Plant Physiol.

(1986) 82, 708-712

0032-0889/86/82/0708/05/$0 1.00/0

Nitrate and Ammonium Induced Photosynthetic Suppression in

N-Limited Selenastrum minutum1
II. EFFECTS OF N03- AND NH4+ ADDITION ON CO2 EFFLUX IN THE LIGHT
Received for publication May 6, 1986 and in revised form June 22, 1986

DOUGLAS G. BIRCH, IVOR R. ELRIFI, AND DAVID H. TURPIN*

Department of Biology, Queen's University, Kingston, Ontario, Canada K7L 3N6

ABSTRACT This model presents several testable hypotheses. One predic-

tion is that N resupply to
The effects of nitrate and ammonium addition on net and gross pho- should result in an increaseNO3-limited Selenastrum minutum
tosynthesis, CO2 efflux and the dissolved inorganic carbon compensation and consequently an increase intricarboxylic
in acid cycle activity
point of nitrogen-limited Seknastrum minutum Naeg. Collins (Chloro- light. This hypothesis can be the rate of CO2 efflux in the
phyta) were studied. Cultures pulsed with nitrate or ammonium exhibited by measuring CO2 efflux in thetested in several ways. The first is
a marked decrease in both net and gross photosynthetic carbon fixation. the specific radioactivity light as reflected by a decrease in
('4DIC/'2DIC)
During this period of suppression the specific activity of exogenous ing the cells. The second is to measure changesof the medium surround-
dissolved inorganic carbon decreased rapidly in comparison to control about by the addition of exogenous nitrogen sources. in the r brought
cells indicating an increase in the rate of CO2 efflux in the light. The In this report we evidence that NO3- or NH4' addition
nitrate and ammmonium induced rates of CO2 efflux were 31.0 and 33.8 to NO3--limited S. present
minutum results in a major increase in the
micromoles CO2 per milligram chlorophyll per hour, respectively, and rate of CO2 effiux in the light. This observation is consistent with
represented 49 and 48% of the rate of gross photosynthesis. Nitrate the mechanism of N-induced photosynthetic suppression pro-
addition to cells at dissolved inorganic carbon compensation point caused posed by Elrifi and Turpin (5).
an increase in compensation point while ammonium had no effect. In the
presence of the tricarboxylic acid cycle inhibitor fluoroacetate, the nitrate-
induced change in compensation point was greatly reduced suggesting the MATERIALS AND METHODS
source of this CO2 was the tricarboxylic acid cycle. These results are
consistent with the mechanism of N-induced photosynthetic suppression Chemostat Culture. Selenastrum minutum Naeg. Collins was
outlined by Elrifi and Turpin (1986 Plant Physiol 81: 273-279). grown axenically in NO3-limited 1.4 L chemostats at 20°C at a
growth rate of 0.3 d-'. Inflow medium was a substantially mod-
ified Hughes medium (5) enriched to 1 mm NaNO3 and 200 ,M
K2HPO4. This ensured that NO3- was the limiting nutrient. The
photon flux density at the culture face was 165 ME m-2s'.
Cultures were buffered at pH 7.0 with 50 mm Hepes and bubbled
rapidly (10 L.-min-') with filter sterilized air. These conditions
In many cases, nitrogen addition to N-limited microalgae or resulted in steady state cell densities of 1.72 Mg Chl ml-'.
natural phytoplankton assemblages results in a transient suppres- DIC Measurement. DIC concentration was determined by GC.
sion of photosynthetic carbon fixation (3-11, 17-19). Experi- Aliquots (100 gl) were removed from the experimental cuvette
ments with the N-limited green alga Selenastrum minutum Naeg. and injected into a modified gas chromatograph (Shimadzu GC
Collins (Chlorophyta) have led to the development of a model 8A) similar in design to that described by Birmingham and
which may explain the mechanism of N-induced photosynthetic Colman (1).
suppression (5). This model suggests that N addition to N-limited DIC Compensation Point (r) Measurement. For r determi-
S. minutum results in rapid N assimilation and an increased nations cells were harvested by centrifugation and resuspended
demand for a-ketoglutarate. In order to maintain the integrity in N-free low DIC medium, buffered at pH 8.0 with 50 mm
ofthe tricarboxylic acid cycle during the period of a-ketoglutarate Hepes (low DIC buffer), at a final density of approximately 4.0
