. Mar. Biol. Ecol. 96: 225-250.
Bruland, K. W. 1989. Complexation of zinc by natural organic ligands in the central North
Pacific. Limnol. Oceanogr. 34: 269-285.
Bruland, K. W. and R. P.Franks. 1983. Mn, Ni, Cu, Zn and Cd in the Western North
Atlantic. p. 395-414. In C. S. Wong, E. A. Boyle, K. W. Bruland, J. D. Burton and E. D.
Goldberg [eds.]I,Trace Metals in Seawater. Plenum Press.
Cutter, G. A. 1991. Trace elements in estuarine and coastal waters- U. S. studies from
1986-1990. Revs. Geophys., Suppl. 29: 639-644.
de Miranda, J. R., M. A. Thomas, D. A. Thurman and A. B. Tomsett. 1990. Metallothion-
ein genes from the flowering plant Mimulus guttatus. FEBS. 260: 277-280.
Evans, I. M., L. N. Gatehouse, J. A. Gatehouse, N. J. Robinson and R. R. D. Croy. 1990. A
gene from pea (Pisumnsativum L.) with homology to metallothionein genes. FEBS Lett.
262: 29-32.
Flegal, A. R. and S. A. Safiudo-Wilhelmy. 1993. Comparable levels of trace metal contamination in two semi-enclosed embayments: San Diego Bay and South San Francisco Bay.
Environ. Sci. Tech. 27: 1934-1936.
Gekeler, W., E. Grill, E.-L. Winnacker and M. H. Zenk. 1988. Algae sequester heavy metals via synthesis of phytochelatin complexes. Arch. Microbiol. 150: 197-202.
Grill, E., S. Loeffler, E.-L. Winnacker and M. H. Zenk. 1989. Phytochelatins, the heavymetal-binding peptides of plants, are synthesized from glutathione by a specific yglutamylcysteine dipeptide transpeptidase (phytochelatin synthase). Proc. Natl. Acad. Sci.
U.S.A. 86: 6838-6842.
Grill, E., E.-L. Winnacker and M. H. Zenk. 1985. Phytochelatins: the principal heavymetal complexing peptides of higher plants. Science. 230: 674-676.
Grill, E., E.-L. Winmnackerand M. H. Zenk. 1987. Phytochelatins, a class of heavy-metalbinding peptides from plants, are functionally analogous to metallothioneins. Proc. Natl.
Acad. Sci. U.S.A. 84: 439-443.
Harrison, G. I. and F. M. M. Morel. 1983. Antagonism between cadmium and iron in the
73
marine diatom Thalassiosira weissflogii. J. Phycol. 19:495-507.
Howe, G. and S. Merchant. 1992. Heavy metal-activated synthesis of peptides in Chlamydomonas reinhardtii. Plant Phys. 98: 127-136.
Hudson, R. J. M. and F. M. M. Morel. 1993. Trace metal transport by marine microorganisms: Implications of metal coordination kinetics. Deep-Sea Res. 40: 129-150.
Lee, J. G., S. B. Roberts and F. M. M. Morel. submitted. Cadmium: a nutrient for the
marine diatom Thalassiosira weissflogii. Limnol. Oceanogr.
Martin, J. H., R. M. Gordon, S. Fitzwater and W. W. Broenkow. 1989. VERTEX: phy-
toplankton/iron studies in the Gulf of Alaska. Deep-Sea Res. 36: 649-680.
Mason, R. P., W. F. Fitzgerald, J. Hurley, A. K. Hanson Jr., P. L. Donaghay and J. M.
Sieburth. 1993. Mercury biogeochemical cycling in a stratified estuary. Limnol. Oceanogr.
38: 1227-1241.
Nimmo, M., C. M. G. van den Berg and J. Brown. 1989. The chemical speciation of dissolved nickel, copper, vanadium, and iron in Liverpool Bay, Irish Sea. Estuarine Coastal
Shelf Science. 29: 57-74.
Parsons, T. R., Y. Maita and C. M. Lalli. 1984. A Manual of Chemical and Biological
Methods for Seawater Analysis. Pergamon Press.
Price, N. M., and others. 1991. Preparation and chemistry of the artificial algal culture
medium Aquil. Biolog. Oceanogr. 6: 443-461.
Price, N. M. and F. M. M. Morel. 1991. Colimitation of phytoplankton growth by nickel
and nitrogen. Limnol. Oceanogr. 36: 1071-1077.
Rauser, W. E. 1990. Phytochelatins. Annu. Rev. Biochem. 59: 61-86.
Reinfelder, J. R. and N. S. Fisher. 1994. The assimilation of elements ingested by marine
planktonic bivalve larvae. Limnol. Oceanogr. 39: 12-20.
Robinson, N. J. 1989. Metal-binding polypeptides in plants. p. 195-214. In A. J. Shaw
[eds.], Heavy metal tolerance in plants: Evolutionary aspects. CRC Press.
Robinson, N. J., R. L. Ratliff, P. J. Anderson, E. Delhaize, J. M. Berger and P. J. Jackson.
1988. Biosynthesis of poly(y-glutamylcysteinyl)glycinesin cadmium-tolerant Datura
innoxia (mill.) cells. Plant Sci. 56: 197-204.
Safiudo-Wilhelmy, S. and A. R. RFlegal.1991. Trace element distributions in coastal waters
along the US-Mexican boundary: relative contributions of natural processes vs. anthropogenic inputs. Mar. Chem. 33: 371-392.
Steffens, J. C. 1990. The heavy metal-binding peptides of plants. Annu. Rev. Plant Phys-
74
iol. Plant Mol. Biol. 41: 553-575.
Wallace, G. T., J. H. Waugh and K. A. Gamrner.
1988. Metal distribution in a major urban
estuary (Boston Harbor) impacted by ocean disposal. p. 67-78. In D. A. Wolfe and T. P.
O'Connor [eds.], Urban wastes in coastal marine environments. Robert E. Kreiger Publishing Co.
Zhang, H., C. M. G. van den Berg and R. Wollast. 1990. The determination of interactions
of cobalt (II) with organic compounds in seawater using cathodic stripping voltammetry.
Mar. Chem. 28: 285-300.
75
Chapter 5
Phytochelatins in Coastal Waters
To be published in collaboration with James Moffett.
76
Abstract
Particulate phytochelatin is measurable in coastal water samples and varies over an
order of magnitude. Dimer concentrations (n=2) range from 0.5 - 17 gmol (g chl a)-l; the
lowest concentrations are below those measured in laboratory studies (Chapter 3) and the
highest concentrations are similar to those measured in laboratory cultures grown in the
synthetic seawater Aquil with picomolar free cadmium (10-12M). A year long study of
phytochelatin concentrations from a transect of Boston Harbor out into Massachusetts Bay
finds that for the most part decreasing seaward concentrations are typical, except in
August when there was no trend and in October when concentrations were very low and
there was a subtle seaward increase. Plotting the data as a function of salinity reveals that
phytochelatin concentrations are not simply a function of freshwater trace metal inputs. A
comparison of phytochelatin concentrations and copper measurements from various harbors on Cape Cod and Providence Harbor is made to assess the role complexation of metals in natural waters may have on phytochelatin production. The results are in accord with
the laboratory result that phytochelatins are a function of the free metal rather than total
metal.
Introduction
The trace metal chemistry of coastal waters is not well understood. Total metal
concentrations are typically higher due to anthropogenic inputs and natural continental
run-off, but coastal waters are also rich in organic compounds that are capable of complexing these metals. Thus the paradoxical result of higher total metal concentrations but
lower free, or available, metal concentrations is possible. It is in fact the high concentration of organic material which has prevented the direct application of electrochemical
techniques utilized in the open ocean to determine metal complexation in coastal waters.
In a unique study, Hering et al (1987) measured copper complexation at two sites
in the New York Bight; one site was at the N. Y. C. sewage sludge disposal sight and the
77
other off Montauk Point, a relatively pristine site. After extensive analytical effort, they
calculated that the free cupric ion concentrations were roughly equal at the two sites. Both
techniques utilized in this study (bacterial bioassay and fixed-potential amperometry)
require substantial calibration and rely on many assumptions to obtain estimates of ambient free copper concentrations. The direct measurement of a biological indicator of trace
metal availability, such as phytochelatin, could greatly advance our understanding of trace
metal distribution and availability in coastal waters.
The objectives of this study were to determine if phytochelatin concentrations in
the field could be measured and to begin unraveling their relation to the complex trace
metal chemistry of natural seawater. Samples from several coastal environments were
measured-
an extensive year long sampling of Boston Harbor and Massachusetts Bay
and a single sampling of several harbors on Cape Cod and in Providence in September of
1993. Three new transects are presented in addition to the transects presented in Chapter
2, and all of these measurements have also been replotted against salinity for further analysis. Measurements of phytochelatin concentrations in samples from Cape Cod and Providence Harbor are compared to electrochemical measurements of free and total copper
concentrations.
