Oct 18, 2018 - When 1 look back six yearç, I'm amazed as to how much 1 have learned ... regulations, the following text is included to inform the external .... Its minute concentration limits primary productivity and ...... phytoplankton were obtained from the Center for Culture of Marine ...... stand in reduction buffer for 4 h.
Iron Acquisition by Marine Phytoplankton
Maria Teresa Maldonado-~areja Department of B i o l o g y , McGill University, ~ o n t r & a l
March 1999
A thesis submitted to the Faculty of Graduate Studies and Research in p a r t i a l f u l f illment of the requirements of the degree of Doctor of Philosophy
O Maria Teresa Maldonado-Pareja
1*1
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A
mi padrino R a f d e l , a l mar verano tras verano
por i n c u l c a m e el amor
Y
a m i s padres, cuyo ejemplo me mantiene a f l o t e .
. . . "The s p r i n g sed belongs a t first t o the dia tcms and t o a l 1 t h e ather microsmpic p l a n t l i f e of t h e plankton . I n the f ierce intensi t y of their qrowth they cover vast d r e d s of medn w i t h d living b h n k e t of their c e l l s . Mile after mile of mter may appear red or brown or g r e e n , the whole surface t a k i n g on t h e mlor of t h e infinicesimal g r a i n s of pigment contained i n e a c h ut* t h e p l a n t c e l l s . "
-
Rachel L Carson, 1951
Table of Contests
Abs tract
ii
~ésumé
iv
Ackowledgements
vi
Thesis Format and Contributions of Co-authors
viii
Statement of ûriqinality
X
Ceneral Introduction
Introductory Remarks 1 Chapter 1
Influence of N substrate on Fe requirements
of marine centric diatoms.
Introductory Remarks I I Chapter II
Nitrate regulation of Fe reduction and
56 57
transport in Fe- Limited Thalassiosira oceanica.
Introductory Remarks III
105
Chapter III
106
Utilization of iron bound t o stronq organic ligands by plankton communities in the subarctic Pacific Ocean.
Introductory Remarks IV Chapter IV
Reduction and transport of orqanically bound Fe by Thalassiosira oceanica .
General Conclusion
156 157
Abstract
Thalassiosira oceanica, a marine centric diatom, possesses an extracellular reductase that reduces iron (Fe(II1)) bound to organic complexes as part of a high-affinity Fe transport mechanism.
A number
of Fe(II1) organic complexes are reduced, including siderophores -eLEective Fe chelates produced by microorqanims in response co Fe stress-. Reduction rates are inversely related to the relative stability constants of the oxidized and reduced Fe chelates (loq KoX/Kred),
and Vary by a Factor of 2.4 in accordance with theoretical
predictions.
Under Fe-Limitinq conditions, reduction rates increase
and the ability of T. oceanica to transport Fe frorn siderophores is enhanced.
Iron bound to the siderophore desferrioxamins B (DFB) is
reduced 2 times faster than it is taken up, suggesting that the reductase is well coupled to the Fe transporter, and can provide a 1 1 the inorqanic Fe to account for the rneasured Fe uptake rates in the presence of excess DFB.
The efficacy of the reductase in providinq
inorqanic Fe for uptake and qrowth is ultimately dependent on the relative concentrations of excess ligands in solution and ce11 surface Fe transporters competinq for inorqanic Fe.
The rates of Fe reduction
and uptake are twice as fast in cells grown in grown in
NHJt,
NO3-
compared to those
suqgesting a link with cellular N rnetabolism and with
NO3- utilization in particular.
Enhanced Fe reductase activity in N 0 3 -
-grown cells enables them to maintain a 1.6-fold higher cellular Fe concentration under low Fe conditions. Experiments conducted in the subarctic Pacific, an Fe-limited oceanic region, demonstrated that even indigenous plankton (both
prokaryotic and eukaryotic plankton) have the ability to acquire Fe bound to stronq organic chelates.
Large p h y t o p l a n k t o n species
p) reduce Fe bound to siderophores extracellularly.
(>
3
Because the
predominant form o f dissolved F e in the sea is bound t o stronq organic complexes, a reductive mechanism as described here may be a critical step in Fe acquisition by p h y t o p l a n k t o n .
iii
Thalassiosira oceanica, une diatomée marine centrique, possède une réductase extracellulaire qui réduit le fer (Fe(II1)) lié aux complexes organiques et qui fait parti d'un mécanisme de transport à
haute affinité par rapport au fer. Un nombre de complexes organiques ferrique sont réduit par la réductase.
Entre autres, les
sidérophores, d e s chélateurs ayant une forte affinité pour le fer et produits par certains microorganismes lors de pénurie de fer, servent de substrat pour cette enzyme.
Les taux de réduction mesurés chez T.
oceanica sont inversement proportionnels aux constantes d'équilibre des chélateurs oxydés et réduits
(
log KOx/Kred), et ces t a u x varient
d'un facteur de 2.4, conformément aux prédictions théoriques. Lors
de pénurie de fer, Les taux de réduction de T. o c e a n i c a augmentent, ainsi que les taux de prise du fer lié aux sidérophores. Le fer lié au sidérophore desferrioxamine B (DFB) est réduit à une vitesse 2
fois plus rapide que le taux de prise du fer. Ceci suggère que La réductase est adéquatement jumelée au récepteur qui transporte le fer à l'intérieur de la cellule, puisque la réductase arrive à procurer
tout le fer inorganique qui correspond aux taux de prise mesurés en présence d'un excès de DFB.
L'efficacité de la réductase dans la
production du fer inorganique est ultimement dépendante de La concentration relative du chélateur en solution et du transporteur à La surface de la cellule. Ces derniers entrent en compétition pour le fer inorganique. Les taux de réduction et de prise du fer sont 2
fois plus élevés chez les cellules cultivées avec du NOj- que celle avec du N H ~ ' , ce qui suggère un lien avec Le métabolisme de L'azote
dans la cellule, l'utilisation du
NO3' en
particulier. L'activité
plus élevée de La réductase chez les cellules nourries de
NO3-
permet
à celles-ci de maintenir une concentration de fer intracellulaire 1.6
fois plus élevée en conditions de pénurie de fer que les cellules dans l e N H 4 + . Des expériences entreprises dans Le Pacifique subarctique, une
région océanique iiini t&
par Le f e r , ont démontré que le plancton
indigène (Les eucaryotes et Les procaryotes) arrive à accumuler Le fer lié à des chélateurs organiques. Les grosses espèces de
phytoplancton
(>
3 p ) réduisent le fer lié aux s i d é r o p h o r e s à La
surface de La cellule. Puisque La forme pr6dominante de fer dissout dans La mer est liée à des complexes orqaniques forts, un mécanisme
de réduction tel que nous décrivons pourrait comporter une é t a p e critique à l'acquisition du fer c h e z le phytopLancton marin.
Ac knowledgements
When 1 look back six yearç, I ' m amazed as to how much 1 have learned, and intellectually grown in the process of this thesis.
owe nearly al1 of that to my supervisor Neil Price. have worked with N e i l and learned from him.
1
1 feel honored to
Neil has taught me that
fun science is risky but rewarding, that meticulous research is difficult but valuable, and that understanding mechanisms is time consuming but worthwhile.
f admire Neil for being an enthusiastic
advisor, a c r i t i c a l and engineering thinker. a storm of fascinatinq ideas, a hard worker, a patient teacher, a believer, a listener, a passionate scientist and most of al1 for his contagious inspiration. 1 want to thank him specially for always believing in me, and
challenging my mind every step of the way.
1 appreciate immensely
every red mark of editing that Neil has put in my manuscripts, and
hope that one day 1 can write as eleqantly and concisely as he does. f also thank my cornmittee members, Joe Rasmussen, f o r sporadic
chats in the hallway, and Raj Dhindsa for his warmth and willingness
to help. 1 treasure three very special peopie: Phi1 Tortell, Julie
Granger and Chris Payne.
I thank Phi1 for makinq me believe in myself
during difficult times, for always qivinq me the strength to continue, for always finding the positive side, and for making me laugh.
I
thank Julie for being such a invaluable friend. Julie has taught me a million things, from the chemistry of siderophores, the tricks of sewing, to the delicacy of sushi-making. Julie has been my most valuable colleaque, especially in the last three years.
I admire her
enthusiasm, her ability to be critical and 'do anything', and her willinqness to help others.
Chris Payne has been the closest to a
family member that f can think of.
1 thank Chris for being patient,
for listening and for keeping me calm.
I'm goinq to miss you,
capullo! Some temporary students in the lab had helped me immensely. Michelle Sirois was such a pleasure to have worked with.
1 love her
positive attitude and her efficiency, and thank her for her very long nights with the Felumen.
Marsha Wysote is my guardizn angel; she did
things quietly before r even asked.
1 loved having my brother,
E s t e b a n t h e s w e e t c o m p l a i n e r , a r o u n d f o r a summer. qrow t h e s e l o v e l y diatoms.
He
a l s o managcd t o
V e r o n i q u e H i r i a r t and Rob S t r z e p e k were my
d e a r p e o p l e a t the b e g i n n i n g of t h i s l o n g j o u r n e y ; t h e y made a n
isolated l a b i n t o a f u n p l a c e .
L a t e r o n , s k i i n g w i t h Zanna C h a s e a t 7
i n t h e m o r n i n g was t h e b e s t way t o wake u p i n the w i n t e r f o r a long day i n t h e lab.
Lisa Nodwell and Graham Peers, t h e n e w e s t a d d i t i o n t o
o u r l a b , have also h e l p m e i n t h e last s t a g e s of t h i s t h e s i s .
The
v e r y l i v e l y L i s a f o r b e i n g t h e srnile o f t h e l a b , a n d t h e v e r y c r a z y Graham, for b e i n g the cheerful one.
E v e r y b o d y i n t h e s i x t h f l o o r h a s made a d i f f e r e n c e o n e way o r another.
1 am e s p e c i a l l y h a p p y t o h a v e J e s s i c a Meeuwig a s a f r i e n d .
1 always loved being w i t h h e r and t a l k i n g to her.
and pushes me t o speak my m i n d .
S h e i n s p i r e s me,
f a d m i r e h e r way o f d o i n g e m p i r i c a l
s c i e n c e , a n d h o p e t o c o l l a b o r a t e i n some wiLd p r o j e c t o n e d a y .
Mark
T r u d e l was a l w a y s a p l e a s u r e t o r u n i n t o i n t h e h a l l , I am g r a t e f u l for h i s s t a t ' s l e s s o n s . 1 t h a n k P a u l for t h e i n s p i r i n g b e a d s a n d o u r ' h a r d h i l l '
rides.
bike
C l a u d i n e h a s b e e n l o t s of f u n a s a f r i e n d .
This t h e s i s would h a v e b e e n d i f f i c u l t t o c o m p l e t e w i t h o u t P i l a r ' s , Todd's, Alfonso's, a n d m i f a m i l i a ( M a r i a , fnma, E s t h e r ,
E s t e b a n , p a p a y mamd) love, support, e n c o u r a g e m e n t , a n d u n d e r s t a n d i n g f r o m miles and miles a w a y .
Thesis Format and contributions to Co-authors
Doctoral candidates at McGill University may submit a thesis based on a series of manuscripts that presents a coherent research programme.
If this option is chosen, in accordance with Faculty
regulations, the following text is included to inform the external examiner of Faculty regulations reqarding the submission of a manuscript-based thesis. Candidates have the option of including, as part of the thesis, the text of one or more papers submitted or to be submitted for publication, or the clearly-duplicated text of one or more published papers. These texts must be bound as an integral part of the thesis. If this option is chosen, connecting t e x t s that provide logical bridges between the different papers are mandatory. The thesis must be written in such a way that it is more than a mere collection of manuscripts; in other words, results of a series of papers must be integrated.
The thesis must still conform to al1 other requirements of the "Guidelines for Thesis Preparation." The thesis must include: A Table of Contents, and abstract in English and French, an introduction which clearly states the rationale and objectives of the study, a comprehensive review of the Literature, a final conclusion and summary, and a thorough biblioqraphy or reference list. Additional material must be provided where appropriate (e.g. in appendices) and in sufficient detail to allow a cLear and precise judgement of the importance and originality of the research reported in the thesis. In the case of manuscripts CO-authored by the candidate and others, the candidate is required to make an explicit statement in the thesis as to who contributed to such work and to what extent. Supervisors must attest to the accuracy of such statements at the doctoral oral defense. Since the task of the examiners is made more difficult in these cases, it is in the candidate's interest to make perfectly elear the responsibilities of a l 1 the authors of the CO-authored papers .
viii
1 have c h o s e n to submit a m a n u s c r i p t - b a s e d t h e s i s which
c o n s i s t s of t h e f o l l o w i n g p a p e r s :
Chapter 1
MALDONADO, M. T. , and N . M . P r i c e . 1996. I n f l u e n c e of N s u b s t r a t e on F e requirements o f marine c e n t r i c
diatoms.
Mar. Ecol. Proq. Ser., 141: 161-172.
Chapter II
MALDONADO, M.T., and N.M. P r i c e . N i t r a t e r e g u l o t i o n o f F e r e d u c t i o n and t r a n s p o r t i n F e - L i m i t e d Thalassiosira o c e a n i c a . Limnol. O c e a n o g r . , i n
Chapter III
review.
MALDONADO, M.T., and N . M . P r i c e . U t i l i z a t i o n of iron
bound t o s t r o n g o r g a n i c l i g a n d s by p l a n k t o n communities
i n t h e subarctic P a c i f ic Ocean.
Deep-Sea Res. I I . , i n
press.
Chapter IV
MALDONADO,
M . T . , and N . M . Price. Reduction and t r a n s p o r t
of o r g a n i c a l l y bound F e by T h a l a s s i o s i r a o c e a n i c a . J . Ph ycol . , submi t t e d .
Statement of Originality
One of the unresolved enigmas in biological oceanography concerns the Fe nutrition of oceanic phytoplankton.
The recent
discovery that the predominant form of dissolved Fe in the sea is organically bound has raised new questions about how the indigenous species acquire the Fe they need for growth since these organic forms
are thought to be inaccessible for direct uptake.
The major
contribution of this thesis is to establish that phytoplankton are indeed capable of utilizing organically bound Fe, in the laboratory and in the field.
Phytoplankton access Fe bound to organic ligands
using a reductive enzyme at the cell surface. The reductase is induced under Fe-deficiency and is linked to nitrogen metabolism.
It
allows phytoplankton to convert organically bound Fe to inorganic Fe, the Labile Fe species, which can be utilized for qrowth.
The specific contributions to original knowledge are stated below in the order that they appear in the t h e s i s :
1) Using a multi-species, multi-habitat comparative approach, 1 demonstrated experimentally the interaction between Fe requirements
and N metabolism in marine centric diatoms of the genus
Thalassiosira: the use of nitrate (NO3-) imparts a higher cellular Fe demand for growth than the use of ammonium ( N H ~ ' ) (Chapter 1). 2)
1 showed for the first time
a link between Fe acquisition and
nitrogen metabolism in phytoplankton.
Nitrate-amended phytoplankton
were able to qrow quickly under Low Fe conditions in the presence of
a variety of strong organic chelators (Chapter I and II).
They
possessed faster non-saturatinq, steady-state (Chapter 1 ) and shortterm (Chapter TI) Fe uptake rates compared with those of N H ~ +-grown
cells. 3)
This thesis reports the first measurements of Fe requirements,
growth rates, steady-state Fe uptake rates and preferences for n i t r o g e n substrates of Thalassiosira spp. isolated from
lirnited equatorial Pacific Ocean.
the Fe-
1 showed that these diatoms are
physiologically distinct from other phytoplankton isolated from coastal or oceanic habitats (Chapter 1). 4)
Measurements of Fe(I1) production using a cherniluminescence assay
directly demonstrated the ability of the marine centric diatom, T. oceanica, to enzymatically reduce Fe bound to
a variety of stronq
organic complexes, includinq siderophores. The reductase was induced by Fe-deficiency (Chapter II), and was somehow linked to nitrogen metabolism.
Nitrate-grown cells reduced Fe(I1I) at faster rates than
N ~ ~ + - g r o cells wn (Chapter II). 5)
In a multi-year survey of five stations in the NE subarctic
Pacific Ocean, I examined acquisition of orqanically complexed Fe by indigenous plankton and measured Fe requirements of heterotrophic bacteria and phytoplankton (Chapter III).
I demonstrated that
heterotrophic bacteria have higher Fe requirements than phytoplankton and are responsible for a large fraction of total community Fe uptake.
This latter result is a consequence of the higher bacterial
C biomass in s i t u (Chapter III). 6) T h i s thesis provides t h e first demonstration that plankton
(heterotrophic bacteria, and autotrophic bacteria and phytoplankton)
are able to acquire organically bound Fe, the predominant form of dissolved Fe in the sea (Chapter III).
1 measured biologically
mediated reduction of organically bound Fe by indigenous phytoplankton in s i t u .
The rates of Fe uptake and reduction of
organically bound Fe were indicative of Fe-limited phytoplankton
(Chapter III). 71
I have shown that 7'. oceanica qrows in Fe-siderophore media,
where inorganic Fe concentrations are well below those necessary to
fulfil the minimum Fe requirements (Chapter II and IV).
Although
phytoplankton are unable to directly use Fe-siderophores, T. oceanica
is a b l e to
access this form of Fe, when Fe-limited, by an
extracellular reductase (Chapter TI and IV) . 8) The thesis research reports the f irst measurements of the half
-
saturation constant of the ferric reductase in Fe-limited phytoplankton for a Fe-siderophore complex (FeDFB).
At
non-
saturating FeDFB concentrations, t h e Fe reduction rates are faster than the Fe uptake rates, demonstrating that the ferric reductase is
able to supply al1 the Fe that is internalized during transport. 9) A highly significant inverse relationship between Fe reduction
rates by T. oceanica and the relative stability constants of the oxidized and reduced Fe chelates (log
(Kox/Kred)
)
is reported. The
relationship can be used to ernpirically determine precise stability constants of Fe chelates.
xii
General ~ntroduction
Fron is the most indispensable trace metal for growth of most organisms
The ferric (Fe(II1)) and ferrous (Fe(I1)) ions are
relatively small and have a marked propensity to form six-coordinate complexes with ligands containing oxygen, nitrogen, and s u l f u r , dre found a t the active senter of many biolcgical molecules.
They
Throiigh
chelation, protein environment and pH control, a remarkable range of redox potentials can be achieved by Fe-containing enzymes (-300 to +700 mV).
