STUDIES
OF NITRATE
REDUCTASE
PHYTOPLANKTON1
R. W. Eppley, J. L. Coatsworth,
Institute
of Marine
Resources,
University
IN MARINE
and Lucia Soldrxano
of California,
San Diego,
La Jolla
92037
ABSTRACT
Certain marine phytoplankton
contain the enzyme nitrate rcductase when growing
on
nitrate, but only low levels of enzyme were found during growth with ammonium or when
the nitrogen source was depleted.
Netted samples of oceanic phytoplankton
contained the
enzyme when taken from waters with nitrate concentrations
2-10 PM. Ammonium
was
assimilated in preference to nitrate in phytoplankton
cultures supplied with both forms of
nitrogen at 5-15 pM. Enzyme synthesis and nitrate use began when ammonium
was
depleted to 0.5-1.0 PM.
Nitrate reductasc assay of phytoplankton
samples is a useful tool in that a positive result
indicates utilization
of nitrate and a negative one implies growth on ammonium, nitrogen
depletion,
or, improbably,
growth with other N-sources such as nitrite, urea, or amino
acids. The enzyme assay seems especially useful for studying the timecourse
of phytoplankton blooms because it provides a sensitive measure of the initiation
and cessation of
nitrate assimilation.
nitro,gen content and rate of nitrogen
assimilation by the phytoplankton
so that
turnover rates can be calculated for cell
nitrogen and for dissolved nitrogenous
nutrients. Because of the different origins
of ammonium and nitrate in the euphotic
zone, methods should allow separate estimates of assimilation of these two ions
( Dugdale 1967).
A fundamental
problem, analogous to
that of estimating phytoplankton
standing
stock, is the contamination of phyto,plankton taken by filtration, netting, or centrifugation with other particulate matter. To
a- degree, certain phytoplankton
enzymes
involved in nitrogen assimilation may serve,
like chlorophyll,
as a specific label for
phytoplankton in the suspended particulate
matter. The most promising of these are
assimilatory nitrate- reducta& and nitrite
reductase. Nitrate may also be used by
certain microorganisms as a terminal electron acceptor in place of oxygen in a
process called “nitrate respiration” or dissimilatory nitrate reduction (Nason 1963).
Glutamic dehydrogenase occurs in animals
as well as in bacteria, fungi, and green
plants (Prieden 1963) and could not be
considered a label for phytoplankton
in
* This research was supported by U.S. Atomic
samples
of
seawater
particulat&
Energy Commission Contract No. AT( 11-l ) GEN
Results of a study of assimilatory nitrate
10, PA 20.
194
INTRODUCMON
Methods for study of nitrogen assimilation by phytoplankton at sea are not abundant. Currently, measurements are usually
limited to 1) analysis of the various forms
of dissolved nitrogen used by phytoplankton for growth (i.e., NOs-, NOz-, NH4+)
and of total particulate nitrogen and dissolved organic nitrogen, 2) rate of uptake
of 15N-labeled substances by particulate
matter (Goering, Dugdale, and Menzel
1966; Dugdale and Goering 1967), and
3) enrichment experiments to measure the
growth response of phytoplankton to added
nutrients (e.g., Menzel, Hulburt, and Ryther
1963; Thomas 1967). A method for estimating phytoplankton
cell nitrogen based
on carotenoid : chlorophyll a ratio,s (Yentsch
and Vaccaro 1958) is not in wide use, but
Manny ( 1969) has reexamined this method
with promising results for cultures. Fitzgerald ( 1968) h as suggested a method for
identifying
N-deficient cells, based on the
rate of ammonium uptake in the dark,
that has yet to be tested at sea.
A minimum set of methods should include means of measuring phytoplankton
NITRATE
REDUCTASE
IN
MARINE
reductase in Chlorella vulgaris, a freshwater green alga (Syrett and Morris 1963;
Morris and Syrett 1963), showed the following: 1) C. vulgaris grew on both ammonium and nitrate but used ammonium
first if both were provided together, 2)
nitrate reductase activity was negligible
(repressed) during growth on ammonium,
3) the enzyme was synthcsizcd (induced)
during growth with nitrate, and 4) intermediate levels of enzyme activity dcveloped in cells grown with other nitrogen
sources, such as urea or amino acids (at
much higher concentrations than occur in
the sea).