Materials and Methods
Boston Harbor - Massachusetts Bay samples were collected as described in Chapter 2, details of the sampling dates, times and the respective tides are listed in Table 1 and
the station positions are listed in Table 2; not all the stations were sampled each cruise.
Chlorophyll a was measured on a Turner designs fluorometer after acetone extraction; the
fluorometer was calibrated spectrophotometrically with a vigorously growing phytoplankton culture (Parsons et al., 1984). The range of chlorophyll concentrations measured for a
given transect are listed in Table 1. Phytochelatin measurements were made as described
in Chapter 3 and Appendix A. Temperature, salinity and nutrient measurements were
made by Battelle Ocean Sciences. All data is listed in Table 3.
Cape Cod and Providence Harbor samples were collected by J. Moffett during
September of 1993; sites are listed in Table 4. Water was collected at the various sites uti-
78
I
-
,V
..
~1f1
)
r t
0
-
.
'+
fIC
v
=
t
C)
+
4.
m
I
9
tn
m
I
$
-
C4
w
iv
V
Iq
'E
&n
-
1
%
to
6
.
I
m
I"
\C
I
1-
--
z
6
CN
.0
Ct
;
k5
v
=
Cl
(5i
la
lz
a
O
N
t
CSi
c
cn
0,
-
v!
_- m
V_:
.
..
t ..
O
C
0)o
V)
-t
m
O
\.
O
..
..
O
.
V)
..
In
-
.1
u
.1
la
0)
*U
0.)
rA
ua
0
t
q)
0V~
c~
Q
C).
oi
In
cr
Cl4
C'o
D
E
c::
C l4
0) N
-
C~
-
$
0
=
D
X
¢ = <
O
u
= >°,
79
2E:
::
an
tb
C)
C.)
C)
0
CA
0
0i
z0
0
cd)
0
4)
i
m
0
4.
0
4e
5(D
la
.)
O
u
to
E I
o.
0
CZ
O
- t
vun
m
00
Q
) LS
C. 00
,,C
C.,,
m
t
'~
0)
N tn
=~~~~3
=
^
m
Ce
C)=
:z
tb
0
to
C6
0
.C
C,,
.z
v
cr)
~~~
*-<~~~~~
~~~~-4
-
80
-4 -4
Z~ ;. -v. v.;I
V
et
u
1-4
-'t
W)
c
=
C
-q
-.
r-..
Co5o
0
4
-
-4
C
-4
t-
o'
-t
lR
l
~c
r- ef)\0
00\0N
ooo
)
= -
00 V
;
Cle
C: \0
(q
V)
Z^
G.
CZ
e-
0
'4
CA
O
L
Ct
= -
z2
o C -_
o
0_
- \0 Cl oX
z4)
D C
O
o
e
a \o 00
ec C
C
S0cn
Co - c'tt
t Co 0 6
o
t- t-
00
= 'I
cq -O
-
Y
-S
C) C,en
-4
- "tD CD C
o>
9
0
-D -I
C). 0. 9,
0
O O
I O O 6
G C
CZ In N
= O C
O)
S
4O
CZ
o
.
.g
E
o
=: C7,4 -o- 0
6oooo
t
O
= -t
)
o
0
-
0C
O5 O O~ O
>
...
f=
00
-,-q
"zr -
.4
-4
C-i _4 Ci C-
CNC.
CN cc C
I
m
en
U0. Un
_
V)
=CZ
CZ _
O U
Cl4
u_
Cl-4\C
00Cl
o6
--,C
=0% tn
6 14 tri
4cl
t-Z
5 -tl
. . .
Men cn
O~
08 c
m r
Cl
W
0>66
= cqNC
00 - j=
~.-
-
emm
00
cr
-
-_4 -_.
C
Cl
cr vU: O m t im
^- - C\
Z0
_ o u) O 4
_* cz
- 4- ,
Vf:
Gl
-
.S
3-
et:: -4.=0
*o
st
.'I
E V
\s C'
O-
=
-
t
C
_-
"
C14 "e
-
OC)
c
00
C
M
=
Hc#O
Cl4\C
-
E
C
.0
.1
(In
o0
tn00 00
C\
C'4
en en
4:1,
-:4 6
Co
4
C
-.- I
Ci
-. 0
-
m C1 eq
N N
,tcn" CN C
crc
tn IR m
-4
N Cl C N t \l
"
.. .. .. ..
=C
o C6 =C
R =o
1-
MS
.
--,~t:~6
0
en
z
en
01
Cl
;.
en
14 c-
4): L1)
Li.
Cl4
Cl
CL. -,9L
't3
Cl4
r-
CIO
\C tn
-" 5E M.
81
L"
4
LL"44
--
u
C..2 --4 C-lt
is [J.
Z Z ",
C
-4
U)
0
cq N0 0 0 - n It
CN
c0
2CZ
CO-
c-
c9
000 00
Ok
WX
'e
ts
U) 0
00
Cl4
= =
65
o
6 6
6O
o1
O
0 00
0
OO
O
0 0 C)
0
C
0
C
Nt
N
c 0
O 0q
O -
-
-
O
Cle
C
N0 0 0 0 0 0
0
coe
_4
C-0,00
O
-I
-4
O)
O
O-
O)
0
C) 6 6
C
5
0
I
0
0
C, C- C140
O>-- -
m
C:=
C .~ .~ .l .
O
O O
c
.
.
.
tl-
OO
Ct
=:
0 0
Cq O
-C
o
CClO6
.-cCZC
COD
oCt
o;-)
=
.
.U o
00
6,
t.-
6o
en
) cO
O
O
14O C
7 =0Nr
=
0
oo
eLq
tn
C_^S:.
00Nc
c
_
-
u:
.
- f -t
n o
en-
Ce
000>_
CO
In
U
LLL
C
N
o6 o6
f4
m
_,
V.
-
- e4 ,
0-
ON -
N
ON 00 00
oc
cn
Cli
r-
CD C
..
On
_,
C
-
V) tr
4 r.
-
.
.
Cf
Lr
N
^
cr-O
N ^f.
Hcl
.1C)
O
,.
a)
.E
O
6666tr
5:
CU)
t:
00
>
C;
6c0tN6 0=0 ,^C-4W
0
cCZ; °~C 0d6Nt° O~
e
; 1.4L
-
-t _-rtn
L Z Z " Z
82
_c r-'t
en
Cl\
'OC
c 4
-
.
o~~~~~o
· o
o,
\Co
q (' ZZc".
". "
o,
Z
.
-,I
,--
,.c
tr
-
t1
Z
u).
_
oo
Y-O6
2
cA L6
G) ;Y
0
C)--66
6
CZ CNn
co
Co
0N O
tM
00 -
0 CON -
N
-
2- 0
z 6
0
66 6
-
u>~ <<O0<0 0 0 0S
-
N
0
0O
-O
0
E
n C
6
6
6
CZ
0
.
O O - i66
0
en
d600t
o~
cn
t
O~O
O4--O -O4
CZ'O- -
O
Cd
-0
n
en
.
.
V
-:
.
r0
.
dw
t
.
m .
t-
lM
D
-t
t
O~ O V
0-4
~ ~
=~
C,
C4a:t C;O
C
<
Kenm n^
rC c
s:- r
N
._
ON
H00
E p
,Nm
I-i "o
oc 0(
tl
N.
--m
0 \C 0
- -", - o -c. 00 N00 _ r, - \c oc 6
O
-
-
_
\,C
.
Ct r'
=
n C)
_r 1
O
-
=
0
00 T.L.-
"T
0O
N r4
tn m
C
=O
O
-
-
N
D.
,:
-,
-
-4
ON\00
N
4 tn
,I.
cr
oo cr
.. .
0
.
-
N
-
Q
_:: V> G
ON
C
0oN mt1
O
-4_-
N
-
-0 z
-
Cn
O
C
-
-
1-O
o.,,
G
VI) V
1-4
0C:
-
_
-
00
-
m r- - oC
t
O~ It
tri
4 r0 6
V) C4
:
. tO
Ok
"t
-1.
L
83
\,D
V,
-J.
\
t--
E: -J.
-OI
CO,
0
0
2
CZ
.u
IC.)
CYO
r44
Cud
C
C).
C4
C CO
U co
CZ
C)
co
ob
z
OC) u
z
.-
ou
oO ;u
,-,
z
E
3
~4-4
~Cu
._
E
0
3u
'
Cu '0
· ,-
o-o C
C
Cu
v <rN
o
*0
E.o
C)
o 0
tca
-^
0
*;E
C)
c)
C
1t'
OC N
a.)O
~C)
E
r-£O.T, r.-
84
V
I
+
u
C.)
