Z - Association for the Sciences of Limnology and Oceanography

Limnol. Oceanogr.. 33(4, part I), 1988, 518-527
0 1988, by the American
Society of Limnology
and Oceanography,
Inc.
Coupling between ammonium uptake and incorporation in a marine
diatom: Experiments with the short-lived radioisotope 13N
Jonathan P. Zehr
Marine
Sciences Research Center, State University
of New York, Stony Brook
11794
Paul G. Falkowski
Oceanographic
Sciences Division,
Brookhaven
National
Laboratory,
Upton,
NY 11973
Joanna Fowler
Chemistry
Department,
Brookhavcn
National
Laboratory
Douglas G. Capone
Chesapeake Biological
Laboratory,
University
of Maryland,
Box 38, Solomons
20688-0038
Abstract
The coupling
between ammonium
transport
and incorporation
in the neritic marine diatom
with the short-lived radioisotope 13N (& = 9.96
min). In ammonium-limited
continuous cultures, growing at rates from 0.2 to 3.2 d-l, no trend
in uptake rates per ccl1 was apparent, but the rate of incorporation
of radiolabeled ammonium
into trichloroacetic
acid (TCA)-insoluble
material, following a saturating pulse of ammonium, was
linearly correlated with growth rate (r2 = 0.93). Calculated growth rates overestimated the measured
rates. The ratio of calculated growth requirements (&) to growth requirements based on incorporation of 13N into TCA-insoluble
material (pq) was nonlinearly
related to growth rate. The
discrepancy between & and pq was explained by isotope dilution in the internal free amino acid
pool. The ratio of uptake to incorporation,
corrected for isotope dilution in the amino acid pool,
showed that uptake and growth were uncoupled at low growth rates but were coupled at P~,,~~.The
ratio of short-term uptake to incorporation
was found to be a direct measure of enhanced uptake
and decreased with increasing growth rate. This ratio may be a useful parameter for determining
growth rates and nitrogen deficiency in natural phytoplankton
communities.
Thalassiosira pseudonana (3H) was investigated
Since the availability
of fixed inorganic
nitrogen is assumed to be a major factor
regulating the abundance and growth rates
of marine phytoplankton
(Ketchum et al.
1958; Ryther and Dunstan 197 1; Thomas
1969, 1970; Thomas and Dodson 1972),
kinetics of nitrogen uptake by marine phytoplankton have provided the focus of many
investigations.
Dugdale (1967) related nitrogen uptake rates to the external concentration of nitrogen by the Michaelis-Menten
equation, V = V,,,[SI(K,
+ S)], where V is
the uptake rate of the nitrogen substrate at
the external concentration S, and K, is the
substrate concentration at which Yis equal
to half the maximum uptake rate, V,,,. The
values of & and V,,,,, could be important
in competitive interactions between species
(Dugdale 1977). The measurement of K, and
V,.,,,, therefore has been the subject of numerous culture (Eppley et al. 1969, 1977;
Horrigan and McCarthy 198 1; Gotham and
Rhee 198 1; Goldman and Glibert 1982) and
field investigations (Maclsaac and Dugdale
1969; Glibert and Goldman 198 1; Wheeler
et al. 1982; Glibert and McCarthy 1984).
Maximum uptake rates (V,,,,,) often exceed nitrogen requirements
for growth
Acknowledgments
(Caper-on
and
Meyer
1972;
Eppley
and RenThis research was supported by NSF grant OCE 85ger
1974;
Conway
et
al.
1976;
McCarthy
15886. Additional
support was provided by the U.S.
and Goldman 1979). Uptake rates deterDepartment of Energy under contract No. DE-AC0276CH00016.
mined from brief incubations
are even
We thank D. Schlyer, and A. Wolf for their help and
higher than rates determined over longer
cooperation in use of the Brookhaven cyclotron facilintervals (Conway et al. 1976; McCarthy
ity. K. Wyman, A. Milligan, and M. Tcdesco provided
and Goldrnan 1979) and have been termed
technical assistance. P. Glibert and an anonymous reviewer provided suggestions on the manuscript.
“surge” (Conway et al. 1976) or “enhanced”
518
13Nammonium uptake
uptake (McCarthy
and Goldman
1979).