Some of the many fundamental biochemical reactions that involve Fe
incLude oxygen transport, detoxification of reactive oxygen species,
NO3'
and dinitroqen reduction, as well as the energy-yielding electron transfer reactions of respiration and photosynthesis (see Guerinot and Yi, 1994; Braun et al. , 1998). In contrast to its high bioloqical demand and its great abundance in the Earth's crust (Taylor, 1964), Fe is one of the most insoluble metals in oxic waters.
Its stable and metastable oxidized forms, ferric
oxides and hydroxides, are only sparingly soluble in seawater and are not directly available for uptake (Wells at al., 1983; Rich and Morel,
1990). Dissolved Fe exists in a variety of chemical species in
seawater, as Fe(II) and Fe(II1) ions, which are complexed to varying deqrees by inorqanic and organic Ligands.
The nature of t h e s e complexes
strongly influences their avaiLability to marine phytoplankton (see Price and Morel, 1998; Morel et al., 1991). One of the most important discoveries in biological oceanography
durinq the last decade is the realization that the concentration of dissolved Fe in the sea is extremely low (average 0.07 nM, Johnson et
al., 1997).
Its minute concentration limits primary productivity and
biomass in three large oceanic regions: the subarctic Pacific (Martin and Fitzwater, 1988; Martin et al., 1989, 1991), the equatorial Pacific (Price et al., 1991; Martin et al., 1 9 9 4 ) , and the Southern Ocean (de Baar et al., 1995).
In essence the open ocean is extremely infertile in
the present interglacial period because of the scarcity of Fe in these regions (Martin, 1992).
This observation has changea aramatically the
way we view how the ocean operates. Traditionally, the absence of fixed N was thought to be the major factor limiting algal growth in the large oligotrophic ocean gyres, where Little N 0 3 - is present.
However, open ocean upwelling regions
remain unproductive, despite high
NO3'
concentrations year round.
They
are far removed from continental Fe sources and t h u s rely exclusively on atmospheric Fe inputs derived £rom land in the form of dust (Duce and Tindale, 1991), and/or upwellinq of Fe-rich deep waters (Coale et al., 1996 ) .
The realization that Fe plays an essential role in oceanic primary production has stimulated much research into the Fe requirements (Sunda
et al., 1991; Sunda and Huntsman, 1997) and physiology of phytoplankton (Rueter and Ades, 1907; Green et al., 1991, LaRoche et al., 1996).
The
electron transfer Fe-S proteins and cytochromes present in the photosynthetic electron transport chains, account for most of the Fe budget of a phytoplankton ce11 (Hewitt, 1983; Raven, 1988).
Iron
limitation reduces the efficiency of light reactions in photosynthesis
(Green et al., 1991), and thus ultimately controls the efficacy of the 'biological C pump', through which atmospheric CO2 is fixed by
phytoplankton in surface waters and is transported to the d e e p sea as particulate organic carbon. The ocean playç a critical role in the global C cycle by releasing and absorbing large quantities of atmospheric carbon dioxide gas (C02). Biologically mediated export of particulate carbon from the surface ocean is a crucial part in the global C cycle because it represents a potential long-term sink of atmospheric
CO2
(Lonqhurst, 1991).
At
steady state, the amount of organic C available for export from the surface ocean is thought to be fueled by the upward vertical flux of -nitrogen (and by nitrogen fixation to some extent).
NO3-
The magnitude of
this C flux depends on the efficiency of N 0 3 - consurnption by phytoplankton (Dugdale and Goering, 1967; Eppley and Peterson, 1979). Nitrate assimilation is an Fe-depecdent pathway.
The reduction of
NO3' to N H ~ +proceeds via two independent steps, which are catalyzed by
two Fe-requiring metallo-enzymes: the reduction of NO3- to NO2- by NAD(P)H-nitrate reductase (NR), and the reduction of N 0 2 - to N H ~ ' by ferredoxin-nitrite reductase ( N i R ) (Galvan et al., 1987). The reducinq power is supplied from the Fe-dependent light reactions of photosynthesis, that the use of
Raven (1988) predicted from theoretical calcuLations NO3-
by autotrophs for growth should impart a 1.5 times
higher cellular Fe demand than the use of N H ~ ' .
Iron-Limited regions are characterized by low phytoplankton biomass and productivity, despite high concentrations of major nutrients s u c h as NOg-. In Fe-limited regions, field studies have demonstrated that
Fe ultimately regulates the utilization of
NO3-
by phytoplankton, and
modulates the phytopLankton community structure (Price at al. 1991, 1994).
Therefore, in these low Fe regions biologically mediated C
export to the deep sea is diminished because N 0 3 - consumption is inefficient. These field studies provided a link between Fe and N nutrition of phytoplankton (Price at al., 1991, 1994), but little is known about how different
N
sources ( N O 3 - or N H ~ ' ) modulate the effects
of Fe-limitation on phytoplankton physiology.
Studies on the dependence of N metabolism on Fe in phytoplankton a r e sparse.
Lron-iimited p h y t o p l a n k t o n have reduced ~ c t i v i t yof
NO3-
assimilatory enzymes (Kessler and Czygan, 1968; Timmermans et al., 1994) which results in slower rates of NO3' uptake (Rueter and Ades, 1987). The Fe:N ratios of Gymnodinium sanguineum, a red tide dinoflaqellate,
are higher when c e l l s are grown in N O 3 - than in N H ~ ' and are coupled to a diminished capacity to acquire and assimilate
NO3-
(Doucette and
Harrison, 1991). While these studies infer a dependence of N metabolism on Fe, the interaction between Fe requirements and N metabolism is not well documented. The initial objective of my thesis was to provide a mechanistic understanding of the interaction between Fe nutrition and N metabolism in marine centric diatomç.
1 examined six species of the genus
Thalassiosira and demonstrated that , as suggested by theoreti c a l calculations, the use of
NO3*
for qrowth imparts a higher cellular Fe
demand t h a n does use of N H ~ ' (Chapter 1). that contrary to expectations,
NO3-
However, my results indicate
use does not reduce fitness under Fe-
limitation. Nitrate-cultured cells are able to grow at similar or faster rates than NHqt cultures. This is a s a result of their unique ability when Fe-limited to take up Fe at a faster rate (Chapter 1). My initial interest on the interaction between Fe and N has l e a d me to investigate mechanisms by which phytoplankton are able to acquire
organically bound Fe. The work of this thesis puts together scattered pieces of the puzzle of Fe utilization from studies published during the last 19 years (Anderson and Morel, 1980, 1982; Jones et al., 1987; Jones and Morel, 1988; Hudson and Morel, 1990, 1993) and presents a synthetic view of how phytoplankton acquire organically bound Fe.
Investigations into Fe acquisition by phytoplankton have been difficult because of the intricate chemistry of Fe in seawater. Much of the progress in this area is attributed to the introduction of thermodynamic and kinetic theory and of model systzms in which the speciation of Fe can be varied systernatically and quantified by chemical equilibrium or kinetic caLculations (Westall et al., 1976). These model systems include trace metal ion buffers, which control free trace metal
ion concentrations in a similar manner to that of the control of [ H + ] by pH buffers. These buffers typically consist of a fixed concentration of
a well-characterized synthetic organic ligand such as ethylene-di-aminetetra-acetic acid (EDTA), and a much lower concentration of specific trace metals.
Most of the trace metals in solution (generally > 95%)
are present as complexes with the added chelator, and only a srnall fraction are present as free ions and inorqanic complexes. These buffers allow for the maintenance of extremely low concentrations of
free metal ions and inorganic complexes (Sunda, 1991).
In the case of
Fe, this situation mirnics that of natural seawater. By
exploiting these chernical properties and four different
synthetic chelators (NTA, DTPA, CDTA, and EDTA), Anderson and Morel (1980, 1982) initiated the study of Fe transport in marine phytoplankton
wi th the centric dia tom, Thalassiosira weissfloqii .
They addressed two
critical questions: the bioavailability of different chernical foms of
Fe in seawater for Fe uptake by phytoplankton, and the mechanism of transport across the membrane.
This work was the first to demonstrate
that Fe uptake is strongly determined by the chemical speciation of Fe. Their work showed that Fe uptake by phytoplankton was a direct function of the free ferric ion concentration ( p F e
- -log [€e3']).
This suqgested
that the extent of Fe binding to ce11 surface transporters (which they called 'phytotransferrin'), was controlled by pseudo-equilibrium with free Fe(II1) in the medium (Anderson and Morel, 1982). Despite the fact that organically bound Fe was thought to be unavailable for direct uptake, Anderson and Morel (1982) showed that Fe uptake by phytoplankton could be enhanced by photochernical reduction of Fe when bound to certain organic ligands.
Photoreduction can increase
the concentration of bioavailable Fe species in solution (Sunda and Huntsman, 1995). This mechanisrn is mediated by Ligand-to-metal electron transfer reactions which result in free, highly soluble Fe(II) (Waite and Morel, 1984). The rapid reoxidation of Fe by 02 and the photochemically produced
Hz02
at seawater pH (Moffett and Zika, 1 9 8 7 )
prevents appreciable build up of Fe(I1).
However, Fe photoreduction
should enhance biological Fe uptake by increasing the concentrations of free ferric ions as a result of the slow oxide aginq kinetics of reoxidized ferric ion (Wells, 1989). Only one expriment with EDTA (Anderson and Morel, 1982) hinted at the possible direct role of phytoplankton in mediating the reductive
dissociation of Fe Erom organic complexes. This supported previous work on the ability of Thalassiosira to reduce Fe bound to EDTA (Anderson and Morel, 1980). Biological Fe reduction can be mediated by plasmalemma bound ferric reductases, which convert Fe(1II) to Fe(I1).
Most of the
organic chelators used in phytoplankton culture studies have a much higher affinity for Fe(II1) than for Fe(II), so reduction of Fe increases the net dissociation of the Fe organic complex (Wilkins, 1991), resulting in higher concentrations of dissolved reactive Fe species .
The work of Anderson and Morel (1980, 1982) was followed by that of Jones et al. (1987) and Jones and Morel (1988). Their investigations demonstrated that phytoplankton cultures have the ability to reduce CU*' complexes (Jones et al., 1987) by an extracellular reaction that they hypothesized was mediated by the diaphorase subunit of pLasmalemma bound
NR (Jones and Morel, 1988).
At
the time when these findings emerqed,
orqanic complexation of Fe in the sea was thouqht to be unimportant. However, in analogy to plant studies (Redinbaugh and Campbell, 1983; Castignetti and Smarelli, 1984; Smarelli and Castignetti, 1988; Ward et
al., 1988, 1989; Tischner et al., 1989; Corzo et al., 1991; ~eyerhoffet al., 1994; Stohr et al., 1993), a possible role of the NAD(P)H:NR on Fe assimilation was suggested. These studies thus provided the first hint as to the role of N03'
use in Fe acquisition.
The research of Hudson and Morel (1990) forms the foundation o f
the current paradigm of Fe uptake by marine phytoplankton.
Its most
substantial addition to our knowledge was the realization of the paramount importance of chernical reaction kinetics in determining both the availability of dissolved Fe and the characteristics of the Fe transport system, and the quantification of these rates. This work established that Fe uptake proceeds via inorganic Fe(I1I) binding to surface ligands and subsequent internalization across
the ce11 membrane (Hudson and Morel, 1990).
The rate of internalization
of Fe is much faster than the rate of dissociation of Fe from the
transporter back into solution. This finding indicates that a true equilibrium does not exist between Fe binding to the surface ligand and Fe in the external medium.
The rate limiting step of the Fe transport
reaction is the rate of binding of Fe to the surface ligand, which is determined by the rate of water Loss and the concentration of the reactive Fe in solution (Hudson and Morel, 1990). Thus, Fe transport appears to be under kinetic control by the concentrations of dissolved inorganic Fe species ([Fe']) which have rapid coordination kinetics and hiqh enough concentrations to permit exchanqe of Fe to the membrane transport ligands (Hudson and Morel, 1990). Ferric organic complexes are impermeable to biological membranes and do not have fast enouqh exchange kinetics to react effic~entlywith Fe transporters: thus they are not directly available for uptake by phytoplankton (Hudson and Morel, 1990). Iron transport is related to the dissolved inorganic Fe species ( [ F e ' ] ) by the saturation kinetic equation
of Michaelis-Menten. The
maximum rate of Fe uptake is dependent on the prior degree of Felimitation experienced by the cell, and can be adjusted up and down by a factor of 20-30 (Harrison and Morel, 1986).
The rate at which Fe reacts
with the transporter provides a measure of its bioavailability. This Fe bioavailabiLity differs from the previous concept of free ferric ion activity controlling Fe uptake
(Anderson and Morel, 1982). However,
because of the proportionality of free metal ion activity and total inorganic P e concentrations (free ions plus inorganic Fe complexes with hydroxide, chloride, carbonate ions) at a given pH and ionic strength,
either one of these parameters can be used to predict or determine bioavailability of Fe in seawater (Hudson and Morel, 1990). Since Fe transport proceeds via inorganic Fe binding to surface
ligands, phytoplankton Fe uptake is ultimately controlled by cornpetition between complex formation and dissociation reactions of Fe in solution (with water, hydroxides, organic chelators), and ligand-exchange reactions at the transport sites (Hudson and Morel, 1990). The experiments of Hudson and Morel (1990) were performed in FeEDTA-based medium, in which the rates of FeEDTA dissociation, both thermal ( =
1 x 10-~
at pH 8, Hudson, 1989) and photochernical (
K =
K
1.7 ~x
1oS6 s'l at 95 (imol quanta m'2 s e l , Anderson and Morel, 1982) contributed
the greatest to the pool of [Fe'1, and thus were thought to be the most important reactions controlLing [Fe']. If biologically mediated reduction of Fe was occurring in the cultures, its relevance could have
been overlooked as its relative importance in providing inorganic Fe could depend on the rates of thermal and photoreductive dissociation of FeEDTA.
On the other hand, if phytoplankton were to grow in a medium
with Fe bound to siderophores (small molecular weight, high-affinity Fe
orqanic ligands that are synthesized and secreted by many microorganisms when grown under Fe-stressed conditions, Neilands, 1974) the relative
importance of the biological reductive dissociation of ferricsiderophores (assuming phytoplankton are able to reduce Fe bound to siderophores) would be greater. This is because siderophores have very slow dissociation constants (see Albrecht-Cary and Crumbliss, 1998) and
are not photolabile (Finden et al., 1984; Allnut and Bonner, 1987). In my thesis I compared
NO3'
or N H ~ ' amended cultures and was thus
able to notice the effect of the ferric reductase. Thalassiosira
~ ~
s p e c i e s were grown i n F e - L i m i t i n g media (FeEDTA-based) w i t h low enouqh i n o r g a n i c Fe c o n c e n t r a t i o n s t o l i m i t F e u p t a k e r a t e s and t h u s growth. However, 1 o b s e r v e d f a s t e r s t e a d y - s t a t e and g r o w t h rates f o r t h e NO3amended c u l t u r e s ( C h a p t e r 1).
With t h e knowledge of t h e mechanism o f F e
u p t a k e b y p h y t o p l a n k t o n a n d t h e work o f Jones a n d More1 ( 1 9 8 8 ) f s u g g e s t e d t h a t plasmalemma bound NR was m e d i a t i n g the r e d u c t i o n o f o r g a n i c a l l y bound Fe p r e s e n t i n the c u l t u r e medium.
I n t h i s way, NO3-
grown c e l l s i n c r e a s e b o t h t h e i n o r g a n i c Fe c o n c e n t r a t i o n i n the medium and t h e i r u p t a k e r a t e s , and t h u s a r e a b l e t o grow f a s t e r . T h e main f i n d i n g o f my s e c o n d c h a p t e r d e m o n s t r a t e d t h a t T.
oceanica, rny mode1 c e n t r i c d i a t o m , c a n indeed c a t a l y z e t h e dissociation o f F e €rom a v a r i e t y of o r g a n i c c h e l a t e s by r e d u c i n g ~ e = a+ t t h e c e 1 1 surface.
I n d i r e c t evidence s u g q e s t e d t h a t u n d e r F e - l i m i t i n g c o n d i t i o n s ,
reduct i o n of o r g a n i c a l l y bound ~e)+ enables NO3
'
qrown c e l l s t o a c q u i r e
Fe f a s t e r a n d s u b s e q u e n t l y a c h i e v e h i q h e r g r o w t h r a t e s t h a n
c u l t u r e s (Chapter I I ) .
grown
In t h i s chapter, 1 investigated other possible
mechanisms by which NOj' grown c e l l s c o u l d a l s o e n h a n c e their F e u p t a k e r a t e s ( i . e . pH i n c r e a s e s a s s o c i a t e d w i t h N 0 3 ' m e t a b o l i s m , Raven and Smith, 1976) As my r e s e a r c h on the role o f the r e d u c t a s e was p r o g r e s s i n g , a
series of c o l l a t e r a l i n v e s t i g a t i o n s on t h e c h e m i c a l s p e c i a t i o n o f d i s s o l v e d F e i n s e a w a t e r demonstrated t h a t i n most m a r i n e s y s t e m s 9 9 . 9 %
of t h e d i s s o l v e d Fe is bound to s t r o n g o r g a n i c l i g a n d s ( G l e d h i l l a n d van d e n Berg, 1 9 9 4 ; Rue and B r u l a n d , 1995; Wu and L u t h e r , 1995).
These
f i n d i n q s , which were u n p u b l i s h e d a t t h e tirne chapter 1 was w r i t t e n , q u e s t i o n t h e paradigm of Fe u p t a k e by p h y t o p l a n k t o n .
W h i l e Fe c h e l a t e s
were t h o u g h t u n a v a i l a b l e t o p h y t o p l a n k t o n , t h e i n o r g a n i c F e
concentrations are inadequate for even the smallest phytoplankton. My study t h u s provided a mechanism by which phytoplankton could indirectly the Fe organic complexes, and thus increase the concentration of
access
inorganic Fe in the vicinity of the cell. As
part of the Canadian Joint Global Ocean Flux Study (JGOFS), 1
participated in a multi-year, rnulti-site research program in the subarctic Pacific Ocean, an Fe-limited reqion. These oceanographic expeditions allowed me to examine the ability of indigenous phytoplankton communities to take up organically bound Fe (Chapter III). Indigenous phytoplankton are able to acquire Fe from orqanic chelates, includinq siderophores. The mechanism by which phytoplankton access organically bound Fe in the field is still uncertain, but field measurements of reduction rates of Fe indicate that at Least Large phytoplankton are able to reduce organically bound Fe. 1 focused my last chapter on the kinetics of uptake and reduction
of Fe bound to the mode1 cheiator desferrioxamine
0
(Chapter IV) because
the naturally occurring organic ligands that bind Fe in the sea are
siderophore-like chelates (i.e. desferrioxamine B, Rue and Bruland 1995).