A study of nitrate and nitrite uptake by
intact cells of the blue-green alga, Anabaena cylindrica,
yielded similar results,
that is, nitrate uptake ability was repressed
during growth on ammonium and was induced by nitrate ( Hattori 196%, b ).
In view of these findings, we measured
nitrate reductase activity in cultures of a
neritic marine diatom, Ditylum
brightwe&, grown on ammonium, nitrite, nitrate,
and in N-deficient cells, and found high
levels of enzyme activity only in cells
growing on nitrate (Eppley and Coatsworth 1968). Thus in algae of diverse
phylogenctic position, the level of nitrate
reductase activity proved an indicator o,f
growth on nitrate as opposed to growth
on ammonium. This seemed sufficient justification for further studies of the enzyme
in phytoplankton
growing with typical
oceanic levels of nutrients,
The study was carried out in three steps:
1) experiments with laboratory cultures
of marine phytoplankton aimed at dcveloping a method for enzyme assay suitable for
many species, 2) a study of variation in
enzyme activity in phytoplankton grown in
cultures with ammonium and nitrate at
concentrations typical of seawater, and 3)
trials at sea,
We are grateful for the help and cooperation o;f Drs. J, R. Beers and W. I-I.
Thomas, scientific cruise leaders, and thank
Capt. F. Heller and Capt. W. Forster and
their crews of the RV Ellen 23. Scripps and
RV David Starr Jordan for their courtesy.
195
PIIYTOPLANKTON
Mr. E. Rcnger kindly supplied the ammonium and nitrate analyses for the EASTROPAC cruise.
METHODS
Lubomtog
cultures
Axenic cultures of Ditylum brightwellii
Coccolithus
huxleyi
(West)
Grunow,
( Lohm ) Kamptner,
Paasche’s strain F;
CycZotelZa nana Hustedt, Guillard’s strain
13-1; and Dunaliella
tertiolecta
Butcher
were maintained in this laboratory. GonyStein was studied in
aulax polyedra
samples of seawater taken at the Scripps
Institution pier during a red-water bloom
in December 1966.
Various culturing
devices. were used.
The D. brightwellii
was grown at 20C in
a 2.8-liter vessel of Pyrex pipe illuminated
by tungsten light passed through a copper
sulfate solution (Eppley and Coatsworth
1966), and C. huxleyi was grown at 20C
with tungsten light in a culture unit described by Paasche ( 1967). Cultures of
D. tertiolecta and G. polyedra were grown
under fluorescent light in a constant-temperature room kept at 20 +- 2°C.
Culture media were prepared with filtered seawater enriched with nitrogen,
phosphate, silicate, trace metals, and the
vitamins B12, biotin, and thiamine. Concentrations elf nutrients other than nitrogen sources were as in IMR medium
(Eppley, Holmes, and Strickland 1967).
The concentrations
of the nitrate and
ammonium were varied in different experiments.
Three cultures of phytoplankton
were
studied during growth in a deep tank (3-m
diam x 10-m depth; capacity, 70,000 liters)
at the hydraulics laboratory here, The use
of the facility, details of culturing, and the
chemical co,mposition of the phytoplankton
were reported earlier (Strickland
et al,
1969). Cachoninu r&i, D. brightwellii,
and
a mixed culture of G. ppolyedra and a
colonial
haptophycean
alga, tentatively
identified as Phaeocystis sp., were studied
in the three experiments.
In each case
ammonium
and nitrate
were initially
present at concentrations between 1 and
13 /.LM.
196
R. W.
EPPLEY,
J.
L.
COATSWORTH,
Studies at sea were undertaken on two
cruises : 1) a cruise of the RV Ellen B.
Scripps between San Diego, California,
and Guadalupe Island and 2) cruise 76
of the EASTROPAC
series on the RV
David Starr Jordan, a ship of the U.S.
Bureau of Commercial Fisheries, from 13”
N lat to 20” S lat on a line approximately
105” W long returning on 112” W long,
On the first cruise, particulate matter retained on a 35-p net and passed by a 102-p
net was collected with an overside pumping system described by Beers, Stewart,
and Strickland
(1967).
On the secoad
cruise, material was collected by 10 consecutive lo,werings to 30 m of a 32-p net
with 0.025-m2 aperture (approx 7.5 m3 of
water passed the net in the 10 hauls).