----q
I
--4
V
0
0
,t
00
;~ 0
0-
.I
1
'
I
.4-I
I
W)-
I
't
NCI C
-
00~~~~~~-
V
t)
-
>
· ,-d
0
0
C-
t
c
C
-
-
_--
_-4
a
oqi
J
:I
)
C\
e4
ci c,
C'
('
N
F
C
xD
ci
=i-
a
0 ~0
'n
Cu m
-
·
:&
=
=,co
o
U
tn
M
V~
-
0(
C
Ni
tn
r,
Ci~
cl
0
-
F- 0
E
0
u-
m
rA
OE
II
":t
I
Ci
00
-
c4
cli
N
c4
--
=
0
-
0
N
o >j;
0U Co
tn
cI "
il*
cl
0,
0
->
to
.=>..0
-
-
-
--
0
0
.0
c1
0
1-4
.bH
GQ "a.-13
>
-
Qn
o
-C
-e
, O v
et
r)
Sw
-
0
l;
C.)
c
0
0
En
85
0
lizing trace metal clean techniques, filtered later in the same day in the laboratory, and
samples for phytochelatin and chl a analysis were stored in liquid nitrogen until analyzed.
Copper measurements were made by J. Moffett using cathodic stripping voltametry.
Results
Phytochelatin measurements from transects on the first three sampling dates exhibited a distinct decreasing seaward trend (Chapter 2). In August and October the data are
significantly different and in February of 1994 concentrations returned to the gradient
observed the previous February (Figure 1). As the flushing of Boston harbor is driven by
the tides (Signell and Butman, 1992), an evaluation of the sampling episodes with regard
to the tidal cycle is necessary. Stations were sampled roughly from east to west, but were
not always sampled in the same order.
For the February, April, and June transects sampling began near low tide (Table 1),
and the ship was generally headed out into an on-shore tidal flow. The phytochelatin measurements display a clear decreasing trend from the harbor seaward as discussed in Chap-
ter2.
In August sampling began -1.5 hours after the high tide, and there is no systematic
trend in the phytochelatin data. This could be due to the fact that the first samples are
taken shortly after hide tide when, presumably, the harbor is fairly well mixed; subsequent
samples were taken as the tide was heading out and the last few as it returned after low
tide. The samples were collected at very different times during the tidal cycle as compared to the other sampling dates. The chlorophyll a concentrations were particularly
variable on this sampling date ranging from 0.5 to 14 Rg 1-1.
October phytochelatin concentrations, on the other hand, shown in detail with the
n=3 & 4 chain lengths included in Figure 2, display an interesting gradient from the coast
seaward different from the previously observed trend. The concentrations start low at the
pier, increase slightly near the outfall at Deer Island, decrease for roughly 25 kilometers,
and then the two farthest data points suggest an increase out into Massachusetts Bay. The
peak at or near Deer Island is seen in many of the transects, including perhaps the August
transect. Again, samples were taken during a different part of the tidal cycle; sampling
86
25
-
February 1993
20
August 1993
_
15
0
I
10
S
I
5
0
I
0
.=
I
I
0
*
.
~
10
20
30
40
S
*
10
0
50
Sq
S
20
30
40
50
25
0
April 1993
October 1993
20
0
15
S
II
10
rC
C)
11
0a)
5
C)
11
0
i..i .
I
0
10
I
el
30
20
40
@.. 20 _x30
*0
I
50
10
40
50
25
June 1993
February 1994
20
15
I -I
10
5
0
.I
I ,- If ,. I
1
I
0
10
-
20
Ip
30
*p
40
50
0
10
20
30
40
50
Km from Black Falcon Pier (Inner Boston Harbor)
Figure 1. Phytochelatin concentrations (n=2) plotted as a function of distance from
harbor. Particulate phytochelatin concentrations are reported in units of tmol
(g chl a)-l; samples were collected on the dates listed in Table 1. Data points are the
average of duplicate samples; error bars represent the two points averaged, and where
not shown the error bar is within the symbol. See Figure 1, Chapter 2 for the actual
location of stations. Points at zero are samples which were below detection, the open
circle indicates a sample (the duplicate of which was below detection) in which a low
phytochelatin measurement was divided by a very low chlorophyll a value.
87
5.0
* n=2
October 1993
v n=3
O n=4
Cdl
0
0
0
So
2.5
1)
0
-f-I
0
0
9
V
V
0
0
--
V
0.0
vV
7
V
I .
0
.
10
.
20
.
30
.I
40
50
Km from Black Falcon Pier (Inner Boston Harbor)
Figure 2. Phytochelatin concentrations (n=2, 3, & 4) plotted as a function of distance
from harbor in October. The scale of the y-axis has been changed to 0 to 5 pmol (g chl
a)-1 to show the detail of the profile. Data points are the average of duplicate samples;
error bars represent the two points averaged, and where not shown the error bar is
within the symbol.
88
began shortly before high tide and continued through the ebb flow.
In February 1994, the decreasing seaward trend of phytochelatin concentrations is
again apparent. Sampling began three hours after low tide, more like the first three sampling dates.
Plots of phytochelatin against surface salinity reveal that phytochelatin concentrations are not simply a function of trace metals associated with fresh water inputs (Figure
3). In February, 1993, the salinity increases as a function of distance from the harbor, thus
plotting the phytochelatin concentrations of a function of either parameter results in a similar profile. The surface salinity in June, August and October is constant from one station
to the next, thus plots of phytochelatin vs. salinity are simply clumps of data at one salinity. Some variability returns to the salinity profile again in February of 1994, but there is
no obvious relationship between phytochelatin concentrations and salinity.
The samples collected from various harbors on Cape Cod and from Providence
Harbor in September 1993, contained phytochelatin concentrations ranging from 2.2 to
115 gmol n=2 (g chl a)-1 (Table 4). Except for the high measurement in Eel Pond, the
range is very similar to the range of concentrations measured in the typical harbor
transects. The concentration in Eel Pond-115 pmol n=2 (g chl a)-1 -exceeded
all previ-
ous concentrations measured in natural samples, but is still well within concentrations
measured in laboratory cultures. It is likely that this concentration of phytochelatin is consuming a significant fraction of the phytoplankton population's intracellular glutathione
(glutathione= ~600 gmol (g chl a)-1 in T. weissflogii), which may be a useful way to
access the physiological implications of phytochelatin measurements.
Total phytochelatin (reported as I (y-glu-cys) = 2(n=2) + 3(n=3)) is plotted in Figure 4 against both total copper and free copper. Total copper concentrations are all
roughly the same, but free copper concentrations vary three orders of magnitude. While
there is no relationship between phytochelatin concentrations and total copper, phytochelatin concentrations are related to free copper concentrations (Figure 4). Although there
are only a few data points, the relationship appears to be much like that observed in the
laboratory study-above free copper concentrations of ~pCu= 11, phytochelatin concentrations begin to increase.
89
25
25
February 1993
20
20
15
15
10
10
5
5
0
30
31
32
0
33
25
30
31
32
33
25
C)
A
0
April 1993
October 1993
20
20
15
15
10
10
5
5
.0
C)
11
,4
ao
VI
eS
0
29
30
31
0
32
25
30
31
32
33
25
i
~
June 1993
February 1994
20
20
15
15
10
10
X
I
31 *30
31
32
32
,
5
5
s
0
30
a
31
32
0
33
30
3
33
Surface Salinity (PSU)
Figure 3. Phytochelatin concentrations (n=2) plotted as a function of salinity. The
same data as in Figure 1(except for the pier samples, data point at distance= 0), replotted as a function of surface salinity in units of psu. Salinity was measured by Battelle
Ocean Sciences at -2 m with a standard CTD mounted on a rosette; data supplied by
the MWRA.
90
Total copper log [Cu]T (
-10
0
0
o)
-8
-6
I
I
10 3
1-
0
o
t;
102
I
0
I
101
*
v
Q0
0
-14
I
-1
-12
-10
Inorganic copper log [Cu+2] (
Figure 4. Phytochelatin concentrations ( y-glu-cys) plotted as a function of copper
concentration. Samples collected from various harbors and a pond on Cape Cod and
from Providence Harbor. Sampling sites listed in Table 4. The top x-axis is log of the
total copper concentration (open circles), and the bottom x-axis is log of the free copper concentration (filled circles).
91
Discussion
Phytochelatin concentrations are roughly 2- 20 pmol (g chl a)-1 , both in the Boston
Harbor/ Massachusetts Bay area and in several harbors on Cape Cod which range from
heavily utilized marinas to fairly pristine bays. In the Boston Harbor/ Mass Bay transects,
phytochelatin concentrations are not simply a function of trace metal inputs via freshwater
sources, but appear to have distinct spatial relationships depending on the mixing regime
due to tides or perhaps some biological factors. A preliminary study comparing copper
concentrations to phytochelatin measurements confirms that, as found in the laboratory,
phytochelatin production in the field depends on the free metal concentration rather than
on the total metal.
Concentrations measured each sampling date were overall very similar to each
other, ranging from 2-20 pmol (g chl a)- 1 , except in October when n=2 concentrations
ranged from 0.5 - 3 gmol (g chl a)-1 . These particular samples were taken during an
intense fall bloom when concentrations of chl a were 8 - 24 gg 1-1. The large amount of
biomass associated with this bloom may have diluted the exposure of individual cells to
available metal concentrations thus resulting in lower phytochelatin concentrations. The
phytochelatin concentrations in October when calculated per liter are actually slightly
higher than other sampling dates (5-35 gM n=2 as compared to 0.6- 14 gM n=2 in February 1994).