Here 1 attempted to reconcile my observations that organically
bound Fe is accessible to phytoplankton with those of previous work on inorganic Fe transport. The results of my thesis build on the work of Anderson and Morel (1980, 1982), and Hudson and Morel (1990, 1993) on the mechanism of Fe uptake by phytoplankton and shows that phytoplankton
can access organically complexed Fe through a reductive mechanism at the ce11 surface. Diatoms are able to reduce and take up Fe bound to some of the strongest Fe ligands, siderophores, and thus grow in their presence. This mechanism is induced under Fe-limitation, and because of
its link to NO3' metabolism, 1 hypothesize t h a t t h e plasmalemma redox enzyme NR mediates the reduction of organically bound Fe.
Reknaxs Albrecht-Gary, A.M.,
a n d A . L . C r u m b l i s s . 1998. C o o r d i n a t i o n c h e m i s t r y o f
s i d e r o p h o r e s : therrnodynamics a n d k i n e t i c s o f i r o n c h e l a t i o n a n d r e l e a s e . I n Metal Ions i n Biologicdl Systems, 3 5 , I r o n t r a n s p o r t and s t o r a g e i n microorganisms, p l a n t s , and animals. A . S i g e l and K. S i g e l ( e d . ) , p p . 239-327. More1 D e k k e r I n c . , New York. A l l n u t t , F.C.T., a n d W . D . B o n n e r Jr. 1987. C h a r a c t e r i z a t i o n o f i r o n u p t a k e f rom f e r r i o x a m i n e B by Chlorella vulgaris. P l a n t P h y s i o l . , 0 5 : 746-750.
A n d e r s o n , M.A., a n d F.M.M. Morel. 1 9 8 0 . U p t a k e of Fe(I1) b y a d i a t o m i n o x i c c u l t u r e medium. Mar. Biol. L e t . , 1: 2 6 3 - 2 6 8 . A n d e r s o n , M.A., a n d F . M . M . Morel. 1982. T h e i n f l u e n c e o f a q u e o u s i r o n c h e m i s t r y on t h e u p t a k e o f i r o n by t h e c o a s t a l d i a t o m
Thalassiosira weissflogii. Limnol. O c e a n o g r . , 27: 789-813. d e B a a r , H.J.W., J.T.M. d e J o n g , D . C . E . U.
B a k k e r , B.M. L o s c h e r , C . V e t h ,
Bathmann, a n d V . Smetacek. 1995. I m p o r t a n c e of iron for
p h y t o p l a n k t o n bloorns a n d c a r b o n dioxide drawdown i n t h e S o u t h e r n Ocean. N a t u r e , 3 7 3 : 412-415. Braun, V., K. H a n t k e , a n d W . Koster. 1 9 9 8 . B a c t e r i a l i r o n t r a n s p o r t : m e c h a n i s m s , g e n e t i c s , a n d r e q u l a t i o n . In M e t a l Ions in Biological Systems, 35, I r o n t r a n s p o r t a n d s t o r a g e i n m i c r o o r g a n i s r n s , p l a n t s , a n d a n i m a l s . A . S i g e l and H . S i g e l (ed.), p p . 67-145. Morel Dekker f n c . , New York.
C a s t i g n e t t i , D . , and J.J. Smarrelli. 1984. S i d e r o p h o r e r e d u c t i o n c a t a l y z e d by h i g h e r p l a n t N A D H : n i t r a t e r e d u c t a s e . Biochem. B i o p h y s . Res. Commun., 1 2 5 : 52-58. C o a l e , K.H., S.E. F i t z w a t e r , R . M . Gordon, K . S .
Johnson, and R.T. Barber.
1 9 9 6 . I r o n l i m i t s new p r o d u c t i o n a n d community g r o w t h a t p i c o m o l a r l e v e l s i n t h e e q u a t a r i a l P i c i f i c o c e a n . N a t u r e , 3 7 9 : 621-624. C o r z o , A . , R . P l a s a , a n d W.R. U u r i c h . 1 9 9 1 . E x t r a c e l l u l a r f e r r i c y a n i d e r e d u c t i o n a n d n i t r a t e r e d u c t a s e activity i n t h e green a l g a
Momoraphidium b r a u n i i . P l a n t S c i . , 7 5 : 221-228. Doucette, G . J . ,
and P . J .
H a r r i s o n . 1 9 9 1 . A s p e c t s of i r o n a n d n i t r o g e n
n u t r i t i o n i n t h e red t i d e d i n o f l a g e l l a t e Gymnodiaium sanguineum. 1. E f f e c t s o f i r o n d e p l e t i o n a n d n i t r o g e n s o u r c e o n b i o c h e m i c a l
c o m p o s i t i o n . Mar. B i o l . ,
1 1 0 : 165-173
Duce, R . A . , and N.W. Tindale. 1991. Atmospheric transport of iron and its deposition in the ocean. Lirnnol. Oceanogr., 36: 1715-1726.
Dugdale, R.C., and J.J. Goering. 1967. Uptake of new and regenerated formç of nitrogen in primary productivity. Limnol. Oceanoqr., 12: 196-206. Eppley, R.W., and B.J. Peterson. 1979, Particulate organic flux and planktonic new production in the deep ocean. Nature, 282: 677680. Finden,
O.A.S..
E. Tipping, G . M . H .
Jaworski, and C.S. Reynolds. 1984.
Light-induced reduction of natural iron(I11) oxide and its relevance to phytoplankton. Nature, 309: 783-784. Galvan, F., L.C. Romero, and A.J. Marquez. 1987. Metalloproteins invalved in the inorganic nitrogen metabolism of Chlamydanonas reinhardtii and other green algae, p . 195-197. In W.R. ULrich
et al. [eds.], inorganic nitrogen metabolisin. Springer. Gledhill, M. and C . M . G . van den Berg. 1994. Determination of complexation of iron ( I I I ) with natural organic cornplexinq Ligands
in seawater usinq cathodic stripping voltammetry. Mar.Chem., 4 7 , 41-54.
Green, R.M., R.J. Geider, and P.G. Falkowski. 1991. Effect of iron limitation on photosynthesis in a marine diatom. Limnol. Oceanog., 36, 1772-1782. Guerinot, M.L., and Y. Yi. 1994. Iron: nutritious, noxious, and not readily available. Plant Physiol., 104: 815-820. Harrison, G.I., and F.M.M. Morel. 1986. Response of the marine diatom Thalassiosira weissflogii to iron stress. Limnol. Oceanoqr., 31: 989-997.
Hewitt, E.J. 1983.
A
perspective of mineral nutrition: Essential and
functional minerals in plants, p . 273-323. In D.A. Robb and W.S. Pierpoint [eds.], Metals and minerals: Uptake and utilization by plants. Academic. Hudson, R.J.M. 1989. The chemical kinetics
of iron uptake by marine
phytoplankton. Ph.D. thesis, Massachusetts Institute of Thechnology .
Hudson, R.J.M., and
F.M.M.
Morel. 1990. Iron transport in marine
phytoplankton: kinetics of cellular and medium coordination reactions. Limnol. Oceanoqr., 35: 1002-1020. Hudson, R.J.M., and F.M.M. Morei. 1993. Trace metal transport by marine microorganisms: implications of metal coordination kinetics. DeepSea
Res. 1, 40, 129-150.
Johnson, K.S., R.M. Gordon, and K.H. Coale. 1997 What controls dissolved iron concentrations in the world ocean? Mar. Chem., 5 7 : 137-161. Jones, G.J., and F.M.M. MoreL. 1988. Plasmalemma redox activity in the diatom Thalassiosira. A possible role for NR. Plant Physiol., 87: 143-147.
Jones, G.J., B.P. Palenick, and F.M.M. Morel. 1987. Trace metal reduction by phytoplankton: the role of plasmalemma redox enzymes. J. Phycol., 23: 2 3 7 - 2 4 4 . Kessler, E., and F.C. Czyqan 1968. The effects of iron supply on the activity of nitrate and nitrite reduction in green algae. Arch. Microbiol., 60: 282-284 LaRoche, J., P.W. Boyd, R.M.L. McKay, and R . J . Geider. 1996. Flavodoxin as an in situ marker for iron stress in phytoplankton. Nature, 3 8 2 : 802-805.
Longhurst,
A,R.
1991. Role of the marine biosphere in the global carbon
cycle. Limnol. Oceanogr., 36: 1507-1526. Martin, J.H. 1992.
Iron as a limiting factor in oceanic productivity.
In Primary productivity and biogeochemical cycles in the sea. P.G. Falkowski and A.D. Woddhead [ e d s . ] . Plenum Press. Martin, J.H., and S.E. Fitzwater. 1988. Iron deficiency limits phytoplankton growth in the north-east Pacific subarctic. Nature, 331: 341-343. Martin, J.H., R.M. Gordon, S.E. Fitzwater, and W.W. Broenkow. 1989. VERTEX: phytoplankton/iron studies in the Gulf of Alaska. Deep-Sea
Res., 36: 6 4 9 - 6 8 0 . Martin, J.H., R.M. Gordon, and S.E. Fitzwater. 1991. The case for iron. Limnol. Oceanogr., 36: 1793-1802. Martin, J.H., K.H. Coale, K.S. Johnson, S.E. Fitzwater, R . M . Gordon, S.J. Tanner, C.N. Hunter, V.A. Elrod, J.L. Nowicki, T.L. Coley,
R.T. Barber, S. Lindley,
A . J.
Watson, K. Van Scoy, C .S. Law,
M.I. Liddicoat, R. Ling, T. Stanton, J. Stockel, C. Collins,
A.
Anderson, R. Bidigare, M. Ondrusek, M. Latasa, F.J. Millero, K. Lee, W. Yao, J. 2. Zhanq, G . Friederich, C. Sakamoto, F . Chavez, K . Buck, 2 . Kolber, R. Greene, P. Falkowski, S.W. Chisholm, F.
Hoge, R. Swift, J. Yungel, S. Turner, P. Nightingale,
A.
Hatton, P.
Liss, and N . M . Tindale. 1994. Testing the iron hypothesis in ecosystems of the equatorial Pacific ocean. Nature, 371: 123-129 Meyerhoff, P.A., T.C. Fox, R.L. Travis, and R.C. Huffaker. 1994. Characterization of the association of nitrate reductase with barley
( Hordeum
v u l g a r e L . ) root membranes. Plant Phys i o l . , 104 :
925-936.
Moffet, J.W., and R.G. Zika. 1987. Reaction kinetics of hydrogen peroxide with copper and iron in seawater. Environ. S c i . Technol., 21: 804-810.
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- 1 7 5 5 .
Neilands, J . B . 1 9 7 4 . Microbial Lron Metabolism. Academic Press, New York, New Y o r k . Price, N . M . , and F.M.M. Morel. 1998. Biological cycling of iron in
the oceans. In Metal Ions in B i o l o g i c a l Systems, 35, Iron transport and storage in microorqanisms, plants, and animais. A.
Sigel and H. Sigel (ed.), pp. 1-36. More1 Dekker Inc., New
York. Price, N . M . , L . Anderson, and F.M.M. Morel. 1991. Iron and nitrogen
nutrition of equatorial Pacific plankton. Deep-Sea Res., 38: 13611378.
Price N.M., B.A. Ahner, F.M.M. Morel. 1 9 9 4 . Ocean:
The equatorial Pacific
Grazer controlled populations in an iron-limited
ecosystem. Limnol. Oceanoqr., 39: 520-534. Raven,
J.A.
1988.
The iron and molybdenum use efficiencies of plant
growth with different energy, carbon, and nitrogen sources. New Phytol . ,
109: 2 7 9 - 2 8 7 .
Raven, J.A., and F.A. Smith. 1976. Nitrogen assimilation and transport in vascular land plants in relation to intracellular pH regulation. New Phytol., 76: 415-431.
Redinbaugh, M.G., and W.H. Campbell. 1983. Reduction of ferric citrate catalyzed by NADH:Nitrate reductase. Biochem. Biophys. Res. Comm., 114: 1182-1188. Rich, H.W., and F.M.M. Morel. 1990.
The availability of well-defined
iron colloids to the marine diatom Thalassiosira weissflogii. Limnol. Oceanogr., 35: 652-662.
Rue, E.L., and K.W. Bruland. 1995. Complexation of Fe(LI1) by natural organic ligands in the central N o r t h Pacific as determined by a new cornpetitive ligand equilibration/absorptive cathodic stripping voltammetric method. Mar. Chem., 50: 117-138. Rueter J., and D. Ades. 1987. The role of iron nutrition in photosynthesis and nitrogen assimilation in Scenedesmus q u a d r i c a u d a (Chlorophyceae). J , Phycol., 23: 452-457.
Smarelli, J., and D. Castignetti. 1988. Iron assimilation in plants: reduction of ferriphytosiderophore by NADH: nitrate reductase from squash. Pianta, 1 7 3 : 563-566.
Stohr, C., R. Tischner, and M.R. Ward. 1993. Characterization of t h e
plasma-membrane-bound nitrate reductase in Chlorella soucharophila (Kruger) Nadson. Planta, 191: 79-85. Sunda, W.G. 1991. Trace m e t a l interactions with marine phytoplankton. Biol. Oceanoqr., 6: 411-442.
Sunda, W.G., and S.A. Huntsman. 1995. Iron uptake and growth Limitation in oceanic and coastal phytoplankton. Mar. Chem., 50: 189-206.
Sunda, W.G., and S.A. Huntsman. 1997. Interrelated influence of iron, Light and ce11 size on marine phytoplankton qrowth. Nature, 390:
389-392.
Sunda W . G . ,
D.G. Swift, and S.A. Huntsman. 1991. Low iron requirement
for growth in oceanic phytoplankton. Nature, 351: 55-57 Taylor, S.R. 1964. Abundance of chemical elements in the continental crust: a new table. Geochim. Cosmochim. Acta, 28: 1273-1285.
Timmermans, K.R., W. Stolte, and H.J.W. de Baar. 1994. Iron-mediated effects on nitrate reductase in marine phytoplankton. Mar. Biol., 121: 389-396.
Tischner, R., M.R. Ward, and R.C. Huffoker. 1989. Evidence for a plasmamembrane-bound nitrate reductase involved in nitrate uptake by C h l o r e l l a sarakiniana. Planta, 178: 19-24.
Waite, T.D., and F.M.M. Morel. 1 9 8 4 . Coulometric study of the redox dynamics of iron in seawater. Anal. Chem., 56: 7 8 7 - 7 9 2 . Ward, M.R., H.D. Grimes, and R.C. Huffaker. 1989. Latent nitrate reductase activity is associated with the plasma membrane of
corn roots. Planta, 177: 4 7 0 - 4 7 5 . Ward, M.R., R. Tischner, and R.C. Huffoker. 1988. Inhibition of nitrate transport by anti-nitrate reductase Ig.
G
fragments and the
identification of plasma membrane associated nitrate reductase in roots of barley seedlings. Plant Physiol., 88: 1141-1145. Wells, M.L. 1989.
The Lability of iron in seawater and its
relatianship to phytoplankton. Ph.D. thesis. University of Maine. Wells, M.L., N.G. Zorkin, and A.G. Lewis. 1983. The role of colloid chemistry in providing a source of iron to phytoplankton. J.
Mar. Res., 41: 7 3 1 - 7 4 6 . 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 çystems. Tech. Note ~
~
1 R.M. 8 Parsons
Lab for Water Resources and Environmental Engineering, MIT, Cambridge, Dept. of Civil Engineering. Wilkins, R.G. 1991. Kinetics and mechanisms reactions of transition metal complexes, 2nd ed., VCH, Weinheim, Cermany. Wu, J., and G.W. Luther. 1995. Evidence for the existence of Fe(II1) organic complexation in the surface water of the Northwest Atlantic ocean by a competitive ligand equilibrium method and a kinetic approach. Mar. Chem., 5 0 : 159-177.
Neil Price suggested that 1 begin my thssis examining the interaction between Fe nutrition and N metabolism in marine centric diatoms of the genus Thalassiosira sp.
The main goal of t h e study
was to experimentally confirm t h e increased Fe demand
of
p h y t o p l a n k t o n when grown in n i t r a t e ( ~ 0 3 - ) - comparod to ammonium ( N H ~ + ) - based medium.
To investigate the generality of t h e response,
1 examined s i x centric diatoms isolated from a variety of habitats:
coastal, oceanic, and an Fe-limited oceanic region. The idea of t h i s paper was Neil's.
1 designed the experiments,
collected and analyzed the data, and wrote the paper.
Neil trained
me in the lab (which makes me feel very lucky), and provided
extensive editing h e l p , insight, and discussion through out the
completion of t h i s p a p e r .
Maria T. Maldonado and Neil M. Price
Marine Ecology Progress Series 141: 161-172.
Ahstract The interaction between Fe requirements and N metabolism in centric diatoms was investigated to determine whether use of nitrate (NOf)
imparts a higher cellular Fe demand for qrowth than use of
ammonium ( N H ~ + ) , and thus reduces fitness under Fe deficiency. species of the genus Thalassiosira
Six
€rom a variety of habitats were
examined. Coastal and central gyre representatives grew faster in Fesufficient media containing N H ~ + , but isolates from the equatorial Pacific, an oceanic hiqh nutrient-low biomass region, achieved maximum
rates with NO3-.
Iron quotas ranqed from 26 to 102 pmol Fe:mol C and
were not affected in a predictable manner by N source or habitat. Relative qrowth rates were diminished in Fe-deficient media, paxticularly in coastal species which grew at Less than 25% of their Al1 oceanic species maintained fast rates of maximum rates (ha,). growth (0.8 ha*) under the same Fe-limiting conditions, despite havinq 4 times Less intracellular Fe than the coastal species. 1ron:C ratios of Fe-deficient Thalassiosira spp. ranged €rom 1.7 to 14 )unol:mol and were significantly greater N source ( p < 0.05).