Enzyme assay
Samples of the cultures and net concentrates were filtered with suction on 4.25cm Whatman GF/C glass-fiber filters. The
cells o,n the filter pad were suspended in
3.3 ml of 0.2 M phosphate buffer, pH 7.9,
containing 1.0 mM dithiothreitol
with 5-20
mg dry polyvinylpyrrolidone
and were homogenized l-2 min in an electrically-driven
Teflon-glass ho,mogenizcr.
The homosgenizer was kept in an ice bath during grinding. The suspension of homogenized filter
and cells was then centrifuged 5-10 min
at about 2,000 x g. The clear supernatant
was decanted and used immediately as the
source of crude enzyme. One ml of the
enzyme extract was incubated for 30 min
with NRDH (final concentration, loo-150
PM),
MgSO (30-100 mM>, and KNOR (510 mM) in a to,tal volume of 1.8 ml at
20-24C. Control tubes lacked only NADH.
The reaction was stopped by adding 5 ml
of cold 95% ethanol and 0.2 ml o,f 1 M
zinc acetate (Medina and Nicholas 1957).
Reaction mixtures were then ccn trifuged.
Nitrite was determined in the alcoholic
supernatants by addition of sulfanilamide
and n- ( I-napthyl) -e thylenediamine
dihydrochloridc solutions (Strickland and Parsons 1965). Appropriate
standard nitrite
solutions were prepared in the same way.
Protein in the ethanol precipitates was
AND
LUCIA
SOL6RZANO
determined by the method of Lowry et al.
(1951) on control tubes lacking NADH.
Enzyme activity of cultures was calculated
on the basis of protein in the extract,
chloro’phyll a, or cell concentration.
With
sea samples, only chlorophyll a was considered a reliable indicator of phytoplankton content of the samples and activity
was so expressed.
At the time of enzyme assay, samples
were also taken for the estimation of chlorophyll a by fluorometry
(Yentsch and
Menzel 1963; IIolm-Hansen
et al. 1965)
and cell concentration (except in the sea
samples) with a model A Coulter counter
or Celloscope model 101 particle counter.
Nitrate, nitrite, and ammonium were dctcrmined (Strickland
and Parsons 1965)
in certain of the culture media after removing the cells by filtration.
The ammonium
method of Richards and Kletsch, as dcscribed by Strickland and Parsons ( 1965))
was used primarily
and a method more
specific for ammonium was used occasionally ( Johns ton 1966).
RESULTS
Development of methods for
enzyme assay
The following account is offered to justify the procedure for enzyme assay already given. It is based primarily on work
with extracts of diatoms, particularly
D.
brightwellii,
since the enzyme from diatoms
appeared to be more difficult to measure
than that from flagellates.
Extraction procedures and reaction mixtures given in the extensive literature on
nitrate reductase vary with the investigator
and no standard set of conditions has been
adopted. Both phosphate and tris bufEers
[ tris (hydroxymethyl) amino methane *IICl]
have been used for extraction, usually with
an added reduced sulfur compound such
as cysteine (Hewitt
and Nicholas 1964)
and sometimes with a reagent to inactivate
phenols,
such as polyvinylpyrrolidone
(Wallace and Pate 1967). In testing various methods, we found that phosphate
buffers ( pH 8) gave active preparations
NITRATE
OV
I
0.2
REDUCTASE
1
I
I
0.4
0.6
0.8
ml ENZYME PREPARATION
IN
MARINE
I
1.0
197
PHYTOPLANKTON
I
1. Nitrate
reductase activity
in extracts
of Ditykm
brightwellii
extracts containing
differcnt concentrations
of phosphate.
FIG.
but that Tris. HCI did not unless phosphate
was also added.
Activity also required a rcduccd sulfur
compound. Cysteine (1 mM)
was
effcctive with the flagellates tested (C. huxleyi
and G. polyedra), but the diatoms were
more demanding, requiring rcduccd glutathione or di thiothrcitol.
The reducing agent used for nitrate reductase assay from different
organisms
again varies. In their classic studies of
the mold, Neurospora crassa, Evans and
Nason (see Nason 1963) dctcrmincd that
the pathway of electron transport was from
NADPE-12 (nicotinamide
adcnine dinucleotide phosphate, reduced form) to FAD
( flavin adenine dinuclcoaide ) to’ molybdenum, a constituent of the enzyme protein. However, the enzyme from some
sources required NADH.
Enzyme from
other organisms was active With either
pyridine
nucleotide.