drain, an increase in activity of the tricarboxylic acid cycle would ug Chl-ml-'. This suspension was placed in a water-jacketed
be expected. This would be supported by increased phosphoen- (20°C) experimental cuvette (20). This cuvette was equipped with
olpyruvate carboxylase and pyruvate kinase activity. One source several serum stoppered sampling ports and was bubbled with
of carbon for these reactions was suggested to be the Calvin CO2 free air (400 ml * min-') to maintain air levels of 02. At this
cycle. This drain of Calvin cycle intermediates would contribute bubbling rate and pH no detectable DIC loss from the medium
to a decrease in the rate of RuBP2 regeneration thus limiting occurred over the duration of the experiment (data not shown).
photosynthetic carbon fixation during periods of N resupply (5). Upon attaining the r the effects of NO3- or NH4' addition in
the presence or absence of various inhibitors were observed.
' Supported by the School of Graduate Studies and Research of Specific Radioactivity (14DIC/`2DIC) Measurement. Cells were
Queen's University and the Natural Sciences and Engineering Research harvested and treated as previously outlined for compensation
Council of Canada. point experiments. Upon reaching compensation point Na-
2Abbreviations: RuBP, ribulose 1,5-bisphosphate; DIC, dissolved in- H '2C03 and NaH'4C03 (Atomic Energy Commission of Canada)
organic carbon; GOGAT, glutamine 2-oxoglutarate aminotransferase were added to bring the DIC concentration to 150,uM and specific
(EC 2.6.1.53); P., gross photosynthesis; r, DIC compensation point; R, radioactivity to 2.4 MACi-,Mmol-'. Samples (750 Ml) were with-
respiration; FA, fluoroacetate; Aza, azaserine. drawn over a period of 40 min and injected into N2 purged,
708
 N-INDUCED CO2 EFFLUX IN SELENASTRUM 709
sealed vials and frozen immediately in liquid N2. Samples were Table I. Effects of Nitrate and Metabolic Inhibitors on the DIC
later thawed and aliquots taken from the vials for analysis of Compensation Point of N03-Limited S. minutum
DIC as previously described. Acid stable radiolabel was deter-
mined by adding 50 Ml aliquots from the sealed vials to 300 ,l Condition Final r Change from
Control
of kill solution (80% aqueous ethanol, 5% HCOOH). Samples
were evaporated to dryness and resuspended in 1.0 ml distilled AM %
H20 and 2.0 ml Scintiverse I (Fisher Scientific). Total radiolabel Control 29.7 ± 2.3
was determined by adding 50 Mul aliquots from the sealed vials to +NO;3 111.7 ± 16.9 +276
1.0 ml of distilled H20 alkalized with 1 drop of saturated KOH. +NO3-+ FAa 59.8 ± 13.8 +101
Two ml of Scintiverse I were added and radiolabel quantitated +NO3- + Azab 30 0
by liquid scintillation counting. The specific radioactivity of DIC a
50 mM fluoroacetate (about 46% inhibition of dark respira-
was determined as (dpmt10 - dpmacid blIe)* DIC-1. The effect of
NO3- or NH4' addition was observed by raising the NaNO3 or tion). b 5 mM Aza.
NH4C1 concentrations to 5 mm at the time of DIC addition. Table II. Estimates of the Rate of CO2 Efflux from N03-Limited
Gross Carbon Fixation at Compensation Point (r). The rate of S. minutum in the Light
gross carbon fixation at the r was measured by short-term
incorporation of '4C into acid stable products. Cultures were CO2 Efflux in the Light
brought to the r in the presence or absence of added NO3- or S.A. '4C-Fixation Net C02
NH4' as previously described. NaH'4CO3 was added to approx-
imately 175 MCi.,umol-' and 0.9 ml samples were withdrawn at experimentsa at rb effluxc
frequent time intervals and placed into 5.0 ml scintillation vials Amol C02.mg-' Chlh' + SE
containing 500 Ml of kill solution. No measurable changes in Control 6.4 ± 2.4 5.3 ± 0.9 0.0
DIC levels resulted from NaH'4CO3 addition. Quantification of +NO3- 37.4 ± 6.9 56.8 ± 14.7 39.2 ± 5.0
radiolabel was as previously described. +NH4+ 40.2 ± 3.5 5.9 ± 1.0 0.0
Metabolic Inhibitors. Aza, an inhibitor of GOGAT, was added NO3-induced
to the experimental cuvette at a concentration of 5 mm. Experi- change in CO2 ef-
ments were initiated following a 60 min preincubation. To fluxd 31.0 51.5 39.2
partially inhibit the tricarboxylic acid cycle cells were preincu- NH4+-induced
bated in the light for 60 min with 50 mM fluoroacetate. change in CO2 ef-