Maximum
nitrogen-specific
uptake rates
were shown to be a function of growth rate
(Eppley and Renger 1974; Dugdale 1977;
McCarthy and Goldman 1979). In steady
state, the maximum
specific uptake rate
(termed V’,,, to indicate that it can vary)
can be related to the growth rate since p =
VQ (P = uptake rate and V = specific nutrient uptake rate normalized to the cell nutrient content, Q): p = V’,,,[SI(K,
+ S)]
(Dugdale 1977). Enhanced V’,,, has been
hypothesized to be an ecological adaptation
that allows phytoplankton
to rapidly sequester transient micropatches of nutrients
(McCarthy and Goldman 1979; Glibert and
Goldman 198 1; Goldman and Glibert 1982)
and maintain high growth rates (Goldman
and Glibert 1982; Goldman et al. 1979).
The importance of enhanced nitrogen uptake to phytoplankton
growth depends on
the coupling of uptake of nitrogen to the
incorporation into new cell material (Collos
1986). If uptake rates respond extremely fast
to short pulses of ambient nitrogen, but the
cells are not able to incorporate that nitrogen on a similar time scale, then growth may
not be limited by the availability of external
nitrogen, but rather by the rate at which the
cell can incorporate
inorganic
nitrogen
(Wheeler et al. 1982).
Models of cellular uptake and growth have
included parameters for uptake into the intracellular inorganic nitrogen pool, as well
as the production of simple organic nitrogen
metabolites (assimilation)
and protein nitrogen (incorporation) (Grenney et al. 1973;
DeManche et al. 1979). It has been suggested that assimilation and incorporation
rates may also vary as a function of nitrogen
deficiency (Wheeler 1983), but most studies
of nitrogen metabolism have focused on uptake. Studies with analogue radiotracers,
such as [ 14C]methylamine as an analogue for
ammonium (Wheeler and McCarthy 1982;
Balch 1986), have been limited to uptake
processes since the analogues are not incorporated efficiently into macromolecules. In
addition, even though methylamine
and
ammonium
are transported by the same
transport system, they are not transported
at the same rate (Wheeler and McCarthy
1982). Studies of incorporation of 15N have
519
been performed
in natural populations
(Wheeler et al. 1982; Glibert and McCarthy
1984), but the investigation of coupling of
short-term uptake to short-term incorporation has been limited by the sensitivity of
this technique. We have used the radioactive isotope 13N (t,,, = 9.96 min) to study
the relationship of short-term ammonium
uptake to incorporation in a marine diatom,
Thalassiosira pseudonana (3H). The use of
a radioactive tracer allowed us to examine
the coupling of ammonium uptake to incorporation
into protein over extremely
short time intervals.
Methods and materials
Cultures of T. pseudonana (3H) were obtaincd from the Culture Collection of Marine Phytoplankton
(Bigelow Laboratory,
Maine) and grown in artificial seawater medium (McLachlan
1964) as modified by
Goldman and McCarthy (1978). The basic
salts were autoclaved in 8- or 16-liter glass
containers and trace metals (f/2), phosphorus, bicarbonate, and ammonium then
added aseptically by sterile filtration. The
bicarbonate (2 mM) maintained pH at 8. l8.2 in actively growing cultures. Chemostat
(0.75-0.85 liter) cultures were grown at different dilution rates under continuous illumination
(100 PEinst m-2 s- I measured
in media in the center of the chemostat).
Temperature was maintained at 18°C by
water jackets. The cultures were mixed continually by a stirring bar and bubbling of
compressed air that had been passed through
a 0.1 N H2SO4 trap. Cell densities were
maintained between 0.3 and 1.5 x 1O6cells
ml-‘.
Samples for cell counts, chlorophyll, ammonium, and particulate carbon and nitrogen analysis were withdrawn from the sample port located at the bottom of the
chemostat. Cells were counted with a hemacytometer or Coulter counter. Samples
were filtered onto glass-fiber filters for particulate carbon, nitrogen, and chlorophyll
determinations.
Particulate carbon and nitrogen were measured on a Perkin-Elmer
CHN analyzer. Filters were extracted in 90%
acetone and absorption spectra scanned on
an Aminco dual-beam spectrophotometer
for determination of Chl a. Ammonium was
520
Zehr et al.
measured with a Technicon autoanalyzer by
the phenolhypochlorite
method (Whitledge
et al. 1981).