Addition
of ionic
molybdenum
was not required
(Nason
1963). The natural cofactor for higher
plants appears to be NADH (Schrader et
al. 1968)) and it has proved effective with
all phytoplankton
species we have tested.
The NADPH2 was inactive with D. brightwellii and G. polyedra extracts but was as
PH
FIG. 2. Variation
with pH of nitrate rcductase
activity in extracts of Ditylum
brightwellii.
Solid
circles, phosphate buffer; open circles and crosses
Tris-phosphate
buffer.
Phosphate precipitated
at
pH 8.9 and 9.5.
effective as NADH for D. tertiolecta extracts. Addition of FAD gave no’ increase
in activity.
Magnesium sulfate additions always increased enzyme activity with our preparations although the degree of activation
varied widely ( 13 to 300% ) . The o’ptimum
concentration was found to bc 30-100 mM
for D. brightwellii
extracts.
The enzyme was rapidly released during
grinding in the homogenizer.
Apparently
the glass fibers of the filter pad facilitate
cell disruption. Maximum, levels of enzyme
were reco,vercd after only 30 set grinding
with D. brightwellii,
a large centric diatom,
but l-2 min were required for small flagcllatcs such as C. huxleyi.
Various incubation
times were tested
with D. brightwellii
extracts. The rate of
nitrite fo,rmation was constant over the first
hour, then declined. It varied directly with
the amount of enzyme extract added to
the reaction mixture (Fig. 1 ), suggesting
that the amount of enzyme limited the
reaction rate.
The effect of concentration of phosphate,
198
R. W.
EPPLEY,
J. L. COATSWORTH,
AND
0
PHOSPHATECONCENTRATION
mM
LUCIA
I
I
200
SOLhZANO
,
I
I
I
400
600
NADH CONCENTRATIONpM
I
I
800
FIG. 3. Variation
in nitrate reductase activity
of DityZum brightwellii
extracts containing
differcnt concentrations
of phosphate.
FIG. 5. Nitrate
reductase activity
in Ditylum
brightwe
extracts vs. concentration
of reduced
nicotinamide
adenine dinucleotide
(NADH)
in
the reaction mixture.
NADH, nitrate, and pH of the reaction
mixture was tested with D. brightwellii
extracts. The pH optimum was 7.9 (Fig.
2) and activity was 50% of maximum between pH 7.3 and 8.6. Fairly high phos-
phate concentration was required for full
activity of the D. brightwellii
enzyme (Fig.
3), but the activity variation with concentration of nitrate (Fig. 4) and NADH
(Fig. 5) was similar to that for enzymes
from other sources. The Michaelis constant
(Km) for nitrate was 1.1 X 1O-4 M. Data
for phosphate and NADH were not good
enough to calculate Km with precision, but
they were probably in the range 50-70 rnM
for phosphate and approximately 20 pM for
NADH.
Activity passed through a maximum with NADH concentrations between
100 and 200 PM.
FIG. 4. Variation
in nitrate reductase activity
of DityZum brightwellii
extracts with concentration
of nitrate in the reaction mixture.
Experiments in the deep tank
1. Ditylum brightwellii
culture.-Timecourse of the events relating to nitrogen
assimilation are shown in Fig. 6. Enzyme
activity at first increased and then declined
as ammonium assimilation was initiated.
The decline continued until ammonium was
depleted to’ approximately 0.5 E,LM on the
8th day when enzyme synthesis began
and nitrate began to disappear from the
medium. The initial increase in enzyme
activity in this centric diatom was not seen
with subsequent cultures of flagellates.
MARINE
PHYTOPLANKTON
-0
6
NO,'
-
DAYS
DAYS
DAYS
FIG. 6. Deep-tank
culture of Ditylum
brightwell%. A.
Timecourse of nitrate and ammonium
concentration.
B. Chlorophyll
a content and nitrate reductase activity
(10” moles NO,- formed
per pug Chl a/hr ) .
Apparently it reprcsen ts enzyme synthesis
induced by nitrate previously accumulated
in the large vacuoles of the D. bright~ellii
cells and carried over from the inoculum.
2. Cactzortina; niei culture.-This
armored dinoflagellate
was o,riginally isolated fro,m the Salton Sea (Loeblich 1968)
where it is important
in dinoflagellate
blooms. The timecourse of changes in ammonium and nitrate and level of enzyme
is shown in Fig. 7. Again ammoaium was
consumed first until its concentration was
reduced to about 1 PM when enzyme syn-
DAYS
7. Deep-tank
culture of Cachoninu
n&i.