The pattern of elevated phytochelatin concentrations near the inner harbor decreasing as one gets farther out into the bay supports the notion that these measurements reflect
elevated metal concentrations due to coastal freshwater sources which are diluted out into
the bay. However, a strict correlation with salinity is only observed in the February 1993
surface salinity-phytochelatin plot. The salinity remains variable for the April transect,
and the profile observed is similar to that as plotted with distance, although the absence of
a pier sample (distance=0) in the salinity profile diminishes the decreasing seaward trend.
The constant surface salinities seen in the next three plots-June, August, and Octoberare likely due to a lens of low salinity water on the surface as a result of increased freshwater runoff during the summer months.
Boston Harbor receives trace metal inputs from a number of anthropogenic
92
sources including industrial and domestic sewage wastes. Together the sewage treatment
plants at Nut and Deer Islands contribute up to 50% of the total fresh water input into Boston Harbor (Wallace et al., 1988). Three rivers make up the rest of the fresh water inputthe Charles, the Mystic and the Neponset-the Mystic River contributing significantly to
trace metal concentrations in the harbor (Wallace et al., 1988). There is also a contribution
by the southern coastal current which brings fresh water and trace metals into Massachusetts Bay from rivers north, such as the Merrimac River. Each freshwater source varies
considerably with respect to trace metal concentration and organic content. Thus it is
somewhat expected that phytochelatin concentrations would not simply be a function of
salinity, but that there would be a complex relationship between the different sources of
metals and the resulting phytochelatin concentrations. Other constituents, such as nutrient
concentrations, may potentially help to distinguish the different fresh water sources of
trace metals. An examination of the nutrient data reveals that some of the higher phytochelatin concentrations do correspond to high ammonium concentrations which would be
indicative of the sewage plume near Deer Island.
Seasonal differences between phytochelatin concentration profiles with respect to
distance from the inner harbor have several possible explanations. The noisiness of the
August sample transect may be due to tidal effects, or could be due to the species composition of the phytoplankton population at this time of year. According to a workshop
report released by MWRA, in August 1993 at least 40% of the total number of phytoplankton cells were microflagellates and at some stations the population was dominated
by this group (Hunt and Steinhauer, 1994). This is in contrast to the other sampling dates
for which phytoplankton species data are available (February, April, June and October of
1993) where the phytoplankton assemblage is typically dominated by diatoms. Included
in microflagellates are primarily unidentifiable small eucaryotic cells. This population
probably contains a much more diverse collection of phytoplankton species than those
samples dominated by diatoms. The production of phytochelatin may be more variable
because of this more heterogeneous population.
Some contribution to total chl a in coastal waters is made by cyanobacteria- particularly Synechococcus sp. The relative abundance of these organisms might contribute
to seasonal variations in phytochelatin measurements because their numbers fluctuate sea-
93
sonally. If there is significant contribution to the total chl a by cyanobacteria, then phytochelatin measurements are artificially lowered by the normalization to total chl a, since
prokaryotic organisms have not been found to make phytochelatins (Olafson et al., 1979).
Waterbury et al (1986) examined seasonal variation of Synechococcus numbers in Woods
Hole Harbor and found that they typically bloom in the early spring as temperatures rise,
peak through April and May, remain fairly constant through the summer, and then as the
temperatures drop in early winter, abundance drops off rapidly. This may explain the
slightly lower concentrations of phytochelatin (per g chl a) in April, but probably does not
explain any of the variability observed in August.
The October transect is distinct in two ways: the relatively low phytochelatin concentrations and the modified seaward trend. The very low phytochelatin concentrations
are probably due to the extremely high biomass present in these samples. The metals are
diluted by the high biomass associated with the bloom. The modified seaward trend may
be a result of sampling at different times with respect to the tidal cycle. In addition, the
resulting profile suggests that the interaction between trace metals and organic material is
an important factor controlling phytochelatin production. The phytochelatin concentration at the pier, the only station within Boston Inner Harbor where metal concentration are
quite high (Wallace et al., 1988), is presumably lowest because of the high concentrations
of organic material complexing the metals. The peak observed at ~12 km is likely due to
trace metal inputs from the sewage outfall on Deer Island which gradually decreases as its
influence is diluted by the ebb tide. At -30 km concentrations begin increasing again perhaps as the organic material is diluted and /or degraded releasing complexed trace metals.
It is clear that phytochelatin measurements need to be compared to measurements
of available metal concentrations. It is however extremely difficult to measure speciation
and collection of trace metal clean samples on a ship requires a great deal of expertise and
equipment. In collaboration with J. Moffett, this is possible for the first time. Though this
correlation is limited by the number of samples, the response with respect to copper is
very similar to what is observed in the laboratory with cultures of Thalassiosira weissflogii in response to copper additions. There is no effect on phytochelatin concentrations
until a threshold of about pCu=l 1, when these concentrations increase.
Sampling locations represent diverse coastal environments. All of the Cape Cod
94
sites are fairly saline environments with little freshwater input, >30 %o/, whereas the
salinity at both Providence Harbor sites was roughly 22 %o (J. Moffett per. comm.). The
different sites on the Cape vary in size and flushing rates. Boating activity also varies
from site to site. Despite these differences, total copper concentrations are roughly the
same at all sampling locations, but free copper varies by three orders of magnitude.
It is however not likely that copper is the only factor controlling phytochelatin
concentrations in these waters. Organic complexation is the parameter that varies from
site to site, consequently other metals, particularly cadmium, will likely be complexed in a
similar manner. Thus the correlation observed between phytochelatin and free copper
concentrations cannot be interpreted as a causal relationship.
The results of these studies are quite encouraging; phytochelatin measurements are
variable in coastal systems and some sense can be made of the various profiles encountered. Measurements do not simply reflect freshwater sources of trace metals in Boston
Harbor, but probably reflect the complicated interaction of organic material and trace metals from various sources, as well as the tidal flushing of the harbor and bay. As techniques
for measuring metal speciation are applied more and more to coastal environments more
information will be available to establish the cause and effect relationship which is
undoubtedly at work.
Acknowledgments
I would like to thank the captain and crew of the R/V Marlin, Batelle Ocean Sciences, and the MWRA for ship time and ancillary data. Thanks to Karina Gin and Keith
Lichten who on several occasions helped collect the transect samples. Special thanks to
Jim Moffett who collected and filtered samples for the Cape Cod/ Providence Harbor phytochelatin measurements, as well as generously providing the copper concentrations and
speciation data.
95
References
Hering, J. G., W. G. Sunda, R. L. Ferguson and F. M. M. Morel. 1987. A field comparison
of two methods for the determination of copper complexation: bacterial bioassay and
fixed-potential amperometry. Mar. Chem. 20:299-312.
Hunt, C. D. and M. Steinhauer. 1994. MWRA Nutrient Indicator Workshop. Battelle
Ocean Sciences.
Olafson, R. W., K. Abel and R. G. Sim. 1979. Prokaryotic metallothionein: Preliminary
characterization of a blue-green alga heavy metal-binding protein. Biochem. Biophys.
Res. Commun. 89: 36-43.
Parsons, T. R., Y. Maita and C. M. Lalli. 1984. A Manual of Chemical and Biological
Methods for Seawater Analysis. Pergamon Press.
Signell, R. P. and B. Butman. 1992. Modeling tidal exchange and dispersion in Boston
Harbor. J. Geophys. Res. 97: 15,591-15,606.
Wallace, G. T., J. H. Waugh and K. A. Garner. 1988. Metal distribution in a major urban
estuary (Boston Harbor) impacted by ocean disposal. p. 67-78. In D. A. Wolfe and T. P.
O'Connor [eds.], Urban wastes in coastal marine environments. Robert E. Kreiger Publishing Co.
Waterbury, J. B., S. W. Watson, F. W. Valois and D. G. Franks. 1986. Biological and eco-
logical characterization of the marine unicellular cyanobacterium Synechococcus. p. 71120. In T. Platt and W. K. W. Li [eds.], Photosynthetic picoplankton. Can. Bull. Fish.
Aquat. Sci.
96
Chapter 6
Phytochelatins in the Equatorial Pacific
To be published in collaboration with Neil M. Price and possibly Kenneth Bruland.
97
Abstract
Particulate phytochelatin concentrations in the Equatorial Pacific, normalized to
total chlorophyll a, are similar to those measured in coastal areas, but are significantly
higher when corrections are made for percentages due to prokaryotic chl a. This is likely
the result of low metal chelation and/or metal limitation and resulting higher uptake rates
of available metals. Surface phytochelatin concentrations are roughly the same at stations
across the Equatorial upwelling from 20 ° S to 10° N, and most of the depth profiles have a
distinct subsurface maxima. Profiles taken at the equator and 2 S had less structure than
those north or south of these stations which may be due to more mixing in the water column within the upwelling. Incubations performed on water sampled at four separate stations induced fairly constant concentrations of phytochelatins, although differences
between metal additions and controls were greater within the upwelling zone where water
presumably has less time to accumulate biogenically derived complexing agents.