Free amino acids were measured in high
and low growth rate cultures with HPLC
(Jones et al. 198 1). Amino acids were derivatized with o-phthaldialdehyde
and separated on a 7.5-cm, reverse-phase (3 pm,
ODS, Phenomenex) column with an acetate
buffer (50 mM, pH 5.9) and methanol gradient. Culture samples (10 ml) were centrifuged and resuspended in 10% methanol (0.5
ml). Samples were boiled for 1.5 min to
extract free amino acids and then derivatized with the procedure of Jones et al.
(1981).
13N was produced on the 60-in. (152.4
cm) cyclotron at Brookhaven National Laboratory with the 160@, CU)I~Nreaction and
a small-volume
(0.5-l .O ml) water target.
Typically, the water target was bombarded
with 18 MeV protons for 10 min at a current
of 15 PA. The 13N (primarily
as 13N03-:
Parks and Krohn 1978) was converted to
l 3NH4+ with DeVarda’s alloy (see Cooper
et al. 1985, and references therein), remotely transferred from the target to the
laboratory by application of gas pressure,
and added to a sealed reaction flask containing 0.5 g NaOH and 0.5 g DeVarda’s
alloy. The reaction flask was connected to
a 1-ml, distilled water trap to collect 13NH3.
The reaction continued for 5-10 min, after
which 2-10 mCi 13NH4+ was recovered.
13NH4+ was pushed through an ion-exchange resin (Ag’ x 8 Cl form, Bio-Rad
Laboratories). The trap and ion-exchange
column were then rinsed with 0.5 ml of distilled water. Stable ammonium
contamination (14NH4+), apparently from the reducing reagents (Cooper et al. 1985),
decreased specific activity, but specific activities > 7 mCi pmol-l were obtained. Purity of the radionuclide was determined by
following its decay through seven half-lives.
Samples from the chemostats (100-l 50
ml) were withdrawn < 15 min before beginning the 13N experiments, and all experiments were started within 1 h so that the
radioactive samples could be counted within seven half-lives of the isotope. Aliquots
of the sample (1 O-l 5 ml) were placed in
clean scintillation vials and kept under con-
stant illumination.
Unlabeled ammonium
chloride was added simultaneously with 35
~1of the 13NH4+ solution. The vial was shaken and a l-ml subsample immediately
filtered through a 25-mm diameter, 0.4-pm
pore-size Nuclepore filter on a filtration
manifold (Hoeffer Instr.). The filter was
rinsed three times with 3 ml of filtered seawater. Filtration of the sample took ~5 s.
Subsamples for uptake were withdrawn and
filtered at 15 s, 30 s, 1 min, 2 min, and 5
min following the spike. Longer times were
sometimes included. Samples for incorporation were filtered and rinsed, the vacuum
was turned off, and 5 ml of 5% cold trichloroacetic acid (TCA) was added to the
filter in the manifold. After 5 min, the vacuum was turned on and the filter rinsed three
times with 5% cold TCA.
Filters were counted in a Packard gamma
counter for 0.5 min. Background counts were
subtracted and all of the sample counts were
decay-corrected to a common time. Filters
and solutions were recounted to check the
half-life of the activity. Nitrogen uptake and
incorporation
rates were calculated from
decay-corrected
counts per minute and
measured specific activities (cpm pmol- I):
p (pg atom N cell-’
min-l)
= R/(X x SA)
where R is the accumulation of radioactivity (cpm ml-’ min-‘),
X the cell density
(cells ml-‘), and SA the specific activity of
ammonium (cpm pg atom N-l). Incorporation rates were calculated assuming that
the specific activity of the internal ammonium pool was equivalent to that of the external ammoniurn pool and that the specific
activity of the free amino acid pool was
equivalent to the specific activity ofexternal
NH,+. This first assumption was based on
the observations that the high initial rate of
ammonium uptake appeared to saturate the
internal ammonium pool and that initial
internal ammonium
concentrations
are
usually low. The validity of the second assumption was evaluated by comparing the
measured protein synthesis rate (pq) to the
known growth requirement
(pQ) as discussed below.
In a few experiments, inhibitors of protein synthesis were added to evaluate the
TCA procedure as a measure of protein syn-
13Nammonium uptake
521
thesis, as well as to compare the relative
synthesis of cytoplasmic to mitochondrial
or chloroplastic
ribosomes. Experiments
with cycloheximide
(inhibitor of cytoplasmic mRNA translation)
and chloramphenicol (inhibitor
of chloroplastic
and
mitochondrial
mRNA translation)
were
performed at final concentrations of 25- 100
and 100 ,ug ml-‘, respectively.