(by = 1.8 times) in a l 1 species when
N03-
was the
Steady-state Fe uptake rates were also faster in
NO3- dependent cells at low Fe. Nitrogen source had different effects on Fe-limited growth rates. Surprisingly, T h a l a s s i o s i r a o c e a n i c a (clone 1 0 0 3 ) and T. weissflogii grew faster with N 0 3 - even though higher Fe
requirements for use of oxidized N were expected to reduce division When total Fe concentrations in the own rates relative to ~ ~ ~ + - g r cells. medium were decreased to 1 nM, growth rates of T. o c e a n i c a (clone 1 0 0 3 ) decreased to 0.2 hax and were significantly faster (25%) in N03'-amended media.
than Ln
Under these more stressful Fe-limiting conditions,
Fe quotas were the same in cells cultured in both N-based media. Our results thus demonstrate that phototrophic phytoplankton require significantly more cellular Fe to grow on NO3- than NH~'. Nitrate-grown cells are able to obtain this extra Fe, even when Fe is limiting, suggesting t h a t Fe acquisition is somehow linked to NO3' metabolism. Under severe Fe deficiency, however, N O 3 - utilization reduces division rates compared to NHq',
because cells are unable to
fulfill their extra Fe requirements.
Surface waters of most oceanic regions contain extremely low dissolved Fe concentrations (Landing and Bruland, 1987; Martin et al., 1989, 1991), t h e cumulative result of low Fe inputs, and high chernical
and biological reactivity.
In p a r t s of the Pacific and Southern oceans,
for exarnple, the concentrations of Fe are so low that they Limit primary
productivity (Martin and Fitzwater, 1988; Martin et al., 1989). Evidence for Fe limitation has been obtained from experiments conducted at different spatial and temporal scales, €rom bottle incubations (Martin and Fitzwater, 1988; Martin et al., 1990, 1991; Price et a l . ,
1991, 1994) to in situ
fertilization experiments (Martin et al., 1994).
Some studies, investigating deficient in Fe,
Fe-N
interactions in waters rich in
NO3-
but
have demonstrated that Fe additions not only enhanced
net phytoplankton growth and biomass, but also N-specific N03' uptake rates of the phytoplankton community (Price et al., 1991, 1994). In contrast, specific N H ~ +uptake rates were unaffected by Fe enrichments. The field results provide indirect evidence of the role of Fe in N03- metabolism.
They are consistent with theoretical calcuLations based
on Fe-use efficiencies and cellular metabolic Fe demands that predict that phytoplankton growing on NO3' need 1.6 times more intracellular Fe than those growing on N H ~ +(Raven, 1988). Ammonium is incorporated directly into amino acids after it is taken up by the cell, in contrast to N 0 3 - which must first be reduced to
N H ~ ' before
assimilation (Syrett
1981). The increased Fe requirement for NO3' use arises because the NOj'
assimilatory enzymes nitrate reductase (NR) and nitrite reductase (NiR) are Fe-containinq redox enzymes (with cytochrome557- and ferredoxinprosthetic groups, respectively) (Cardenas et al., 1974; Zumft, 1971; Guerrero et al., 1901; Galvan et al., 1986).
Additional reductant is
also necessary for NO3' reduction (8 mol c'/mol
N)
and is derived from
Fe-dependent photosynthetic redox reactions. Phytoplankton Fe requirements should thus reflect the N source used for growth: N03- users are predicted to need higher intracellular Fe concentrations. Such high Fe requirements should impede NO3* consumption when Fe is scarce so that
in NO3- rich-Fe deficient waters, new production w i l l be significantly restrained . Despite the field investigations (Price et al., 1991, 1994) and a theoretical prediction (Raven, 1988), the interdependence of Fe and
N
metabolism in phytoplankton has received Little attention. Activity of the NO3' assimilatory enzymes, for example, is inhibited when Fe limits ce11 growth (Kessler and Czygan, 1968; Timmermans et al., 1994) and NO3'
uptake rates are correspondingly reduced (Rueter and Ades, 1987). Consistent with the role of Fe in NO3- metabolism, Doucette and Harrison (1991) measured higher Fe:N ratios in Fe-limited Gymnodinium sanguineum
grown on
N03-
than in those grown on NH~'.
While these studies infer a
relationship between Fe and NO3- metabolism, direct proof of the higher intracellular Fe concentrations required for phytoplankton growth on N03-
compaxed with that on N H ~ +is still lacking.
The main goal of this study was to experimentally confirm the results of Price and others
(
1991, l994), and the theoretical
predictions of Raven (1988), suggesting a higher Fe demand for phytoplankton growth on NO3- compared with that on N H ~ + . To investigate the generality of a higher Fe requirement for NO3- use by phytoplankton, we examined a variety of centric diatoms isolated from coastal and
oceanic regions, includinq the low Fe waters of the equatorial Pacific, an oceanic h i g h nutrient-low biomass region (HNLB, Chisholm and Morel,
1991). S i x species of the g e n u s Thalassiosira
habitats were cultivated in Fe-sufficient (8.4 and Fe-Limiting media (12.5 nt4 Fe and 100 NO3- or N H ~ +as a N source.
from these different pl4
Fe and 100 p.hl EDTA)
EDTA) enriched with either
Experiments tested the effects of N source
( N H ~ +v s . N 0 3 - ) on growth rates, biochemical composition, and cellular Fe
requirements as a function of Fe nutritional s t a t e .
Six species of centric diatorns of the genus Thalassiosira were
examined. Two species, T. pseudonana (clone 3H) and T. weissflogii
(clone Actin), were isolated from coastal waters (coastal isolates), and two others, T. oceanica (clones 13.1 and 1003), were oceanic species
fram the Sargasso
Sea (oceanic central gyre isolates).
These
phytoplankton were obtained from the Center for Culture of Marine Phytoplankton (Bigelaw Laboratory for Ocean Sciences, West Boothbay Harbor, ME).
Thalassiosira subtilis (clone 50 Ait) and T. partheneia
(clone Thal 9 ) were isolated (by N. M. Price) from Fe-enriched (1nM) water samples from the equatorial Pacific ocean ( 1 4 2 9 ,
3OS)
in August
1991 (EQPAC isolates).
Culture Media
Al1 phytoplankton were grown in the artificial seawater medium Aquil (More1 et al., 1979; Price et al., 1988/89) containing standard enrichments of phosphate (pod3-)and silicate ( s i o j 2 - ) ,with either 50 H03- or 50 pM NH~'.
)iH
Synthetic ocean water (SOW, pH 8.2) and nutrient
enrichment stock solutions (NOg- and NH~')
were purified of trace metals
using Chelex 100 ion exchange resin (Bio-Rad Laboratories, Richmond,
CA)
according to the procedure of Price et al. (1988/89). Media were sterilized by microwaving in acid-washed Teflon bottles (Keller et al., 1988), and enriched with filter-sterilized (0.2 p Acrodisc) EDTA-trace
metal and vitamin (812( thiamine and biotin) solutions. Trace metal concentrations were buffered with 100 pl4 EDTA so that Cu, Mn, Zn, Co free-ion concentrations were 10-l39'-
M, 1 0- 2 ~ 7 MI ~ L O - ~ M,~ 10-1° - ~ -~8 8 M,
respectively. These concentrations were calculated using the chernical
equilibrium program MINEQL (Westall et al., 1976). Total Mo and Se concentrations in the media were
M,
and 1 o m 8 M, respectively.
Premixed Fe-EDTA (I:l) was added separately at a total concentration of 8.4 ph¶, or 12.5 nM to achieve free ferric ion concentrations of 10-18-18 M,
and
M,
corresponding to estimated inorganic Fe concentrations
(Fe') of 41 nM and 20
PM.
The estimated Fe' concentrations were those
computed for Aquil seawater medium in the dark by the chemical equilibrium proqram MINEQL (Westall et al., 1976), iqnorinq possible precipitation of Fe hydroxides at high Fe concentrations (Sunda and Huntsman, 1995), and photoreduction of FeEDTA chelates (Hudson and Morel, 1990 ) . In some experiments Fe was not added to the medium.
In these
cases, phytoplankton were able to grow using the low Levels of Fe contamination in our medium, estimated from final biomass yield of T.
oceanica 13.1 to be 1-2 nM.
To minimize contamination, al1
manipulations were performed in a Laminar flow hood.
The medium was
allowed to equilibrate chemically overnight before use and was stored in sterile, acid-washed polycarbonate bottles. Al1 bottles and tubes used in our experiments were acid-washed (soaked for at least 24 hours in 10% HC1 solution), followed by repetitive washes with Q-H2O (Millipore, > 18
Mn cm-1).
Phytoplankton were grown in acid-washed, 28 ml polycarbonate tubes a t 200C under a continuous, saturating photon flux density of 200 pE m - 2 s'l,
using the semi-continuous batch culture technique described by Brand
et a l . (1981). Although sterile techniques were used for al1 culture work to minimize bacterial contamination, the cultures were not axenic. Using the Coulter counterB (mode1
ZM)
however, we estimated that
b a c t e r i a l b i a v o l u m e s i n o u r c u l t u r e s were n e g l i g i b l e c o m p a r e d t o t h o s e of phytoplankton.
F u r t h e r m o r e , i n most o f t h e e x p e r i m e n t s , c u l t u r e s
were c o l l e c t e d o n f i l t e r s w i t h p o r e s i z e o f 1 pm o r 3 p, s u f f i c i e n t t o c a p t u r e a l 1 t h e p h y t o p l a n k t o n , b u t v e r y few b a c t e r i a . Growth rates
Biomass was d e t e r m i n e d d a i l y by m e a s u r i n g i n vivo f l u o r e s c e n c e o f c u l t u r e s u s i n g a T u r n e r D e s i g n s mode1 1 0 f l u o r o r n e t e r .
S p e c i f i c growth
r a t e s were d e t e r m i n e d f r o m L i n e a r r e q r e s s i o n s o f l n i n v i v o f l u o r e s c e n c e v s . time d u r i n g t h e e x p o n e n t i a l p h a s e o f g r o w t h .
A c c l i m a t i o n was
c o n s i d e r e d c o m p l e t e when g r o w t h r o t e s o f s u c c e s s i v e t r a n s f e r s d i d n o t V a r y b y more t h a n 1 0 % . U n l e s s o t h e r w i s e s p e c i f i e d , a l 1 t h e d a t a r e p o r t e d t h r o u q h o u t a r e e x p r e s s e d a s mean
+ standard
error.
Fe quota measuremen t s
I r o n quotas ( p o l Fe:mol C ) w e r e m e a s u r e d u s i n g t h e r a d i o t r a c e r
5 5 ~ e ~ (specific ls a c t i v i t y 2 5 - 40 m C i mg-'.
DuPont C a n a d a ) .
After
c u l t u r e s were a c c l i m a t e d , a p h y t o p l a n k t o n i n o c u l u m was t r a n s f e r r e d i n t r i p l i c a t e t o a n i d e n t i c a l medium i n w h i c h 1 0 % o r 1% o f t h e t o t a l F e was a d d e d w i t h the 5 5 ~ e - r a d i o t r a c e r s o l u t i o n ( @ F e 2 1 a n d pFe 1 8 , respectively).
To e n s u r e c o m p l e t e L a b e l l i n g o f c e l l u l a r F e , 8 o r more
c e l l u l a r d i v i s i o n s were a l l o w e d b e f o r e h a r v e s t i n g .
D u r i n g mid-
e x p o n e n t i a l p h a s e , s a m p l e s w e r e f i l t e r e d o n t 0 3 p or lpm p d y c a r b o n a t e
Poretics f i l t e r s ( d e p e n d i n g o n ce11 s i z e ) , a n d a l l o w e d t o s t a n d f o r 10 m i n . i n T i ( I L I ) E D T A - c i t r a t e r e d u c i n g s o l u t i o n t o d i s s o l v e Fe h y d r o x i d e s and d e a b s o r b f e r r i c i o n s bound t o ce11 s u r f a c e s (Hudson a n d
Morel, 1 9 8 9 ) . dry.
T h e f i l t e r s were washed w i t h 1 0 m l o f SOW b e f o r e running
F l u o r ( E c o l i t e ) was a d d e d t o t h e samples, a n d then 5 5 ~ vea s
m e a s u r e d by liquid s c i n t i l l a t i o n counting w i t h a Beckman mode1 LKB
scintillation counter. Cellular 5 5 ~ values e were corrected for filter adsorption using culture media without cells. Total cellular Fe was computed using the specific activity of 5 5 ~ e in the medium (dpm mol-')
(dpm), correcting for quenching and decay. and the particulate 5 5 ~ e Culture samples were preserved with Lugol's solution and ce11 density measurements made by microscapic counting (minimum 600 cells) Palmer-Maloney chamber.
A
in a
single sample was counted twice.
Relative Fe quotas of Fe-limited phytoplankton grown in medium e the medium. with no Fe additions were measured by adding 0.2 nH 5 5 ~ to
These quotas are uncertain because the 5 5 ~ specific e activity was calculated using an estimated background Fe contamination of 1-2 nM. The relative difference between Fe content of cells in the NO3- and N H ~ + cultures however is not biased, since the same medium was used for both. (mol Fe cell'l) to Fe:C ratios To convert the cellular 5 5 ~ values e and intracellular Fe concentrations (M), measurements of mol C tell-L and ce11 volumes (fl
tell-l) were
determined in parallel cultures with non-
radioactive Fe additions. C and N intracellular c o n c e n t r a t i o n s
Cellular C and N were measured in mid-exponential phase cultures. Samples (25 ml) were filtered ont0 glass fiber filters (Whatman CF/C, pre-combusted at 450°c
for 4.5 hr.), and rinsed with 25 ml of SOW
before filtration was completed. The filters were then oven dried at 50°c, pelletized, and analyzed for elemental C and N using a Carlo Erba
1106 elemental analyzer. An aliquot of the phytoplankton culture was
reserved for immediate ce11 volume determinations using a Coulter ~ountefl(mode1 ZM) previously calibrated with polystyrene beads. An
a d d i t i o n a l s u b s a m p l e was p r e s e r v e d w i t h L u g o l ' s f o r s u b s e q u e n t ce11 counts .
Habitat Classification F o r c o m p a r a t i v e p u r p o s e s , p h y t o p l a n k t o n were g r o u p e d a c c o r d i n g t o
their s p e c i f i c growth r a t e s i n F e - d e f i c i e n t (pFe 2 1 ) medium. S t a t i s t i c a l a n a l y s i s showed t h a t t h e six s p e c i e s f o r m e d t h r e e d i s t i n c t h a b i t a t g r o u p s : c o a s t a l , oceanic c e n t r a l g y r e , a n d EQPAC.
The k o a s t a l n
g r o u p , c o m p r i s i n g T. pseudonana ( c l o n e 3 H ) and T. weissflogii ( c l o n e A c t i n ) , grew s l o w e s t , t h e " e n t r a 1
g y r e " group, c o m p r i s i n g T. o c e a n i c a
( c l o n e s 13.1 a n d 1 0 0 3 ) , grew f a s t e s t , a n d t h e "EQPAC" g r o u p , comprising T . s u b t i l i s ( c l o n e 50 A i t ) a n d T. partheneia ( c l o n e T h a l 9 ) , grew a t
i n t e r m e d i a t e r a t e s ( 2 - w a y ANOVA, p < 0 . 0 0 1 , B a n f e r r o n i t - t e s t , p < 0.05).
Results Fe s u f f i c i e n c y Under F e - r e p l e t e c o n d i t i o n s ( p F e 18) maximal s p e c i f i c growth rates ( r e g a r d l e s s of t h e N s o u r c e i n the medium) r a n g e d € r o m 1 . 1 4 t o 2 . 1 6 d''
( F i . 1).
Thalassiosira pseudonana h a d s i g n i f i c a n t l y f a s t e r r a t e s of
g r o w t h than a n y of t h e o t h e r s p e c i e s ( p < 0 . 0 5 ) .
The o t h e r coastal
r e p r e s e n t a t i v e , T. weissflogii , g r e w f a s t e r on a v e r a g e t h a n the c e n t r a l g y r e i s o l a t e s ( T , oceanica LOO3 a n d 1 3 . 1 ) , but t h e d i f f e r e n c e b e t w e e n t h e rates was n o t s i g n i f i c a n t ( p > 0 . 0 5 ) .
subtilis 0.05).
The EQPAC s p e c i e s ( T .
a n d T. partheneia) had t h e slowest growth rates a t pFe 18 ( p
0.05).
F i a u r e 1. Mean growth rates ( d - l ) for T. oceanica 13.1 ( n (n
=
IO), T. p a r t h e n e i a T h a l 9 ( n
pseudonana 3H ( n
sufficient N H ~ +(shaded
=
=
9 ) and 1 0 0 3
IO), T. subtilis 50 Ait ( n
15), and T . weissflogii Actin ( n
=
=
1 3 ) , T.
2 5 ) , grown on Fe-
(pFe 18) Aquil medium enriched with 50 pM NO3'
bars) as N source.
=
(open bars) or
Error bar represents t h e standard e r r o r
around the mean, and was too small to be resolved i n cases where it is not visible. Species' growth rates with same l e t t e r above bars were not significantly
different from o n e another ( 2 - w a y ANOVA ( N source and habitat g r o u p ) p < 0 . 0 5 , Bonferroni t-test p c 0 . 0 5 ) .
*significantly d i f f e r e n t ( p a i r e d t - t e s t : N O 3 * v s . N H ~ +growth rates, p < 0.01)
+*significantly different (paired t-test: ~ 0 3 -v s . NHq* growth r a t e s , p c
Nitrogen substrate significantly affected the maximum growth rate attained by the species (Fig. 1) except in T. oceanica 13.1. The EQPAC isolates grew faster
(t-test,p < 0.01) in NO3' than in N H ~ +-amended
media, while the central gyre clone, T. oceanica 1003, and the coastal isolates, T. pseudonana
and T. weissflogii, grew significantly faster
with N H ~ +as the N substrate (t-test, p < 0.01). Neither N source nor habitat had predictable effects on the intracellular chemical composition of the Fe-replete phytoplankton species.
Intracellular Fe, C, and N concentrations ranged from 0.4
2.8 mM, 7.8 :N
-
27 M, and 1.16 - 3.32
M,
respectively (Table 1).
-
Carbon
ratios were in good agreement with Redfield proportions, indicative
of healthy cells. Fe:C ratios ranged from 26 to 102 junol Fe:mol C and in three species ( T . partheneia, T. oceanica 1 3 . 1 , and 1003) were similar in
N H ~ +and N 0 3 -
-amended cultures.
Thalassiosira
s u b t i l i s and
T. pseudonana had higher Fe:C ratios when growing w i t h NO3'.
Thalassiosira weissflogii
showed the opposite trend with higher Fe:C
ratios in the N H ~ +-qrown cells. With the exception of T. oceanica 13.1, ce11 volumes were always greater in N H ~ + than in
N03'
grown cells (Table
1) Fe
deficiency
In general, growth rates of al1 the species were significantly reduced under Fe-limiting conditions (pFe 21) (Fig. 2).