Nitrate Analysis. One ml culture samples were filtered through fluxd 33.8 0.6 0.0
Whatman 934-AH glass fiber filters. Nitrate content of the filtrate a Calculated as the rate of '2C02 release required to give the initial rate
was determined using the method of Strickland and Parsons (12)
modified for flow-through sample injection. of decrease in exogenous specific activity. b Calculated from the rate
Photosynthetic Kinetics. Aliquots of chemostat cultures were of gross '4CO2 fixation occurring at the r assuming that Pg = R. c The
initial net rate of DIC efflux upon pulsing with N at the r. d The rate
centrifuged and resuspended in low DIC medium in the experi- of CO2 efflux in the presence of NO3- or NH4' less the control rate.
mental cuvette (20). Photosynthetic kinetics with respect to DIC
were measured by monitoring 02 evolution with a Clarke-type
electrode (Yellow Springs Instruments) (20). was observed when NO3- was added to N03-sufficient cultures
Other Measurements. Chl and cell numbers were measured as (data not shown).
previously described (4). The rate of gross carbon fixation at the r was linear over the
time it was measured (Fig. 3). At compensation points in the
RESULTS
absence of added N03 and NH4', the rates of gross carbon
fixation were low (i = 5.3 ± 0.9 MAmol C02-mg-' Chl-h-'; Table
Photosynthetic Kinetics. Cells harvested from the chemostat II). Following N03 addition and the establishment of a higher
vessels exhibited half-saturation constants for photosynthesis compensation point, gross carbon fixation had increased (i =
with respect to [DIC] (KxDf2c ± SE) of 98 ± 22 Mm. The maximum 56.8 ± 14.7 Amol C02.mg-' Chl-h-'; Table II). Intermediate
carbon saturated rate of net photosynthetic 02 evolution was 298 rates of gross carbon fixation were observed at the intermediate
37 umol 02-mg ' Chl-h-' (data not shown). compensation points obtained following NO3- addition to fluo-
Effect of N03- and NH4' on r. In measuring the response of roacetate treated cultures (17.3 Amol C02-mg-' Chlh-').
the F to N addition, a great deal of day to day variability was Effects of N on Photosynthesis and CO2 Efflux. Addition of
observed. Table I represents the mean and SE as determined from 150 ,AM HCO3 to control cells at compensation point resulted
all measurements, whereas Figures 1 and 2 illustrate representa- in initial rates of gross C fixation of 124.1 ,mol mg-' Chl-h-'
tive experiments. Cells resuspended in low DIC medium reduced (Fig. 4A). The initial net rate of C fixation was 120.0 Amol * mg-'
[DIC] to an average compensation point of 29.7 ± 2.3 Mm (i ± Chl-h-' (Fig. 4B). Over the duration of the experiment there was
SE; Table I). Following NO3- addition the level of DIC in the little change in the specific activity of exogenous DIC (Fig. 4C).
medium increased nearly 4-fold until a new compensation point Cells pulsed with NO3-, however, exhibited lower initial rates of
was reached (111.7 ± 16.9 uM; Table I). The initial net rate of gross C fixation (62.8 ,mol-mg-' Chl-h-'; Fig. 4A) and greatly
DIC efflux following NO3- addition was 39.2 ± 5.0 Mmol-mg-' reduced rates of net C fixation (5.9 ,umol-mg' Chlh-'; Fig.
Chl- h-' (Table II). During this time NO3- uptake was constant 4B). Over the duration of the experiment there was a major
at a rate of 107.1 Mmol NO3-Tmg' Chl.h-' (Fig. 1). (52.4%) decrease in the specific activity of the exogenous DIC
Nitrate addition following partial inhibition of the tricarbox- pool (Fig. 4C). Addition of NH4' to NO3-limited cells yielded a
ylic acid cycle with fluoroacetate resulted in a lower rate of net similar response (Table II).
CO2 efflux in response to NO3- addition (9.8 ± 1.7 Mmol -mg-'
Chl- h-'; Fig. 2) and the establishment of an intermediate com- DISCUSSION
pensation point (59.8 ± 13.8 Mm; Table I). Inhibition of N03
assimilation by the GOGAT inhibitor Aza alleviated any effect Consistent with our previous results the addition of NO3- or
of N03 on the r (Table I; Fig. 2). No increase in the r was seen NH4' to N-limited S. minutum resulted in_a suppression in
when NH4+ was added to N03--limited cultures and no change photosynthetic C-fixation- (Fig. 4, A and B). Recently, we have
710 BIRCH ET AL. Plant Physiol. Vol. 82, 1986