Results and discussion
Time-courseofammonium
uptake-Am-
monium uptake rates after adding a 10 PM
NH4+ pulse were constant for up to 2 min,
after which they usually decreased (Fig. la).
Negligible amounts of 13N were adsorbed
by heat-killed cells or formaldehyde-preserved cells (2% of control after 2 min). Since
ammonium was not depleted below 8-10
PM during the first few minutes of incubation (2-10% of label taken up within 2
min) and uptake rates per cell were constant
during the first l-2 min of incubation, the
data indicate that rates at saturating concentrations measured over a 2-min interval
are good approximations
of the maximal
transport rate for T. pseudonana (3H).
The decrease in uptake rates with time
(Fig. la), following an ammonium
pulse,
has been suggested to be the result of filling
an internal pool (Parslow et al. 1985; Dortch
et al. 1984). The amount of nitrogen taken
up per cell (0.3 1 pg-atom cell-‘) during a
3-min pulse would result in an average internal ammonium concentration of 37 mM
(assuming a cell volume of 25 pm3, Table
1). This value is close to the maximum of
40 mM determined by Dortch for this
species (pers. comm. cited by Parslow et al.
198 5). Uptake rates decreased after 3-5 min
(Fig. la), as the maximal internal concentration was attained, or as a result of feedTable 1. Cellular characteristics
0.5-0.8
0.9-l .5
1g-2.2
3.0
(10 2 pg-atoms
4.67
5.43
6.83
9.79
-7 0.8
?I
0
1
(b)
TIME (min)
Fig. 1. a. Time-course of uptake of 13NH,+ in cultures of Thalassiosira pseudonana (3H) at high and low
growth rates. Uptake rates (dashed line) were calculated
from the uptake curve. b. Time-course of incorporation
of 13NH,+ into cold TCA-insoluble
material in cultures
at high and low growth rates (0-p = 0.5 d-l; x -p =
3.0 d-l).
back inhibition.
Uptake rates at low concentrations (~0.3 PM) measured with 13N
showed that uptake rates decreased over
even shorter time intervals when substrate
additions were low (data not presented); the
substrate concentration
was significantly
depleted during the experiment (up to 77%
of the added label being taken up within 2
of Thalassiosira pseudonana (3H) at different
c
N
P (d-7
TIME(min)
cell-‘)
54.0
53.6
43.8
85.0
C:N*
11.6
9.9
9.0
8.7
Chl a cell ’
(PP)
0.059
0.08 1
0.130
ND
growth rates.
Protein cell-’
(Pi3)
4.3
6.4
9.3
12.2
Cell volumet
29.8
31.7
28.6
ND
522
Zehr et al.
min with submicromolar additions). Therefore, uptake rates at low substrate concentrations decrease only if the concentration
of the substrate is reduced (Parslow et al.
198 5). When external concentrations of ammonium are saturating, uptake rates decrease with time due to feedback inhibition
or the filling of an internal ammonium pool.
SpeciJic uptake rates and the cell quotaNitrogen, Chl a, and protein per cell in ammonium-limited
chemostats of T. pseudonana increased with growth rate, but the
C : N ratio decreased with increasing growth
rate (Table 1). The fit of the data to the
linearized form of the Droop model (p =
PQ - Kp: Goldman 1977) gave values of
3.67 d-l and 3.38 x 1O-2 pg-atoms N cell-’
for E and k,, respectively. Cell volume and
carbon per cell were not correlated with
growth rate (Table 1).
The cell nitrogen quota (Q) increased with
growth rate (Table 1). Nitrogen-specific
uptake rates are linearly related to the cellular
nitrogen quota since I”,,,aX = p,,,/Q, where
Q is the internal concentration of nitrogen
(Dugdale 1977; McCarthy and Goldman
1979). McCarthy (198 1) suggested that v’,,,
would vary in a hyperbolic fashion with respect to growth rate as a result of an enhancement in uptake rates per cell at low
growth rates. The nitrogen quota in this
study varied by about 2.5-3 between the
lowest and highest dilution rates (Table 1).
Therefore, nitrogen-specific
uptake rates
should vary by a factor of 3 if pmaxis constant with growth rate.