The exceptions
were T. p a r t h e n e i a ( ~ ~ ~ + - a m e n cultures) ded and T. o c e a n i c a 1003
(NO3'-
âmended cultures), which had slower rates of growth in pFe 21 tlian in
pFe 18, but the difference was not statistically significant ( p > 0.05). Coastal species showed the greatest growth rate reduction (mean p / h a x 0.21), whereas al1 oceanic isoLates grew at rates near their maximum
=
(mean p/hax
=
0.8). Regardless of N source, the specific growth rates
of the coastal, the central gyre, and the EQPAC species were significantly different at pFe 21 (2-way ANOVA, p < 0.001, Bonferroni ttest, p e 0.05) and increased in the order of coastal, EQPAC, and central gyre species. Intracellular Fe concentrations of cells in Fe-limited cultures ranged from 14 to 154
and were greatly decreased by an average of 10-
fold compared with those of Fe-sufficient cells. Except for T. pseudonana, C (11-25 M) and
N
(1.88-3.23 M) concentrations were not
significantly affected by Fe limitation (Table 2). Fe:C ratios of coastal species (mean
=
9.35 f 4.1 p 0 1 Fe:mol C) were approximately
four times those of al1 other species (mean
=
2.46
+
1.3 pmol Fe:mol C,
t-test, p < 0.05) (Table 2), a consequence of higher intracellular Fe (mol Fe fl-l), and lower C (mol C fl-l) concentrations. Coastal isolates were smaller when grown on N H ~ + , whereas oceanic isolates were srnaller when grown in
NO3-
cultures (t-test,p
c
0.05). Ce11 volumes of T.
oceanica 13.1 were the same in both media.
Under Fe-limiting conditions, half of the species examined had
similar specific growth rates in
N03-
and ~~~'-arnended media (Fig. 2).
The effects of N source on those species with dissimilar growth rates in the two media v a r i e d . rates in
Thalassiosira partheneia achieved faster growth
N H ~ +(t-test,p
0.05). Nitrate-grown cells were thus
unable to acquire the extra Fe needed for
NO3'
metabolism under these
very limiting conditions and consequently grew slower than ~ ~ ~ ' - q r o w n cells.
Fe quotas and qrowth rates
The results of this study show clearly that N substrate influences both Fe uptake and Fe requirements for phytoplankton growth. Under Fe deficiency, al1 species examined contained significantly more Fe cell-l
volume and mol Fe:mol C when using N03- as opposed to NH~'.
Carbon and N
composition of the cells was unaltered by Fe limitation, consistent with previous studies (Doucette and Harrison, 1991; La Roche et al., 1993).
The average increase in cellular Fe content (1.6 and 1.8 times, mol Fe cell'l and mol Fe:mol C, respectively) agrees remarkably well with the increase in metabolic iron requirements predicted from first principles (Raven, 1988; More1 et al., 1991).
Thus, the greater Fe quota of
NO3'-
grown cells can be accounted for by the iron contained in the NOgassimilatory enzymes and in the redox proteins that supply reductant for NO3-
reduction.
Higher Fe demand for NO3- use might easily be fulfilled when Fe concentrations are saturating for qrowth, as in the case of pFe 18
medium.
Changes in the Fe transport system could accommodate increases
or decreases in cellular requirements affected by the N sources. Such modulation of Fe transport is one means by which phytoplankton maintain near maximal rates of growth over a wide range of Fe concentrations. Iron uptake is a function of the rate constant of metal binding to the transport ligand, the transport ligand density and the concentration of reactive Fe ( F e i ) (Hudson and Morel, 1990).
When phytoplankton are
grown in low Fe media, Fe uptake capacity increases by several-fold (Harrison and Morel, 1986). This adaptive strategy compensates for Louer Fe concentrations, but it has a limit. The limit is ultimately
set by the maximum density of iron transport ligands that can fit in the ce11 membrane.
In Thalassiosira weissflogii, this corresponds to about
5 pmol c m - 2 (Hudson and Morel, 1990).
As Fe concentrations decrease
further, the enhanced transport system can no longer acquire Fe at a sufficient rate, and cellular growth rates are correspondingly reduced. Thus, under growth rate limiting conditions, both N 0 3 - - and NH~+-grown
cells should equally enhance their Fe transport systems to maximize Fe acquisition. Nitrate-grown phytoplankton should not have a greater own ability to take up more Fe than ~ ~ ~ + - g r cells. Another way for phytoplankton to achieve hiqher Fe quotas under limiting conditions is to decrease ce11 size.
Indeed, a l 1 Thalassiosira
species were observed to be significantly smaller in Fe-limitinq than in Fe-sufficient media.
The benefit of size reduction exists because
transport is proportional to surface area and Fe quota is proportional to volume. ratios
Small cells, which have greater surface area to volume
(SAD)
than Large ones, may thus transport Fe at faster rates
relative to volume and hence accumulate larger quotas.
Nitrate-grown
cells could theoretically obtain this same advantage if they were o w n however, their reduction in size would have smaller than ~ ~ ~ + - g r ones, to be very substantial for this to be an effective mechanism.
The effect of size reduction can be illustrated in the following example by considering two phytoplankton cells, one growing on NO3' and the other on N H ~ + .A
t
steady state, Fe quota (Q; mol celle')
is simply
the ratio of the uptake rate (p; mol celle= h - l ) to the growth rate (k; h-1):
This equation can also be rewritten as a function of cellular volume: Q/v
where
&A
21
QSA( SA/V)/P
is the transport rate per membrane surface area.
If
PSA
is the
same regardless of N source and we assume for the moment that growth
rates are also equal, then the relative increase in quota is equal to the relative increase in S A P of the cell:
31
( Q / V N O ~ - I / ( Q / V N H=~ + (SAflN03) )/(sA/VNH~+)
To account for a 1.6 times greater Fe quota, N03--grown phytoplankton would thus have to have a 1.6 times greater
SA/V
than N H ~ + -
grown cells. However, the mean (sA/VNO~-)/(SA/VNH~+) at pFe 21 for al1 species examined in this study was only 1.04 f 0.08, so size reduction alone cannot account for the increased Fe quota in the NOf
cultures. If
under Fe limitation, volume reduction were the only means by which N 0 3 - n then a qrown cells could increase Fe uptake compared to ~ ~ ~ ' - g r o wcells,
hypothetical ce11 of a 1000 pn3 would have to reduce its size to 298 m3
when grown i n
Nol-. T h i s e x t e n t o f volume r e d u c t i o n was n e v e r o b s e r v e d
d u r i n g o u r s t u d y , a n d i n t w o c a s e s (2'.
weissflogii
and T. pseudonana)
h a d h i g h e r Fe c e l i s were a c t u a l l y smaller when g r o w i n g o n N H ~ +but , q u o t a s w i t h N03'. A c c o r d i n g t o e q u a t i o n 1, N03'-grown
c e l l s m u s t e i t h e r grow slower
a n d / o r t r a n s p o r t Fe a t a f a s t e r r a t e t h a n ~ ~ ~ + - g r ocw e lnl s i f t h e y c o n t a i n more Fe when F e is l i m i t i n g . t r a n s p o r t rate i n N03'-grown
A
p r e f e r e n t i a l i n c r e a s e i n t h e Fe
cells is i n c o n s i s t e n t w i t h our u n d e r s t a n d i n g
of F e t r a n s p o r t r e q u l a t i o n , a s d i s c u s s e d a b o v e .
T h e low c o n c e n t r a t i o n
o f Fe i n the pFe 2 1 medium was c l e a r l y l i m i t i n g t o a l 1 s p e c i e s t o some d e g r e e so t h a t Fe t r a n s p o r t rates s h o u l d b e maximized i n b o t h N treatments. The g r o w t h r a t e r e s u l t s a r e a l s o u n a b l e t o a c c o u n t f o r t h e d i f f e r e n c e s t h a t were s e e n i n t h e Fe q u o t a s . NOf-grown
I n al1 but one species,
c e l l s g r e w a s f a s t o r f a s t e r t h a n ~ ~ ~ + - g r oc w e lnl s ( F i g . 2 ) ,
which s h o u l d t e n d t o d e c r e a s e n o t i n c r e a s e t h e i r Fe q u o t a s .
This
observation and t h e preceding analyses suggest t h a t o u r assumptions
regardfng Fe t r a n s p o r t may be i n c o r r e c t or t h a t NOf-grown
cells have
o t h e r means t o i n c r e a s e c e l l u l a r Fe c o n t e n t . B e c a u s e o f t h e h i g h e r q u o t a s and f a s t g r o w t h r a t e s , c a l c u l a t e d s t e a d y - s t a t e u p t a k e r a t e s of N03--grown c e l l s were s i g n i f i c a n t l y faster t h a n i n ~ ~ ~ + - g r ocw e lnl s i n pFe 2 1 medium ( T a b l e 3 ) .
Under t h e s e F e -
l i m i t i n g c o n d i t i o n s NO3--grown c e l l s were t h u s a b l e t o t a k e u p F e a t
faster r a t e s t h a n ~ ~ ~ + - g r oc w e lnl s , a r e s u l t t h a t was true r e g a r d l e s s o f how t h e rates were n o r m a l i z e d ( p e r u n i t volume, s u r f a c e area or c e l l u l a r C).
T h i s o b s e r v a t i o n is s u r p r i s i n g , b e c a u s e it s u g g e s t s t h a t NOf-grown
c e l l s h a v e a u n i q u e a b i l i t y t o i n c r e a s e t h e i r u p t a k e rates for F e .
Experiments that compared the effects of N substrate and Fe requirements used Aquil medium enriched with al1 nutrients except N. The different treatments were established by adding either NOg- or NH~'
to subsamples of the same medium so that any variations in Fe concentration in the base medium would be the same for both N substrates. We note that any variation in the levels of Fe contamination in the two N-based media cannot simultaneously explain the growth and quota difference that we observed. Preferential Fe contamination of the N07' media, for example, would decrease the measured Fe quota by increasing phytoplankton growth rates (Eq.1) and by diluting of the 5 5 ~ tracer. e The most obvious difference between the phytoplankton in the cultures is the additional biochemical pathways required for N O j - assimilation and transport. Ammonium produced from NO3' or taken up directly from the medium was assumed to be incorporated into amino acids by the same mechanism; although, the use of glutamate dehydrogenase instead of the glutamine synthetase pathway for NH~' assimilation (Syrett, 1981) may possibly have differed between the cult u e s . A
decrease in Fe concentration in the medium to the background
fevel reduced the growth rate of maximum.
2'.
oceanica
1003 to 20% of the
In this experiment, Fe quotas wexe not significantly different
between the
NO3-
and the N ~ ~ + - g r o w cultures. n The qrowth rates of the
NO3'-grown cultures were slower than those in N H , ~ ' , indicating that if the N03' cells are unable to acquire the extra Fe they need, they grow slower. Our results show that there is a great deal of variability in phytoplankton response among species. Some species grew slower on even at pFe 21. We predict that al1 species in this study should
NO3'
eventually grow slower on ~ 0 if~ the - F e concentration was reduced to still lower levels. The link between Fe acquisition and NO3' metabolism that our
results suggest has been observed in other taxa. Bacteria can reduce Fe(I1I) bound to organic complexes by a process involving NO3- reductase
(Ottow, 1968). Mutants Lacking this enzyme are deficient in the Fe reduction pathway (Ottow, 1970). Field studies (Sfrensen, 1982; Jones et al., 1983) also suggest that facultative NO3- reducing heterotrophs
are involved in Fe reduction. Purified NR from higher plants is able to reduce a variety of Fe complexes in vitro, such as ferric citrate (Redinbaugh and Campbell, 1983), cytochrome c (Solomonson and Vennesland, 1972), ferricyanide (Solomonson and Vennesland, 1972), and Fe bound to siderophores (Castignetti and Smarrelli, 1984).
Plasmalemma
bound NR might be involved in the acquisition of Fe by root cells since it reduces Fe-containing molecules such as cytochromes (Stohr et al., 1993; Meyerhoff et al., 1994).
Although none of these studies
demonstrate that NR reduction of Fe could enhance Fe acquisition during Fe-deficient conditions, they stronqly support the link between Fe acquisition and N O j - metabolism. phytoplankton cultures are also able to reduce cu2' and ~ e ~ + complexes (Anderson and Morel, 1980; Jones
et al., 1987) by an
extracellular reaction that has been hypothesized to be mediated by the diaphorase subunit of plasmalemma bound NR (Jones and Morel, 1988). For Fe-lirnited phytoplankton growing in chelated medium, most of the Fe is present as FeEDTA complexes and is not directly available for uptake. Reduction of this organic Fe shoulb result in higher inorganic Fe concentrations and faster phytoplankton growth rates.
Nitrate-grown
c e l l s rnay t h u s h a v e a u n i q u e mechanism t o i n c r e a s e t h e c o n c e n t r a t i o n s o f d i s s o l v e d i n o r g a n i c F e n e a r the ce11 s u r f a c e , e i t h e r b y r e l e a s e o f s o l u b l e r e d u c i n g compounds ( A n d e r s o n a n d Morel, 1 9 8 0 ; J o n e s e t a l . , 1 9 8 7 ) w h i c h r e d u c e Fe bound t o o r g a n i c c o m p l e x e s , o r by e x t r a c e l l u l a r
r e d u c t i o n o f o r g a n i c F e by plasmalemma bound r e d o x p r o t e i n s ( J o n e s e t a l . , 1987).
Iron Use Efficiencies I r o n - u s e e f f i c i e n c y ( I U E ) is d e f i n e d a s t h e C a s s i m i l a t i o n r a t e p e r u n i t o f c e l l u l a r Fe ((mol C : m o l F e ) h - l ) , c a l c u l a t e d from q u o t i e n t o f t h e s p e c i f i c g r o w t h r a t e a n d t h e Fe:C ratio.
I t t h u s provides a
c o m p a r a t i v e measure o f t h e a b i l i t y o f p h y t o p l a n k t o n t o grow u n d e r low Fe t h a t Fe q u o t a a l o n e c a n n o t c o n v e y .
O c e a n i c p h y t o p l a n k t o n a r e w e l l known
t o grow f a s t e s t h a n c o a s t a l s p e c i e s u n d e r l o w F e c o n c e n t r a t i o n s ( R y t h e r
a n d Kramer, 1 9 6 1 ; B r a n d e t a l . , 1 9 8 3 ) .
T h e i r lower Fe r e q u i r e m e n t s are
t h o u g h t t o b e a n e v o l u t i o n a r y a d a p t a t i o n t o t h e low a m b i e n t levels o f F e t h a t characterize t h e i r oceanic habitats.
I r o n use e f f i c i e n c i e s a r e
c o n s i d e r a b l y h i g h e r f o r o c e a n i c t h a n c o a s t a l s p e c i e s ( S u n d a and Huntsman, 1995), a t r e n d t h a t i s a l s o a p p a r e n t i n o u r d a t a ( T a b l e 4 ) . The t w o EQPAC i s o l a t e s a r e a l s o i n d i s t i n g u i ç h a b l e f r o m t h e c e n t r a l g y r e s p e c i e s i n t h i s r e g a r d : t h e i r a v e r a g e I U E for b o t h N s u b s t r a t e s w a s 7 . 0 I 4 . 4 x 105
(mol C:rnol F e ) d - l .
The a v e r a g e I U E f o r c o a s t a l a n d o c e a n i c p h y t o p l a n k t o n was 0 . 4 4 k
0 . 1 4 a n d 5 . 5 f 3 . 6 x 105 (mol C : m o l ) F e h - L ( T a b l e 4 ) , r e s p e c t i v e l y , i n a g r e e m e n t w i t h t h o s e v e c a l c u l a t e d from o t h e r s t u d i e s (IULcoastal
f 0 . 2 8 a n d IUEoceanic
= 4.62 k 1.19,
Table 5).
=
0.66
Nitrogen s u b s t r a t e had a
c o n s i d e r a b l e e f f e c t on IUE a s e x p e c t e d from t h e l a r g e d i f f e r e n c e s i n q u o t a s i n NO3- and ~ ~ ~ + - a m e n dc ue ldt u r e s .
Theoretical calculations of
-le
4. Fe-use efficiencies ((mol C:mol Fe) d - l ) of Fe-limited
(pFe 21)
T h a l a s s i o s i r a s p p . grown under either 50 pi N 0 3 - or N H ~ +amended media.
Fe-use efficiencies Thalassiosira s p p .
(
lo5 ( m o l C:mol Fe) d - l ) NO3 -
N H ~ +
T. p a r t h e n e i n
( T h a l 9)
T. subtilis (50 Ait)
3.8 13.0
+ + +
0.05
2.1 2 0.04
3.6
5.1
0.2
6.4
+ 0.2 + 0.05 + 0.01
T. oceanica (13.1)
7.6
T. oceanica (1003)
3.6 f 0.03
T. pseudonana ( 3 H )
0 . 5 4 0.01
0.34 k 0.01
T. weissflogii (Actin)
0.6 +, 0.03
0.3 .t 0.01
2.5
TableS.C a l c u l a t e d d-')
and meaaured F e - u s e efficicncies
((mol C:mol Fe)
of Fe-iimited phytoplankton species.
Pe:C (prnol mol-')
Crowth rale Id-')
Fe-use efficlency
N soiuce
tu IOs mol C mol-' Fe dm')
NH,' NO,' Raven (1988, 19!lO), Pe-sulfideol ceUsb
Nlit
N0,Irboratory mersriremonls Coastal spedes , nialassiosira pseudonana (3fi)' Thslassioslra weissfiogtt (AcUn)' Prorocrrntnim minimum (ENv)' Thalassiostca w e i ~ n ~( Ai ~i J I ) * Th&siosirb weisstlogii (Actin)' nialassiosirapseudonana (314)'
,-
12.5
NO
13.1
NOa-
1O.?