-t204
-
1500 FIG. 1. A representative experiment show-
ing the effect of NO3- addition (2 mM) on
- 15( s the r of N03-limited chemostat grown cells
0- :t of S. minutum; DIC concentration (0); NO3
z 1000r concentration (0). Addition of 2 mm N03
° 10( 0 (t = 40 min) to cells at compensation point
.E. caused a net increase of culture DIC at a rate
o of 53.0 umol-mg7' Chl-h-'. A new DIC com-
z -
500 pensation point of 220 Mm was eventually
w 5C reached. Over the duration of the experiment
NO3- uptake was linear at a rate of 107 Amolk
. mg-' Chl-h-'.
C)
0 250
2mM NO3 TIME (min)

* CONTROL
050mM FA
- X 50 mM azoserine
0Z 75
70
z
0.
a 0 ---O FIG. 2. The effect of metabolic inhibitors on the N03-
induced increase in F in N03-limited chemostat grown cells
j 50 l- 0
of S. minutum. The initial compensation point was 25 AM
while the addition of NO3- (5 mM, t = 30 min) resulted in a
Ce) final of 80 Mm (0). Nitrate addition to cells treated with 50
z
w 0 mM fluoroacetate (0) resulted in a final of 49.8 Mm. Nitrate
X 25
0
)
,..Oa addition to cells treated with 5 mm Aza exhibited no change
in DIC compensation point (x).
c) 5 mM NO3

30 60 90
TIME (min)

* - N03 0
o + N03
01
8 0
E
FIG. 3. An example of '4CO2 incorporation at the DIC
0
compensation point as a function oftime in a control culture
with a compensation point of 8.3 Mm DIC (0); and a nitrate-
E
4
pulsed culture with a compensation point of 288 Mm DIC
(0).
0o
.00, o
1 1 1 1 I--]~~~~~
jj::°O *--O.
I
G.
2 4 6 8 10
TIME (min)
proposed that this suppression is due in part to the competing triose-P, the depletion of which would decrease RuBP regenera-
demands for carbon skeletons between the processes of N assim- tion thereby limiting carbon fixation. This hypothesis was con-
ilation and CO2 fixation (5). We suggested that sudden N resup- sistent with the increases in dark carbon fixation and respiration
ply to NO3--limited cultures of S. minutum resulted in increased observed in response to N addition as well as the observed effects
tricarboxylic acid cycle activity associated with the supply of on photosynthetic 02 evolution (5).
carbon skeletons for N assimilation. These increased demands A testable prediction of this model is that the N-induced
on tricarboxylic acid cycle intermediates would require anapler- increase in tricarboxylic acid cycle activity should result in an
otic reactions to maintain cycle activity. One source of carbon increase in CO2 efflux in the light as a result of increased carbon
for these reactions was suggested to be Calvin cycle generated flow through isocitrate dehydrogenase, pyruvate dehydrogenase
 N-INDUCED CO2 EFFLUX IN SELENASTRUM 711