A. Nitrate
and ammonium
concentration.
B.
Chlorophyll
a content and nitrate reductase activity (units of activity defined in Fig. 6). Ammonium was determined
by two methods:
Richards
and Kletsch, which includes some amino acid nitrogen (NEL+ + a.a. ), and rubazoic acid (NJ&+),
FIG.
thesis began and nitrate utilization
commenced. On the 8th day of the experiment,
the tank was partially drained and refilled
with seawater without much loss of cells
since they had migrated to the surface of
the tank-aeration
and mixing having been
discontinued the previous evening. From
the 9th to 12th day amImonium, nitrate, and
200
R. W.
I
I
I
I
1
EPPLEY,
,
A.
,
,
J.
L.
(
,
COATSWORTH,
,
(
NH;
Added
AND
LUCIA
SOL6RZANO
1. R&e of nitrate
assimilation
measured
by three methods with deep-tank cultures of murine phytoplankton
TABLE
Rate NO,- assimilation as ,umoles/liter
of culture/hr
Expt
1
2
3
DAYS
NADH-nitrate
reductase
0.009
0.016
0.013
FIG. 8. Deep-tank
culture of Gonyaulux polye&a and Phaeocystis sp. A. Nitrate and ammonium. B. Chlorophyll
n concentration
and nitrate
reducta,se activity
( units as in Fig. 6 ).
nitrite were essentially depleted (<0.2 PM)
and enzyme concentration fell to a nearly
undetectable level. On the 13th day, nitrate was added to a final concentration
of 5 PM and a rapid synthesis of nitrate
reductase took place.
3. Mixed culture of Gonyaulux polyedra
and Phneocytiis sp.-Results in this experiment were similar to those of expt 2, but
Increase in
particulate-N
0.042
0.054
0.058
0.031
0.063
0.05.8
enzyme assays were less frequent.
Ammonium was consumed first while low
enzyme levels were measured (Fig. 8).
Nitrate began to disappear when ammonium was reduced to about 0.5 ~,CM and
nitrate reductase activity
simultaneously
increased. On the 8th day, ammonium reappeared in the medium, apparently released by the phytoplankton.
By the
10th day both ammonium and nitrate were
undetectable and enzyme level declined.
Limit
DAYS
Nitrate disappcarante from medium
of detection of NADHnitrate reductase
From the limit of spectrophotometric
detection of nitrite, taken as 0.02 absorbance units with a IO-cm cuvette, a visually
detectable pink color, and the specific
enzyme activity per unit chlorophyll a observed in the three cultures provided with
typical seawater levels of nitrate, we can
estimate the detection limit of the enzyme
assay in terms of the amount of plant
material required, expressed as chlorophyll
a. The nitrite detection limit above corresponds to’ 0.4 x 1O-Dmoles NOs- formed in
the 30-min enzyme reaction or 0.8 x 10 -O
moles NOa- formed/hr. In the three experiments, enzyme activity during growth with
nitrate was about 1 x 10 D moles NOzformed per pg Chl a/hr. The required
amount of plant material, as Chl a/ml of
enzyme extract, is then 0.8 pg. All the
extracts from the deep-tank experiments
contained greater amounts of chlorophyll a
than this. The very low enzyme activity
observed during ammonium assimilation
and after N-depletion cannot be attributed
to insufficient
sample, as will be seen
below with some of the seawater samples.
NITRATE
TABLE
2.
Activity
REDUCTASE
(lat)
.025
.029
.035
.053
.062
.071
.079
.083
,120
.130
.152
8”
6”
4”
0”
1”
3”
5”
8”
7”
4”
0”
* Number
31’ N
9’N
17’ N
42’ N
11’S
16’S
26’ S
3’S
9’S
10’S
30’N
of replicate
Recove y
MARINE
Nitrogen in O-30 m
water c0hm-m
(PM)
201
PIIYTOPLANKTON
of NADH-nitrate
reductase from seawater samples
cruise 76 in the tropical Pacific Ocean
Location
Sta.