NO,-
18
NO3-
12.5
NO,-
7.5
NO,-
9.1
NO,'
NO,NH4' NOs-
NO,' NO,.Our calcu)ated fieoretical marima IüEm for Fe-lhiled phytoplanktoni assuming 8 mol Pe pet 'mol' eledron transport &sin, crilnilaled uslng ~hl:P~1;PSIl:cyl.b-f-PeS:ferredo~n:cyIc rnotar ratlos 8s 250:0.25;0.5;0.25;0,1;1 {Glover 1973, S a n d m a ~& M d k h 1983, Terry 1983, Sandmann 1985, Green el al. 1092), and Infedng tliat Ienedorln 1s repiaced by tlavododn and that Iha ATP neccrssry forblosynlhesis b gcncrated dlrectly lrom pholophosphorylailoo bnieorsticil minim. IUE tor Fe-sulflcisnl c e h na calculalad by Raven (1960,1990)assumlng 0.45 g C 9'' dry weighi, and growth rmter o12.6 ci'' sowcu: CSunds& Iiunismaii (1995). Syedfic growtli rates were mul(ipUed by 1.71 to adjusl llisir 14 h Uyhl:lO b dark cycle to ahe con. Uniiow Ught cycle usad in i U ihe otber .tudia d ~ n d s r &~ Morel n (1982), sssumlng s C quola of 12 pmol C cell-' .Hudson & Morel I W O ) , assumlng a C v o t a of 12 end 10 pmol C cell-' for 'I: weissnogii and II caiierae, respectlvely 'Sunda et al. (1091) @
I U E s (Raven 1988, 1990) a r e c o n s i d e r a b l y l o w e r t h a n t h e measured v a l u e s
for o c e a n i c p h y t o p l a n k t o n , by a f a c t o r o f 6 .
These t h e o r e t i c a l I U E s
were c a l c u l a t e d u s i n g m a x i m a l growth r a t e s , a n d o p t i m a l r e l a t i v e r a t i o s
of F e r e d o x c a t a l y s t s n e c e s s a r y f o r t h e p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t c h a i n for non F e - L i m i t e d a l g a e a n d p l a n t s .
These ratios
include Fe-containing redox c a t a l y s t involved i n photosynthetic e l e c t r o n transfer,
a n d t h e Fe-containing ccmponents of P S I and PSII.
Because
t h e r e l a t i v e r a t i o s of t h e s e Fe r e d o x c a t a l y s t s a r e a l t e r e d u n d e r F e -
stress before t h e rates o f p h o t o s y n t h e t i c e l e c t r o n t r a n s p o r t a n d g r o w t h are a f f e c t e d , w e h a v e r e v i s e d t h e c a l c u l a t i o n s l i g h t l y u s i n g r e c e n t l y p u b l i s h e d d a t a o n t h e s t o i c h i o r n e t r y of F e - c o n t a i n i n g redox c a t a l y s t s i n Fe-limited phytoplankton (Table 5 ) .
T h e new e s t i m a t e s a r e twice a s h i g h
a s t h e p r e v i o u s v a l u e s , b u t s t i l l c a n n o t e x p l a i n t h e high XUEs t h a t are measured.
T h e g r e a t e s t u n c e r t a i n t y i n our c a l c u l a t i o n e x i s t s i n t h e
r e l a t i v e r a t i o s of Fe r e d o x c a t a l y s t s f o r o c e a n i c and c o a s t a l phytoplankton under F e - l i m i t a t i o n .
Even t h o u q h r e p l a c e m e n t s of F e -
c o n t a i n i n g redox c a t a l y s t s by n o n - m e t a l l i c m o l e c u l e s a n d s u b s t i t u t i o n o f Fe by other m e t a l s w i t h i n c e r t a i n c a t a l y s t s h a v e been d o c u m e n t e d ,
p h y t o p l a n k t o n m i g h t have Fe s u b s t i t u t i o n s unknown t o u s p r e s e n t l y . F u r t h e r r e s e a r c h o n p o s s i b l e Fe s u b s t i t u t i o n s by p h y t o p l a n k t o n m i q h t e n l i g h t e n the discrepancy b e t w e e n c a l c u l a t e d a n d m e a s u r e d f U E s . EQPAC
isola tes T h e o c e a n i c s p e c i e s of p h y t o p l a n k t o n for w h i c h we h a v e t h e b e s t
i n f o r m a t i o n r e g a r d i n g F e r e q u i r e m e n t s have been isolated from t h e c e n t r a l q y r e s o r t h e G u l f Stream.
I r o n c o n c e n t r a t i o n s i n some o f t h e s e
h a b i t a t s a r e u n d o u b t e d l y l o w a n d h a v e b e e n s e l e c t e d for t o l e r a n t g e n o t y p e s , b u t t h e major n u t r i e n t s ( N a n d P ) are b e l i e v e d t o b e t h e
resources in shortest supply in these regions. The Fe requirements of the EQPAC isolates examined in this paper are the first r e p o r t e d for centric diatoms from Fe-limited waters (equatorial Pacific ocean). Both of these species were isolated at 0°, 140%
from incubation bottles that
had been enriched with 1 nM Fe. Their growth rates under Fe-limitation were indistinguishable from one another, and were siqnificantly slower than the T. oceanica clones from the Sarqasso Sea.
At
first blush, it
might appear that the EQPAC species are not as well adapted to live in low Fe waters as the oceanic central gyre species. However, considering that the equatorial Pacific is a reqion of intermittent upwelling (Murray et al., 1994), the phytoplankton in these waters might encounter sporadic Fe inputs lnstead of constantly low concentrations. The ability of species to sequester and store Large amounts of Fe when concentrations are high (Sunda and Huntsman, 1995) would thus seem to be advantageous. we evaluated the ability of phytoplankton ta grow under Fe-limiting conditions with sporadically high Fe concentrations (ie. scarce aeolian Fe deposition in HNLB regions, or intermittent upweliinq) by calculating the ratio between Fe-sufficient (pFe 18) and Fe-Limiting (pFe 21) Fe:C quotas (pl01 Fe: moi C) .
This ratio (QPemax/QFemin)
reflects the ability of t h e diatoms to store high intracellular Fe concentrations when
dissolved Fe concentrations are hiqh, and lower
their Fe requirements to a minimum when Fe concentrations are Limitinq. The r a t i o of the quotas was significantly higher in the EQPAC species ( 4 5 . 5 I 3.5) compared to the coastal (9.33 2 7.54) and central gyre
clones (15 2 9.9) (2-way ANOVA, p < 0.05; Bonferroni t-test, p < 0.05). Thus, T. partheneia and T. subtilis, would be able to maintain positive rates of growth for longer periods of time during Fe starvation than any
of the other species. Maximizing iron storage and lowering Fe requirements may thus represent unique strategy for phytoplankton living in low Fe environments with sporadic Fe inputs. The two EQPAC phytoplankton were also unique in their preference
for
NO3'
at high Fe. Ammonium was not likely toxic to these species
because lower concentrations in the growth media yielded the same slow growth rates (results not shown).
If the increase in Fe quota that we
observed in the N03--amendedcultures is realized in the field, then
N@3'
consumption rnay not be an obvious disadvantage, although the extent of
Fe deficiency will be important. The in situ growth rates of 2'. subtilis and T. partheneia can be estimated, usinq the measured
Fe-
limited quotas. Assuming that the inorganic dissolved Fe concentration is 10% of the total dissolved Fe (Wells et al., 1994) and that phytoplankton can take up 2/3 of the diffusive flux (Kd, Hudson and Morel, 1993), growth rates can be determined from:
where Kd
- 4xr
diffusion coefficient ( 9 + 1 0 - ~cm2 s'l) , and Fe1
-
3 pM.
The calculation suggest that both species should grow at about 50 and 75% of their maximum rates in the equatorial Pacific ocean.
Until now phytoplankton species have been either cateqorized into coastal or oceanic habitat groups based on their physiology and isolation sites (Ryther and Kramer, 1961; Tortell and Price, 1996). Our results suggest that phytoplankton from Fe-limited HNLB regions, such as the equatorial Pacific ocean, may be distinctly different from other oceanic species. This conclusion is based on their unique growth rates
under Fe-sufficient and Fe-deficient conditions, their high ratios of Q
F
~
~
~
/and Q
their F ~ particular ~ ~ ~ ~ preference for growth in N03--based
media.
The results of this study demonstrate that phytoplankton require 1.6 times more Fe f o r growth on NOj' than N H ~ ' .
Nitrate-grown cells can
obtain their extra Fe even when Fe is limiting, suggesting that Fe acquisition is somehow linked to nitrate metabolism. Under severe Fe stress, nitrate-grown cells grow slower t h a n t h o s e using N H ~ +because they are u n a b l e to obtain the extra Fe needed for growth. The deqree of Fe limitation of phytoplankton in the ocean may thus strongly influence
their use of different N substrates.
Acknowledcrments We thank Philippe Tortell and Chris Payne for invaluable help throughout out the completion of this study. We are also grateful to Maureen Soon
(U.
of British Columbia) for the CHN analysis, and to G.A.
Frywell (Texas A and M University) for kindly identifying the EQPAC
isolates T. subtilis Ostenfeld Gran. (50 Ait) and T. partheneia Shrader (Thai 9). This work was funded by grants from the Natural Sciences and Research Council of Canada, the NSERC/DFO Science
Subvention program, and by a McGill University Faculty of Graduate Studies and Research Equipment Grant.
References Anderson, M.A., and F.M.M. Morel. 1900. Uptake of Fe(I1) by a diatom
in
oxic culture medium. Mar. Biol. Lett., 1: 263-268. Anderson, M.A., and F.M.M. Morel. 1982. The influence of aqueous iron chemistry on the uptake of iron by the coastal diatom Thalssiosira weissflogii. Limnol. Oceanogr., 27 : 789-813.
Brand, L.E., R.R.L. Guiilard, and L.S. Murphy. 1981. A method for the rapid and precise determination of acclimated phytoplankton reproductive rates. J. Plankton Res., 3: 193-201. Brand, L.E., W.G. Sunda, and R.R.L. Guillard. 1983. Limitation by marine phytoplankton reproductive rates by zinc, manganese, and iron. Limnol. Oceanogr., 28: 1182-1198. Cardenas, J., J . Rivas, A. Paneque, and
M.
Losada. 1974. Effects of Fe
supply on the activities of nitrate-reducing system from Chlorella. In: Cardenas J (ed) Bioenergetics and metabolism of green algae. 1. MSS Information Corp, New York, NY, p 10-13.
Castignetti, D., and J.J. Smarrelli. 1984. Siderophore reduction catalyzed by higher plant NADH:nitrate reductase. Biochem. Biophys. Res. Commun., 125: 52-58. Chisholm, S.W., and F.M.M. Morel. L991. What controls phytoplankton production in nutrient-rich areas of the open sea? Limnol. Oceanogr . , 36 : preface . Doucette, G.J., and P.J. Harrison. 1991. Aspects of iron and nitrogen nutrition in the red tide dinoflagellate Gymnodinium sanguineum. 1.
Effects of iron depletion and nitrogen source on biochemical composition. Mar. Biol., 110: 165-173. Galvan, F., L.C. Romero, and A.J. Marquez. 1986. Metalloproteins involved in the inorganic nitrogen metabolism of Chlamydomonas reinhardtii and other green algae. In: Ullrich WR, Aparicio PJ,
Syrett PJ, Castillo F (ed) Inorganic nitrogen metabolism. SpringerVerlag, Berlin, p 195-197. Glover, H. 1977. Effects of iron deficiency on Isochrysis Galbana (Chrysophyceae) and Phaeodactylum tricornutum (Bacillariophyceae). J. Phycol., 13: 208-212.
Green, R.M., R.J. Geider, 2. Kolber, and P.G. Falkowski. 1992. Ironinduced changes in Light harvesting and photochemical energy
conversion processes in eukaryotic marine algae. Plant Physiol., 100: 565-575.
Guerrero, M.G., J.M. Vega, and M. Losada. 1981. The assimilatory nitrate-reducing system and its regulation. A. Rev. Pl. Physiol.. 32: 169-204.
Harrison, G.I., and F . M . M . M o r e l . 1986. Response of the marine diatom Thalassiosira weissflogii to iron stress. Limnol. Oceanogr., 31: 989-997.
Hudson, R., and F.M.M. Morel. 1989. Distinguishing between extra- and intra-cellular iron in marine phytoplankton. Limnol. Oceanogr., 3 4 : lll3-112O.
Hudson, R., and F.M.M. Morel. 1990. Iron transport in marine phytoplankton: kinetics of cellular and medium coordination reactions. Limnol. Oceanogr., 3 5 : 1002-1020. Hudson, R., and F.M.M. Morel. 1993. Trace metal transport by marine microorganisms: Fmplications of metal coordination kinetics. Deep Sea Res. I . , 40: 129-150. Jones,
J.G.,
S. Gardner, and B.M. Simon. 1983. Bacterial reduction of
ferric iron in a stratified entrophic Lake. J , Gen. Microbiol., 129: 131-139.
Jones, G.J., and F.M.M. Morel. 1988. Plasmalemma redox activity in the diatom Thalassiosira weissflogii , A possible role for nitrate
reductase. Plant Physiol., 8 7 : 143-147. Jones, G.J., B . P . Palenick,
F.M.M.
Morel. 1987, Trace metal reduction by
phytoplankton: the role of plasmalemma redox enzymes.
3.
Phycol.,
23: 237-244.
Keller, M.D., W.K. Bellows, and R.R.L. Guillard. 1988. Microwave treatment for sterilization of phytoplankton culture media. J. Exp.
Mar. B i o l . Ecol., 117: 2 7 9 - 2 8 3 . Kessler, E., and F.C. Czygan. 1968. The effects of iron supply on t h e activity of nitrate and nitrite reduction in green algae. Arch. Microbiol., 60: 282-284. Landing, W.M., and K.W. Bruland. 1987. The contrasting biogeochemistry of iron and manganese in the Pacific Ocean. Geochim. Cosmochim. Acta, 5 7 : 29-43.
LaRoche, J., R.J. G e i d e r , L.M. Graziano, H. Murray, and K. Lewis. 1993. Induction of specific proteins in eukaryotic alqae grown under iron-
, phosphorus-, or nitrogen-deficient conditions. J. Phycol., 29: 767-777.
Martin, J.H., K.H. Coale, K.S. Johnson, S.E. Fitzwater, R.M. Gordon, S.J. Tanner, C.N. Hunter, V.A. Elrod, J.L. Nowicki, T.L. Coley, R.T. Barber, S. Lindley, A.J. Watson, K. Van Scoy, C.S. Law, M.I. Liddicoat, R. Ling, T. Stanton, J. Stockel, C. Collins,
A.
Anderson, R. Bidigare, M. Ondrusek, M. Latasa, F.J. Millero, K. Lee, W. Yao, J.Z. Zhang, G. Friederich, C. Sakamoto, F. Chavez, K. Buck,
2.
Kolber, R. Greene, P. Falkowski, S.W. Chisholm, F.
Hoge, R. Swift, J. Yungei, S. Turner, P. Nightingale,
A.
Hatton, P.
Liss, and N.M. Tindale. 1994. Testing the iron hypothesis in ecosystems of the equatorial Pacific ocean. Nature, 371: 123-129. Martin, J.H., and S.E. Fiztwater. 1988. Iron deficiency limits phytoplankton growth in the north-east Pacific subarctic. Nature, 331: 341-343.
Martin, J.H., R.M. Cordon, S. Fitzwater, and W.W. Broenkow. 1969. VERTEX: phytoplankton/iron studies in the Gulf of Alaska. Deep Sea Res., 36: 649-680. Martin, J.H., S. FLtzwater, and R . M . Gordon. 1990. Iron deficiency lirnits phytoplankton growth in Antarctic waters. Global Bioqeochem. Cycles, 4: 5-12. Martin, J.H., R . M . Gordon, and
S.
Fitzwater. 1991. The case for iron.
Limnol. Oceanoqr., 36: 1793-1802. Meyerhoff, P.A., T.C. Fox, R.L. Travis, R.C. Huffaker. 1994. Characterization of the association of nitrate reductase with Barley (Hordeum v u l g a r e L. ) root membranes. Plant Physiol . , 104 : 925-936.
Morel, F.M.M., R.J. Hudson, and N.M. Price. 1991. Limitation of productivity by trace metals in the sea. Limnol. Oceanogr., 36:
1742-1755.
Morel, F.M.M., J.G. Rueter, D. M. Anderson, and R.R.L. Guillard. 1979. Aquil: a chemically defined phytoplnakton culture medium for trace metal studies. J. Phycol., 1 5 : 135-141.
Murray, J.W., R.T. Barber, M.R. Roman, M.P. Bacon, and R.A. Feely. 1994. Physical and biological controls on carbon cycling in the equatorial Pacific. Science, 266: 58-65. Ottow, J.C.G. 1968. Evaluation of iron reducing bacteria in soi1 and the physiological mechanism of iron reduction in Arthrobacter aerogenes. Zeitsch. für allg. Mikrobiol., 8: 441-443. Ottow, J.c.G. 1970. Selection characterization and iron reducing capacity of nitrate reductaseless (nit-) mutants of iron reducing bacteria. Zeitsch. für allg. Mikrobiol., 1 0 : 55-62. Price, N.M., L, Anderson, and F.M.M. Morel. 1991. Iron and nitrogen nutrition of equatorial ~acificplankton. Deep-Sea Res., 38: 13611378.
Price,
N.M.,
B.A.
Ahner, and
F.M.M.
Morel. 1994. The equatorial Pacific
Ocean: Grazer controlled populations in an iron-limited ecosystem. Limnol. Oceanogr., 39: 520-534. Price, N . M . , G.I. Harrison, J.G. Hering, R.J. Hudson, P.M.V. N i r e i , B. Palenik, and F.M.M. Morel. 1988/89. Preparation and chemistry of the artificial algal culture medium Aquil. Biol. Oceanogr., 6: 443-461. Raven, J. 1988. The iron and molybdenum use efficiencies of plant growth
with different energy, carbon and nitrogen sources. New Phytol., 109: 279-287.
Raven, J., 1990. Predictions of Mn and Fe use efficiencies of phototrophic growth as a function of light availability for growth and of C assimilation pathway. New Phytol., 116: 1-18. Redinbaugh, M.G., and W.H. Campbell. 1983. Reduction of ferric citrate catalyzed by NADH:nitrate reductase. Biochem. Biophys. Res. Commun., 114: 1182-1188.
Rueter J., and D. Ades. 1987. The role of iron nutrition in photosynthesis and nitroqen assimilation in Scenedesmus quadricauda (Chlorophyceae). J. Phycol., 23: 452-457. Ryther, J.H., and D.D. Kramer. 1961. Relative iron requirement of some coastal and offshore plankton algae. EcoLogy, 42: 444-446. Sandmann,
G.
1985. Photosynthetic and respiratory electron transport in
CU^+-deficient
Dunalieila.
Physiol. Plant., 65: 4 8 1 - 4 8 6 .