No trend between pmaxper cell and growth
rate was apparent (Fig. 2a). The mean uptake rate per cell at growth rates < 1 d-l was
not significantly different than the mean uptake rate at growth rates > I d- I [0.3 1 -to. 14
(SD) and 0.32kO.09 (SD) fmol N cell- ’
min-‘, respectively]. Uptake rates may be
reduced in severely nitrogen-deficient
cells,
giving rise to a bell-shaped curve of uptake
rate vs. growth rate (Goldman and Glibert
1982; Morel 1987). The mean uptake rate
of cells growing at 0.5-1-O d-l was only
O-38+0.13 (SD) fmol N cell- I min-‘, however, still not significantly different from the
mean uptake rate at p > 1 d- ’ .
Since hax per cell was not correlated with
growth rate, variation in the specific uptake
rate in N-limited chemostats was due mainly to the changes in cell quota Q. The relationship of maximal nitrogen-specific uptake rates (V’,,,,
where Q was estimated
from linear regression of Q on CL)to growth
rate was similar, however, to that found by
Goldman and Glibert (1982) (Fig. 2b). The
largest values of this parameter were found
at dilution rates of 0.4-0.6 d -I and were
greater than four times the value at the highest dilution rate (Fig. 2b). This variation is
somewhat greater than that predicted by the
range in Q values (2.5-3.0 in this study), but
indicates that. pmaxvaried by no more than
1.5.
There is variability in the Q data of Goldman and McCarthy ( 1978), as there is in the
results of this study. Although the C.V. of
particulate
nitrogen determinations
was
usually < LO%, we found that variability in
Q was often related to errors associated with
counting clumped cells. Clumping of cells
was more evident at low growth rates than
at high, which may result in greater variability of $/‘max and pmaxat growth rates < 1
d-l. Therefore, the variability
in Q probably explains the variability
in specific uptake rates found in our study (Fig. 2b).
Incorporation of nitrogen into proteinsThe incorporation
of a saturating pulse of
ammonium
into TCA-insoluble
material
(primarily protein) was nonlinear, with a 23-min lag before incorporation
began (Fig.
1b). Incorporation rates into TCA-insoluble
material were correlated with growth rate
(r2 = 0.93, n = 10) (Fig. 3a). About 88-94%
of the incorporation
of nitrogen into TCAinsoluble material was inhibited by cycloheximide, indicating that incorporation
is
primarily due to cytoplasmic protein synthesis. Chloramphenicol
inhibited an additional 6% of the incorporation,
indicating
that only a small fraction of the total proteins are synthesized on chloroplastic or mitochondrial rRNA.
Radioactivity
was detected in macromolecules within a few minutes (Fig. 1b),
but the maximal rate of incorporation
of
nitrogen proceeded at the protein synthesis
rate set by the growth rate. This finding is
consistent with the suggestion that uptake
over long incubations (> 15 min) is limited
by protein synthesis or growth rate (Conway
523
13N ammonium uptake
-
0.6 1
Z.?
‘h
4 40-
(al
0
0
(a)
0
T=
8 30z
B
D
0
0
0
5
j) 20a
N
b
c IOU
a.
0
0
0
II
O-i
0
I
I
GROWTH
I
3
I
2
I
4
RATE (day-‘)
p (day-‘)
(b)
(b)
01
0
I
I
GROWTH
I
2
I
3
I
4
RATE (day”)
0~
0
2
3
4
pL(day-‘1
Specific uptake rates of 13NH4-fi in cultures
of Thalassiosira pseudonana (3H) at high and low
growth rates. a. Uptake rates per cell. b. Nitrogen-specific uptake rates.
Fig. 3. a. 13N-incorporation
rates (~q) as a function
of growth rate, and calculated protein synthesis rates
(pQ) as a function of growth rate of Thalassiosira pseudonana (3H). (Details of calculations given in text.) b.
Ratio of q : Q as a function of growth rate.
et al. 1976; Wheeler ct al. 1982). Nitrogen
taken up following a saturating pulse appeared to be incorporated at the rate at which
proteins were synthesized before addition
of the spike, since incorporation
rates were
correlated with growth rate (Fig. 3a). We
found no evidence of increased incorporation rates with increasing nitrogen deficiency. The rate of protein synthesis appeared
to continue at the growth rate of the organism, at least over 20-30 min. Cells apparently continue to grow at the growth rate
attained before the nitrogen pulse.