Introduction
In recent years considerable interest, among trace metal chemists and biologists
alike, has arisen regarding the Equatorial Pacific upwelling region. It is included in a
group of several areas regarded as "high nutrient low chlorophyll" or "HNLC" areas.
Martin et al (1989; 1990; 1991) demonstrated that, by adding iron to water samples collected in these areas, biomass increased and the nutrients were drawn down. Many ships
have thus made the pilgrimage to one of the most pleasant of these "HNLC" areas, namely
the Equatorial Pacific, to test out the many forms of the "Iron Hypotheses" and the counter
hypotheses. During August of 1991, aboard the R/V Moana Wave, I sampled depth profiles from several stations across the Equator and performed several incubation experiments, not to test the Iron Hypothesis, but to look into another hypothesis regarding areas
of newly upwelled water.
In the late 1960's Barber and others performed many experiments with newly
98
upwelled water from the Cromwell current upwelling (Barber and Ryther, 1969) and off
the coast of Peru (Barber et al., 1971). They found that newly upwelled water supported
less biomass than expected given the high concentrations of the major nutrients. Addition
of organic chelators to the water increased the biomass significantly in bottle incubations.
They hypothesized that the chelators either reduced availability of toxic metals (copper
and cadmium) or enhanced the availabilty of nutrient metals such as iron and manganese
(Barber and Ryther, 1969). As these studies were done well before the advent of the "Ultraclean" sampling techniques, in hindsight there are many contamination scenarios that
could explain some of these results.
Nonetheless, it is certainly true that the deeper water below the photic zone contains higher concentrations of trace metals (Bruland and Franks, 1983) and these metals
are less complexed by organic ligands (Coale and Bruland, 1988; Bruland, 1989; Bruland,
1992), thus the inorganic metal concentrations are orders of magnitude higher in water
below the photic zone. Organisms seeding newly upwelled water experience higher free
metal concentrations than those typical of euphotic surface water. Given that phytochelatin concentrations are dependent on free metal ion concentrations (Chapter 3), particularly
cadmium and copper, phytochelatin measurements could indicate the inorganic metal concentration or the degree of metal complexation in surface waters. The equatorial
upwelling is a shallow upwelling; water is estimated to derive from a depth of less than
225 meters (Quay et al., 1983), thus this water will not be entirely characteristic of deep
sea water, but it is enriched with many nutrients relative to average surface seawater.
Depth profiles of phytochelatin concentrations were measured from 20° S to 100 N
to determine if phytoplankton exposure to trace metals, particularly cadmium and copper,
within the upwelling region was significantly different from phytoplankton exposure to
trace metals outside of this region. Incubations of water collected using trace metal clean
techniques were performed with various additions of cadmium and copper to determine if
phytochelatin production could be stimulated and to again determine whether there was
any difference between additions made to newly upwelled water and to water which has
had time to accumulate biogenically derived complexing agent.
99
Materials and Methods
Particulate samples were collected during from August 5 - 29, 1991 aboard the R/
V Moana Wave from 20 ° S to 10° N between 135° and 1520W (Figure 1). Depth profiles
were sampled with 10 liter Go-fio bottles (not trace metal clean), and samples (-4 liters)
were filtered with gentle vacuum pressure (< 5 psi) immediately onto Whatman 25mm
GF/F glass fiber filters; smaller aliquots from the same bottles were filtered similarly for
chlorophyll a analysis (Parsons et al., 1984). Samples were stored in liquid nitrogen dewars until analyzed for either phytochelatin or chl a. Sampling times are as noted in Figure
2.
Seawater for on board incubations was obtained using trace metal clean techniques
(Bruland et al., 1979); acid-cleaned 1 liter polycarbonate bottles were filled with seawater
from morning casts taken with thirty-liter Teflon-coated Go-flo bottles suspended on Kevlar line at a depth of roughly 50% Io (-15 mn).Additions of either cadmium or copper were
made in a laminar flow hood and then the bottles were tripled bagged and left in an on-
deck plexiglass incubator cooled by with surface seawater and covered with neutral density screening to achieve -50% Io . After two days, two one-liter duplicate bottles were
pooled for each treatment and were filtered onto to 25 mm filters; a small subsample (-200
mls) of each treatment was filtered for chl a analysis. Samples were stored in liquid nitrogen until analyzed.
Sample treatment and analytical method for measuring phytochelatin is detailed in
Chapter 3 and Appendix A. Improvements in analytical sensitivity were made part way
through analyzing these samples, thus some stations have more data points at depth than
others.
Results
The first measurements of particulate phytochelatin from open ocean blue-water
samples are shown in Figure 2. The chlorophyll concentrations used to normalize phytochelatin concentrations are plotted against depth to the right of each phytochelatin depth
profile. The phytochelatin concentrations measured ranged from -2- 50 gmol n=2
100
50
40
20
00
20
40
50
160 °
1 40
120 °
100
80O
60°
40
°
20
'
Figure 1. Location of stations occupied during the August 1991 cruise aboard the R/V
Moana Wave.
101
Ug cl L'
jg chl L'
0
I
r
--Oj
0
o.o 0.2 0.4
0.0 0.2 0.4
1- I
T
St.4 5.00 S
50
8:00
100
150
0
200
0
.
,
0.0 0.2 0.4
o.o 0.2 0.4
0.0 0.2 0.4
0.0 0.2 0.4
,
21:30
50
100
0
1.57 S
Xs
150
200
0
.N
50
100
150
1...... .
200
0 10 20 30 40 50
0.0 0.2 0.4
0
50
100
150
200
0
10 20 30 40 50
Phytochelatin (n=2) mol/ g chl a
Figure 2. Depth profiles of phytochelatin concentrations (umol (g chl a)-1 ) at Stations
1, 4, 5, 6, 7, 8 and 10. Latitude of each station is noted on the individual graph, as well
as the time that the samples were collected. Open circles represent samples which
were below detection limits. Symbols represent the average of two measurements
made upon the same sample, the ends of the error bar being the two measurements
(roughly the analytical error). I was not able to run replicates from the samples at Station 6. Chlorophyll a measurements from the same cast are plotted to the right of each
phytochelatin profile.
102
(g chl a)-1 . Surface concentrations are remarkably constant at 12-18 gmol n=2 (g chl a)'1 ,
except for the surface sample at Station 7 which contained ~30 pmol (g chl a)' l . Five of
the seven depth profiles exhibit distinct subsurface maxima somewhere between 60 and
100 m (concentrations roughly two times higher than surface concentrations). These subsurface maxima are most pronounced at stationsjust to the north (Stations 1&4) and south
(Stations 7&8) of the upwelling zone, but there is a slight hint of a phytochelatin concentration maximum at 100 meters in the Station 5 profile in the midst of the upwelling. For
the most part, nondetectable data points are due to the low concentrations of phytoplankton at depth. The upwelling zone is delimited roughly by high surface nitrate concentrations as plotted in Figure 3. Depth profiles of nitrate (Figure 4) at each station reveal the
absence of a nutricline at the equator (Station 6) demonstrating that upwelling is most
intense at the equator.
The single point maximum at 60 m in Station 7 is not simply an analytical anomaly
since both of the other oligomers (n= 3 and n=--4)also increase significantly at this depth
(Figure 5). The sample may have been contaminated with metals during sampling or filtering, but given the relatively short time between sampling and freezing the filter (2
hours), a fairly significant metal addition would have to had occurred (1 nM Cd by comparison to incubation experiments). The longer chain peptides (n=3 and n=4) were measured at some of the other stations as well, and for the most part concentrations were
significantly lower than the n=2 concentrations.
These maxima are not artifacts due to the normalization to total chlorophyll a. At
the surface chlorophyll a to biomass ratios are less and gradually increase with depth, thus
the normalization would artificially enhance a surface maximum not the reverse.
Incubation experiments demonstrate that the algal population is able to synthesize
more phytochelatin in response to cadmium and copper additions (Figure 6). In each
experiment the addition of 1 nM cadmium stimulates phytochelatin production over that
of the control, and the addition of more cadmium (5 nM or 10 nM) stimulates still greater
concentrations. The addition of lnM Cu induces a small amount of phytochelatin in incubations at Stations 4 and 6, but at Station 9 no effect is seen for either addition of 1 or 5
nM. A large increase is observed for the 5 nM copper addition at Station 4 and 6.
Control bottles, for the most part, contained roughly the same concentrations as
103
10
I
I
I
I
I
5
,iI
I
I
I
I
I
I
0
5N
1ON
Station number
8
Q)
I
6
6
ci
0)
uz
*0
f
.ta:
Vd
4
&I
2
0
I
I
20S
15S
I
I
10 S 5S
Latitude
Figure 3. Surface nitrate concentrations (pM) as a function of latitude courtesy of L.
Miller and K. Bruland.