Sandmann, G., and R. Malkin. 1983. Iroa-sulfur centers and activities of the photosynthetic electron transport chain in iron deficient
cultures of the blue-green alqa Aphanocapsa. Plant Physiol., 73: 724-726.
Solomoson, L.P., and B. Vennesland. 1972. Properties of nitrate reductase of C h l o r e l l a . Biochem. Biophys. Acta, 267: 544-557. Sfrensen, J. 1982. Reduction of ferric iron in anaerobic, marine sediment and interaction with reduction of nitrate and sulfate. Appl. Env. Microbiol., 43: 319-324. Stohr, C., R. Tischner, and M.R. Ward. 1993. Characterization of the plasma-membrane-bound nitrate reductase in Chlorella saccharophila (Kruger) Nadson. Planta, 191: 79-85. Sunda, W.G., and S.A. Huntsman. 1995. Iron uptake 3nd growth limitation in oceanic and coastal phytoplankton. Mar. Chem., 50: 189-206. Sunda, W.G., D.G. Swift, and S.A. Huntsman. 1991. Low iron requirement for growth in oceanic phytoplankton. Nature, 351: 55-57. Syrett, P.J. 1981. Nitrogen metabolism in microalgae. In: Platt T ( e d ) Physiological bases of phytopLankton ecology. Canadian Department of Fisheries and Oceans, Bull 210, Ottawa, Ontario p. 182-210. Terry, N. 1983. Limiting factors in photosynthesis. IV Iron stressmediated changes in light-harvesting and electron transport capacity and its effects on photosynthesis in v i v o . Plant. Physiol., 71: 8 5 5 860.
Timmermans, K.R., W. Stolte, and H.J.W. de Baar. 1994. Iron-mediated
effects on nitrate reductase in marine phytoplankton. Mar. Biol., 121: 389-396.
Wells, M.L., N.M. Price, and K.W. Bruland. 1994. Iron limitation and the cyanobacterium Synechococcus in equatorial Pacific waters. Limnol. Oceanogr., 39: 1481-1486. Westall, J.C., J.L. Zachary, and F.M.M. Morel. 1976. MINEQL: a computer program for the calculation of chemical equilibrium composition aqueous systems. Tech. Note
NO.
18. R.M. Parsons tab for WatGr
Resources and Environmental Engineering, M.I.T., Cambridge, Dept. of Civil Engineering.
Zumft, W.G., and
H.
Spiller. 1971. Characterization of a flavodoxin
from the green alga Chlorella, Biochem. Biophys. Res. Commun., 4 5 : 112-119,
Chapter II is a continuation of the initial work (Maldonado and Price, 1996), which showed t h a t t h e use of NO^' imparts a higher cellular Fe demand for phytoplankton growth than the use of N H ~ . Contrary to expectations, I observed that under Fe-limiting conditions al1 six species of marine centric diatoms have a superior ability to acquire Fe when instead of NH~'. which
NO3'
NO3-
is used as t h e N source for growth
In this chapter 1 thus investiqated the mechanism by
grown cells were able to maintain faster rates of Fe uptake
and growth.
1
chose Thalassiosira oceanica as a mode1 centric diatom
for t h e s e experiments. The idea of the reductase was Neil's.
1 designed the
experiments, measured Fe reduction rates of T. oceanica by modifyinq the cherniluminescence assay (not an easy task!), collected and
analyzed the data, and wrote the paper.
NeiL provided help by
editing, and providing insight and discussion throughout the completion of this paper.
NITRATE REGULATION OF
FE REDUCTION AND TRANSPORT I N F E -LIMITEDTHALASSIOSIRA OCEANICA
Maria T. Maldonado and Neil M. Price Limnology and Oceanography, in review.
Abstract Under Fe-limiting conditions, nitrate (NO3')-grown phytoplankton have higher intraceLlular Fe requirements, but divide as
fast or faster
than ammonium ( ~ ~ ~ + ) - q r ocells w n by maintaininq faster steady state Fe uptake rates. Here w e report that Thaiassiosira oceanica 1003 possesses
an Fe reductase that reduces Fe(II1) bound to a v a r i e t y of organic ligands, including the siderophore desferrioxamine B, an Fe(II1)specific, high affinity ligand. phytoplankton (1.61 k rate than when
NOp-
.2
Iron reduction occurs in Nil4'-grown
am01 Fe fl-l h - l ) , but at a significantly slower
is used f o r growth ( 3 . 2 3 t . 2 am01 Fe fl-l h-l).
Reduction is mediated extracellularly by an enzyme resembling nitrate reductase (NR) and is induced by Fe deficiency. Short-tem and long-tem
Fe uptake rates are faster (by 1.42 and 2.7 times, respectively) in N O j - than in ~ ~ ~ + - g r phytoplankton own when Fe concentrations are
subsaturating, in agreement with calculated steady state Fe uptake rates. These results suqgest that faster rates of reduction and transport allow
more rapid growth of phytoplankton in
NOj'
than in lWr'-amended
media
when Fe is limiting. Thalassiosira oceanica is able to grow with
nanomolar additions of Fe even in the presence of excess desferrioxamine B (FeDFB). Under this Fe-limiting condition N03--amendedcultures still
achieve faster doubling rates than ~ ~ ~ + - a m e n d cultures, ed and have faster short-term uptake rates of Fe bound to F e D F B .
The implications of these
finding could be significant for understanding phytoplankton Fe nutrition in oceanic waters where organic complexation dominates the speciation of
Fe. We hïpothcsize t h a t t h e reductive Fe transport pathway may enable phytaplankton to directly utilize Fe bound to strong organic ligands in the sea. ctron Iron plays a catalytic role in many biochemical reactions as a cofactor of enzymes and proteins involved in chlorophyll synthesis, detoxification of reactive oxygen species, respiratory and photosynthetic electron transport, and N assimilation. Changes in the activity of these reactions or their replacement by functionally equivalent Fe-deficient pathways can greatly influence cellular Fe requirements of organisms. Because the NO3- assimilatory pathway is highly Fe dependent, utilization of N03- by marine centric diatoms (Thalassiosira spp.) imparts a hiqher metabolic demand for Fe than use of
(Maldonado and Price, 1996).
The demand for Fe is such that cellular Fe quotas (Fe:C) increase to a
level 1.8 times higher than those observed for phytoplankton using NH~'. Nitrogen source clearly modulates Fe requirements, but in seawater containing Low Fe concentration, has little or no effect on phytoplankton growth rates (Maldonado and Price, 1996).
Nitrate-grown cells apparently
compensate for their e x t r a Fe requirement by sustaining faster steady state Fe uptake rates (Maldonado and Price, 1996). The mechanism by
which the
NO3'
-dependent cells maintain these faster rates is presently
unknown . Iron uptake by phytoplankton is largely determined by dissolved inorganic Fe (Fe') concentration (Anderson and Morel, 1982; Hudson and Morel 1990, 1993).
In Fe-poor culture medium containing cheiators, Fe'
concentration can be extremely low as most of the Fe is orqanically complexed.
These Fe complexes, including FeEDTA
(ethylenediaminetetraacetic acid) species, are kinetically less labile than Fe', are impermeable to ce11 membranes and are thus not thought to be directly available for uptake (Hudson and Morel, 1993).
Photochemical
reactions, however, can catalyze the dissociation of Fe from these organic ligands by a reductive reaction. Photoreduction of ferricchelates typically involves intramolecular charge transfer reactions in
which Fe(II1) is reduced to Fe(I1) and the ligand is oxidatively deqraded (i.e. in EDTA by a decarboxylation reaction, Budac and Wan, 1992). The
photo-degradation of the ligand substantially contributes to the photodissociation of ferric chelates, thus increasing [Fe']. This effect may have important biological consequences.
Indeed, photoreduction of
Fe(II1)EDTA increases Fe uptake rates by phytoplankton (Anderson and Morei, 1980, 1982) and presumably could partially ameliorate Fede€iciency .
Biologically mediated reduction of Fe may be an alternative means to increase the chernical reactivity of organic Fe complexes. This strategy is used by hiqher plants, yeast, and bacteria to obtain Fe when it is limiting (Guerinot, 1994; Guerinot and Yi, 1994).
In higher plants
(dicots and nongraminaceous monocots) Fe reduction is facilitated by two pathways: acidification at the root surface that enhances the release of
reducing compounds, and transplasmalemma bound enzymes that reduce Fe at the m o t surface (Bienfait, 1987).
Excretion of reducing biochemicals is
t00 slow to account for the Fe reduction rates meaçured at the roots of Fe-deficient plants (Romheld and Marschner, 1983) and is thus of minor importance. Iron reduction appears to be mediated primarily at the ce11 surface by plasmalemma bound redox enzymes which are induced in Felimited plants (Bienfait, 1987). Since most organic Fe chelators have a hiqher affinity for ferric (Fe(II1)) than ferrous ion (Fe(II)), reduction of complexed Fe(II1) results in a n e t dissociation of Fe from the Ligand,
increasing [Fe']. A
variety of enzymes are able to reduce Fe, and in some studies the
reduction has been linked to N 0 3 - metabolism.
Heterotrophic bacteria,
for example, reduce Fe(II1) bound to organic complexes. The redox protein mediating the reduction appears to be nitrate reductase (NR), since mutants lacking this enzyme are deficient in the reductase pathway (Ottow, 1968, 1970). In Fe-Limited higher plants, the role of NR in this process is inferred from experiments which show that NR can reduce a variety of organic complexes (Redinbaugh and Campbell, 1983; Castignetti and Smarrelli, 1984, 1986; Smarelli and ~astiqnetti,1988), and from measurements of NR associated with ce11 surface (Ward et al., 1988, 1989; Tischner et al., 1989; Corzo et al., 1991).
Direct determination of Fe
reduction by a plasmalemma bound NR has recently been demonstrated
(Meyerhoff et al., 1994; Stohr et al., 1993).
Phytoplankton also
extracellularly reduce trace metals ( s u c h as Cu and Fe) bound to organic liqands (Anderson and Morel, 1980; Jones et al., 1987). Indeed, plasmalemma bound
NR
was hypothesized to mediate the reduction of Cu
complexes by T. weissflogii (Jones and Morel, 1988).
In the present study, we examined the influence of N substrate on Fe reduction by Fe-limited Thalassiosira oceanica 1003. This oceanic
marine centric diatorn was known to grow f a s t e s and to have a higher Fe quota in N03- than in
based medium under Fe-limiting conditions
(Maldonado and Price, 1996). Reduction rates were measured with BPDS, an organic ligand with high affinity for Fe(II), and by direct determination of Fe(1I) concentrations using an automated, flow injection analysis system with luminol-based cherniluminescence detection (King et al., Iron uptake rates and growth rates were measured concurrently.
1995).
Iron-Lfmitinq Culture Media
Thalassiosira oceanica (clone 1003), a small 6 pm diameter, centric
diatom isolated from the Sarqasso Sea, was obtained from the ProvasoliGuillard Center for Culture of Marine Phytoplankton (West Boothbay Harbor, ME) and grown under Fe-Limiting conditions in the artificial seawater medium Aquil (More1 et al., 1979; Price et al., 1988/89).
The
seawater, containing the major salts
(50
I 3 pm made up about 44 i 16 phytoplankton assemblage (Table 2).
%
of the biomass of the total
Their contribution to the
community was most variable at the inshore stations where it differed by more than a factor of 2 during the cruises.
Biomass of bacteria was 2 to 3-fold less variable than phytoplankton among stations and years (Table 2).
In 1 9 9 5 and 1997, it
CO-varied with phytoplankton biomass so that the highest and lowest bacterial biomasses occurred at stations with the highest and lowest chlorophyll a concentrations. This relationship, however, was not apparent during the 1996 cruise. Acquisi tion of Fe bound in siderophore complexes Heterotrophic bacteria and phytoplankton took up
5 5 ~ ebound
to
the siderophores, DFB and DFE (Fig. 1). The rates of Fe transport were constant during 24 h incubations and thus independent of Light.
Measurements of sulfoxine-reactive Fe (Hudson et al., 1992) in seawater samples amended with ~ ~ F ~ D verif F B ied that al1 the added 5 5 ~ was e bound by the desferrioxamines (DF) (see methods) and that the 5 5 ~ e ~ ~ complexes were not exchangeable nor photochemically labile (data not shown), at least under the natural photon flux densities prevalent in the sub-arctic Pacific at the time of our experiments (winter, 30-125
III Table 2 .
P a r t i c u l a t e carbon concentrations ( w o l C 1 - l ) i n samples
from 1 0 m depth along l i n e P derived £rom measurernents of POC and from c a l c u l a t i o n s based an empirical r e l a t i o n s between C and b a c t e r i a , and chlorophyll a .
T h r e e size f r a c t i o n s are i d e n t i f i e d : t o t a l , Total represents the
heterotrophic b a c t e r i a , and phytoplankton.
unfractionated community (POC c o l l e c t e d on a combusted GF/F glass f i b e r
filter). Phytoplankton
carbon ( > 3 p ) was
estimated from s i z e -
fractionated chlorophyll a , assuming a C:chla r a t i o of 50 (see Methods).
Bacterial c d 1 abundance was converted t o C biomass assuming
20 f g C per ce11 (Lee and Fuhrman, 1 9 8 7 ) .
Chlorophyll concentrations
a r e also reported i n parentheses ( t o t a l , and >
3 p
s i z e - f r a c t i o n , pg
chla 1 - l ) .
Station
S i z e Fraction 1995
p o l C 1 - I (pg chld 1 - l ) 1996 1997
1
Total
(0.2-Lpm) Beterotrophic Bacteria
(>3CLm)
Phytoplankton
O. 204 (O.049) 0.087 0.229 1.420 0.858
(0.021) (0.055) (0.341) (0.206)
4.17 (1.00) 0.292 (0.070) 0.417 (0.100) 1.38 (0.330) 0.333 (0.000)
0.393 (0.104)
0.975 (0.223) 0.625 (0.150) 0.663 (0.159)
III
e
1. Time course of accumulation of particuîate
5 5 ~ e [pmoi. ~e
(mol C)*l] during Fe-siderophore uptake experiments by heterotrophic bacteria ( A and C, 0.2-1 p size-fraction), and greater than 3 pm phytoplankton
(8
and D) at stations P4 and P26 during September 1996.
Iron was added as a premixed compiex bound to DFB
Fe and 10 nM siderophure (DF).
(O)
and DFE ( e ) : 2 nM
III m o l quanta m e 2 s ' l , and summer, 50-900 p o l quanta
pers. comm.).
s - l , P.W. Boyd
Accumulation of Fe by plankton in the control samples
poisoned with glutaraldehyde was less than 5% of the total Fe uptake by the living samples. At three stations, P12, P20 and P26, metabolic inhibitors of procaryotes (streptomycin) and eucaryotes (cyclohexirnide) were added during the uptake experiments in 1995.
These additions
reduced rates of Fe uptake by 35% and 50% on average, respectively, demonstrating that the accumulation of siderophore-bound Fe on the filters was biologically mediated.
Identical FeDF uptake experiments were conducted at two sites in the California coastal upwelling (Big Sur and ARo Nuevo) during July of 1996. The dissolved Fe concentration in theçe two sites was 0.06 nM
(DPCSV reactive Fe; Rue and Bruland, 1997) and 1 n M (ACSV-labile Fe;
K.W. Bruland pers. comm.), respectively. Only the biota at Big Sur accumulated siderophore bound Fe at a significant rate (Fig. 2). 5 5 ~ uptake e
The
measured at ~iioNuevo was identical to that observed in the
glutaraldehyde controls, and thus negliqible. In the vast majority of the siderophore experiments, more than 70% (73 f 15%) of the Fe was taken up by the bacteria six-fraction
(Table 3).
Phytoplankton greater than 3 p n were at times responsible
for accumulating the larqest portion of the siderophore-bound Fe, but this occurred in only 3 out of 14 experiments and 2 of these were at the near shore station (P4) where chlorophyll biomass was the highest recorded during the study.
Iron uptake by the 1-3 pin size fraction
never amounted to more than 20% of the total flux, averaging 12 2 7%
for al1 experiments, although similar amounts of chlorophyll a were present in this and the large size fraction.
III
20
1O
Time ( h )
m
e 2.
Time course of accumulation of particulate s 5 ~ e(pnol Fe
(mol C)-l] by > 3 p n phytoplankton during Fe-siderophore uptake experiments in the California coastal upwelling, at Big Sur (35Ot4, lSl%,
closed symbols) and
Ai50
Nuevo ( 3 7 . 0 4 O ~1 2 2 . 2 1 q , open çymbols)
during J u l y 1996 aboard the Point Sur.
Iron was added a s a premixed
complex bound to DFE (O) and DFB (A): 1 nM Fe and 10 nM siderophore (DF). The arnbient dissolved Fe concentration at Big S u r and Afio Nuevo
was 0 .O6 nM (Hutchins and Bruland, 1998; DPCSV reactive Fe, Rue and Bruland, 1 9 9 7 ) and 1 nM (ACSV-labile Fe, K.W. Bruland p e r s . c o m m . ) , respectively.
Voliimetric (frnol ~e 1-1 h - l ) and C-speclfic () 3.3, which was sufficient for the Fe to be completely coordinated and very stable with respect to dissociation (Monzyk and Crumbliss, 1982).
~
)
Desferrioxamines B and E were dissolved in sterilized MilliQ-H2O and 50% diçtilled methanol, respectively. Total complexation of Fe by the desferrioxamines was verified using the sulfoxine method (Hudson et al., 1992).
Thirty minutes after the premixed organically complexed Fe
(i.e. 2 nM Fe:lO nM DF) was added to the seawater samples, inorganic Fe
could not be detected by sulfoxine measurement (detection limit
[Fet] =
0 . 1 PM).
Effect of Pt(1I) on Fe uptake
The role of Fe reduction in uptake of inorganic (70 nM FeC13) and otganically bound Fe (140 nM Fe: 1 pl4 DFB) was investigated using Pt(II), a competitive inhibitor of ferric reductase activity (Eide et al., 1992);
and ascorbate, an Fe(II1) reducing agent (Anderson and Morel, 1 9 8 2 ) . Platinum (11) and ascorbate were added singly and in combination to the
Fe uptake media at concentrations of = 0.1 pi and ImM, respectively. The addition of Pt(I1) had no affect on pH (8.1) but ascorbate addition decreased the pH of the media to 6.95. The stock solution of Pt(I1) (5 m ~ was )
prepared in = 1 M
HC1.