Our results suggest that cells can maintain
a steady growth rate even in a patchy environment. Eppley (198 1) suggested that if
phytoplankton
are growing in a patchy environment, then their growth may not be
balanced (i.e. all cellular constituents are not
produced at the same rate). If protein syn-
thesis rates do not respond immediately to
saturating pulses, particularly when the pulse
rapidly diffuses, again lowering the external
concentration (Jackson 1980), then phytoplankton could be in steady state and balanced growth in spite of patchy nutrient environments.
The lag before 13N was incorporated into
TCA-insoluble
material was due to the time
required for the isotope to pass through the
free amino acid pool. Therefore, the increase in incorporation rate with time is related to the time needed to reach isotopic
equilibrium
in the precursor (free amino
acid) pool. The first derivative of this function (obtained from estimating rates between consecutive points on the incorporation curve, e.g. Fig. lb) can be used to
calculate the half-time of the pool. The halftime is related to the turnover time of the
Fig. 2.
524
Zehr et al.
0
-I
I5
Fig. 4. Change of 13N-incorporation
rate into TCA-insoluble
material with time. Calculation
amino acid pool is shown. x -0.5 d-l; V-O.77 d-l; O-O.93 d-l; #--3.2 d-l.
pool, but the turnover time cannot be calculated without making assumptions regarding the reaction order. The change in
rate of incorporation
vs. time shows that
the half-time is similar (on the order of minutes) for cultures growing at different growth
rates (Fig. 4). The average calculated halftime (determined from half the time from
the end of the lag to the time when half the
maximal rates were attained, Fig. 4) of the
amino acid pool was about 2 min. This halftime is short compared to the hours required for amino acids to reach isotopic
equilibrium
in 14C experiments (Lohrenz
and Taylor 1987a,b).
The estimated l”N-incorporation
rate (pq)
underestimated the actual N-incorporation
rate (pQ) (Fig. 3a). The ratio of the estimated cell quota (q, calculated from PqIp)
to the measured cell quota: (Q) was nonlinearly related to growth rate (Fig. 3b). The
ratio q : Q varies by a factor of 5 from high
to low growth rates. This ratio is a measure
of isotope dilution by the internal free amino acid pool. In this study, the sizes of the
measured cellular free amino acid pools were
5.8 times greater at high (1.6 d--l) compared
to low (0.2 d-l) growth rates (Table 2), consistent with the fivefold variation in q : Q
over the range of growth rates. The amino
acid data show that certain amino acid pools
(e.g. glutamine, asparagine) vary more than
of half-time
of
others (Table 2). The estimated isotope dilution reflects the average difference in amino acid pools that are in greatest abundance
or turn over rapidly.
Coupling between uptake and incorporation -The implications of nonlinear uptake
at trace and saturating concentrations can
be seen from a conceptual model of uptake,
assimilation, and incorporation
(Fig. 5). At
saturating concentrations,
ammonium
is
taken up at the maximal transport rate,
which is independent of growth rate. Ammonium in the internal ammonium pool is
rapidly assimilated into amino acids. The
amino acids are then incorporated into protein at V = p (Fig. 5). Only I’ of incorporation is necessarily related to growth rate
in this case. The ammonium taken up in
excess of IA reaches a maximum concentration (determined by the maximum concentration gradient or feedback inhibition).
If
the higher ammonium
concentration
is
maintained by continued exposure to high
concentrations of ammonium, it will lead
to higher free amino acid concentrations
which eventually allow higher rates of protein synthesis and growth.
In contrast, at trace concentrations, the V
of uptake must equal p. Therefore, the internal ammonium pool, and hence the free
amino acid pool, is maintained at low concentrations. When the concentration of in-
13Nammonium uptake
PROTEIN
Incorporation
PROTrA
t V=p
AMINO
525
ACID
C-
Dilution of 13N
by14N aminoacids
f
-
V=p
AMINO
ACID’
’
Assimilation
h
cI?Izl
AMMONIUM
AMMONIUM
Saturating
Fig. 5.
Conceptual
,
Trace
model of uptake, assimilation,
ternal ammonium is low, the capacity to
take up ammonium in excess of growth requirements is greater. At both trace and saturating concentrations,
the initial ammonium pool size is small, and therefore 13N
activity is not substantially diluted by the
internal ammonium pool, but is diluted by
the larger amino acid pool.
Since the protein synthesis rate (v of incorporation) is set by the growth rate, regardless of transient fluctuations
in the
internal ammonium
pool (Fig. 5), the
measurement of the protein synthesis rate
is a better index of growth rate and nitrogen
deficiency than are maximal transport rates.