IN
taken
EASTROPAC
during
NADH-nitrate
reductase
activity as 10-D moles NOz
formed per pg Chl alhr
(long 1
NO,-
NH,+
pg Chl u/ml
enzyme extract
105” 09’W
104” 59” W
105” 0’ W
105” 02’ W
105” 6’w
104” 58’W
104” 57’ W
105” O’W
lll”57’W
112” 3’W
111” 51’W
0
0
0
2-3
5-6
8-10
&7
7
7
6-7
7-8
0.39
0.50
0.37
0.40
0.81
0.77
0.60
0.56
0.61
0.75
0.48
0.73
0.39
0.42
2.97
3.48
1.84
0.51
1.26
2.02
2.10
1.69
Found
0
0
0
3.8
1.3
3.5
0
0
0.5
1.5
2.1
(l)*
(1)
(1)
(2)
(2)
(2)
(1)
(2)
(1)
(2)
(2)
Dctcction
limit
1.1
2.0
1.9
0.3
0.2
0.4
1.6
0.6
0.4
0.4
OS
samples,
of
enzyme activity
The rate of nitrate use in the three decptank experiments was calculated from the
rate of increase of phytoplankton
particulate nitrogen during NOs- utilization using
data of Strickland et al. (1969) and from the
rate of decline of nitrate concentration in
the medium for comparison with the measured activity of NADH-nitrate
reductase
( Table 1). Enzyme activity in vitro averaged 24% of the other estimates of nitrate
assimilation rates. This is not an unusual
recovery of enzyme activity, assuming that
all NO,- assimilated is processed by this
enzyme, but it is not oabvious why recovery
was so nearly constant between the three
experiments.
Calculation of the turnover
rate of nitrate in the sea from an enzyme
assay would be of doubtful significance
unless this recovery were truly constant.
Enzyme assay with seawater samples
Planning for the first cruise was based
on the assumption of enzyme activity per
pg chlorophyll a in the range 5-50 x 19”
moles NOa- formed/hr; our experience at
that time was limited to work with cultures
grown with 75-50 pM nitrate, so we were
unaware of the effect o,f nitrate concentration on the specific activity of the enzyme. Consequently, sufficient sample was
taken for enzyme assay on only eight oc-
casions. Seven of these were from water
containing less than 0.5 PM nitrate, so no
activity was expected and noae found.
Maximum possible undetected activity was
as low as 0.1 x lo-” moles NOa- formed per
lug Chl a/hr in these samples. In the single
sample with adequate chlorophyll a from
water with measurable ( 5 PM) nitrate,
the corresponding value was <OS in the
same units.
WC were better prepared for the second
cruise and found activity in six stations out
of eight (11 samples out of 14, including
replicates) in the high nitrate waters of
the equatorial Pacific Ocean ( Table 2).
The active samples contained more than 1.5
,ug Chl a/ml enzyme extract although the
two inactive samples from waters with
nitrate contained less pigment, Predominant organisms taken by the 32-p-mesh net
were Coscinodiscus spp,, Planktoniella: sol,
and Ceratium spp. Both nitrate levels in
the water and enzyme activity per unit
chlorophyll a were similar to those in the
deep-tank experiments. Where no activity
was detected, the upper limit of possible
undetected activity was calculated.
It was difficult to get sufficient sample
for assay in waters with undetected nitrate
even with 10 tows of the net because of the
low standing stock and the fact that most
of the phytoplankton
probably
passed
through the 32-p apertures of the net. The
202
R. W.
EPPLEY,
J.
L.
COATSWORTII,
problem was greatly eased in waters containing nitrate where the standing stock
was greater.
Results of the two cruises were as expected in that enzyme activity was low in
waters lacking nitrate and activity was
essentially identical to that in the deeptank experiments when nitrate was >2 PM.
Calculation of the time required by the
phytoplankton
crop to deplete the nitrate
present was not undertaken for the reasons
mentioned and because the 32-p net captured an unknown fraction of the crop,
AND
LUCIA
extracts of flagellates appeared to be less
sensitive to loss of activity during extraction and less demanding in their requirement for a reduced sulfur compound. We
have not attempted to purify the enzyme
to study in detail its cofactor requirements,
but this seemed unwarranted by our purpose. Our method gave estimates of nitrate
assimilation about one-fourth that of more
direct methods.
However, the foreseen
work at sea requires only a semiquantitative procedure.