-J
~DI
0
x
0
7-
a-

(.-) FIG. 4. Effects of NO3- addition on gross, and net

C] photosynthesis and the specific activity of exogenous
DIC. Nitrate-limited, chemostat grown cells of S.
minutum at a density of 7.1 Ag Chl ml-' were placed
in a culture cuvette and brought to 150 AM NaHCO3
in the absence (0) or presence of 5 mM NO3- (O). A,
Decrease in 14C in the external media; B, decrease in
exogenous DIC; C, resulting changes in specific activ-
-t ity.
E
0.

t
75QL
1 w
E
0~
Cl

TIME (min)

complex and a-ketoglutarate dehydrogenase complex. Earlier NH4' addition, respectively. These fluxes represent approxi-
work by Syrett (13-16) showed that addition of NO3- to N- mately a 6-fold increase in the rate of CO2 efflux in the light in
starved cells of Chlorella resulted in an increase in both CO2 response to NO3- or NH4+ (Table II). During the period of NO3-
production and 02 consumption in the dark. If our model is to or NH4' induced photosynthetic suppression observed in these
account for the process of photosynthetic suppression, a similar experiments (Fig. 4), these rates were equivalent to 49 and 48%
response must occur in the light. One method of evaluating CO2 of gross photosynthesis, respectively.
efflux in the light is to follow the change in the specific activity Another indication of the magnitude of CO2 efflux in the light
of exogenous DIC using the technique of Birmingham et al. (2). is the response of the r to NO3- orNH4' addition. Compensation
If exogenous DIC is enriched with `4C, the fixation of both point is achieved when the rate of gross carbon fixation equals
species of carbon (14C and '2C) will be in direct proportion to the rate of CO2 release. If NO3- orNH4' addition were to enhance
their relative abundance. In the short term, however, any CO2 CO2 efflux in the light, NO3- or NH4' addition should result in
efflux from the cells will be primarily unlabeled '2C02. Conse- an increase in the compensation point. Our results for N03-
quently, '2C02 efflux in the light can be detected as a decrease addition are consistent with this prediction (Fig. 1). The initial
in the specific activity of exogenous DIC over time. The relative net rate of CO2 efflux in reponse to NO3- addition was 39.2 +
constancy of the specific activity in control cells (Fig. 4C) indi- 5.0 ,umol CO2 mg-' Chl * h-' (Table II). Cells treated with 50 mM
-