104
IM
0F)
0
CN
CD
4
(0
CD
CD4
0n
CD5
. I
I
.
.~~~~~~~
D
rn)
CO
rn
I~~~~
0F)
K,,M
0
F)
n
N
CM
co
CD
N
GO
CD
CD
0
0
co
K)
F)
(D
0
to)
0
fe)
mv
i
N
CN
co
V
CD4
.-
CD
z
CN
CD
0
CD
0
K)
C2
I
I
I
CD
-
I
(N
CN
CD
CN
CN
CD
co
0
- -
I
!
J
!
J
0
J
to
CD
C)
0
F)
4
-4;
0
K)
M
CD4
CD
N
-I.
m
.4:
CD4
CD4
0D
0 0 0 0 0 0
-
0
' -N
Cm0 N
C)
00 0LO
F)
F)
0 LI
0 00(L 0 0
N
0
0000
N
0F) 0
n
(ui) qldoa
Figure 4. Depth profiles of nitrate concentrations (IM) at Stations 1-10, data courtesy
of L. Miller and K. Bruland.
105
chl a Ag / 1
0.2
0.0
0
Station
0.4
s
X
.
I
I
7
chl a
50 _
1-11,
5
7
100 _
At
CU
P
v
150
-0
0
200
0
10
20
30
40
50
Phytochelatin ,umol / g chl a
Figure 5. The depth profile of phytochelatin concentrations at Station 7 from Figure 2,
including all three chain lengths. Axes are as labelled in Figure 2.
106
100
St.4 5°S
8/14/91
_
80
5n
Cd
6 u Cu
60
40
a5
i
N
N
1 nM Cd
20
N
ControlA
I nu
u
N
11
[1
.I
0
,§
a9
,I.
.e
St. 9 8N
100
8/25/91
80
i .X
l nCd5 n Cd
60
_
K
40
i
Control
20
U
50
lnuCu
Incubation Condition
Figure 6. Phytochelatin measurements(umol (g chl a)-1 ) after 48 hour incubations with
the various metal additions. Incubations were performed at Stations 1, 4, 6, and 9.
There was only one sample per treatment and the average analytical error for these
samples was 10%.
*The chlorophyll sample for the control for Station 6 was lost, so an average of the
final chl a concentrations of the four other incubation bottles was used to normalize
the phytochelatin measurement, adding additional error of ±50%.
107
measured in the surface samples from the depth profiles, confirming that our sampling and
incubation procedures were trace metal clean. The control concentration for Station 1,
however, was over three times that measured in the surface water at that same station.
This bottle was possibly contaminated or the water collected for the incubation was quite
different from that sampled for the depth profile.
Discussion
Phytochelatin values in the Equatorial Pacific are remarkably similar to concentrations measured in coastal waters. Along the transect from 20° S to 10° N, no real difference in surface concentration is observed, and most profiles exhibit a subsurface
maximum, although this structure appears to be less pronounced at the stations within the
upwelling. Addition of cadmium to incubation bottles of surface seawater from various
stations stimulated phytochelatin production above ambient levels, and the ability of copper to stimulate production above concentrations in controls was limited to stations within
the upwelling zone.
The range of phytochelatin concentrations measured on this cruise are remarkably
(and unexpectedly) similar to those measured in the Boston Harbor - Massachusetts Bay
transects where concentrations range from 2- 20 mol (g chl a)-1 (Chapter 2 and Chapter
5). Phytochelatin concentrations are on average higher in the Equatorial Pacific than in
Boston Harbor. This difference is even more pronounced when corrections are made for
the fraction of the total chlorophyll due to prokaryotic vs. eucaryotic organism. In Boston
Harbor, diatoms dominate the flora throughout the year, as discussed in Chapter 5. In
these open ocean samples up to 46% of the total chlorophyll a is due to divinyl chl a from
prochlorophytes (DiTullio, per comm). Prokaryotic organisms synthesize metallothionein-like metal-binding proteins (Olafson et al., 1979), and have not been reported to make
phytochelatin, thus the normalization to total chl a underestimates the amount of phytochelatin produced by the eucaryotic population. A rough doubling of these phytochelatin
concentrations (yielding concentrations -4- 100 pmol (g chl a)
'
is consequently better for
comparison to other field measurements and laboratory studies with pure cultures.
Prominent features of this data set include the higher than expected phytochelatin
108
concentrations and the recurring subsurface maxima at most stations. As in any field study
there is always a chance that differences are due to variable species composition, and one
cannot rule this out. Another plausible explanation is that free metal concentrations are
higher here than in coastal waters, and that these concentrations are varying over depth. In
addition the extremely low concentrations of some essential metals, such as iron and zinc,
may enhance the effects of the other phytochelatin inducing metals such as cadmium and
copper.
The high phytochelatin concentrations could simply reflect the species composition present in these samples. The two oceanic isolates examined for phytochelatin production in Chapter 3 were Thalassiosira oceanica (13-I) and Emiliana huxleyii (BT6). At
the lowest free cadmium concentrations (pCd=13 and 12), phytochelatin concentrations in
both organisms are very similar to the neritic species, but at pCd=1l1T. oceanica contains
much lower concentrations whereas E. huxleyii produces a large amounts of phytochelatin
(Chapter 3, Table 2). Thus the variability in species composition might well contribute to
overall concentration differences, as well as to changes in concentration over depth.
The subsurface maximum observed in the depth profiles is not directly correlated
with total or free metal concentrations in the euphotic zone. Measurements of cadmium in
the central north Pacific (both total and calculated available inorganic concentrations) do
not have much structure within the photic zone (Bruland, 1992). Likewise, total and free
ion concentrations of copper have no particular subsurface maxima (Coale and Bruland,
1988; Moffett et al., 1990). Moffett et al (1990) actually reports a surface maxima of free
copper in the Sargasso Sea, putatively due to photolysis of organic chelators.
Another explanation for the relatively high phytochelatin concentrations in the
Equatorial Pacific is that the extremely low concentrations of essential metals stimulates
the uptake of all available metals. Low iron concentrations result in enhanced maximum
iron uptake rates due to increased numbers of transport ligands on the cell surface (Hudson and Morel, 1990). Harrison and Morel (1983) established the competitive relationship
between iron and cadmium: cadmium toxicity could be reversed by addition of iron and
lowering cadmium concentrations enabled cells to increase intracellular iron. Enhanced
transport of iron may result in enhanced transport of cadmium if these metals are in some
way competing for the same transport ligand. Likewise zinc limitation enhances cadmium
109
uptake rates in Thalassiosira weissflogii (Lee et al., submitted). Phytochelatin concentrations in this species are higher at the same cadmium concentrations under conditions of
low zinc (data not shown). It is thus conceivable that under conditions of metal limitation,
the uptake of all metals is enhanced and thus the same or lower concentrations of cadmium or copper may be accompanied by higher phytochelatin concentrations.
Incubation experiments involving the addition of cadmium and copper to on-deck
incubation bottles, though limited, do suggest some conditioning of the seawater by the
phytoplankton over time. The upwelling at the equator is wind driven Eckman transport
(Gargett, 1991).The shallow upwelling at the equator is accompanied by divergence of
water masses north and south and with downwelling at roughly 10 ° of latitude. Meridional or poleward advection is very slow, thus water outside of the upwelling is predominantly old surface water.
The greatest difference between the phytochelatin concentrations measured in the
control and in the metal addition bottles is seen at the two stations within the upwelling.
The addition of 1 nM copper stimulates significant phytochelatin production at these stations, whereas at 8 ° N, Station 9, addition of 5 nM copper does not stimulate any phytochelatin production above the control. Again this may be due to differences in the
phytoplankton populations (some species seem to produce more phytochelatin in response
to copper than others (Chapter 4)), but it could also be due to the presence of organic chelators (or lack there of) which would make the added metals less available to the phytoplankton. Likewise in Boston Harbor additions of 5nM cadmium had no effect of
phytochelatin concentrations (Chapter 2), thus demonstrating that excess chelators are
available to complex the added metal in this coastal system.
Strong copper chelators have been measured in oligotrophic waters (Coale and
Bruland, 1988; Moffett et al., 1990). A strong ligand, roughly 2 nM, and a weaker ligand,
estimated to be about 80 nM in surface waters, are measured in surface water (Moffett et
al., 1990). The concentration of these chelators decreases below the chlorophyll maximum, and it is assumed that they are biogenically derived. Moffett et al (1990) also measured a similar chelator in cultures of Synechococcus sp. Stations within the upwelling
have higher concentrations of chlorophyll (initial chl a concentrations for incubations at
Stns. 4 & 6 were 0.19 and 0.60 gg chl a 1-1, whereas initial chl a at Stns. 1 &9 were 0.09
110
and 0.10
g chl a 1-1), it is possible that the newly upwelled water has lower concentra-
tions of these chelators. However, the first measurements of copper chelators at the Equator made by Miller and Bruland (1994) indicate that ligand concentrations are not
significantly different from those outside the upwelling.