Origin
DISCUSSION
Results of the experiments in the deep
tank, provided with nitrate and ammonium
levels comparable to natural seawater, confirm the earlier observations on C. vulgaris
( Morris and Syrett 1963) in that 1) both
ammonium
and nitrate were used for
growth and ammonium was used first when
both forms were given together, 2) nitrate
reductase activity was repressed during
growth on ammonium, and 3) the enzyme
was synthesized during growth on nitrate.
Enzyme levels in cells gro,wn with urea
or amino acids remain to bc tested, but
there was no indication in these experiments that the unidentified
components of
the dissolved organic nitrolgen fraction
were used for growth or influenced enzyme activity (Strickland et al. 1969).
Measurements of enzyme activity in the
particulate
matter of seawater samples
confirm the expectation that the enzyme is
present only if the water contains nitrate
and suggest that the phytoplankton in these
samples were using nitrate at the time of
sampling,
Lack of activity
in samples
without nitrate may imply growth on ammonium or other nitrogen source that does
not induce nitrate reductase, such as nitrite
(Eppley and Coatsworth 1968), or that
the organisms had depleted their nitrogen
source.
Enzyme assay
Suitable conditions for NADH-nitrate
reductase assay were based on experiments
with laboratory cultures of D. brightwellii.
The diatom was chosen for study because
SOL6RZANO
of enzyme activity
seawater samples
in
NADH-nitrate
reductase was found in
particulate matter from seawater samples
containing nitrate. The particulate matter
included animals, phytoplankton,
and detritus large enough to be retained by the
32-p net. Conceivably the enzyme activity
is due to heterotrophic
microorganisms
associated with detritus or animal gut
contents as well as phytoplankton.
The
bacterial and fungal floras of detritus particles, fecal pellets, and zooplankton gut
contents are, of course, unknown as are the
nitrogen sources available to these floras.
The excretion of ammonium. by zooplankton suggests that it, rather than nitrate, is
a primary source of inorganic nitrogen for
the flora of the guts and fecal pellets, but
it is not known whether ammonium assimilation represses nitrate reductase synthesis
in these microorganisms or indeed if they
have the genetic capacity to synthesize
nitrate reductasc. Evidence that activity in
our samples was due primarily to’ phytoplankton is only indirect. 1) The specific
activity of the enzyme per unit chlorophyll
a was similar to that seen in the deep-tank
cultures although the bacterial content was
likely to be different. There were no animals in the experiments in the deep tank
and much of the detritus was removed
when the water was initially filtered. 2)
Bacteria in the homogenates would probably remain intact during grinding and the
intact cells would not be expected to require exogenous NADH to reduce NOsand to release NOa- to the medium. In
NITRATE
REDUCTASE
IN
MARINE
these experiments, activity was calculated
frosm the difference in nitrite formation
in samples incubated with and without
NADH.
There was no measurable NOaformation in the tubes without NADH although there was some absorption at 543
nm owing primarily
to carotenoid pigments. 3) Adding animals (a mixture of
unidentified
zooplankton)
collected using
a net with large mesh to D. brightwellii
extracts gave no increases in enzyme
activity.
Levels of ammonium required for
enzyme repression
It is clear from the three experiments in
the deep tank that the occurrence of nitrate
in seawater does not necessarily mean that
it is the nitrogen source for phytoplankton
growth if ammonium is also present. Ammonium and nitrate assimilation in cultures
appear to be mutually
exclusive events,
sequential in time, as shown earlier for
C. vuZ~ar2s (Morris and Syrett 1963) and
recently for the diatom Cylindrothecu closterium (Grant, Madgwick, and Dal Pont
1967). The use of ammonium in preference to nitrate may be fairly common
among algae and can be explained by the
repression of nitrate reductase during ammonium assimilation with subsequent induction of enzyme by nitrate when the
ammonium is depleted. The current data
show a synthesis of nitrate reductasc
during ammonium assimilation
at sufficicntly low concentrations of ammonium,
These levels were approximately
0.5, 1.0,
and 0.5 pM in the three experiments. Ammonium is apparently recycled rapidly in
the sea and is formed in the euphotic zone
as a result of mineralization
by bacteria
and especially by excretion of zooplankton
(cf. Dugdale 1967; Martin 1968). The
above NHd+ concentrations are probably
typical of oceanic water (cf. Beers and
Kelly 1965; Thomas 1966; Martin 1968) so
it appears unlikely that ammonium assimilation commonly represses nitrate rcductase synthesis in the sea. This may account
for the simultaneous
uptake of nitrate
203
l?IIYTOPLANKTON
and ammonium observed by Dugdale
Gocring (1967) in seawater samples.