cates low rates of CO2 efflux in the light (6.4 ± 2.4 smol CO2- fluoroacetate and subsequently pulsed with 5 mM NO3- exhibited
mg-' Chl-h-'; Table II). This is in general agreement with the a lower initial net rate of CO2 efflux and a correspondingly lower
rates reported for green algae by Birmingham et al. (2). In the r (Table I; Fig. 2). The inhibitory effect of fluoroacetate on this
present study both NO3- and NH4I addition decreased net and increase in compensation point was taken as evidence that a
gross C-fixation. Over the same period there was a major decrease major source of this CO2 was from the tricarboxylic acid cycle.
in the specific activity of DIC. The rates of CO2 efflux required The alleviation of the NO3--induced increase in compensation
to produce the observed changes in specific activity were 37.4 ± point by the GOGAT inhibitor, Aza, demonstrated that this
6.9 and 40.2 ± 3.5 Amol C02-mg-' Chl-h-' upon NO3- and increase in CO2 efflux was dependent upon the process of N
712 BIRCH ET AL. Plant Physiol. Vol. 82, 1986
assimilation (Fig. 2). These observations are in agreement with of 51.5 Mmol C02.mg-' Chl h-' and net rate of CO2 efflux at
our model ofN-induced photosynthetic suppression in N-limited compensation point of 39.2 Amol C02-mg ' Chl h-' (Table II).
microalgae. These yielded an average rate of 40.6 ± 6.0 Amol CO2 mg:'
Suprisingly, ammonium addition to cells at r failed to induce Chl h-'. Given a respiratory quotient of 1 it would be expected
the predicted increase in the rate of net CO2 efflux (Table II). that gross 02 consumption in the light would increase by a
The observation that both NO3- and NH4I caused CO2 efflux at similar value upon NO3- addition. It is interesting to note that
high DIC, as determined from the specific activity experiments NO3- addition in the dark resulted in an increase in the rate of
(Table II), yet NH4I had no effect at compensation point may 02 consumption by a similar magnitude (61.0 Mmol 02.mg'
point to an important difference in carbon partitioning during Chlh-') (5).
NH4' assimilation at low and high CO2 levels. In the specific In summary these results show that NO3- addition to N-limited
activity experiments, DIC levels were relatively high (- 150 JM) cells resulted in a major increase in the rate of CO2 efflux in the
and photosynthesis following N addition was probably limited light. Similarly, NH1+ addition at relatively high levels of DIC
by RuBP regeneration (5). At the F, however, photosynthesis is produced nearly identical results. These observations are consist-
limited by CO2 supply. This difference may result in newly ent with the suggestion that N-induced photosynthetic suppres-
assimilated ammonia moving into alanine and aspartate when sion results in part from competition for carbon skeletons be-
cells are at compensation point rather than into glutamine and tween the processes of N-assimilation and photosynthetic carbon
glutamate. If most of the carbon flux was into alanine and fixation. It also shows that under conditions of transient N
aspartate there would be no tricarboxylic acid cycle mediated resupply to S. minutum in the light, dark respiration may be of
increase in CO2 efflux. However, it is still unclear as to why N03 a similar magnitude to photosynthetic carbon fixation.
and NH4' addition invoke such different responses. Although
we are currently evaluating these discrepancies, our results show LITERATURE CITED
that at relatively high levels of DIC both NO3- and NH4' addition 1. BIRMINGHAM BC, B COLMAN 1979 Measurement of carbon dioxide compen-
stimulate CO2 efflux in the light. sation points of freshwater algae. Plant Physiol 64: 892-895
At the F gross photosynthesis equals respiration (Pg = R). 2. BIRMINGHAM BC, JR COLEMAN, B COLMAN 1982 Measurement of photores-
piration in algae. Plant Physiol 69: 259-262
Consequently, an additional estimate of the rate of CO2 efflux at 3. COLLOS Y, G SLAWYK 1979 `3C and 'IN uptake by marine phytoplankton. I.
the r can be obtained by measuring the rate of gross carbon Influence of nitrogen source and concentration in laboratory cultures of
fixation. The rate of CO2 release from control cells at the r was 4. ELRIFIdiatoms. J Phycol 15: 186-190
calculated to be 5.3 ± 0.9 Amol C02-mg ' Chl-h-' (Table II). IR, DH TURPIN 1985 Transient photosynthetic responses of nitrogen
limited microalgae to nitrogen addition. Mar Ecol Prog Ser 20: 253-258