These first open ocean particulate phytochelatin concentrations provide new
insights into the possible interactions of trace metals with the biota and the extent to which
metals are complexed in seawater. Phytochelatin concentrations are higher than might be
expected, higher in fact than initial measurements in a fairly polluted coastal area. This
may be due to the competitive nature of essential and toxic metals in an area which
receives very limited eolian input of all trace elements. Most of the phytochelatin profiles
have some structure (a subsurface maximum) which suggests some variation in metal
availability with depth. One possible interpretation of the few incubation experiments is a
chemical "conditioning" of seawater by release of metal chelators from the plankton community.
Acknowledgments
A special thanks to the captain and crew of the R/V Moana Wave, and to Ken Bruland, the chief scientist, and the members of his laboratory who made our participation on
this cruise and the collection trace metal clean samples possible. Also a special thanks to
Neil Price who spent many hours helping to filter many liters of water and to Jack DiTullio
for the divinyl chl a measurements. Funding of this project was provided by NSF, ONR,
EPA, Sea Grant and the MWRA.
111
References
Barber, R. T., R. C. Dugdale, J. J. MacIsaac and R. L. Smith. 1971. Variations in phy-
toplankton growth associated with the source and conditioning of upwelled water. Inv.
Pesq. 35: 171-193.
Barber, R. T. and J. H. Ryther. 1969. Organic chelators: Factors affecting primary production in the Cromwell Current upwelling. J. Exp. Mar. Biol. Ecol. 3: 191-.
Bruland, K. W. 1989. Complexation of zinc by natural organic ligands in the central North
Pacific. Limnol. Oceanogr. 34: 269-285.
Bruland, K. W. 1992. Complexation of cadmium by natural organic ligands in the Central
North Pacific. Limnol. Oceanogr. 37: 1008-1017.
Bruland, K. W. and R. P. Franks. 1983. Mn, Ni, Cu, Zn and Cd in the Western North
Atlantic. p. 395-414. In C. S. Wong, E. A. Boyle, K. W. Bruland, J. D. Burton and E. D.
Goldberg [eds.], Trace Metals in Seawater. Plenum Press.
Bruland, K. W., R. P. Franks, G. A. Knauer and J. H. Martin. 1979. Sampling and analyti-
cal methods for the determination of copper, cadmium, zinc and nickel at the nanogram
per liter level in seawter. Anal. Chim. Acta. 105: 223-245.
Coale, K. H. and K. W. Bruland. 1988. Copper complexation in the Northeast Pacific.
Limnol. Oceanogr. 33: 1084-1101.
Gargett, A. E. 1991. Physical processes and the maintenance of nutrient-rich euphotic
zones. Limnol. Oceanogr. 36: 1527-1545.
Harrison, G. I. and F. M. M. Morel. 1983. Antagonism between cadmium and iron in the
marine diatom Thalassiosira weissflogii. J. Phycol. 19: 495-507.
Hudson, R. J. M. and F. M. M. Morel. 1990. Iron transport in marine phytoplankton:
Kinetics of cellular and medium coordination reactions. Limnol. Oceanogr. 35: 10021020.
Lee, I. G., S. B. Roberts and F. M. M. Morel. submitted. Cadmium: a nutrient for the
marine diatom Thalassiosira weissflogii. Limnol. Oceanogr.
Martin, J. H., S. E. Fitzwater and R. M. Gordon. 1990. Iron deficiency limits phytoplankton growth in Antarctic waters. Global Biogeochem. Cycles. 4: 5-12.
112
Martin, J. H., R. M. Gordon, S. Fitzwater and W. W. Broenkow. 1989. VERTEX: phy-
toplankton/iron studies in the Gulf of Alaska. Deep-Sea Res. 36: 649-680.
Martin, J. H., R. M. Gordon and S. E. Fitzwater. 1991. The case for iron. Limnol. Oceanogr. 36: 1793-1802.
Miller, L. A. and K. W. Bruland. 1994. Determination of copper speciation in marine
waters by competitive ligand equilibration/liquid-liquid extraction: an evaluation of the
technique. Anal. Chim. Acta. 284: 573-586.
Moffett, J. W., R. G. Zika and L. E. Brand. 1990. Distribution and potential sources and
sinks of copper chelators in the Sargasso Sea. Deep-Sea Res. 37: 27-36.
Olafson, R. W., K. Abel and R. G. Sim. 1979. Prokaryotic metallothionein: Preliminary
characterization of a blue-green alga heavy metal-binding protein. Biochem. Biophys.
Res. Commun. 89: 36-43.
Parsons, T. R., Y. Maita and C. M. Lalli. 1984. A Manual of Chemical and Biological
Methods for Seawater Analysis. Pergamon Press.
Quay, P. D., M. Stuiver and W. S. Broecker. 1983. Upwelling rates for the equatorial
Pacific Ocean derived from the bomb14 C distribution. J. Mar. Res. 41: 769-792.
113
Chapter 7
Future Work
114
The phytochelatin measurements reported in this thesis suggest environmental
applications and generate new hypotheses that could be tested in further laboratory and
field experiments. Using measurements of phytochelatin to assess metal availability or
toxicity in natural waters is an obvious and worthwhile pursuit. Study of other factors
influencing phytochelatin concentrations such as the interaction of multiple metals and
further characterization of species variability should be pursued. Possible alternative
mechanisms of metal detoxification in particular algal species and their evolutionary
implications is worth examining. Many aspects of the physiology of phytochelatins
remain to be elucidated, such as their stability and location in the cell and the mechanisms
that control both the synthesis and elimination of phytochelatin.
Can we determine whether a certain concentration of phytochelatin represents a
deleterious amount of metal? If phytochelatin concentrations become a significant fraction
of the total -glu-cys in the cell, depleting the pool of glutathione needed to perform essential roles, then one could imagine that there would be some threshold beyond which one
could say that the health of the cell was compromised. Before this can be done, however,
much more information about glutathione concentrations in phytoplankton needs to be
collected and an evaluation of the extent to which cells can compensate for the loss of glutathione which has been incorporated into phytochelatin needs to be addressed.
Phytochelatin measurements are a long way from providing quantitative estimates
of water quality, but a comparison of the field and laboratory measurements shows that the
concentrations are in the same range and provide the beginning of some sort of calibration
(Table 1). Many factors may control the magnitude of phytochelatin production in natural
communities of phytoplankton- the consortium of species in a given population, and less
obviously the concentrations of other metals interacting either synergistically or antagonistically. An obvious extension of this thesis is further testing of other phytoplankton
species under various trace metal conditions. Although little difference was observed
between coastal and oceanic species further testing would be instructive, particularly with
the simultaneous manipulation of various metals. Limitation of phytoplankton growth by
a particular metal induces a high maximum uptake rate for that particular metal and for
any other metal that may be transported by the same uptake ligands (Lee et al., submitted).
115
Table 1. Phytochelatin measurements in marine waters
n=2
gmol (g chl a) 1
I -glu-cys
Wmol(g chl a)-1
2 - 50
7- 150*
Boston Harbor /Mass. Bay
0.5 - 20
1 - 60
Cape Cod/Prov. Har.
2 - 115
6 - 350
Open ocean
Equatorial Pacific
Coastal areas
Laboratory cultures**
No added cadmium
3 - 40
pCd=12 (30 pM Cd')
43- 87
pCd=1 (300pM Cd')
59 - 890
*Calculated from Station 7 depth profile including all chain lengths
**The range of concentrations from the species which had a significant
phytochelatin response from Table 2, Chapter 3 (excluding P. lutheri and
H. pygmaea).
116
Phytochelatin production may thus also be enhanced by metal limitation if another phytochelatin inducing metal is also taken up. This has been observed in preliminary experiments with zinc limited cultures of Thalassiosira weissflogii with added cadmium (data
not included). Our data in the Equatorial Pacific suggest that such a mechanism may be
important in nature and worth studying.
The dinoflagellate, Heterocapsa pygmaea, does not respond to high cadmium concentrations by synthesizing phytochelatins, yet cadmium is accumulated in the cell (Chapter 3). Phytochelatin measurements ought to be made in other dinofiagellate species to see
if this result can be generalized. It is possible that dinoflagellates have an alternate detoxification mechanism.Perhaps they synthesize metallothioneins to chelate intracellular metals. One could possibly isolate this protein and then determine whether a bacterial
symbiont or the algae is responsible for its synthesis.
As we learn more about detoxification mechanisms in various species, we can
begin to hypothesize about evolutionary reasons for particular differences. It is believed
that procaryotic organisms dominated the early oceans when reduced conditions existed.