Due to its Limited solubility, the
Pt(II)C12 was allowed to dissolve for two weeks in the dark prior to use.
Uptake rates were measured as described above. Reduction rates of organicall y bound F e ( I I 1 )
Extraceilular reduction of Fe(II1) was determined by directly measurinq Fe(I1) production in the dark, using a fully automated, flow injection a n a l y s i s system with Luminol-based cherniluminescence detection (King et al., 1995). Celis were concentrated on an in-line filter (3 p polycarbonate, Poretics) and continously fLushed with seawater to which orqanically bound Fe(II1) complexes were added (Maldonado and Price, submitted).
This method allowed rapid, sub-nanomolar detection of Fe(I1)
concentrations in seawater. Our detection Limit of Fe(1I) was 0.2 nM (Maldonado and Price, submitted). Reduction rates of organically bound Fe by Fe-limited T. oceanica were measured in N-free, Aquil (minus the trace-metal stock and vitamins, pH 6.6) as described (Maldonado and Price, submitted).
Iron was bound to
a variety of synthetic chelators: diethylenetriaminepentaacetate (DTPA), ethylenediaminetetraacetate (EDTA), and nitrilotriacetate (NTA); and the fungal siderophores, deferrioxamine B (DFB) and deferrioxarnine E (DFE).
The media for the reduction rate measurements were enriched with the premixed organic Fe complexes (see above for preparation of organically bound Fe), and allowed to equillbrate chemically for a week in the dark. Concurrent determination of uptake and reduction of organically bound Fe A
series of experiments were designed to determine the amount of Fe
taken up during the reduction rate measurernents.
In these experiments,
reduction rates were determined as described above, except radioactive Fe (10 p i 5 5 ~ e )vas bound to 100 p î EDTA and added to the reduction rate
solution [N-free,Aquil (minus the trace-metal stock and vitamins), pH 6.6).
At
the end of the reduction rate measurement
(a
1 h), the cells on
the in-line filter were rinsed with Ti(I1I) citrate EDTA reagent (Hudson and Morel, 1989). and collected for detemination of particulate 5 5 ~ e .
The total Be uptake rate was calculated frorn the particulate 5 5 ~ eretained on the filter, after normalizing to ce11 density and time. To determine uptake rate of Fe(II1). the 5
BPDS.
5
~ solution e ~ ~was ~amended ~ with 100 pM
Because of its high affinity for Fe(fI), bioloqically reduced
Fe(II1) was instantly complexed by BPDS, and no Fe(Z1) was detected in solution. The Fe(I1)BPDS complex is impermable to ceil membranes
(Anderson and Morel 1980) so the reduced Fe(II1) could n o t be taken up by
e on the filter the phytoplankton. The amount of particulate 5 5 ~ collected
following the BPDS addition was used to calculate the rate of Fe(If1) uptake. Uptake of Fe(I1) was calculated from the difference between the total Fe uptake rate (using 5 5 ~ particulate) e and the Fe(II1) uptake rate (using 5 5 ~ particulate e of cells f lushed with PeEDTA-enriched, N-free Aquil, amended with BPDS).
Although the rates calculated in this way are
reported as Fe(II) uptake r a t e s , we have no direct evidence for Fe(1I)
transport per se because the Fe(I1) could be oxidized prior to or during transport, The absolute rate of Fe(II1) reduction by T. oceanica was calculated by summing the rate of Fe(I1) uptake and the measured Fe(I1I) reduction rate.
Results Effects of Fe-limitation on Fe uptake Short-term uptake rates of Fe were greatly enhanced in Fe-limited Thalassiosira oceanica (0.85 ha,)at al1 Fe concentrations regardless of the Fe-chelate complex that was added (Table 1).
Uptake of Fe from FeDFB
was undetectable in Fe-sufficient cells at the low concentration and was 32 times faster at the high concentration when cells were Fe-Limited. Less
of an enhancement in Fe uptake was observed when Fe was supplied as
an EDTA complex. The transport rates were directly proportional (1.2:1, pFe:[Fe]) to the concentration of FeEDTA and the corresponding Fe' for both Fesufficient and -1imited cells. With DPB, however, the same range in total Fe concentration elicited only a 3-fold faster uptake rate in the low Fe cells. The most remarkable observation was that the uptake rates
were only slightly slower with FeDFB than
FeEDTA despite a 6 order of
D b l e 1.
Short-term Fe uptake rates
mol Fe p-2 h-l) of Fe-
(*
limited (12.9 nM Fe) and Fe-sufficient (8.6 JîM Fe) T. oceanica (mean f range).
Rates were determined at sub-saturating Fe concentrations in
the presence of 100 pl4 EDTA or 1 pM DDIB. and 35.9
Cell densities were 8.0
10'
104 cells ml-l in the Fe-limited and Fe-suf ficient treatments.
Iron-limited cells grew at 85% of Chiax.
(b.d.
- measuroment
below detection limit)
-
e [Fe'] calculated with the equation: [Fe'] = (FeDFB]/((Lq ] where KpelLCond 1016.5 (Hudson et al., 1992). #
p i
*
~
~
~
[Fe'] calculated assuming [Fe'J/[FeEDTAl = 0.00119 in the dark with 100 EDTA (Sunda and Huntsman, 1995).
[Fej:[ligandj
[Fev1
Fe- Limited
* 0.036
Fe-sufficient
10 nM Fe : 1 pl DFB
0.3 aMd"
0.552
10 n M Fe:lOO p EDTA
12.0
0.697 k 0.043
0.049 f 0.003
100 nM Fe:l phf DFB
3.2 abfa
1.767 f 0.110
0.054 î 0.004
100 n M : l O O ~ E D T A
118.9p~'
8.424
* 0.252
b.d.
0.491 f 2.8
l
~
~
magnitude difference in Fe' concentration between the two media.
Rates
of FeDFB uptake were considerably faster than those expected based on Fe' concentration alone. To establish the relationship between Fe uptake from FeDFB and its chemical speciation a series of experiments were conducted in which [Fe'] was manipulated by varying [Fe] and [ D F B J . Because the sulfoxine method
(Hudson et al., 1992) lacked the necessary sensitivity to measure Fe' in these experiments, we computed its equilibrium concentration for each of the media.
When the [Fe'] was varied by 100-fold, by keeping the
concentration of Fe constant (experiment A , Table 2), the uptake rates were practically identical (0.25 f 0.06
10*21 mol Fe p-2 h*l).
Uptake
of Fe from FeDFB at constant Fet concentration, however, was variable, ranging from 0.29
-
2.50
*
10-21 mol Fe p - 2 h'.
In some of these
experiments we observed variations in uptake rate from day to day and from culture to culture, although the chemical speciation of the medium remained constant (Table 2, 10 nM Fe). Differences in physiological state of the Fe-limited cells is likely to have accounted for some of this variation.
Nonetheless, the data stronqly suggest that rates of
Fe
uptake from FeDFB are not a function of [Fe']. Uptake
of Fe bound to desferrioxamine B
To examine the pattern of Fe uptake from FeDFB more thoroughly, the data from al1 the FeDFB uptake experiments were combined. In these experiments, DFB concentration was always added in excess of that of Fe ( F i . 1).
In 85% of the uptake experiments, [DFB] was at least 10 t i m e s
higher than the [Fe], and in the rest of the experiments the [DFB] was at
Least twice as hiqh as that of [Fe]. The [DFB) ranged from 1000 nM (for 19 data points) to 100 n M (for 10 data points).
Only one data point had
Table 2 .
Short-term Fe uptake rates of Fe bound to DFB
(*
1oa2l mol Fe
jlm-2 h - l ) of Fe-limited (12.9 nM Fe) T. oceanica (mean f range) . were
Rates
determined at sub-saturating Fe concentrations by either keepinq the
concentration of Fe constant and varying the concentration of DFB or by adding both in constant ratio so as to maintain a constant inorganic Fe concentration ([Fe']).
Experiments identified by the same letter used
cells fron the same culture:
* 104
d * ' ) , B. (10.2
cells mls1,
-
A.
(1.7
cells ml-l, p
=
104 cells ml-',
- 1.41 doublings
1.37 doublings del) C. (6.8
cells mld1, p
1.3 doublings d-l), D. ( 3 . S a 10'
doublings d-l), and E. (2.09
P
10' cells ml-l, p
-
=
* 104 1.7
1 - 6 5 doublings d - I l
.
e [ F e t ]calculated with the equation: [Fe'] a [FeDPB]/([L1] * KFelLcond)l where 1016m5 (Hudson et al., 1992). ~~~l~~~~~
-
[FetI
~ M (K
Fe uptake rate 1od21mol Fe h'l)
r
.
Short-term F e u p t a k e r a t e s ( l o g ( m o l F e cell-1 h ' l ) ) of F e -
l i m i t e d T . oceanica a s a f u n c t i o n of l o g (FeDFB] ( A ) , or Log [Fe'] ( 8 ) . The [DFB] ranged from 1000 nM ( f o r 19 d a t a p o i n t s ) to 100 nM (for 10 d a t a
points).
Only o n e data p o i n t had a ( D F B ] o f 10000 nM (also i n Table 2 ) .
The Fe c o n c e n t r a t i o n ranged from 1 nM t o 500 nM. The p l o t t e d l i n e was o b t a i n e d by l e a s t - s q u a r e s r e g r e s s i o n : A ,
tell-?
h-?)
a
-15.73
i
0.523
*
log ( F e D F B ] , r2 - 0 . 8 ; F - r a t i o
d i . p sO.OOO1). and B, [ l o g (mol
r2
=
0 . 4 3 ; F-ratio
=
tell-1 h-l)
=
1 9 . 3 , 2 5 df, p ~ 0 . 0 0 0 1 ) .
-11.58
+
0.403
[Log (mol
-
104 . O %
25
Log [Fe'].
a [DFB] o f 10000 nM ( a l s o i n T a b l e 2 ) .
The Fe c o n c e n t r a t i o n r a n g e d f r o m
1 nM t o 500 nM. T h e t r a n s p o r t rate o f Fe bound t o DFB was more s t r o n g l y related t o
t h e c o n c e n t r a t i o n of o r g a n i c a l l y bound F e ( r 2 = 0 . 8 , F i g . 1 A ) t h a n t o t h e c o n c e n t r a t i o n o f i n o r g a n i c Fe
( r2 = 0 . 4 7 ,
c o n c l u s i o n d r a w n from t h e d a t a i n T a b l e 2 .
F i g . 10). s i m i l a r t o t h e The u p t a k e rates were
expressed a s m o l F e c e l l - l h-l (Fiq. 1) s i n c e t h e v a r i a t i o n i n u p t a k e w a s
s i g n i f i c a n t l y s m a l l e r when the r a t e s were n o r m a l i z e d to ce11 ( r 2 = O . 8 ) t h a n when they were n o r m a l i z e d p e r ce11 s u r f a c e a r e a ( r 2
-
0.57).
R a t e s o f F e u p t a k e f r o m FeDFB by T. oceanica a r e s t r o n g l y r e l a t e d t o t h e [FeDFB], s u g g e s t i n g t h a t c e l l s a r e a c q u i r i n g Fe d i r e c t l y from t h e FeDFB c o m p l e x .
O n e p o s s i b l e mechanism f o r a c c e s s i n g F e bound
to DFB is
r e d u c t i o n of t h e DFB-bound f e r r i c i o n t o t h e f e r r o u s s t a t e , w h i c h c o n v e r t s t h e t i g h l y bound f e r r i c - D F B complex t o t h e l o o s e l y bound f e r r o u s compiex ( T u f a n o a n d Raymond, l98l), t h u s i n c r e a s i n q t h e r a t e of d i s s o c i a t i o n o f t h e FeDFB c h e l a t e .
Saturation kinetics of reduction of organically bound Fe(II1) The d e p e n d e n c e of F e ( I I 1 ) r e d u c t i o n rates o n t h e c o n c e n t r a t i o n o f o r g a n i c a l l y b o u n d Fe was e v a l u a t e d i n media c o n t a i n i n g e i t h e r FeEDTA (100 EDTA) o r FeDFB (10 phf DFB).
Ligands exceeded t h a t of Fe.
I n al1 cases t h e concentration of orqanic R e d u c t i o n r a t e s by F e - l i m i t e d T . oceanica
e x h i b i t e d s a t u r a t e d k i n e t i c s expected of an e n z y m a t i c a l l y mediated process (Fig. 2 ) .
A l t h o u g h t h e c o n c e n t r a t i o n o f EDTA was 1 0 times h i g h e r
t h a n t h a t of DFB, t h e s a t u r a t i o n curves were very s i m i l a r for b o t h Fe-
complexes.
A
Hanes-Woolf t r a n s f o r m a t i o n o f t h e M i c h a e l i s - M e n t e n e q u a t i o n
( S e g e l , 1 9 7 6 ) y i e l d a s i m i l a r h a l f s a t u r a t i o n c o n s t a n t for feDFB ( K m 0.680
pi) and FeEDTA ( K m
=
1.18 p) r e d u c t i o n by T. oceanica.
The
=
P
Reduction rates of Fe(1II)
2.
(*
EDTA (100 pl, open symbols) or DFB ( 1 0
10-21 mol Fe ~ ( m -h ~- l ) bound to
p4,
c l o s e d symbols) as a f u n c t i o n
of F e concentration (mean k standard error, n
oceanica.
-
3-6) by Fe-limited T.
The concentration of DFB was kept constant (10
varied from 10 nM to 8 pM.
pf),and Fe
A Hanes-Woolf t r a n s f o r m a t i o n was used to
calculate t h e h a l f saturation constant (Km) and maximum reduction rate
( v a ) . The d a t a fit the Hanes-Woolf Linear mode1 the FeDFB and 0.84 for the FeEDTA plot.
-
,,V
57.9
i:
reduction (r2 Km
-
1.18
8.2
-
* 10-~'
mol Fe
0 . 9 4 ) , and ,V ,
pM for FeEDTA ( r 2
-
in-^
0.84 )
h'l
with an r2 of 0 . 9 4 f o r
The calculated parameters wexe:
and Km
-
0 . 6 8 0 pM for PeDFB
4 7 . 9 k l . 8 Ç10'~l mol Fe pe2 h'' and
.
The growth rates of T. oceanica f o r
the FeEDTA and FeDFB experiments were 1 . 6 k 0 . 9 and 1 . 3 f 0 . 5 doublings d l , respectively .
c a l c u l a t ê d maximum r e d u c t i o n r a t e waç 5 7 . 9 k 8 . 2 and 4 7 . 9 k 1 . 8
1 0 ' ~ ~
mol F e p n - 2 h-' f o r F e b o u n d t o DFB a n d EDTA, r e s p e c t i v e l y . T h e e x p e r i m e n t s d e s i g n e d t o d e t e r m i n e t h e amount of F e t a k e n u p d u r i n g t h e r e d u c t i o n ( T a b l e 3 , see m e t h o d s ) i n d i c a t e d that on a v e r a g e , 5 0 % o f t h e r e d u c e d F e was i m m e d i a t e l y i n t e r n a l i z e d by t h e cells.
I n the
t w o i n d e p e n d e n t e x p e r i m e n t s we c o n d u c t e d , t h e measured rates of Fe reduction were significantly d i f f e r e n t .
gowever, when t h e rates were
corrected for u p t a k e o f r e d u c e d F e , the c a l c u l a t e d t o t a l r e d u c t i o n r a t e s were i d e n t i c a l f o r b o t h experiments (310 f 7.8
*
10-2i mol F e C(rnm2 h * ' ) .
Effect of Pt(II) and ascorbate on t h e u p t a k e of Fe U p t a k e rates were n o t a f f e c t e d by P t ( 1 I ) a d d i t i o n when F e w a s s u p p l i e d a s i n o r g a n i c s p e c i e s , b u t were s i g n i f i c a n t l y s l o w e r t h a n t h e c o n t r o l rates w i t h FeDFB ( 8 5 v s . 1 1 2 * 10-~' mol
tell-i
h-')
(Fig. 3).
In
the p r e s e n c e of t h e reductant, a s c o r b a t e , Fe t r a n s p o r t decreased by 80% i n t h e i n o r g a n i c F e t r e a t m e n t a n d t o a lesser e x t e n t w i t h F e D F B ( 5 5 % ) . P a r t of t h i s i n h i b i t i o n w a s d u e t o pH, s i n c e ascorbate made t h e u p t a k e medium more a c i d i c (pH 6 . 9 5 ) .
I n d e e d , C f i x a t i o n r a t e s d e c l i n e d by 30%
following ascorbate addition.
T h e r e m a i n i n g i n h i b i t i o n i n F e u p t a k e was
a t t r i b u t e d t o c h a n g e s i n F e r e d o x s t a t e compared t o the c o n t r o l c u l t u r e s . U p t a k e r a t e s o f F e from FeDFB were n o t a f f e c t e d by Pt(I1) when a s c o r b a t e was a d d e d ( F i g . 3 ) .
Reduction of organically bound Fe as a f u n c t i o n of stability constants of
Fe-chelator complexes T h e c h e m i c a l s p e c i f i c i t y o f t h e F e r e d u c t i o n pathway i n F e -
L i m i t e d T. oceanica was i n v e s t i g a t e d u s i n g a v a r i e t y o f F e chelators, i n c l u d i n g s y n t h e t i c o r g a n i c Ligands (NTA, DTPA, a n d EDTA), and fungal s i d e r o p h o r e s (DFB a n d D F E ) .
R e d u c t i o n rates were f a s t a n d w e r e
l
e 3.
Simultaneous determination of uptake and reduction of Fe
bound to EDTA (10 pM Fe:100 pt4 EDTA) by Fe-limited T. oceanica
mol Fe p n - 2 h-')
(see methods) .
cells ml-l, 8 pm diam, p
=
=
1.47 doublings d -
1.48 doublings d-l).
Experiment A
B
Measured total Fe uptake rate
189.9
70.7
Measured F e ( I I 1 ) uptake rate
19.7
5.85
Calculated Fe(I1) uptake rate
179.2
64.9
Measured Fe(II1) reduction rate
136.2
239.4
Absolute Fe(I1I) reduction rate
315.4
304.3
-
-
-
10-21
Two independent experiments were
performed A. (50,000 cells ml-l, 7.5 prn diam., and L( l ) and B. (41,410
(*
- -
-
--
-
Organic Fe (FeDFB)
Inorganic Fe(II1)
al trc
0.8.
80.
4
60 ..
O
E
0.4 b
+ ascorbace
+ ascorbate
0.6
rd
b
30 20
-
O 0
1
2
3
4
5
0
6
1
2
Pi