The rate of CO2 efflux (determined as gross C-fixation) obtained 5. ELRIFI IR, DH TURPIN 1986 Nitrate and ammonium induced photosynthetic
at the high compensation points following NO3- addition was suppression in N-limited Selenastrum minutum. Plant Physiol 81: 273-279
much greater (56.8 ± 14.7 Amol C02-mg ' Chl-h-'). The differ- 6. FALKOWSKI PG, DP STONE 1975 Nitrate uptake in marine phytoplankton:
energy sources and the interaction with carbon fixation. Mar Biol 32: 77-84
ence between these two rates (51.5 Amol C02-mg ' Chl-h-') is 7. GOLDMAN JC, MR DENNETT 1985 Photosynthetic response of 15 phytoplank-
an indication of the magnitude of NO3--induced CO2 efflux ton species to ammonium pulsing. Mar Ecol Prog Ser 20: 259-264
(Table II). The rate of NO3--induced CO2 efflux in the presence 8. HEALY FP 1979 Short term responses of nutrient deficient algae to nutrient
of fluoroacetate as determined by this method was substantially 9. LEAN addition. J Phycol 15: 289-299
DRS, FR PICK 1981 Photosynthetic response of lake plankton to nutrient
reduced (9.8 ± 1.7 Amol C02-mg ' Chl-h-'). This increase in enrichment: a test for nutrient limitation. Limnol Oceanogr 26: 1011-1019
14C incorporation upon NO3- addition to cells at compensation 10. LEAN DRS, TP MURPHY, FR PICK 1982 Photosynthetic response of lake
point may at first seem contrary to the observation of N-induced 11. plankton toFR combined nitrogen enrichment. J Phycol 18: 509-521
photosynthetic suppression at higher DIC. At compensation OHMORI M, WOLF, JA BASSHAM 1984 Botryococcus braunii carbon/
nitrogen metabolism as affected by ammonia addition. Arch Microbiol 140:
point it is CO2 supply which limits the rate of carbon fixation. 101-106
Consequently, any increase in compensation point will result in 12. STRICKLAND JDH, TR PARSONS 1972 A practical handbook of seawater
an increased rate of carbon fixation. At high DIC however, the analysis, Ed 2. Fish Res Bd Can Bull 167, p 71
N-induced suppression of photosynthesis is due to RuBP limi- 13. SYRErr PJ 1955 The assimilation of ammonia and nitrate by nitrogen-starved
cells of Chlorella vulgaris. I. The assimilation of small quantities of nitrogen.
tation (5). NH4' addition unlike NO3-, failed to produce any Physiol Plant 8: 924-929
increase in the rate of CO2 efflux as determined from these 14. SYREIT PJ 1956 The assimilation of ammonia and nitrate by nitrogen-starved
carboxylation rate measurements (Table II). This is consistent cells of Chlorella vulgaris. II. The assimilation of large quantities of nitrogen.
with the lack of an NH4' induced increase in compensation 15. SYRETT Physiol Plant 9: 19-27
PJ 1956 The assimilation of ammonia and nitrate by nitrogen-starved
point. It also rules out the possibility that NH4+ may have caused cells of Chlorella vulgaris. III. Differences of metabolism dependent on the
an increase in CO2 efflux, with its effect on the compensation nature of the nitrogen source. Physiol Plant 9: 28-37
point being masked by a simultaneous increase in carbon fixa- 16. SYRErT PJ 1956 The assimilation of ammonia and nitrate by nitrogen-starved
cells of Chlorella vulgaris. IV. The dark fixation of carbon dioxide. Physiol
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Our measurements of CO2 effiux from unperturbed cells were 17. TERRY KL 1982 Nitrate uptake and assimilation in Thalassiosira weisflogli
low and account for less than 4% of the measured rate of and Phaeodactylum tricornutum: interactions with photosynthesis and with
photosynthesis. This is in agreement with observations made by 18. THOMAS the uptake of other ions. Mar Biol 69: 21-30
other workers (2). The addition of NO3- to N03 -limited S. RJ, CR HIPKIN, PJ SYRETT 1976 The interaction of nitrogen assimi-
lation with photosynthesis in nitrogen deficient cells of Chlorella. Planta
minutum resulted in a major increase in this rate. The three 133: 9-13
independent estimates of the rate of CO2 efflux resulting from 19. TURPIN DH 1983 Ammonium induced photosynthetic suppression in am-
NO3- addition were in reasonable agreement. Estimates from 20. TURPIN monium limited Dunaliella tertiolecta (Chlorophyta). J Phycol 19: 70-76
DH, DB LAY-ZELL 1985 A culture system enabling in situ determina-
specific activity experiments indicated a CO2 effiux of 31.0 ,mol tion of net and gross photosynthesis, 02 evolution, N assimilation and
C02- mg' Chl- h-', gross photosynthesis at compensation point C2 H2 reduction in cyanobacteria. Can J Bot 63: 1025-1030