These organisms probably utilized a metallothionein-like protein for detoxification or
metal homeostasis, similar to that employed by the modem day cyanobacteria. As photosynthetic cells evolved and began oxygenating the air and sea, shifting the oceans to predominantly oxidized conditions, these organisms had to evolve mechanisms to maintain
reduced intracellular conditions. The mechanisms utilizing glutathione were presumably
put in placce to maintain reduced sulfhydryls in proteins and scavenge toxic peroxides, the
harmful byproducts of aerobic life, particularly in photosynthetic organisms. As various
symbiotic relationships evolved into what are now eucaryotic organisms those which were
photosynthetic probably needed more glutathione and perhaps extended the use of glutathione to include metal detoxification by synthesizing phytochelatins. Non-photosynthetic eucaryotes did not have the added pressure of internal oxygen evolution and
continued using a gene transcription product-metallothionein-to
regulate and detoxify
intracellular metals. Examination of phytochelatin production (or lack thereof) by various
phytoplankton species with respect to their phylogeny may allow us to refine speculations
regarding the evolutionary origin of the different detoxifcation mechanisms.
Another logical extension of this work is to study the mechanisms of control and
117
the dynamics of phytochelatin in the cell. Ratios of intracellular cadmium to phytochelatins do not always follow simple stoichiometric relationships and are sometimes highly
variable (Chapter 3). If phytochelatin production is not simply controlled by internal
metal concentrations, how is it regulated? The rapid adjustment of pools of phytochelatin
in T. weissflogii to reflect changes in the external medium (Chapter 4), particularly the loss
of phytochelatin upon alleviation of metal exposure, raises intriguing questions regarding
the fate the phytochelatin. Intracellular cadmium remains constant upon alleviation of
cadmium exposure (Harrison and Morel, 1983) but the phytochelatin pool is substantially
decreased. Upon resuspension of cells in medium containing more or the same amount of
cadmium, I observed considerable exchange of intracellular cadmium with external cadmium (data not included - rates of exchange measured to be -50% of the uptake rate). It
would be interesting to know if this is this accompanied by release of phytochelatins into
the medium.
Are phytochelatins actively excreted from some species of phytoplankton? A
mechanism for long term detoxification could be the excretion of metal binding ligands.
Moffett et al (1990) measured strong copper ligands in a culture of Synechococcus sp. and
McKnight and Morel (1979) measured weak copper binding ligands in cultures of many
freshwater eucaryotic algae. The interest in finding specific metal binding ligands
excreted by phytoplankton is high in view of the fact that a large percentage of many metals in surface waters are organically complexed. Calculations in Chapter 2 argue against
intracellular phytochelatin as a source of these ligands, and even if average open ocean
water column concentrations of phytochelatin are as high as 20 mol (g chl a)- 1 as in the
Equatorial Pacific (instead of the 2 gmol (g chl a)-1' used in the previous calculation), the
turnover time would still need to be upwards of -50 days to achieve ligand concentrations
of -100 pM as measured by Bruland (1992). If organisms were actively excreting these
ligands turnover times could be significantly decreased and the accumulation of these peptides in the water column would be more likely.
A recent study has identified a vacuolar membrane transport protein which enables
the yeast Schizosaccharomyces pombe to accumulate sulfide containing phytochelatinmetal complexes (Ortiz et al., 1992). The authors hypothesize that this transporter functions analogously to the common glutathione-S-transferase export pumps which transport
118
glutathione and various toxic agents in the liver. A similar pump has been identified in the
vacuolar membrane of plants (Martinoia et al., 1993). It is tempting to hypothesize that
such a pump exists in phytoplankton cells, enabling the organism to excrete unwanted
metal ions either to the vacuole or outside of the cell.
Finally, further measurements of phytochelatin in the field should be made and
compared to speciation measurements of various trace metals, particularly cadmium and
copper. Additional incubation type experiments should be performed in and out of
upwelling areas. In various bodies of water metal titration type experiments could be done
to determine concentrations of metal binding ligands similar to the bacterial bioassay of
Hering et al (1987). Alternatively incubations with the addition of synthetic chelators may
be instructive.
119
References
Bruland, K. W. 1992. Complexation of cadmium by natural organic ligands in the Central
North Pacific. Limnol. Oceanogr. 37: 1008-1017.
Harrison, G. I. and F. M. M. Morel. 1983. Antagonism between cadmium and iron in the
marine diatom Thalassiosira weissflogii. J. Phycol. 19: 495-507.
Hering, J. G., W. G. Sunda, R. L. Ferguson and F. M. M. Morel. 1987. A field comparison
of two methods for the determination of copper complexation: bacterial bioassay and
fixed-potential amperometry. Mar. Chem. 20: 299-312.
Lee, J. G., S. B. Roberts and F. M. M. Morel. submitted. Cadmium: a nutrient for the
marine diatom Thalassiosira weissflogii. Limnol. Oceanogr.
Martinoia, E., E. Grill, R. Tommasini, K. Kreuz and N. Amrhein. 1993. ATP-dependent
gluathione S-conjugate 'export' pump in the vacuolar membrane of plants. Nature. 364:
247-249.
McKnight, D. M. and F. M. M. Morel. 1979. Release of weak and strong copper-complexing agents by algae. Limnol. Oceanogr. 24: 823-837.
Moffett, J. W., R. G. Zika and L. E. Brand. 1990. Distribution and potential sources and
sinks of copper chelators in the Sargasso Sea. Deep-Sea Res. 37: 27-36.
Ortiz, D. F., L. Kreppel, D. M. Speiser, G. Scheel, G. McDonald and D. W. Ow. 1992.
Heavy metal tolerance in the fission yeast requires an ATP-binding cassette-type vacuolar
membrane transporter. EMBO J. 11: 3491-3499.
120
Appendix A
Methodological Details of Phytochelatin Analysis
121
The following outlines the exact recipe for preparation of algal phytochelatin samples for
HPLC. Details of HPLC method are outlined in Chapter 3.
Step 1. Place cells on filter into 2.5 mL 10 mM methanesulfonic acid (MSA) at 70 ° C in
Wheaton glass grinding tube. Maintain at 70 ° for 2 minutes with hot water bath on a hot
plate; transfer immediately to ice bath.
Step 2. Grind samples for 3 minutes with Wheaton telfon pestle and grinding tube, holding tube in an ice bath.
Step 3. Transfer into two 1.5 mL Eppendorf centrifuge tubes and centrifuge for 10 minutes.
Step 4. Transfer 0.8 mL of supernatent into another Eppendorf tube for reaction. Save
rest of supernatent for future analysis.
Step 5. Add 30 pL of 15mM dithiothreitol (DTT)1 , 84 pL 100mM sodium borate
(pH=9) & 10 mM diethylenetriaminepentaacetic acid (DTPA). Shake and wait 10 minutes.
Step 6. Add 30 gL of 50 mM monobromobimane (mBrB) dissolved in acetonitrile.2
Shake and wait 10 minutes.
Step 7.
Add 15 AL 60mM reduced cysteine. 1 Shake and wait 5 minutes.
Step 8. Add 60 AL 1M acetic acid. Final volume roughly 1 mL for autosampler on
HPLC.
1. Prepared fresh daily.
2. Prepared ahead, add 1.9 mL acetonitrile to anew 25 mg bottle of mBrB and stored in freezer.
122
The chromatogram below provides further confinrmationthat the peaks in the natural samples are indeed phytochelatin peaks. Amino acid analysis of peaks collected from
the cell extract have confirmed their identity. A cell extract is diltued and then added to a
natural sample. The natural sample peaks coelute with the Thalassiosira weissflogii phytochelatin peaks.
0.015
0.010
0.005
0.015
0
0.010
a)
0C,
Th
V
0.005
0
0.015
0.010
0.005
50
60
70
80
Time (minutes)
Figure 1. An example of a blank (top), field sample chromatogram (middle) and the same
field sample + 10 pL of a 100 times diluted high cadmiumT. weissflogii sample (bottom).
Each phytochelatin peak increases upon addition of the cell extract The n=2, n=3, & n=4
peak areas of cell extract added to the blank (not shown) are 2.8, 4.2, & 1.8 respectively.
The field sample peak areas are 2.2, 0.8, & 4.3 and the addition of cell extract to this field
sample resulted in peak areas of 4.6, 3.8, & 6.2, roughly the sum of the individual chromatograms for n=2 and n=4.
123
Table 1. Amino acid analysis of peaks collected off HPLC from a run
injected with high cadmium T. weissflogii cell extract. Values are
normalized to glycine concentration. Subtraction of blanks (background
concentrations of amino acids in eluant) yields ratios Glu:Cys:Gly of
and 4.4:4.0:1.0 for n=2, n=3 and n--4 respectively.
3.1:2.6:1.0,
1.6:1.3:1.0,
Analysis was performed by the MIT Biopolymers Laboratory.
n=2
n=3
n=4
Ala
0.13
0.05
0.05
Arg
0.08
0.03
0.03
Asp
0.18
0.06
0.15
Cysa
0.9b
2.1
3.7
Glu
1.1
2.5
3.4
Gly
1.0
1.0
1.0
His
0.12
0.12
0.10
Leu
0.14
0.02
0.03
Lys
0.50
0.02
0.02
Ser
0.30
0.09
0.11
Thr
0.08
0.03
0.03
Tyr
0.06
0.05
0.05
Val
0.08
0.04
0.04
Cys was measured as cysteic acid.
b Values shown in bold are the amino acids found in phytochelatins.
a
124