Loss of nitrate reductme
and
activity
In an earlier experiment, G. polyedru
was grown first on nitrate and subsequently
on ammonium
(Holmes, Williams,
and
Eppley 1966). Total enzyme activity per
unit volume of culture reached a moderate level during growth on nitrate and
remained constant during further growth
with ammo,nium, but activity per unit chlorophyll a declined at the same time-the
enzyme being diluted out by new cell synthesis. As in the current experiments, this
result supports the notion that the enzyme
is not synthesized during growth on ammonium. The decline in enzyme activity
per unit chlorophyll w may in general reflect, during growth on ammonium, either
the rate of intracellular
enzyme degradation or the rate of enzyme dilution by new
growth. In the G. poZyedra experiment, the
dilution effect was predominant although
in the first experiment in the deep tank
the enzyme activity was degraded between
day 3 and 5 since enzyme activity declined
per unit culture volume as well as per
chlorophyll a. When growth stopped due
to depiction of nitrogen, as in the second
and third experiments in the deep tank,
the decline of enzyme activity must represent degradation of the protein.
The
enzyme of corn plants is degraded with a
half-life of only 3.5-4.,2 hr (Schradcr and
Hageman 1967). The half-time for loss of
enzyme activity in the deep-tank expcriments was apparently l-2 days and may
vary with species.
Potential uses of nitrate
reductase assays
The primary value of the enzyme assay
at sea is confirmation, by a positive assay,
that the phytoplankton
are using nitrate
for growth. Conversely, a very low activity
implies either assimilation of ammonium
(or possibly other reduced forms of nitrogen) or that the phytoplankton
have
depleted their nitrogen supply.
If the
latter were true one would expect dcclin-
204
R. W.
EPPLEY,
J.
L.
COATSWORTII,
ing physiological
activity and changes in
chemical composition as could bc infer&l,
for example, by changes in the carotenoid : chlorophyll ratio in the phytoplankton (Yentsch and Vaccaro 1958; Manny
1969). Decline of enzyme activity was
noted in deep-tank experiments 2 and 3
before any decline in the photoassimilation
rate of carbon (expressed per unit chlorophyll a) or increase in the carotenoid : chlorophyll a ratio. Thus the enzyme assay
provided a particularly
sensitive measure
of incipient nitrogen deficiency for a phytoplankton community grown on nitrate.
Blooms of red-water dinoflagellates
off
La Jolla, California, appear to result from
enrichment of surface waters with nitrate
owing to upwelling.
Nitrate was the primary nitrogen source for growth of bloom
organisms as indicated by a high NADHnitrate reductase titer (12 x 1O-gmoles NOzformed per rug Chl a/hr) (Holmes et al.
1966). It would be interesting to assess
enzyme activity in Gymnodinium
brevis
blooms off the west coast of Florida where
nitrate enrichment by upwelling of deep
water is not a likely stimulus. The enzyme
assay would help to identify the source of
nitrogen for development of such blooms as
are associated with land drainage rather
than upwelling.
General use of the enzyme assay depends on the ability of phytoplankton
to
assimilate both NIL+ and N03-, the induction of enzyme synthesis by nitrate, and its
repression during ammonium assimilation.
The ability to use amino acids or urea as
a nitrogen source, the possibility of repression of NADH-nitrate
reductase during
such assimilation, the concentration of free
amino acids and urea in seawater, and the
use of such concentrations by phytoplankton have not been examined in detail and
some caution is required until this is done.
An extreme example of behavior different
-from that seen in phytoplankton
is given
by cultures of isolated tobacco plant cells.
Here, the enzyme was induced by nitrate
and repressed by certain amino acids
.when added to the culture medium, but
not by ammonium. The cells used neither
AND
LUCIA
SOLhZANO
ammonium nor these amino acids for
growth and some of the latter inhibited
growth
with nitrate.
Moreover,
other
amino acids did not block growth with
nitrate and in fact relieved the inhibitory
effects of the first group of amino acids
(Filner 1966). Should this situation find a
parallel in phytoplankton
growth in the
sea, the use of the enzyme assay would
be meaningless. We assume, on the basis
of our current results, that this is not
the cast.
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