Our findings suggest that the rate of protein
synthesis is equivalent to the growth rate,
even after a saturating pulse of nitrogen.
Therefore, the incorporation
of 14C into
protein or specific protein amino acids could
be measured after a saturating pulse of nitrogen, to avoid artifacts associated with
containment and nitrogen depletion (Goldman et al. 198 1; Lohrenz and Taylor 1987a).
Since transport rates per cell were essentially constant across all growth rates and
incorporation
rates were correlated with
growth rate, the ratio of the two rates was
correlated with growth rate (Fig. 6a). Enhanced uptake is the difference between the
maximal potential transport rate and the
calculated nitrogen incorporation rate (pQ>.
The ratio of short-term maximal uptake rate
to incorporation
rate is a direct measure of
the difference between uptake and incorporation, i.e. “enhanced” uptake. Values for
this ratio were about 10 times higher at low
growth rates than at high ones (Fig. 6a), and
the ratio was correlated with q : Q (Fig. 6b).
A correction for the change in isotope di-
and incorporation
of ammonium.
lution in the amino acid pool with growth
rate can be derived from the variation of
q: Q with h (Fig. 3b). Values of q : Q at all
growth rates were normalized to the lowest
value of q : Q found at p = 0.2 d-l. This
analysis allows the correction of the incorporation data for isotope dilution, presumably resulting from the difference in amino
acid pool sizes at different growth rates. This
correction lowers the uptake-incorporation
curve (Fig. 6a) so that the derived uptake :
incorporation ratio at high dilution rates approaches 1 (1.2 at p = 3.2 d-l), which is
predicted if uptake and growth are coupled
uYtlax = p). This analysis strongly suggests
that uptake and growth are uncoupled at low
growth rates and coupled at high growth
rates in T. pseudonana (3H).
Table 2. Free amino acid concentrations (fmol cell-l)
in nitrogen-limited
cultures of Thalassiosira pseudonana
(3H) at high (1.6 d-l) and low (0.2 d-l) growth
rates.
0.2
1.6
Ratio
(high to
low)
0.19
0.38
0.02
0.05
0.04
0.03
0.07
0.19
0.02
0.14
0.10
0.03
0.03
0.03
0.01
0.02
1.3
0.96
2.6
0.55
1.43
0.25
0.15
0.12
0.36
0.05
0.30
0.43
0.13
0.10
0.06
0.05
0.19
7.7
5.1
6.8
34
31
5.8
4.5
1.8
1.9
3.3
2.2
4.3
4.2
3.1
2.4
3.6
11.2
5.8
Growth
Aspartate
Glutamate
Asparagine
Glutamine
Lysine
Arginine
Threonine
Serine
Tyrosine
Glycine
Alanine
Valine
Leucine
Isoleucine
Phenylalanine
Methionine/tryptophan
Total
rate (d-l)
Zehr et al.
was determined only at the end of the experiment. Eventually, all nitrogen that is
taken up will be incorporated, although it
will be incorporated slower at low growth
rates and faster at high growth rates. Therefore, the uptake rate should be determined
over a brief interval. The ratio of short-term
uptake to incorporation is a measure of the
uncoupling between uptake and incorporation and appears to be a sensitive index
of nitrogen deficiency and growth rate (Fig.
6).
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Fig. 6. a. Ratio of 13N uptake per cell to 13N incorporation
per cell as a function of growth rate.
q b-llncorrected
for isotope dilution by the intracellular amino acid pool; V-corrected
for isotope dilution. b. Correlation of q: Q to the ratio of 13N uptake
to 13N incorporation.
We found that the ratio of maximal transport rates to incorporation
rates is related
to phytoplankton
growth rates. Since it is
difficult to estimate pool sizes in natural assemblages, the use of this ratio measured
with traditional
stable isotope techniques
(short-term 15N uptake to 15N incorporation
into TCA-insoluble
material) may provide
a useful index of growth rate and nutritional
status. The ratio of uptake to incorporation
is a direct measure of enhanced uptake (or
uncoupling) and is unitless. This ratio is not
dependent on measurements of cell number, biomass, or Q. The percentage of nitrogen taken up that is incorporated into
TCA-insoluble material has previously been
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natural populations (Glibert and McCarthy
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Submitted: 10 July 1987
Accepted: 8 February I988
Revised: 4 April I988